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

Abstract

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

Description

[Technical field]

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

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

[0003] Mannan, a polysaccharide derived from yeast cell walls, consists of a backbone of predominantly β-(1,4)-linked mannose with a small number of α-(1,6)-linked glucose and galactose side chain residues. Furthermore, a protein content of approximately 5% has been detected in conventional mannan preparations. As an important component of fungal cell walls, mannan is widely used as a component in carbohydrate-based vaccines for candidiasis (Han and Rhew, Arch Pharm Res 2012, Vol 35, No 11, 2021-2027; Cassone, Nat Rev Microbiol. 2013 Dec; 11(12): 884-91; Johnson and Bundle, Chem. Soc. Rev., 2013, 42, 4327). Furthermore, various examples of vaccines based on mannan carrier-antigen complexes / conjugates have been developed, including mannan-mucin 1 (MUC1) fusion protein conjugation for tumor therapy, or conjugates of mannan with model allergens such as ovalbumin (OVA), papain, or Betv1.

[0004] Mucins are highly glycosylated proteins expressed on the cell surface. MUC1 is the prototypic mucin that has been found to be overexpressed on a wide range of tumor cells. Along these lines, MUC1 fusion proteins containing human MUC1 (containing the immunodominant epitope: APDTRPAPGSTAPPAHGVTS) and five tandem repeats of a peptide (Cpl3-32) were generated and, when conjugated to mannan under oxidizing or reducing conditions, induced dramatically different immunological responses, i.e., oxidized mannan-MUC1 stimulated a Th1-type response mediated by CD8+ T cells, resulting in IFN-γ secretion and a predominantly IgG2a antibody response, whereas reduced mannan-MUC1 stimulated a Th2-type response, resulting in IL-4 production and a high IgG1 antibody response. The fusion proteins used represented a single protein displaying T and B cell epitopes.

[0005] Recently, protein-carbohydrate / mannan conjugates of papain and OVA were also made to analyze their allergenic potential. It was found that the binding and cross-linking of IgE antibodies to papain could be reduced when mannan was coupled to the protein surface. Interestingly, the conjugation of either mannan, dextran, or maltodextrin only reduced the allergenic potential of papain, but not that of OVA in these experiments, indicating the importance of carbohydrate selection for vaccine design (Weinberger et al. J.Control.Release 2013; 165:101-109). These experiments also showed that mannan conjugation resulted in the development of increased IgG titers against OVA after intradermal immunization.

[0006] Similar to the neoglycoconjugate vaccine used for MUC1, Ghochikyan et al. (DNA AND CELL BIOLOGY, Volume 25, Number 10, 2006, Pp.571-580) and Petrushina et al. (Journal of Neuroinflammation 2008, 5:42) applied amyloid beta (Aβ) 28, a 28aa residue peptide with binding B-cell and T-cell epitopes of human Aβ 42 peptide, linked to mannan, and was able to induce low-level anti-Aβ responses in mice. These responses were also found to attenuate amyloid deposition in the cortical and hippocampal regions of APP transgenic mice after subcutaneous immunization. Immunization also led to the induction of increased anti-mannan titers in Aβ 28-mannan- and BSA-mannan-treated animals. However, the treatment was not developed further due to the occurrence of increased microbleeds in the brains of treated animals, likely due to the potential adverse effects of mannan as a trigger of adverse vascular events, highlighting the importance of carbohydrate selection for designing an efficient and safe vaccine.

[0007] Conjugates using individual B-cell or T-cell epitope peptides linked to mannan or other related CLECs are not known so far.

[0008] β-Glucans comprise a group of β-D-glucose polysaccharides. These polysaccharides are the major structural components of the cell walls of fungi and are also found in bacteria, yeasts, algae, lichens, and plants such as oats and barley. Depending on the source, β-glucans differ in bond type, degree of branching, molecular weight, and tertiary structure.

[0009] β-glucan is a source of soluble, fermentable fiber, also called prebiotic fiber, that provides substrate for the microflora in the large intestine, increases stool bulk, and produces short-chain fatty acids as by-products with a wide range of physiological activities. For example, dietary intake of cereal β-glucan from oats in a daily dose of at least 3 grams reduces total cholesterol and low-density lipoprotein cholesterol levels by 5-10% in people with normal or elevated blood cholesterol levels.

[0010] β-glucans usually form a linear backbone with one to three β-glycosidic bonds, but vary with respect to molecular mass, solubility, viscosity, branching structure, and gelling properties. Yeast and fungal β-glucans are usually based on a β-(1,3) backbone and contain β-(1,6) side branches, while cereal β-glucans contain both β-(1,3) and β-(1,4) backbone bonds, with or without side branches.

[0011] β-glucans are recognized by the innate immune system as pathogen-associated molecular patterns (PAMPs). The PRR dectin-1 has emerged as the major receptor for these carbohydrates, and binding of β-glucans to dectin-1 induces various cellular responses via the Syk / CARD9 signaling pathway, including phagocytosis, respiratory burst, and secretion of cytokines. In addition, complement receptor 3 (CR3, CD11b / CD18) has also been implicated as a receptor for β-glucans. Stimulation via dectin-1 has been reported to stimulate Th1 responses, Th17 responses, and cytotoxic T lymphocyte responses.

[0012] Members of the β-glucan family include: Beta-glucan peptide (BGP) is a high molecular weight (approximately 100 kDa) branched polysaccharide extracted from the fungus Trametes versicolor. BGP consists of a highly branched glucan moiety containing a beta-(1,4) backbone and beta-(1,3) side chains, with the beta-(1,6) side chains covalently linked to polypeptide moieties rich in aspartic acid, glutamic acid, and other amino acids.

[0013] Curdlan is a high molecular weight linear polymer of β-(1,3) linked glucose residues derived from Agrobacterium species.

[0014] Laminarin, derived from the brown alga Laminaria digitata, is a linear β-(1,3)-glucan with β-(1,6) linkages. Laminarin is a low molecular weight (5-7 kDa), water-soluble β-glucan that can act as either a dectin-1 antagonist or a dectin-1 agonist. Laminarin can bind to dectin-1 without stimulating downstream signaling and can block dectin-1 binding of particulate β-(1,3)-glucans such as zymosan.

[0015] Pustulan is a medium molecular weight (20 kDa), linear β-(1,6)-linked β-d-glucan from the lichen Lasallia pustulata that can also bind to dectin-1 as its primary receptor and activate signaling through dectin-1.

[0016] Lichenan is a high molecular weight (approximately 22-245 kDa) linear β-(1,3)β-(1,4)-β-D glucan from Cetraria islandica with a structure similar to that of barley and oat β-glucans. Lichenan has a much higher ratio of 1,3-β-D to 1,4-β-D bonds than the other two glucans. The ratio of β-(1,4)-β-D to β-(1,3)-β-D bonds is approximately 2:1.

[0017] β-glucans from oats and barley are linear β-(1,3)β-(1,4)-β-D glucans that are commercially available in a range of molecular weights (from a medium molecular weight fraction of 35.6 kDa up to a high molecular weight fraction of up to 650 kDa).

[0018] Schizophyllan (SPG) is a gel-forming β-glucan derived from the fungus Schizophyllum commune. SPG is a high molecular weight (450 kDa) β-(1,3)-D-glucan with β-(1,6) monoglucosyl branches every three β-(1,3)-glucosyl residues on the backbone.

[0019] Scleroglucan is a high molecular weight (>1000 kDa) polysaccharide produced by fermentation of the filamentous fungus Sclerotium rolfsii. Scleroglucan consists of a linear β-(1,3)D-glucose backbone with one β-(1,6)D-glucose side chain for every three main residues.

[0020] Whole glucan particles (WGP) are β-glucans known for their ability to modulate immune responses. WGP dispersion (WGP® dispersion from Biothera) is a microparticle of Saccharomyces cerevisiae β-glucan preparation. It consists of hollow yeast cell wall "ghosts" composed mainly of long polymers of β-(1,3) glucose obtained after a series of alkaline and acidic extractions from Saccharomyces cerevisiae cell walls. In contrast to other dectin-1 ligands such as zymosan, WGP dispersion lacks TLR stimulating activity. In contrast, soluble WGP binds to dectin-1 without activating this receptor. It can also significantly block the binding of WGP dispersion to macrophages and its immunostimulatory effect.

[0021] Zymosan is an insoluble preparation of yeast cells that activates macrophages via TLR2, which cooperates with TLR6 and CD14 in response to zymosan. Zymosan is also recognized by dectin-1, a phagocytic receptor expressed on macrophages and dendritic cells, which cooperates with TLR2 and TLR6 to enhance the immune response triggered by the recognition of zymosan by each receptor.

[0022] As major components of fungal cell walls, various β-glucans have been used as antigens to generate anti-glucan antibodies against fungal infections (e.g., Torosantucci et al. J Exp Med. 2005 Sep 5; 202(5): 597-606.; Bromuro et al., Vaccine 28 (2010) 2615-2623; Liao et al., Bioconjug Chem. 2015 Mar 18; 26(3): 466-76).

[0023] Torosantucci et al. (2005) and Bromuro, et al. (2010) disclosed conjugates of the branched β-glucan laminarin and the linear β-glucan curdlan linked to the diphtheria toxoid CRM197. These conjugate vaccines induced high IgG titers against β-glucan and provided protection against fungal infection in mice. Furthermore, using such conjugates, high titers against CRM197 could also be detected (Donadei et al., Mol Pharm. 2015 May 4;12(5):1662-72). The authors also produced β-glucan-CRM197 vaccines using synthetic linear β-(1,3)-oligosaccharides or β-(1,6) branched β-(1,3)-oligosaccharides formulated with the human-tolerable adjuvant MF59. All conjugates induced high titers of anti-β-(1,3)-glucan IgG and / or anti-β-(1,3)-glucan IgG as well as anti-β-(1,6)-glucan antibodies, demonstrating the immunogenicity of the different glucans in combination with classical carrier proteins. Interestingly, Torosantucci et al. were unable to demonstrate superior anti-CRM titers after immunization with CRM-glucan conjugates compared to unconjugated CRM alone.

[0024] Donadei et al. (2015) also analyzed conjugates of the diphtheria toxoid CRM197 linked to the linear β-(1,3) glucan curdlan or to synthetic β-(1,3) oligosaccharides. The conjugates were immunogenic and showed comparable antibody responses to CRM197. Interestingly, the authors showed that CRM-curdlan conjugates, when delivered intradermally, led to higher antibody titers compared to intramuscular (im) immunization. However, intradermal administration of CRM-curdlan did not show different immunogenicity compared to subcutaneous administration. Moreover, the in vivo efficacy was comparable between CRM-curdlan and curdlan-unlinked CRM with Alum adjuvant. Thus, no additional benefit of CLEC linkage on the overall immune response could be detected in this system.

[0025] Liao et al. (2015) disclosed a series of linear β-(1,3)-β-glucan oligosaccharides (hexa-, octa-, deca-, and dodeca-β-glucans) that were linked to KLH to generate glycoconjugates. These conjugates were shown to elicit robust T cell responses, were highly immunogenic, and induced high anti-glucan antibody levels. Mice immunized with such vaccines also elicited protective immune responses against the lethal pathogen Candida albicans. A comparison of anti-KLH titers to unconjugated KLH was not performed, and therefore, no information regarding the potential benefits of β-glucans is available in this experimental setting.

[0026] These findings are crucial for the applicability of glucan-based neoglycoconjugates as novel vaccines. That is, potential anti-glucan antibodies induced upon initial glucan conjugate immunization may lead to rapid clearance of the same β-glucan vaccine in subsequent booster immunizations or may attenuate immune responses to novel neoglycoconjugate vaccines for other indications, an effect well known from vector vaccines. Thus, as demonstrated above for mannan and β-glucan (Petrushina et al. 2008, Torosantucci et al. 2005, Bromuro et al., 2010, Liao et al., 2015), the presence or even (re)stimulation of high levels of anti-glucan antibodies may reduce or eliminate potential immune responses elicited by conjugate vaccines. Therefore, ensuring that the glucan antibody-inducing capacity of the polysaccharides / oligosaccharides used is very low or non-existent will be important for novel and sustainable platforms using CLECs, especially β-glucans, as scaffolds for immunization.

[0027] Glucan particles (GPs) are highly purified 2-4 μm hollow porous cell wall microparticles composed primarily of β-(1,3)-D-glucan with minor amounts of β-(1,6)-D-glucan and chitin, typically isolated from Saccharomyces cerevisiae using a series of hot alkaline, acid, and organic extractions. Glucan particles interact with their receptors dectin-1 and CR3 (with evidence also implicating interactions with toll-like receptors and CD5 as additional factors for GP function), upregulating cell surface presentation of MHC molecules, leading to altered expression of costimulatory molecules, as well as inducing the production of inflammatory cytokines. GPs have been investigated for vaccine delivery due to their immunomodulatory properties.

[0028] There are three general approaches to applying GPs in vaccines: (i) as adjuvants co-administered with antigens to enhance T- and B-cell mediated immune responses, (ii) chemically cross-linked to antigens, and most frequently (iii) used as physical delivery vehicles for antigens entrapped within hollow GP cavities to provide targeted antigen delivery to APCs.

[0029] Regarding (i): Antigen-specific adaptive immune responses can be enhanced by co-administration of GP with the antigen. In this conventional adjuvant strategy, both innate and adaptive immune responses are activated to exert a protective response against the pathogen. For example, Williams et al. (Int J Immunopharmacol. 1989;11(4):403-10) adjuvanted a killed Trypanosoma cruzi vaccine by co-administration of GP. The immune response elicited using this formulation resulted in 85% survival of mice challenged with Trypanosoma cruzi. In contrast, controls administered dextrose, glucan, or vaccine alone had 100% mortality.

[0030] Regarding (ii): The carbohydrate surface of GPs can also be covalently modified using NaIO4 oxidation, carbodiimide cross-linking, or 1-cyano-4-dimethylaminopyridinium tetrafluoroborate-mediated conjugation of antigens to the GP shell. Using this approach, the conjugation efficiency is very low (e.g., approximately 20%, as described in Pan et al. Sci Rep 5, 10687 (2015)), i.e., significantly limiting the applicability and number of vaccine candidates compared to antigen encapsulation in GPs or the proposed platform technology provided in this application. Such covalently linked antigen-GP conjugates have been used in cancer immunotherapy and infectious disease research. For example, Pan et al. (2015) used OVA cross-linked to periodate-oxidized GP and immunized mice subcutaneously with this vaccine. A significant reduction in tumor size was observed when mice were challenged with OVA-expressing E.G7 lymphoma cells. GP-OVA increased the number of DCs (CD11c) in lymph nodes 12 and 36 hours after subcutaneous injection. + MHC-II + Tumor protection was associated with increased total anti-Ova immunoglobulin (Ig)G titers, enhanced expression of MHC-II and costimulatory molecules (CD80, CD86), and elevated cytotoxic lymphocyte responses.

[0031] Regarding (iii): The most effective approach to apply GPs in vaccines is to use them for encapsulation of vaccines / antigens into hollow cores. GPs can encapsulate one or multiple antigens / DNA / RNA / adjuvants / drugs / combinations thereof with high loading efficiency determined by the type of payload and the intended mode of delivery.

[0032] Antigens can be encapsulated in the hollow cavities of GPs using polymer nanocomplexation methods such as loading and complexing the payload using bovine serum albumin or mouse serum albumin and yeast RNA / tRNA, or adding alginate-calcium or alginate-calcium-chitosan mixtures. Using these strategies, for example, Huang et al. (Clin. Vaccine Immunol. 2013; 20:1585-91) reported that mice vaccinated with GP-OVA showed strong CD4+ T cell lymphoproliferation, Th1 and Th17 skewed T cell-mediated immune responses, along with high IgG1 and IgG2c specific antibody responses against ovalbumin. The non-covalent encapsulation strategy induced a stronger immune response compared to GPs co-administered with antigens.

[0033] An example of a GP-encapsulated subunit vaccine is a GP encapsulating a soluble alkaline extract of a Cryptococcus neoformans acapsular strain (cap59), which protected mice (60% survival) challenged with a lethal dose of virulent Cryptococcus neoformans by inducing an antigen-specific CD4+ T cell response (positive for IFN-γ, IL-17A) that reduced fungal colony forming units (CFU) more than 100-fold above the initial challenge dose (Specht CA et al. Mbio 2015; 6:e01905- e1915. and Specht CA et al., mBio 2017; 8:e01872- e1917.). Furthermore, vaccination of mice with GP encapsulating antigens was found to be effective against Histoplasma capsulatum (Deepe GS et al., Vaccine 2018; 36:3359-67), F. tularensis (Whelan AO et al., PLOS ONE 2018; 13:e0200213), Blastomyces dermatitidis (Wuthrich M et al., Cell Host Microbe 2015; 17:452-65), and C. posadasii (Hurtgen BJ et al., Infect.Immun.2012; 80:3960-74).

[0034] In addition to applications in cancer and infectious diseases, a limited number of studies have also been performed using self-antigens using GP as an encapsulating agent for vaccine delivery. Along these lines, Rockenstein et al. (J.Neurosci., January 24, 2018; 38(4):1000 -1014) describe the application of recombinant human a-synuclein protein (containing both B and T cell epitopes suitable for the induction of anti-aSyn immune responses) and rapamycin-loaded GP, known to induce antigen-specific regulatory T cells (Tregs) in mouse models of synucleinopathy. As expected from previous studies using full-length a-synuclein as an immunogen, application of aSyn-containing GP results in the induction of robust anti-a-synuclein antibody titers and attenuates the pathological changes caused by a-synuclein in animals to a similar extent as previously published. The addition of rapamycin efficiently induced the formation of iTreg (CD25 and FOXP3+) cells, as the number of such Treg cells was significantly increased after rapamycin exposure. Thus, GP loaded with the antigen a-synuclein and rapamycin elicited both neuroprotective humoral and iTreg responses in a mouse model of synucleinopathy, and the combined vaccine (aSyn+rapamycin) was more effective than humoral immunization (GP aSyn) or cellular immunization (GP rapamycin) alone. No information on the equivalence of efficacy to conventional a-synuclein-containing non-GP immunization has been reported.

[0035] β-glucan neoglycoconjugates efficiently target dendritic cells via the C-type lectin receptor Dectin-1 and enhance their immunogenicity. Specifically, certain β-glucans have also been used as potential carriers for vaccination with model antigens such as OVA (Xie et al., Biochemical and Biophysical Research Communications 391 (2010) 958-962; Korotchenko et al., Allergy.2021;76:210-222.) or MUC1-based fusion proteins (Wang et al., Chem.Commun., 2019, 55, 253).

[0036] Xie et al. and Korotchenko et al. used the branched β-glucan laminarin as a backbone for OVA conjugate formation. These glyconeoconjugates were then administered to mice either epidermally or via subcutaneous routes. Xie et al. showed that laminarin / OVA conjugates, but not the unconjugated mixture of compounds, induced an increase in anti-OVA CD4+ T cell responses compared 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, OVA / laminarin conjugates significantly enhanced the antibody response. Similarly, Korotchenko et al. demonstrated that laminarin conjugate formation to OVA significantly increased BMDC uptake and induced their activation and secretion of pro-inflammatory cytokines. These properties of the LamOVA conjugate also led to enhanced stimulation of OVA-specific naive T cells co-cultured with BMDCs. In preventive immunization experiments, the authors were able to confirm that immunization with LamOVA reduced its allergenicity and induced approximately three-fold higher IgG1 antibody titers compared to OVA after two immunizations. However, this effect was lost in all groups treated after the third immunization, when all groups showed similar antibody titers. Lam / OVA and OVA / Alum conjugates showed comparable therapeutic efficacy in a mouse model of allergic asthma. Thus, these experiments failed to provide a clear superior efficacy of glucan-based conjugates compared to conventional vaccines.

[0037] Wang et al. (2019) analyzed the efficacy of a β-glucan-based MUC1 cancer vaccine candidate. The MUC1 tandem repeat sequence GVTSAPDTRPAPGSTPPAH, a well-studied cancer biomarker, was selected as a peptide antigen providing T-cell and B-cell epitopes within the repeat sequence. 1,1'-carbonyl-diimidazole (CDI)-mediated conditions were applied to link β-glucan and MUC1 peptide to yeast β-(1,3)-β-glucan polysaccharide using an ethylene glycol (i.e., PEG) spacer. The diameter of the β-glucan-MUC1 nanoparticles was in the range of 150 nm (actual average 162 nm), while unmodified β-glucan formed particles of approximately 540 nm. The β-glucan-MUC1 conjugates induced high titers of anti-MUC1 IgG antibodies, which were significantly higher compared to the control group. Further analysis of the isotype and subtype of the antibodies produced showed that IgG2b was the predominant subtype, indicating the activation of a Th1-type response, since the IgG2b / IgG1 ratio was greater than 1. The significant amount of IgM antibodies observed indicates the involvement of the C3 component of the complement system, which often induces cytotoxicity, which can be problematic for the use of such scaffolds for vaccines, e.g. for chronic or degenerative diseases, where the occurrence of cytotoxicity should be avoided.

[0038] US 2013 / 171187 A1 discloses an immunogenic composition comprising glucan and a pharma- ceutically acceptable carrier for eliciting protective anti-glucan antibodies. Metwali et al. (Am. J. Respir. Crit. Care Med. 185 (2012), A4152; poster session C31 Regulation of Lung Inflammation) investigate the immunomodulatory effects of glucan derivatives in lung inflammation. WO 2021 / 236809 A2 discloses a multi-epitope vaccine comprising amyloid-beta and tau peptides for treating 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-amyloidogenic peptides homologous to amyloid beta, alone or conjugated, for treating AD. US 2013 / 171187 A1 discloses anti-glucan antibodies used as protective agents against fungal infections caused by Candida albicans. WO 2004 / 012657 A2 discloses particulate β-glucan as a vaccine adjuvant. CN 113616799 A discloses a vaccine vector consisting of oxidized mannan and a cationic polymer. CN 111514286 A discloses a Zika virus E protein conjugate vaccine containing glucan. US 4,590,181 A discloses a viral antigen mixed in solution with pustulan or mycodextran. Lang et al. (Front. Chem. 8 (2020):284) review carbohydrate conjugates in vaccine development. Larsen et al. (Vaccines 8 (2020):226) show that pustulan activates chicken bone marrow-derived dendritic cells in vitro and inhibits ex vivo CD4+ expression against infectious bronchitis virus. +It has been reported that it promotes T cell recall responses. US 2010 / 266626 A1 discloses glucans, preferably laminarin and curdlan, as antigens conjugated to adjuvants for immunization against fungi. Mandler et al. (Alzh.Dement.15 (2019), 1133-1148) report the effect of mono- and combination immunotherapy approaches targeting amyloid-beta protein and α-synuclein in a Lewy body dementia-like model. Mandler et al. (Acta Neuropathol.127 (2014), 861-879) report a next-generation active immunization approach for synucleinopathies using short immunogenic (B cell response) peptides that are too short to induce T cell responses (autoimmunity), do not have natural epitopes, but rather have sequences that mimic the original epitopes (e.g., oligomeric α-synuclein), and its impact on Parkinson's disease (PD) clinical trials. Mandler et al. (Mol. Neurodegen. 10 (2015), 10) report that active immunization against α-synuclein ameliorates degenerative pathology and prevents demyelination in a model of multiple system atrophy (MSA). Jin et al. (Vaccine 36 (2018), 5235-5244) review β-glucans as potential immune adjuvants, mainly with regard to adjuvanticity, structure-activity relationships, and receptor recognition properties. WO 2022 / 060487 A1 discloses vaccines comprising specific α-synuclein peptides for treating neurodegenerative diseases. WO 2022 / 060488 A1 discloses multi-epitope vaccines comprising amyloid-beta and α-synuclein peptides for treating AD. US 2009 / 169549 A1 discloses conformers of modified versions of alpha-synuclein made by introducing cysteines into the alpha-synuclein polypeptide and scrambling disulfide bonds to form stable immunogenic isomers. WO 2009 / 103105 A2 discloses vaccines with mimotopes of the alpha-synuclein epitope stretching from amino acid D115 to amino acid N122 in the native alpha-synuclein sequence.

[0039] So far, there have been no published reports demonstrating the construction or use of individual B-cell or T-cell epitope peptides linked to β-glucans, in particular linear β-glucans and / or pustulan, with high binding specificity / capacity for Dectin-1, thereby forming novel gluconeoconjugates as proposed in the present application. Summary of the Invention [Problem to be solved by the invention]

[0040] It is therefore an object of the present invention to provide an improved vaccine as a conjugate vaccine composed of a vaccinating antigen conjugated to a carbohydrate-based CLEC adjuvant, in particular a vaccine that provides an improved immune response in vaccinated individuals compared to current prior art conjugate vaccines, in particular carbohydrate-based CLEC-peptide / protein conjugate vaccines.

[0041] It is a further object of the present invention to provide vaccine compositions that immunize against short, easily exchangeable, highly specific B-cell / T-cell epitopes using CLEC scaffolds with efficacy, specificity and affinity previously unmet by conventional vaccines.

[0042] A particular object of the present invention is to provide a vaccine with improved selectivity and / or specificity of CLEC-based vaccines for the dermal compartment.

[0043] Another object of the present invention is to provide a vaccine that induces, as exclusively as possible, a target-specific immune response, but does not induce a CLEC- or carrier protein-specific antibody response, or only an extremely limited CLEC- or carrier protein-specific antibody response.

[0044] It is a further object of the present invention to provide a vaccine composition that immunizes against short, easily exchangeable, highly specific B-cell / T-cell epitopes of alpha-synuclein using a CLEC scaffold with efficacy, specificity and affinity previously unmet by conventional vaccines to adequately prevent and treat synucleinopathies.

[0045] A specific object of the present invention is to provide an alpha-synuclein vaccine with improved selectivity and / or specificity of CLEC-based vaccines for the dermal compartment.

[0046] Another object of the present invention is to provide a vaccine that induces, as exclusively as possible, an alpha-synuclein-specific immune response, but does not induce a CLEC- or carrier protein-specific antibody response, or only a very limited CLEC- or carrier protein-specific antibody response.

[0047] Another object of the present invention is to provide peptide immunogenic constructs of alpha-synuclein protein (aSyn) and formulations thereof for the treatment of synucleinopathies. [Means for solving the problem]

[0048] The present invention therefore provides a conjugate consisting of or comprising at least a β-glucan or mannan and at least a B-cell or T-cell epitope polypeptide, the β-glucan or mannan being covalently linked to the B-cell and / or T-cell epitope polypeptide to form a conjugate between the β-glucan or mannan and the B-cell and / or T-cell epitope polypeptide, the B-cell and / or T-cell epitope polypeptide being an α-synuclein polypeptide. Alternatively, the conjugate according to the present invention consists of or comprises at least a β-glucan or mannan and at least an α-synuclein B-cell epitope polypeptide, the β-glucan or mannan being covalently linked to the B-cell epitope polypeptide to form a conjugate between the β-glucan or mannan and the B-cell epitope polypeptide.

[0049] Preferably, the β-glucan is pustulan, lichenan, laminarin, curdlan, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particle (WGP), zymosan, or lentinan, preferably pustulan, laminarin, lichenan, lentinan, schizophyllan, or scleroglucan, in particular the β-glucan is pustulan.

[0050] According to a preferred embodiment of the present invention, a β-glucan is provided for use as a C-type lectin (CLEC) polysaccharide adjuvant for a B cell and / or T cell epitope polypeptide, in particular a β-glucan that is covalently bound to a B cell and / or T cell epitope polypeptide to form a conjugate between the β-glucan and the B cell and / or T cell epitope polypeptide, and the β-glucan is a predominantly linear β-(1,6)-glucan having a ratio of β-(1,6)-linked monosaccharide moieties to non-β-(1,6)-linked monosaccharide moieties of at least 1:1, preferably at least 2:1, more preferably at least 5:1, in particular at least 10:1.

[0051] The present invention successfully solves one or more of the above listed objectives, which was unexpected for a person skilled in the art, since until now, there have been no reports published in the art demonstrating the construction and applicability or efficacy of compounds similar to the novel, small, modular gluconeoconjugates according to the present invention.

[0052] Surprisingly, it has been shown in the present invention that conjugation (i.e. covalent linkage; used synonymously herein) of peptides / proteins to the selected CLEC-carriers according to the present invention (linkage may be based on prior art chemistry) results in superior pharmaceutical formulations for generating immune responses. A significant number of different linkage methods are available in the art. In the course of establishing the present invention, linkage via hydrazone formation or heterobifunctional linkers was identified as a particularly preferred method. In general, activation of the CLEC prior to conjugation (e.g. formation of a reactive aldehyde on the vicinal OH group of the sugar moiety) and the presence of a reactive group on the selected peptide / protein (e.g. N- or C-terminal hydrazide residue, SH group (e.g. via N- or C-terminal cysteine)) are required. The reaction may be a one-step reaction (e.g., mixing an activated CLEC with a hydrazide-peptide resulting in hydrazone formation) or a multi-step process (e.g., reacting an activated CLEC with a hydrazide from a heterobifunctional linker and then linking the peptides / proteins via their respective reactive groups).

[0053] Thus, the components of the conjugate according to the invention may be directly linked to each other, for example by linking the B-cell epitope and / or the T-cell epitope to the β-glucan or mannan and / or the carrier protein, or by linking the β-glucan or mannan to the carrier protein (in all possible orientations). Reference to a "B cell epitope polypeptide" or a "T cell epitope polypeptide" herein by default means the B cell epitope or T cell epitope of the "B cell epitope polypeptide" or "T cell epitope polypeptide", and not the B cell epitope or T cell epitope of the carrier protein (if present), unless the B cell epitope or T cell epitope of the carrier protein is explicitly mentioned. According to a preferred embodiment, the B-cell epitopes and / or T-cell epitopes are preferably linked via a linker, more preferably a linker comprising a cysteine ​​residue or a cysteine ​​or glycine residue, a linker resulting from hydrazide-mediated linkage, a linker resulting from linkage via a heterobifunctional linker such as N-β-maleimidopropionic acid hydrazide (BMPH), 4-[4-N-maleimidophenyl]butyric acid hydrazide (MPBH), N-[ε-maleimidocaproic acid) hydrazide (EMCH) or N-[κ-maleimidoundecanoic acid] hydrazide (KMUH), The β-glucan or mannan and / or carrier protein are linked by a linker resulting from imidazole-mediated coupling, a linker resulting from reductive amination, a linker resulting from carbodiimide coupling, a -NH-NH2 linker; a peptide linker such as a NRRA linker, a NRRA-C linker, or a NRRA-NH-NH2 linker, a dimeric, trimeric, tetrameric (or higher) peptide group such as CG or CG, or a cleavage site such as a cathepsin cleavage site; or a combination thereof, in particular a cysteine ​​linker or a NRRA-NH-NH2 linker. It is clear that "a linker resulting from (for example) hydrazide-mediated coupling" refers to the resulting chemical structure in the conjugate after coupling, i.e., present in the conjugate obtained after coupling.The amino acid linker may be in conjugate form through a peptide bond (eg, with a glycine-containing linker) or through a functional group of the amino acid (such as a disulfide bond in the case of a cysteine ​​linker).

[0054] The novel class of conjugates according to the invention has been found to confer immunity to short, easily exchangeable, highly specific B / T cell epitopes by using the CLEC scaffold of the invention, which show potency, specificity and affinity previously unmet by conventional vaccines. Indeed, the conjugates according to the invention are the first example of the use of short B / T cell epitopes in a CLEC-based vaccine, which circumvents the need to present α-synuclein epitopes in the form of fusion proteins involving the formation of tandem repeats of the epitope or the fusion of different tandem repeats to form a stable and effective immunogen.

[0055] The present invention also avoids the need to use full-length proteins (i.e. payloads in glucan particles (GPs)) for use in CLEC vaccines. Furthermore, when using CLECs, the problem of autoimmune responses induced by (undesirable) T cell epitopes present in immunogens such as self-proteins, such as alpha-synuclein, can also be avoided.

[0056] According to the present invention, for the first time, short epitopes (B-cell and / or T-cell epitopes, mainly peptides, modified peptides) can be integrated into functional CLEC-based scaffolds using covalent linkages based on well-established chemistries, where possible linkage methods can be adapted to the requirements of a particular epitope based on methods well known in the art.

[0057] The presentation of short peptides according to the invention can be done as individually linked moieties in combination with individual foreign T cell epitopes (as short peptides or long proteins) or as complexes / conjugates with larger carrier molecules that provide the T cell epitopes to induce a sustainable immune response. The design of the vaccine according to the invention allows the preparation of multivalent conjugates as a prerequisite for efficient immune response induction by highly efficient B cell receptor (BCR) cross-linking.

[0058] Moreover, the present invention allows providing CLEC-based vaccines with excellent selectivity / specificity for the dermal compartment. Indeed, the conjugate design according to the present invention is based on CLECs as carriers for target-specific α-synuclein epitopes that show high binding specificity for dermal APCs / DCs, in particular PRRs on Dectin-1 (or MR and DC-SIGN in case of mannan), to enable skin-selective / skin-specific uptake and receptor-mediated uptake (=targeted vaccine delivery).

[0059] The CLEC polysaccharides used as carriers according to the present invention are preferably used to concentrate the carrier-peptide conjugates in dermal / skin DCs and initiate an immune response. This is due, among other things, to the epidermal or dermal (but not subcutaneous) specificity. The CLEC scaffolds according to the present invention and the efficient dermal immune response initiation also help to avoid the mandatory use of adjuvants (e.g., the use of alum, MF59, CFA, PolyI:C, or other adjuvants) that are typical of conventional vaccines and are also used in the exemplary CLEC-based vaccines. According to preferred embodiments of the present invention, the use of adjuvants can be significantly reduced or omitted, for example in situations where the addition of adjuvants is not indicated.

[0060] Although several CLECs have been used in previous applications, none of the proposed conjugate structures were able to confer skin selectivity (i.e., high Dectin-1 binding capacity, highly efficient dermal DC targeting, and superior immunogenicity for dermal administration compared to all other routes (i.e., subcutaneous, intramuscular, and ip)).

[0061] The selection of CLEC according to the present invention was made to provide a novel solution to target skin-specific DCs and skin-specific immunization with high efficacy. The conjugate according to the present invention also shows limited activity in other classical tissues for immunization, such as muscle or subcutaneous tissue, which is in contrast to previous CLEC-based vaccines / vaccine candidates that have been administered im or sc. As a result of the experiments carried out in the course of the present invention, the vaccine according to the present invention, in particular the vaccine using pustulan as CLEC, was surprisingly identified as selective for skin immunization.

[0062] The present invention allows to focus on the induction of α-synuclein-specific immune responses that induce no or only very limited CLEC- or carrier protein-specific antibody responses. The conjugates of the present invention therefore solve the problem posed by classical conjugate vaccines, which must rely on the use of foreign carrier proteins to induce a sustainable immune response. Current prior art conjugate vaccine development is strongly based on carrier molecules such as KLH, CRM197, tetanus toxoid, or other suitable proteins, complexed with target-specific short antigens that deliver substrates for immune responses against a-synuclein for synucleinopathies such as Parkinson's disease.

[0063] A preferred polypeptide immunogen construct according to the invention contains a B cell epitope derived from alpha-synuclein (aSyn, alpha Sny) and a heterologous T helper cell (Th) epitope linked to a CLEC. The present invention provides novel conjugates that are surprisingly superior to conventional vaccines in immunogenicity, cross-reactivity to aSyn, selectivity for aSyn species / aggregates, affinity, affinity maturation, and inhibitory potency.

[0064] Covalent conjugation of α-synuclein polypeptides to β-glucan or mannan according to the invention allows for a surprising and unexpected enhancement of the immune response to such polypeptides, which is particularly impressive in direct comparison with traditional vaccine formulations such as those described by Rockenstein et al. (2018), as also demonstrated in the Examples section below.

[0065] Rockenstein et al. (2018) disclose the application of yeast whole glucan particles (GPs) non-covalently complexed with aSyn and rapamycin as immunotherapeutic agents for Parkinson's disease. These GPs are produced following a series of hot alkaline, organic, and aqueous extraction steps from Saccharomyces cerevisiae that result in a final product consisting of a highly purified yeast cell wall preparation, 3-4 μm in diameter, free of cytoplasmic contents and bound by a porous, insoluble shell of β-glucan (mainly β1-3 β-glucan).

[0066] Importantly, the vaccine compositions disclosed by Rockenstein et al. (J. Neurosci., January 24, 2018; 38(4):1000-1014) consisted of GP non-covalently complexed with either ovalbumin and mouse serum albumin (MSA), human aSyn and MSA, or human aSyn, MSA and rapamycin. This complexation method relies on the co-incubation of different payloads with GP and their subsequent diffusion into the hollow GP cavity without covalent binding, and is therefore similar to the set of vaccines disclosed in Example 5 provided within the present application, which formulated the vaccine using only mixing of the components and without covalent binding, and proved to be inefficient and inadequate compared to the vaccines according to the present invention.

[0067] 1) Rockenstein et al. show that non-covalent mixing of aSyn with GP results in a detectable immune response to aSyn, thus indicating that GP can act as an adjuvant. However, Rockenstein et al. also show that non-covalent addition / co-complexation of rapamycin is necessary to induce significantly enhanced functionality of such vaccines compared to controls. In this respect, a mixture of different adjuvants (GP and the mTOR inhibitor rapamycin) is necessary to provide a fully functional vaccine, such as the vaccine disclosed by the present invention.

[0068] 2) The vaccine disclosed by Rockenstein et al. is active in this aSyn-overexpression model because it provides aSyn-specific T cell epitopes (among other T cell epitopes, such as MSA-derived epitopes) to exert its full functionality, i.e., induction of neuroprotective anti-aSyn-directed cellular (i.e., T cell-mediated) and humoral (i.e., antibody / B cell-based) immune responses. This is in direct contrast to the teaching of the present invention, where it is already sufficient if only aSyn-specific B cell responses are elicited by the selected vaccine.

[0069] 3) The use of full-length aSyn also poses the risk of inducing / boosting autoreactive aSyn-specific T cells that have the potential to exacerbate the underlying neuropathology of PD and other synucleinopathies. Therefore, the GP-aSyn-rapamycin vaccine proposed by Rockenstein et al. is also not suitable for human use with this issue.

[0070] 4) As shown in Example 5, non-covalent mixing of aSyn-derived peptides (e.g., SEQ ID NO:2, i.e., B cell epitopes) and promiscuous T cell epitopes (e.g., SEQ ID NO:7) with β-glucan particles (e.g., non-oxidized pustulan) can also induce low-level antibody responses against aSyn, similar to Rockenstein et al. However, vaccines according to the invention based on the covalent attachment of such peptides to the appropriate glucan exert a significantly different and superior immune response (see also FIG. 5).

[0071] Moreover, as also disclosed in Example 6 and Figure 7, such covalently bonded vaccines also show a highly beneficial lack of anti-glucan antibody responses compared to non-covalently bonded combination vaccines based on glucan particles and peptides disclosed by the present invention.

[0072] Thus, the prior art disclosure by Rockenstein et al. does not suggest the claimed subject matter disclosed by the present invention.

[0073] Any alpha-synuclein polypeptide comprising a B-cell epitope and / or a T-cell epitope can be used in the context of the present invention, including the polypeptide vaccine candidates proposed in the prior art, e.g. WO 2004 / 041067 A2, WO 2006 / 045037 A2, WO 2009 / 103105 A2, WO 2011 / 020133 A1, or Mandler et al. (Mol.Neurodeg.10 (2015), 10; Acta Neuropath.127 (2014), 861-879). Thus, alpha-synuclein polypeptides having a native amino acid sequence (as per human alpha-synuclein), or aSyn-derived polypeptides comprising a B-cell epitope and / or a T-cell epitope of aSyn, such as mimetics or mimotopes thereof, can be used as the alpha-synuclein polypeptide component in a conjugate according to the present invention.

[0074] Particularly preferred aSyn polypeptides to be conjugated in the present invention are those that have the amino acid sequence of native alpha-synuclein or the following native human alpha-synuclein: [ka] or 126-135, preferably comprising or consisting of amino acid residues 1-8, 91-100, 100-108, 102-108, 102-109, 103-129, 103-135, 107-130, 109-126, 110-130, 111-121, 111-135, 115-121, 115-122, 115-123, 115-124, 115-125, 115-126, 118-126, 121-127, 121-140, or 126-135 of a polypeptide comprising or consisting of: DQPVLPD, DQPVLPDN, DQPVLPDNE, DQPVLPDNEA, DQPVLPDNEAY, DQPVLPDNEAYE, DSPVLPDG, DHPVHPDS, DTPVLPDS, DAPVTPDT, DAPVRPDS, and YDRPVQPDR.

[0075] Weihofen et al. (Neurobiology of Disease 124 (2019) 276-288) disclosed N-terminal residues aa1-10 (MDVFMKGLSK) as a suitable aSyn epitope. PRX002 (disclosed in Masliah et al., (PLoS One 6:e19338);Games et al., (2014, J Neurosci 34:9441-9454);Schenk et al., (Mov Disord.2017 Feb;32(2):211-218.);Jankovic et al., (JAMA Neurol.2018 Oct 1;75(10):1206-1214)) is directed against the C-terminal region aa118-126 (VDPDNEAYE). Schofield et al. (Neurobiol Dis.2019 Dec;132:104582.) disclosed an aSyn-specific monoclonal Ab MEDI-1341 against a similar epitope in the C-terminal region of aSyn (aa103-129, NEEGAPQEGILEDMPVDPDNEAYEMP). The MEDI Ab (aa121-127; DNEAYEM) was disclosed by Nordstrom et al., (Neurobiology of Disease 161 (2021) 105543). Other suitable epitopes for antibody / immunotherapy in aSyn known to those of skill in the art include epitopes from autoantibodies (Heinzel et al., (PLoS ONE 9(12):e114566.); Rabenstein et al., (Neurosci Lett.2019 Jun 21;704:181-188); US 2014 / 0377271 A1, and Li et al., (Acta Neuropathologica 137, 825-836 (2019)), including aa60-100, aa60-95, aa73-82, aa91-100, aa74-79, and aa92-97, aa121-140, and aa111-121. Additionally, Games et al. al. (2014) also disclose suitable epitopes aa91-99 and aa118-126.US 2005 / 0037013 A1 discloses immunogenic α-synuclein fragments capable of inducing immune responses against specific epitopes within residues aa70-140. WO 2009 / 103105 A2 and WO 2011 / 020133 A1 disclose molecular mimetics against aa102-108 and aa115-121, respectively. WO 2016 / 062720 A1 provides modified VLPs containing Th cell epitopes representing the middle region (aa102-109) and N-terminal (aa1-8) or C-terminal sequences (i.e., aa131-140) that were able to induce high anti-peptide responses. Middle- and C-terminal-derived epitopes were also able to induce aSyn-specific titers that recognize different aSyn species (e.g., monomers and oligomers). Interestingly, all the vaccines provided failed to reduce aSyn burden or behavioral changes in in vivo PD models. US 2015 / 0232524 A1 discloses polypeptide immunogens, specifically aa10-14 and aa36-69, with epitopes shown at aa85-99, aa109-126, and aa126-140. Interestingly, all constructs initiate anti-aSyn immune responses, although epitope aa109-126 seems less effective. WO 2018 / 232369 A1 shows the application of target-specific B cell epitopes (10-25aa, from the C-terminus of aSyn; region: aa111-135).

[0076] In principle, the present invention can improve all suggested aSyn vaccination polypeptides, but selected epitopes were specifically evaluated for their compatibility with the present platform. For example, aa1-8 (SEQ ID NO:12 + SEQ ID NO:13) was shown to be superior to KLH-based vaccines.

[0077] The nature of the alpha-synuclein polypeptide is not critical for the production of β-glucan or mannan conjugates, but there are preferred and less preferred alpha-synuclein epitopes depending on the purpose the conjugate must solve (specificity, selectivity, affinity for monomers or aggregates, generation of antibodies, etc.).

[0078] For some embodiments of the invention, B cell epitopes aa91-100, aa100-108, aa107-114, and aa131-140 are less preferred due to reduced anti-peptide response and reduced reactivity to alpha-synuclein (aa91-100); low cross-reactivity (CR) to alpha-synuclein (despite high anti-peptide titers), low selectivity for aggregates (aa100-108); low potency for inhibiting aggregation (aa107-114); and altered selectivity (for monomers instead of aggregates), reduced selectivity, and low efficacy for reducing alpha-synuclein aggregates (aa131-140). Furthermore, epitope polypeptides having 5 or fewer amino acid residues (or having 6 or fewer amino acid residues) are also typically less preferred due to the lower immune response elicited by such shorter forms.

[0079] Particularly preferred alpha-synuclein epitopes according to the invention include the above mentioned epitopes comprising or consisting of amino acid residues 1-5, 1-8, 1-10, 60-100, 70-140, 85-99, 91-100, 100-108, 102-108, 102-109, 103-129, 103-135, 107-130, 109-126, 110-130, 111-121, 111-135, 115-121, 115-122, 115-123, 115-124, 115-125, 115-126, 118-126, 121-127, 121-140, or 126-135 of the amino acid sequence of native human alpha-synuclein.

[0080] For example, the above mentioned aa115-126 as well as shorter sequences and mimotopes thereof are particularly suitable with regard to immunogenicity, selectivity, aggregate inhibition etc.; in general, polypeptides with 7 or more amino acid residues show good immune responses and cross-reactivity against alpha-synuclein; for aa115-121 (and C-terminally extended polypeptides, e.g. up to aa126), excellent immune reactivity and cross-selectivity (in contrast to monoclonal LB509 (Jakes et al., Neurosci Lett. 1999 Jul 2;269(1):13-6), which binds to epitope 115-122); and surprisingly, the conjugates according to the invention show significantly improved selectivity against aggregates compared to "classical constructs" using CRM197 or KLH as carrier proteins (e.g. CRM-based vaccines frequently lack selectivity against aggregates or are even selective for monomers, whereas the conjugates according to the invention show clear and significant activity against aggregates).

[0081] This is even more surprising in light of published experiments where LB509 binds to α-synuclein aggregates but fails to inhibit aggregation (Breydo et al. Mol Neurobiol 53, 1949-1958 (2015)), indicating that not all antibodies from this epitope are biologically active as is the case in the present invention.

[0082] It is well accepted in the art that PD patients suffer from various alterations in the T cell compartment compared to healthy controls (e.g. Bas et al., J Neuroimmunol 2001; 113:146-52 or Gruden et al., J Neuroimmunol 2011; 233:221-7). Such phenotypic changes of T cells in PD are, for example, a decrease in the absolute number of lymphocytes, a decrease in the absolute and relative number of total T cells, a decrease in the absolute and relative number of CD4+ and sometimes CD8+ lymphocytes, an increase in the Th1 / Th2 and Th17 / Treg ratios, and an increase in the expression of inflammatory cytokines. However, most of these changes are also seen during healthy aging, making it difficult to discern the impact of a disease such as PD, which shows a very wide range of onset (approximately 30-90 years) and variable progression rates. With regard to absolute cell numbers, there seems to be a consensus of a net decrease in CD3+CD4+ T cells in PD. This decrease in CD4 is supported by the changes in the CD4:CD8 ratio described.

[0083] Along these lines, for example, Bhatia et al. (J Neuroinflammation (2021) 18:250) show an overall decrease in total CD3+ T cells in PD associated with disease severity (e.g., measured using H+Y stage). This suggests a progressive systemic T cell dysfunction with ongoing disease, likely reflecting the combined effects of ongoing inflammation, medications, and lifestyle changes. Also, Lindestam Arlehamn et al. (2020) show that the highest T cell activity is detectable in PD patients in the prodromal or early clinical stage (<10 years duration and H+Y stages 0-2).

[0084] It is therefore a preferred embodiment of the present invention to provide a treatment for enhancing or maintaining T cell numbers, particularly T effector cell numbers and T cell function in PD patients, preferably comprising a checkpoint inhibitor or vaccine combination using anti-immune checkpoint inhibitor epitopes to induce an anti-immune checkpoint inhibitor immune response in combination with a target-specific vaccine of the present invention to enhance or maintain T cell numbers, particularly T effector cell numbers and T cell function in PD patients.

[0085] Patients adapted / suitable for treatment are characterized by a global decrease in CD3+ cells at all disease stages, especially CD3+CD4+ cells typical of PD patients. The preferred disease stages defining the patient population suitable for this combination are H+Y stages 1-4, preferably H+Y1-3, most preferably H+Y2-3, respectively.

[0086] Also, affinity maturation of target-specific responses induced upon repeated immunization with carrier conjugates is impaired due to over-representation of carrier-specific epitopes in the conjugates. Affinity maturation in immunology, as used and understood herein, refers to the process of targeting a target specific antigen by a target antigen. FH Cellular activation is the process by which B cells produce antibodies with increasing affinity for antigens during the course of an immune response. Repeated exposure to the same antigen causes the host to produce antibodies of successively higher affinity. Secondary responses can elicit antibodies with several-fold higher affinity than the primary response. Affinity maturation occurs primarily in the surface immunoglobulins of germinal center B cells, through somatic hypermutation (SHM) and T FHIt occurs as a direct consequence of selection by cells (see also https: / / en.wikipedia.org / wiki / Affinity_maturation). Affinity maturation according to Segen's Medical Dictionary (https: / / medical-dictionary.thefreedictionary.com / affinity+maturation”>affinity maturation) is the increase in the average affinity of antibodies for an antigen following immunization. Affinity maturation results in an increase in specific, more homogeneous IgG antibodies, following a less specific, more heterogeneous initial response by IgM molecules.

[0087] All current prior art CLEC vaccines induce high titers against the carrier proteins used (e.g. CRM197 or OVA). However, this high immunogenicity as well as the structural complexity and heterogeneity of the carrier protein components may result in the induction of high levels of carrier / protein-specific antibodies at the expense of target-specific responses that may be under-represented compared to the induced carrier responses.

[0088] Moreover, high anti-carrier responses also pose the risk of immunological rejection and related safety issues.

[0089] Therefore, the identification of effective constructs with low or non-existent carrier reactivity (i.e. against protein carriers), high immunogenicity, high target specificity, and high tolerability / safety according to the present invention successfully addresses this challenge with an innovative solution. Moreover, it is important that the novel vaccine according to the present invention provides an immunotherapeutic agent that induces no / very weak immune response against the sugar backbone. This is particularly important since high anti-CLEC antibody levels induced upon immunization may inhibit or reduce the efficacy of repeated immunizations with the same CLEC-based vaccine due to vaccine neutralization or may negatively affect the use of this type of vaccine for sequential immunization against various different targets.

[0090] The vaccine platform according to the invention also meets the need to combine different epitopes against one or several targets in one formulation without the risk of reducing efficacy due to unintended epitope spreading as reported for classical vaccines. The modular design of the platform according to the invention allows for easy exchange of B- and T-cell epitopes without the adverse effects of carrier-induced responses.

[0091] The present invention is based on CLECs that exert highly specific binding to their cognate receptors. This binding is important as only strong binders are efficient as vaccine carriers / scaffolds.

[0092] According to the present invention, the CLEC conjugate formation allows an efficient immune response with novel characteristics. The conjugate formation according to the present invention prevents the formation of anti-CLEC antibodies, especially against pustulan, which can be impressively demonstrated in the course of the present invention. This lack of induction of anti-CLEC antibodies is of great importance for the reusability and re-booster stability of individual vaccines designed on the platform according to the present invention, whether with the same or different antigens.

[0093] In contrast to the conjugate embodiment of the present invention, simply mixing the CLEC polysaccharide adjuvant with B cell epitope peptide or T cell epitope peptide does not produce the same effect in vivo. However, when conjugated, the orientation of the peptide does not significantly affect the performance of the compound according to the present invention; therefore, CLEC conjugate formation is substantially independent of the peptide orientation in the construct. In the course of the present invention, it can be shown that CLEC conjugate formation, especially to pustulan, leads to novel peptide immunogens / antigens of α-synuclein and improvement of existing peptide immunogens / antigens: this improvement is achieved by higher, more target-specific, and more affine antibody responses (as can be shown by antibody selectivity and functionality). This effect was most pronounced with pustulan or similar β-glucans, which are primarily linear β-(1,6)-glucans having a ratio of β-(1,6)-linked to non-β-(1,6)-linked monosaccharide moieties of at least 1:1, preferably at least 2:1, more preferably at least 5:1, especially at least 10:1, which surprisingly performed significantly better than KLH or CRM in direct comparisons, and even better than conjugates of mannan or lichenan, or conjugates with barley β-glucan.

[0094] As used herein, the term "predominantly linear" β-(1,6)-glucan refers to a β-(1,6)-D-glucan in which cross-linked sugar monomer entities are absent or nearly absent, i.e., less than 1%, preferably less than 0.1%, in particular less than 0.01%, of the monosaccharide moieties have three or more covalently linked monosaccharide moieties.

[0095] As already mentioned above, pustulan is the most preferred CLEC according to the invention. Since pustulan is generally free of cross-linked sugar moieties and is predominantly β-(1,6) linked, a typical pustulan preparation used for preparing a conjugate according to the invention will contain less than 1%, preferably less than 0.1%, in particular less than 0.01% of monosaccharide moieties having three or more covalently linked monosaccharide moieties and will contain up to 10% impurities including β-(1,3) linked monosaccharides or β-(1,4) linked monosaccharides.

[0096] The fact that pustulan was found to be the most effective CLEC in the course of the present invention was unexpected, since various references have shown that pustulan is not very effective in binding dectin-1 (e.g., Adams et al., J Pharmacol Exp Ther. 2008 Apr;325(1):115-23), and the literature has reported that linear 1,3 and branched (1,3 backbone and 1,6 branches) are the most effective dectin-1 binders. For example, Adams et al., 2008 reported that mouse recombinant dectin-1 recognizes and interacts only with polymers containing a β-(1,3)-linked glucose backbone. Dectin-1 did not interact with glucan (pustulan) composed exclusively of a β-(1,6)-glucose backbone, nor did it interact with non-glucan carbohydrate polymers such as mannan.

[0097] Thus, according to a preferred embodiment of the present invention, the β-glucan of the conjugate is a dectin-1 binding β-glucan. The ability of any compound, particularly a glucan, to bind to dectin-1 can be easily determined by the methods disclosed herein, particularly in the Examples section. In case of doubt, a "dectin-1 binding β-glucan" is a β-glucan that binds to the soluble mouse Fc-Dectin-1a receptor with an IC50 value of less than 10 mg / ml, as determined by competitive ELISA, for example as disclosed in the Examples.

[0098] The Dectin-1-binding β-glucans according to the present invention (such as linear β-(1,6)-glucans) are advantageous compared to other glucans, such as DC-SIGN β-glucans (such as β-(1,2)-glucans), because such Dectin-1-binding glucans can address a wider range of DCs (immature, mature, myeloid, plasmacytoid; also: APCs), significantly increasing the likelihood of inducing an effective immune response in vivo compared to non-Dectin-1-binding glucans (immature DCs, myeloid DCs), which have limited applicability.

[0099] WO 2022 / 060487 A1 and WO 2022 / 060488 A1 disclose conjugates that link peptide immunogens to immunostimulatory polymeric molecules (e.g., β-(1,2) glucans). β-(1,2) glucans, including cyclic variants, have been previously suggested as potential adjuvants (Martirosyan A et al., doi:10.1371 / journal.ppat.1002983). They are a class of glucans that primarily bind to a specific PRR, DC-SIGN (Zhang H et al., doi:10.1093 / glycob / cww041), and specifically bind to N-linked high mannose oligosaccharides and branched fucosylated structures. Importantly, β-1,2-glucans are unable to bind to Dectin-1 (Zhang H et al., doi:10.1093 / glycob / cww041), thereby restricting their activity to DC-SIGN positive cells.

[0100] DC-SIGN (CD209) was the first SIGN molecule identified and found to be highly expressed only on a restricted subset of DCs, including immature (CD83-negative) DCs, as well as specialized macrophages in the placenta and lungs (Soilleux EJ et al., doi:10.1189 / jlb.71.3.445). At peripheral, e.g., skin or mucosal sites, expression, and thus the potential for biological activity as a receptor according to the present invention, is only detectable in a subset of immature DCs. Mature plasmacytoid DCs and other APCs, such as epithelial DC-like Langerhans cells, do not express DC-SIGN (Engering A, et al., doi:10.4049 / jimmunol.168.5.2118).

[0101] In contrast, the target receptor of the β-glucan-based immunogens provided herein is Dectin-1, which is expressed by a variety of different DC types, including immature DCs, myeloid DCs, but also plasmacytoid DCs, which express Dectin-1 at both the mRNA and protein levels, and DC-like Langerhans cells of the skin (Patente et al., doi:10.3389 / fimmu.2018.03176; Joo et al., doi:10.4049 / jimmunol.1402276).

[0102] Thus, the biological activity of DC-SIGN targeting polymers such as β-(1,2) glucan is limited to a specific DC target cell population, whereas the Dectin-1 targeting polymers applied in the present invention can exert their function on a variety of different additional DC types. Thus, these novel conjugates can exert significantly different and superior immune responses compared to other conjugates. Thus, the disclosure of the prior art does not suggest the claimed subject matter disclosed by the present invention.

[0103] According to a particularly preferred embodiment, the conjugate of the invention comprises a potent Dectin-1 binding β-glucan, preferably a β-glucan that binds to soluble mouse Fc-Dectin-1a receptor with an IC50 value of less than 10 mg / ml, more preferably an IC50 value of less than 1 mg / ml, even more preferably an IC50 value of less than 500 μg / ml, in particular an IC50 value of less than 200 μg / ml, as determined by competitive ELISA, e.g. as disclosed in the Examples. Particularly preferred are conjugates that bind to soluble mouse Fc-Dectin-1a receptor with an IC50 value of less than 1 mg / ml, more preferably an IC50 value of less than 500 μg / ml, even more preferably an IC50 value of less than 200 μg / ml, in particular an IC50 value of less than 100 μg / ml, as determined by competitive ELISA; and / or

[0104] β-glucan that binds to the soluble human Fc-Dectin-1 receptor with an IC50 value of less than 10 mg / ml, more preferably less than 1 mg / ml, even more preferably less than 500 μg / ml, and especially less than 200 μg / ml, as determined by competitive ELISA; and / or

[0105] The conjugate binds to the soluble human 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, and especially with an IC50 value of less than 100 μg / ml, as determined by competitive ELISA, e.g. as disclosed in the Examples.

[0106] Furthermore, the conjugates according to the invention also showed a proportionally higher increase in the ratio of antibodies reacting with the target polypeptide rather than the carrier molecule, as is the case with CLEC-free vaccines, especially pustulan-free vaccines, which significantly increases the specific focusing of the antibody immune response against the target rather than the carrier, thereby increasing the efficacy and specificity of the response.

[0107] CLEC conjugation according to the present invention, particularly to pustulan, also results in increased affinity maturation (AM) towards the target protein (largely increased AM, whereas KLH / CRM conjugates show only limited AM upon repeated immunization).

[0108] In the field of vaccines, suitable vaccines have been disclosed that have only B cell epitopes or only T cell epitopes. There are certain situations where it is appropriate and preferable to have a vaccine that has exclusively T cell epitopes or exclusively B cell epitopes. However, most of the vaccines available on the market contain both types of epitopes, i.e. T cell epitopes and B cell epitopes.

[0109] For example, vaccines containing only B cell epitopes are in most cases less effective, even though they produce a detectable antibody immune response. In most cases, however, this immune response is usually much less effective than vaccines containing B cell and T cell epitopes. This is also consistent with the examples given in the Examples section of the present invention, where lower levels of responses were detectable.

[0110] On the other hand, vaccines containing only T cell epitopes (e.g. in vaccines where a specific T cell response is the active component of the response) are of particular interest for certain applications, especially cancer, where only cancer-specific cytotoxic T lymphocyte and T helper cell epitopes or CTL epitopes are combined with the vaccine platform according to the invention. In this case, the T cell epitopes with the CLEC polysaccharide adjuvant according to the invention are provided with only T cell epitopes. This is particularly preferred when, for example, somatic mutations in cancer affect protein-coding genes that may give rise to potential therapeutic neoepitopes. These neoepitopes can guide adoptive cell therapy and peptide (and RNA-based) neoepitope vaccines to selectively target tumor cells using the patient's autologous cytotoxic T cells. The use of vaccines with only T cell epitopes may also be preferred for specific autoimmune diseases. The therapeutic effect of the respective T cell epitope-only conjugates is the depletion of effector T cells and the suppression of the respective autoimmune disease (e.g., multiple sclerosis or similar diseases) by the regulatory T cells (T reg Related to the development of a population of cells.

[0111] Since most of the conventional vaccine settings contain both B and T cell epitopes, the CLEC conjugates according to the invention also preferably contain both individual B and T cell epitopes (at a minimum: at least one B cell epitope of alpha-synuclein and at least one T cell epitope) for sustained B cell immune response. However, a weak effect may be demonstrated for T cell independent immunity if required.

[0112] Thus, the conjugates according to the invention are not limited with respect to possible vaccine antigens. Thus, the alpha-synuclein vaccine according to the invention may further comprise additional antigens to provide a bispecific, trispecific, tetraspecific, pentaspecific, hexaspecific (etc.) or multispecific vaccine.

[0113] It is preferred that the vaccine antigen (ie, B cell and / or T cell epitope polypeptide) has a length of 6 to 50 amino acid residues, preferably 7 to 40 amino acid residues, and particularly 8 to 30 amino acid residues.

[0114] Using the vaccine according to the invention, cross-linking of B cell receptors is also possible. According to a specific embodiment, the conjugate according to the invention is used for T cell-independent immunization. T cell-independent responses are well known for polysaccharide vaccines. These vaccines / polysaccharides generate an immune response by direct stimulation of B cells, without the assistance of T cells. T cell-independent antibody responses are short-lived. Antibody concentrations for pneumococcal capsular polysaccharides usually decline to baseline in 3-8 years, depending on the serotype. Usually, polysaccharide vaccines do not constitute immune memory, so additional doses cannot be used to boost the vaccine response. In children under 2 years of age, polysaccharide vaccines are poorly immunogenic. Here, the reason for direct stimulation may be because B cells express a molecule called CR3 (complement receptor type 3). Macrophage-1 antigen or CR3 is a human cell surface receptor found on B and T lymphocytes, polymorphonuclear leukocytes (mainly neutrophils), NK cells, and mononuclear phagocytes such as macrophages. CR3 also recognizes iC3b when bound to the surface of foreign cells and β-glucan, meaning that direct uptake of vaccines by B cells via Pus-CR3 interactions can result in stimulation of the cells and the generation of low-level TI immune responses.

[0115] The adjuvants, conjugates and vaccines according to the invention are capable of fixing complement and can be opsonized. Opsonized conjugates according to the invention can have increased B cell activation capacity which can lead to higher antibody titers and affinity. This effect is known for C3d conjugates (Green et al., J.Virol.77 (2003), 2046-2055) and can unexpectedly be used in the course of the present invention.

[0116] Another unexpected advantage of the present invention is that the CLEC structure of the present invention allows modular design of vaccines. For example, epitopes can be freely combined and the platform is independent of conventional carrier molecules. Although the main emphasis of the present invention is on peptide-only vaccines, the present invention also works with independent linkage of proteins and peptides, as well as linkage of peptide-protein conjugates to the CLEC scaffold according to the present invention, in particular pustulan. As shown in the Examples section with pustulan, significantly superior immune responses are obtained according to the present invention compared to classical vaccines.

[0117] As already outlined above, the conjugates according to the invention, when provided in a pharmaceutical preparation (e.g. as a vaccine intended to be administered to a (human) subject in order to induce an immune response against a specific polypeptide epitope bound to the CLEC scaffold against which an immune response should be elicited), can be administered without the need to use (by co-administration) a (further) adjuvant in this preparation. According to a preferred embodiment, the pharmaceutical formulation comprising the conjugates according to the invention does not comprise an adjuvant.

[0118] A particularly preferred class of CLEC polysaccharide adjuvants according to the present invention are β-glucans, especially pustulan. Another preferred CLEC polysaccharide adjuvant is mannan. In contrast to the present invention, pustulan has only been used in antifungal vaccines in the prior art (pustulan was used as an antigen, not as a carrier as in the present invention). Pustulan also presents a different backbone, since it is composed only of β-(1,6) linked sugar moieties.

[0119] Pustulan is a medium-sized linear β-(1,6) glucan. The synthetic forms of pustulan and linear β(1,6) glucan are different from all other glucans used, since β-glucans usually consist of branched glucan chains (preferably β-(1,3) backbone with β-(1,6) side chains, such as yeast extract, GP, laminarin, schizophyllan, scleroglucan, etc.) or linear glucans that rely only on β-(1,3) glucans, such as synthetic β-glucans, curdlan, Saccharomyces cerevisiae β-glucan (150 kDa), or linear β-(1,3:1,4) glucans, such as barley β-glucan and oat β-glucan, and lichenan.

[0120] As shown for the first time in this invention, in vitro binding of glucan conjugates to the Dectin-1 receptor is a surrogate for subsequent in vivo efficacy: low binding molecules are only able to elicit a low immune response, moderate binders are better, and high efficiency binders induce a highly efficient response (oat / barley BG<licanan<pustulan).

[0121] According to the present invention, CLECs are linked (e.g., by standard techniques) to individual alpha-synuclein polypeptides that are not crosslinked, aggregated, and typically in the size range of greater than 100 nm (typical range (diameter; 150-500 nm, e.g., Wang et al. (2019) provided particles with a diameter of 160 nm (assessed by DLS) and a size of about 150 nm as assessed by TEM; Jin et al. (2018) provide β-glucan particles (nanoparticles of aminated β-glucan-ovalbumin) with a size of 180-215 nm (assessed by DLS and SEM, respectively), characterized by the creation of small nanoparticles with low polydispersity (hydrodynamic radius (HDR) range: 5-15 nm) that do not form larger particles similar to conventional CLEC vaccines such as glucan particles (2-4 μm) or β-glucan particles disclosed in the literature. These conjugates preferably contain at least one T cell epitope, in particular a promiscuous T cell epitope, a linear T cell epitope, or a carrier peptide T cell epitope (e.g., derived from CRM197 or KLH).

[0122] By definition, the DLS measured hydrodynamic radius is the radius of a hypothetical hard sphere diffusing at the same rate as the particle under test. The radius is calculated from the diffusion coefficient, assuming a spherical shape of the molecule / particle and a given viscosity of the buffer. The HDR is also called the Stokes radius and is calculated from the diffusion coefficient using the Stokes-Einstein equation (see https: / / en.wikipedia.org / wiki / Stokes_radius).

[0123] The preferred diameter range of the nanoparticles according to the invention may be that usually provided in the prior art, i.e. diameters of 1-5000 nm, preferably 1-200 nm, in particular 2-160 nm, as determined as hydrodynamic radius (HDR) by dynamic light scattering (DLS). According to a preferred embodiment of the invention, the particle size is smaller, for example 1-50 nm, preferably 1-25 nm, in particular 2-15 nm, as determined as HDR by DLS. Thus, these preferred particles, including peptide-only conjugates (average HDR of about 5 nm) and CRM-pustulan conjugates (average HDR of about 10-15 nm), are smaller. Thus, the preferred particles according to the invention are less than 100 nm, which would separate us from Wang et al.

[0124] Thus, the present invention also relates to a vaccine product designed to vaccinate an individual against a specific alpha-synuclein antigen comprising a compound comprising β-glucan or mannan as a C-type lectin (CLEC) polysaccharide adjuvant covalently linked to a specific antigen.

[0125] Preferably, the vaccine product according to the invention comprises a conjugate disclosed herein or obtainable or obtained by a method according to the invention.

[0126] According to a preferred embodiment, the vaccine product according to the invention comprises an alpha-synuclein antigen comprising at least one B cell epitope of alpha-synuclein and at least one T cell epitope, preferably the antigen is a polypeptide comprising one or more B cell epitopes and T cell epitopes.

[0127] According to a preferred embodiment, the covalently linked antigen and CLEC polysaccharide adjuvant in the vaccine product according to the invention are present as particles having a diameter of 1-5000 nm, preferably 1-200 nm, in particular 2-160 nm, as determined as the hydrodynamic radius (HDR) by dynamic light scattering (DLS). As used herein, all particle sizes are median particle sizes, the median being the value that separates the half of the particles having a larger diameter from the half of the particles having a smaller diameter. The median particle size is the determined particle size at which half of the particles are smaller and half are larger.

[0128] According to a preferred embodiment, the covalently linked antigen and CLEC polysaccharide adjuvant in the vaccine product according to the invention are present as particles having a diameter of 1-50 nm, preferably 1-25 nm, in particular 2-15 nm, determined as HDR by DLS.

[0129] Preferably, the covalently linked antigen and CLEC polysaccharide adjuvant in the vaccine product according to the invention are present as particles having a diameter, determined as HDR by DLS, of less than 100 nm, less than 50 nm, preferably less than 70 nm, especially less than 50 nm.

[0130] The vaccine product according to the invention exhibits high storage stability: it can be determined that the particle size does not increase significantly (i.e. by more than 10%) upon storage, so that virtually no aggregation occurs when stored as liquid or frozen material (storage temperatures: -80°C, -20°C, 2-8°C or at room temperature for extended periods, at least 3 months).

[0131] The extremely high efficacy of such small particles made by using the medium molecular weight component pustulan according to the invention is surprising: for example, according to Adams et al. (J Pharmacol Exp Ther. 2008 Apr;325(1):115-23), the best dectin-1 substrates are linear β(1,3) glucan phosphates (about 150 kda), and branched glucans (containing a β(1,3) backbone and β(1,6) side chains) such as scleroglucan, or a glucan derived from Candida albicans, or laminarin. Furthermore, data from Adams et al., Palma et al. (J Biol Chem.281(9) (2006) 5771-5779) and Willment et al. (J Biol Chem.276(47) (2001), 43818-23) indicate that dectin-1 does not interact or only weakly interacts with pustulan, and that dectin-1 also does not interact with non-glucan carbohydrate polymers such as mannan. In fact, various references report pustulan as being less effective at binding dectin-1. However, in general, linear 1,3 and branched (1,3 backbone and 1,6 side branches) are the most effective dectin-1 binders; Adams et al. (2008) showed that only mouse recombinant dectin-1 recognized and interacted with polymers containing a β(1,3)-linked glucose backbone. Dectin-1 did not interact with glucans composed exclusively of a β(1,6)-glucose backbone (such as pustulan), nor did it interact with non-glucan carbohydrate polymers such as mannan.

[0132] In contrast to these findings, it was shown in the course of this invention that pustulan-based conjugates can bind strongly to dectin-1 and induce cellular responses in vitro.

[0133] According to a preferred embodiment of the present invention, β-(1,6)-glucan is used. Since the prior art has reported that larger particles are usually more effective at activating PRRs than smaller ("soluble") monomeric formulations, particles containing larger glucans are superior (and therefore preferred), and small, soluble glucans can be used to block DC activation, thereby preventing the intended effect. It is well accepted that particulate β-glucans, such as the widely used yeast cell wall fraction zymosan, bind to and activate Dectin-1, thereby inducing cellular responses. In contrast, the interaction of soluble β-glucans with Dectin-1 has been the subject of debate. However, the general consensus is that soluble β-glucans such as the small branched glucan laminarin (β-(1,3) and β-(1,6) side chains) bind to Dectin-1 but are unable to initiate signaling and induce cellular responses in DCs (Willment et al., J Biol Chem.276(47) (2001), 43818-23; Goodridge et al. Nature.2011, 472(7344):471-475.).

[0134] According to the present invention, conjugates using high molecular weight glucans (10 times the size of pustulan; e.g. oat / barley 229 kDa / lichenan 245 kDa) can be shown to be less effective than pustulan particles (20 kDa). Korotchenko et al. show that OVA / Lam conjugates have a diameter of about 10 nm, bind to dectin-1 and induce DC activation in vitro, but are branched glucans, are not skin specific and are not superior in terms of efficacy in vivo compared to OVA administered to the skin or OVA / alum administered sc. Wang et al. provide β-glucan particles with a diameter of more than 100 nm (mean diameter: 160 nm). Jin et al. (2018) show aminated β-glucan-ovalbumin nanoparticles with a diameter of 180-215 nm.

[0135] According to the present invention, pustulan-based particles have been shown to be potent Dectin-1 binders, activating DCs in vitro (changes in surface marker expression) and inducing very strong immune responses, a) superior to other routes, b) comparable to KLH / CRM conjugate vaccines (usually much larger particles) and C) larger glucans and mannans. This is true for Pep+Padre+pustulan (5 nm diameter) and Pep+CRM197+pustulan conjugates (11 nm diameter).

[0136] For an optimal immune response, the degree of activation of CLECs, especially pustulan, and the peptide / sugar ratio resulting from this activation degree are crucial. Activation of the respective CLECs is achieved by mild periodate oxidation. The degree of oxidation is therefore determined based on adding periodate solution at a defined molar ratio, i.e., periodate:sugar subunit; 100% = 1 Mol of periodate per Mol of sugar monomer.

[0137] According to a preferred embodiment, the conjugate according to the invention comprises activated CLEC in a ratio of periodate to β-glucan or mannan (monomer) moieties of 1 / 5 (i.e. 20% activated) to 2.6 / 1 (i.e. 260% activated), preferably 60% to 140%, in particular 70% to 100%.

[0138] The optimal range of oxidation degrees from low / medium to high oxidation degrees (directly proportional to the number of epitope polypeptides in the final conjugate) can be defined as the reactivity with Schiff's Fusine reagent similar to the reactivity of an equivalent amount of a given carbohydrate (e.g., pustulan) oxidized with periodate at molar ratios (sugar monomer:periodate) of 0.2-0.6 (low / medium), 0.6-1.4 (optimum range), and 1.4-2.6 (high), respectively.

[0139] Preferred glucan to peptide ratios, in particular the pustulan to peptide ratio, are in the range of 10:1 (w / w) to 0.1:1 (w / w), preferably 8:1 (w / w) to 2:1 (w / w), in particular 4:1 (w / w) (however, if the conjugate comprises a carrier protein, the preferred ratio of β-glucan or mannan to B-cell-epitope-carrier polypeptide is 50:1 (w / w) to 0.1:1 (w / w), in particular 10:1 to 0.1:1, i.e. a molar ratio of sugar monomer to peptide of 24:1), which are lower than those for effective vaccines reported elsewhere (e.g. Liang et al., Bromuro et al.).

[0140] The degree of oxidation, and the amount of reactive aldehydes available for sugar conjugation, is determined using prior art methods such as: 1) gravimetric measurements, which allow the determination of the total mass of the sample; 2) the anthrone method (according to Laurentin et al. 2003) for the determination of the concentration of intact, non-oxidized sugars in the sample; in this case, glucans are dehydrated with concentrated H2SO4 to form furfural, which is condensed with anthrone (0.2% in H2SO4) to form a green complex, which can be measured colorimetrically at 620 nm; or 3) the Schiff assay: the oxidation state of the carbohydrates used in the conjugation is evaluated using Schiff's fuchsin-sulfite reagent. Briefly, the fuchsin dye is decolorized by sulfur dioxide. Reaction with aliphatic aldehydes (on the glucans) restores the purple color of the fuchsin, which can then be measured at 570-600 nm. The resulting color reaction is proportional to the degree of oxidation (amount of aldehyde groups) of the carbohydrates. Other suitable analytical methods are also possible. Peptide ratios can be assessed using appropriate methods including UV analysis (205 nm / 280 nm) and amino acid analysis (aa hydrolysis, derivatization, and RP-HPLC analysis).

[0141] The conjugates according to the invention can be used to induce a target-specific immune response but not a CLEC or carrier protein specific antibody response or only a very limited CLEC or carrier protein specific antibody response. The conjugates according to the invention can further be used to induce an alpha-synuclein specific immune response but not a CLEC or carrier protein specific antibody response or only a very limited CLEC or carrier protein specific antibody response. As also shown in the Examples section below, the invention also allows for an improved and focused alpha-synuclein specific immune response, since it induces an immune response away from the response to the carrier protein or CLEC (e.g. in conventional peptide-carrier conjugates or in a non-conjugate comparative setting, especially the application of non-oxidized CLEC such as pustulan).

[0142] Unless indicated to the contrary, as used herein, "peptide" refers to shorter polypeptide chains (2-50 amino acid residues long) and "protein" refers to longer polypeptide chains (more than 50 amino acid residues long). Both are referred to as "polypeptides". The B-cell and / or T-cell epitope polypeptides bound to the CLEC according to the invention include, in addition to polypeptides having amino acid residues naturally used in normal gene expression and protein translation, all other forms of such polypeptide-based B-cell and / or T-cell epitopes, in particular their natural or artificially modified forms such as glycopeptides, and all other post-translationally modified forms thereof (e.g. the pyro-Glu form of Aβ as disclosed in the Examples). Furthermore, the CLEC according to the invention is particularly suitable for presenting structural epitopes, e.g. structural epitopes that are part of larger natural polypeptides, mimotopes, cyclic polypeptides, or surface-bound constructs.

[0143] According to a preferred embodiment, the conjugate according to the invention comprises a CLEC polysaccharide scaffold and a B-cell epitope. A "B-cell epitope" is a part of the α-synuclein antigen to which an immunoglobulin or antibody binds. B-cell epitopes can be divided into two groups: structural or linear. There are two main methods of epitope mapping: structural or functional studies. Methods to structurally map epitopes include X-ray crystallography, nuclear magnetic resonance, and electron microscopy. Methods to functionally map epitopes often use binding assays such as Western blots, dot blots, and / or ELISA to determine antibody binding. Competitive methods determine whether two monoclonal antibodies (mAbs) can bind to an antigen simultaneously or compete with each other for binding at the same site. Another technique involves high-throughput mutagenesis, an epitope mapping strategy developed to improve the rapid mapping of structural epitopes on structurally complex proteins. Mutagenesis uses random / site-directed mutations at individual residues to map epitopes. B cell epitope mapping can be used for the development of antibody therapeutics, peptide-based vaccines, and immunodiagnostic tools (Sanchez-Trincado et al., J. Immunol. Res. 2017-2680160). For many antigens, B cell epitopes are known and can be used in the present CLEC platform.

[0144] According to a particularly preferred embodiment, the conjugate according to the invention comprises a CLEC polysaccharide backbone and a promiscuous T cell epitope and / or an MHC II epitope, known to act at several / al MHC alleles of a given species and other species.

[0145] According to another aspect, the present invention also relates to the use of the present CLEC technology to improve known T cell epitopes. Thus, the present invention also encompasses β-glucan or mannan for use as a C-type lectin (CLEC) polysaccharide adjuvant for T cell epitope polypeptides, covalently linked to the T cell epitope polypeptide to form a conjugate of β-glucan or mannan and the T cell epitope polypeptide.

[0146] A single T cell epitope that binds to more than one HLA allele is called a "promiscuous T cell epitope". Preferred promiscuous T cell epitopes bind to more than 5, preferably more than 10, in particular more than 15 HLA alleles. Promiscuous T cell epitopes are suitable for various species, most importantly for several MHC / HLA haplotypes of a given species (referring to both MHC I and MHC II epitopes known to function at some / all MHC alleles) and other species. For example, as mentioned in the Examples section, the MHC II epitope PADRE (=non-natural pan-DR epitope (PADRE)) functions at several human MHC alleles and in mice (C57 / Bl6, but less effective in Balb / c). According to a preferred embodiment, the conjugate of the invention comprises a T cell epitope, preferably a T cell epitope comprising the amino acid sequence AKFVAAWTLKAAA ("PADRE (polypeptide)") or a PADRE (polypeptide) variant.

[0147] Preferred PADRE polypeptides or PADRE polypeptide variants include linkers such as a cysteine ​​residue or a linker containing a cysteine ​​residue ("-C" or "C-"; particularly for maleimide linkages), NRRA, NRRA-C, or NRRA-NH-NH2 linkers (also preferred for other polypeptide epitopes used herein).Preferred PADRE polypeptide variants include those disclosed in the prior art (e.g., Alexander et al., Immunity 1 (1994), 751-761; US ​​9,249,187 B2), preferably a truncated variant having no C-terminal A residue (AKFVAAWTLKAA), a variant in which the first residue alanine is replaced by an aliphatic amino acid residue (e.g., glycine, valine, isoleucine, and leucine), a variant in which the third residue phenylalanine is replaced by L-cyclohexylalanine, a variant in which the thirteenth (last) amino acid residue alanine is replaced by an aliphatic amino acid residue (e.g., glycine, valine, isoleucine, and leucine), and a variant in which the thirteenth (last) amino acid residue alanine is replaced by an aliphatic amino acid residue (e.g., glycine, valine, isoleucine, and leucine). a variant comprising aminocaproic acid, preferably linked to the C-terminus of a PADRE variant, or a variant having the amino acid sequence AX1FVAAX2TLX3AX4A (X1 is selected from the group consisting of W, F, Y, H, D, E, N, Q, I, and K; X2 is selected from the group consisting of F, N, Y, and W, X3 is selected from the group consisting of H and K, and X4 is selected from the group consisting of A, D, and E) (with the proviso that the oligopeptide sequence is not AKFVAAWTLKAAA; US 9,249,187 B2); in particular, T cell epitopes include AKFVAAWTLKAAANRRA-(NH-NH2), AKFVAAWTLKAAAN-C, AKFVAAWTLKAAA-C, AKFVAAWTLKAAANRRA-C, aKXVAAWTLKAAaZC, aKXVAAWTLKAAaZCNRRA (SEQ ID NOs: 7, 8, 87, 88, 89, 90, 91, 92), aKXVAAWTLKAAa, aKXVAAWTLKAAaNRRA, aA(X)AAAKTAAAAa, aA(X)AAATLKAAa, aA(X)VAAATLKAAa, aA(X)IAAATLKAAa, aK(X)VAAWTLKAAa, and aKFVAAWTLKAAa (Alexander et al., 1994 sequences 760.5, 760.57, 906.09, 906.11, 965.10, 1024.03), in which X is L-cyclohexylalanine, Z is aminocaproic acid, and a is an aliphatic amino acid residue selected from alanine, glycine, valine, isoleucine, and leucine.

[0148] T cell epitopes are presented on the surface of antigen-presenting cells, where they are bound to major histocompatibility complex (MHC) molecules. In humans, most nucleated somatic cells present MHC class I peptides, while professional antigen-presenting cells are specialized to present MHC class II peptides. T cell epitopes presented by MHC class I molecules are typically peptides of 8-11 amino acids in length, while MHC class II molecules present longer peptides of 13-17 amino acids in length, and non-classical MHC molecules also present non-peptide epitopes such as glycolipids. MHC class I and MHC class II epitopes can be reliably predicted by computational means alone, but not all in silico T cell epitope prediction algorithms are equal in their accuracy. There are two main methods to predict peptide-MHC binding: data-driven and structure-based. Structure-based methods model peptide-MHC structures and require large computational power. Data-driven methods have higher predictive performance than structure-based methods. Data-driven methods predict peptide-MHC binding based on the peptide sequence that binds to the MHC molecule (Sanchez-Trincado et al., 2017). Identifying T cell epitopes allows scientists to track, phenotype, and stimulate T cells. For many antigens, including α-synuclein, T cell epitopes are known and can be used in the present CLEC platform.

[0149] Interestingly, recent breakthrough studies have demonstrated that α-synuclein-specific T cells are increased in PD patients, possibly in association with HLA risk haplotypes, suggesting autoimmune involvement of T cells in PD (Sulzer et al., Nature 2017;546:656-661 and Lindestamn Arlehamn et al., Nat Commun.1875;2020:11). The causal role of α-synuclein-reactive T cells has also been strengthened in recent years by animal model studies (Williams et al., Brain.2021;144:2047-2059). The occurrence of α-synuclein-reactive T cells increased many years before motor onset in case studies, and their frequency was highest near and immediately after motor onset in a larger cross-sectional cohort of PD patients (Lindestam Arlehamn et al.). After motor onset, T cell responses to α-synuclein declined with increasing disease duration. Thus, anti-aSyn T cell responses are highest before or shortly after diagnosis of motor PD and decline thereafter (i.e., maximal activity detectable <10 years after diagnosis; and favor Horne-Yarle (H+Y) stages 1 and 2) (Lindestamn Arlehamn et al. 2020).

[0150] Thus, there are commonly known T cell epitopes contained within the sequence of human alpha-synuclein. Examples are provided in Benner et al. (PLoS ONE 3(1):e1376.60), Sulzer et al., (2017) and Lindestam Arlehamn et al. (2020).

[0151] Benner et al (Benner et al., (2008) PLoS ONE 3(1):e1376.) disclosed the α-synuclein T cell epitope aa71-86 (VTGVTAVAQKTVEGAGNIAAATGFVK) using a 60 aa long nitrated (at Y residues) polypeptide containing the C-terminal portion of aSyn emulsified in an equal volume of CFA containing 1 mg / ml Mycobacterium tuberculosis as an immunogen in a PD model.

[0152] Sulzer et al. (Nature 2017;546:656-661) identified two T cell antigenic regions in the N- and C-terminal regions of α-synuclein in human PD patients. The first region is located near the N-terminus and consists of MHCII epitopes aa31-45 (GKTKEGVLYVGSKTK) and aa32-46 (KTKEGVLYVGSKTKE), and also contains the 9-mer polypeptide aa37-45 (VLYVGSKTK) as a potential MHC I class epitope. The second antigenic region disclosed by Sulzer et al. is near the C-terminus (aa116-140) and required phosphorylation of amino acid residue S129. Three phosphorylated aaS129 epitopes, aa116-130 (MPVDPDNEAYEMPSE), aa121-135 (DNEAYEMPSEEGYQD), and aa126-140 (EMPSEEGYQDYEPEA), produced significantly higher responses in PD patients than in healthy controls. The authors also demonstrate that the naturally occurring immune response to PD-associated α-synuclein has both MHC class I and MHC class II restriction components.

[0153] Furthermore, Lindestam Arlehamn et al. (Nat Commun. 1875;2020:11) also disclosed α-synuclein peptide aa61-75 (EQVTNVGGAVVTGVT) as a T cell epitope (MHCII) in PD patients.

[0154] Thus, preferred T cell epitopes according to the invention include the 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).

[0155] Regulatory T cells ("Treg cells" or "Treg") are a subpopulation of T cells that regulate the immune system, maintain tolerance to self-antigens, and prevent autoimmune diseases. Treg cells are immunosuppressive and generally suppress or downregulate the induction and proliferation of effector T cells. Tregs produced by the normal thymus are called "natural". Selection of natural Tregs occurs in radioresistant hematopoietic-derived MHC class II expressing cells in the medulla oblongata or in Hassall's corpuscles in the thymus. The process of Treg selection is determined by the affinity of interaction with self-peptide-MHC complexes. Selection to become a Treg is a "Goldilocks" process - i.e., not too high, not too low, just right - T cells that receive very strong signals undergo apoptotic death, while cells that receive weak signals survive and are selected to become effector cells. T cells become regulatory cells when they receive intermediate signals. Due to the stochastic nature of the process of T cell activation, every T cell population bearing a given TCR ends up with a mixture of Teff and Treg (the relative proportions are determined by the affinity of the T cells for self-peptide MHC).Tregs formed outside the thymus, i.e., by differentiation of peripheral naive T cells or in cell culture, are called "adaptive" or "inducible" (i.e., iTregs).

[0156] Natural Tregs are characterized by the expression of both the CD4 T cell coreceptor and CD25, a component of the IL-2 receptor. Thus, Tregs are CD4+CD25+. Expression of the nuclear transcription factor forkhead box P3 (FoxP3) is a defining characteristic that determines the development and function of natural Tregs. Tregs are thought to suppress the activation, proliferation, and cytokine production of CD4+ and CD8+ T cells, as well as B cells and dendritic cells, thereby dampening autoimmune responses.

[0157] Along these lines, several studies have shown that Treg numbers and function are reduced in PD patients. For example, Hutter Saunders et al. (J Neuroimmune Pharmacol (2012) 7:927-938) and Chen et al. (MOLECULAR MEDICINE REPORTS 12:6105-6111, 2015) showed that regulatory T cells (Tregs) from PD patients exhibited impaired ability to suppress effector T cell function, with increased proportions of Th1 and Th17 cells, while decreased proportions of Th2 and Treg cells. Thome et al. (npj Parkinson's Disease (2021) 7:41) showed that reduced PD Treg function correlated with increased proinflammatory T cell activation that may directly result in subsequent increases in proinflammatory signaling by other immune cell populations. Treg suppression of T cell proliferation was significantly correlated with peripheral proinflammatory immune cell phenotype. The suppressive capacity of PD Tregs against T effector cell (e.g., CD4+) proliferation decreased with increasing PD disease burden using the H&Y disease scale, with the highest activity in stages H+Y 1 and 2. Importantly, Lindestam Arlehamn et al. (2020) showed that anti-aSyn T cell responses were highest before or immediately after diagnosis of motor PD and decreased thereafter (i.e., maximal activity detectable <10 years after diagnosis; and favoring Horne-Yarr (H+Y) stages 1 and 2) (Lindestamn Arlehamn et al. 2020).

[0158] Therefore, the vaccine of the present invention and 1) vaccines containing alpha-synuclein-specific Treg epitopes (e.g., CD4 epitopes such as those disclosed by Brenner et al., Sulzer et al., and Lindestam Arlehamn et al. (aa31-45 (GKTKEGVLYVGSKTK), aa32-46 (KTKEGVLYVGSKTKE), aa61-75 (EQVTNVGGAVVTGVT), aa71-86 (VTGVTAVAQKTVEGAGNIAAATGFVK), aa116-130 (MPVDPDNEAYEMPSE), aa121-135 (DNEAYEMPSEEGYQD), and aa126-140 (EMPSEEGYQDYEPEA)); and / or 2) Treg inducers such as rapamycin, low-dose IL-2, TNF receptor 2 (TNFR2) agonists, anti-CD20 antibodies (e.g., rituximab), prednisolone, inosine pranobex, glatiramer acetate, and sodium butyrate. Combination with aSyn is preferred in the early stages of disease (i.e., less than 10 years after diagnosis; and Horn-Yahr stages 1 and 2 are preferred) to enhance the waning / decline in Treg numbers and activity, thereby reducing the autoimmune reactivity of aSyn-specific T effector cells and attenuating the autoimmune response in PD patients.

[0159] In addition to this, Tregs have been found to be decreased and / or dysfunctional in several diseases, especially in 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), among others (ALS: Beers et al., JCI Insight 2, e89530 (2017); AD: Faridar et al., Brain Commun. 2, fcaa112 (2020); ALS: Beers et al., JAMA Neurol.75, 656-658 (2018); MS: Haas et al., Eur.J.Immunol.35, 3343-3352 (2005); T1D: Lindley et al., Diabetes 54, 92-99 (2005):AID:Putnamet al., J.Autoimmun.24, 55-62 (2005);Autoimmune diseases: Ryba-Stanislawowska et al., Expert Rev.Clin.Immunol.15, 777-789 (2019);aSLE:Valencia et al., J.Immunol.178, 2579-2588 (2007);MS:Vigliettaet al., J.Exp.Med.199, 971-979 (2004);sLE:Zhang et al., Clin.Exp.Immunol.153, 182-187 (2008);AD+MS:Ciccocioppo et al., Sci.Rep.9, 8788 (2019)).

[0160] Therefore, it is also preferred to provide suitable T cell epitopes as Treg epitopes or Treg inducers in diseases with reduced or dysfunctional Treg populations in combination with a vaccine according to the invention in order to enhance the number and activity of depleted / reduced Tregs, thereby reducing the autoimmune reactivity of disease-specific T effector cells and attenuating the autoimmune response in patients. Suitable Treg epitopes are defined as self-MHC epitopes (MHC type II) characterized by their ability to induce intermediate signals during the T cell selection process.

[0161] According to a preferred embodiment, the conjugate according to the invention comprises a polypeptide comprising or consisting of the amino acid sequence(s) SEQ ID NO: 7, 8, 22-29, 87-131, GKTKEGVLYVGSKTK, KTKEGVLYVGSKTKE, ​​EQVTNVGGAVVTGVT, VTGVTAVAQKTVEGAGNIAAATGFVK, MPVDPDNEAYEMPSE), DNEAYEMPSEEGYQD, EMPSEEGYQDYEPEA, or combinations thereof.

[0162] Thus, preferred T cell epitopes are: [Table 1]

[0163] [Table 2]

[0164] According to another preferred embodiment, the conjugate according to the invention comprises a B-cell epitope and a T-cell epitope of alpha-synuclein, preferably a pan-specific / promiscuous T-cell epitope, independently linked to a CLEC polysaccharide scaffold according to the invention, in particular pustulan.

[0165] According to another preferred embodiment, the conjugate according to the invention comprises a B-cell epitope of alpha-synuclein linked to a "classical" carrier protein such as CRM197, this construct being further linked to a CLEC carrier according to the invention, in particular pustulan.

[0166] For example, in the first step, the formation of the CRM conjugate can be performed by activation of CRM, such as via GMBS or sulfo-GMBS; then the maleimide group of the activated CRM is reacted with the SH group of the peptide (cysteine). The CRM conjugate is then treated with DTT to reduce the disulfide bond and generate the SH group on the cysteine. A one-pot reaction may then be performed to mix the reduced CRM conjugate with BMPH (N-β-maleimido-propionic acid hydrazide) and activated pustulan (oxidized) to produce a CLEC-based vaccine. The mechanism in the one-pot reaction may be to react (for pustulan) oxidized pustulan with BMPH (bearing a hydrazide residue) to form BMPH-hydrazone. The reduced CRM conjugate is then reacted with the maleimide of BMPH-activated pustulan via the SH group on the CRM conjugate.

[0167] According to another preferred embodiment, the conjugate according to the invention comprises a "classical" carrier protein, such as CRM197, containing multiple T cell epitopes. The conjugate according to the invention also comprises a B cell epitope covalently linked to the polysaccharide moiety. In this embodiment, both polypeptides (B cell epitope and carrier molecule) are independently linked to the CLEC carrier according to the invention, in particular to pustulan.

[0168] According to another preferred embodiment, the conjugate according to the invention also comprises a "classical" carrier protein, such as CRM197, containing multiple T cell epitopes. The conjugate according to the invention also comprises a B cell epitope covalently linked to the "classical" carrier protein. The peptide-carrier conjugate according to the invention is also covalently linked to the polysaccharide moiety. In this embodiment, both polypeptides (B cell epitope and carrier molecule) are linked as one conjugate to the CLEC carrier according to the invention, in particular to pustulan. The carrier protein therefore represents the link between the β-glucan or mannan and the B cell and / or T cell epitope polypeptide in the conjugate according to the invention. The covalent conjugate formation between the β-glucan or mannan and the B cell and / or T cell epitope polypeptide is therefore carried out by the carrier protein (as a functional linking moiety).

[0169] A preferred conjugate according to the invention may comprise a B cell epitope linked to CRM197, this construct being further linked to a CLEC polymer according to the invention, in particular a β-glucan, which β-glucan is pustulan, lichenan, laminarin, curdlan, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particle (WGP), zymosan or lentinan, preferably pustulan, laminarin, lichenan, lentinan, schizophyllan or scleroglucan, in particular pustulan.

[0170] According to the present invention, a novel B cell epitope-CRM197 conjugate linked to pustulan has been shown to be a potent Dectin-1 binder and induces a very strong immune response, superior to conventional CRM conjugate vaccines.

[0171] According to the present invention, the formation of CLEC conjugates to novel B cell epitope+CRM197 conjugates, in particular the creation of B cell epitope+CRM197+β-glucan, more preferably B cell epitope+CRM197+linear β-(1,6)-glucan or B cell epitope+CRM197+pustulan conjugates, has been shown to be essential for the induction of superior immunogenicity as 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 pustulan conjugates, in comparison to conventional CRM conjugate vaccines, adjuvanted or unadjuvanted, by mixing with β-glucan / pustulan.

[0172] It is therefore a preferred embodiment to provide a B cell epitope+CRM197 conjugate covalently linked to β-glucan, more preferably a peptide+CRM197+linear β-(1,6)-glucan conjugate or a peptide+CRM197+linear pustulan conjugate.

[0173] The present invention therefore also relates to the improvement and / or optimization of a carrier protein (which already contains one or more T cell antigens (optionally in a post-translationally modified form) as part of its polypeptide sequence) by covalently linking the carrier protein to a CLEC polysaccharide adjuvant according to the present invention, i.e. a β-glucan or mannan, preferably pustulan, lichenan, laminarin, curdlan, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particle (WGP), zymosan or lentinan. The present invention therefore relates to a β-glucan or mannan for use as a C-type lectin (CLEC) polysaccharide adjuvant for B-cell and / or T-cell epitope polypeptides of alpha-synuclein, wherein the β-glucan or mannan is covalently linked to the B-cell and / or T-cell epitope polypeptide to form a conjugate of β-glucan or mannan and the B-cell and / or T-cell epitope polypeptide, and the carrier protein is covalently linked to the β-glucan or mannan.

[0174] This improvement / optimization results in a significant reduction or elimination of the B cell response against the CLEC and / or carrier protein and / or an enhancement (or at least maintenance) of the T cell response against the T cell epitopes of the carrier protein, which allows a reduction or elimination of the antibody response against the CLEC and / or carrier (which then results in only a T cell response) and a specific enhancement of the antibody response against the actual target polypeptide bound to the carrier and CLEC.

[0175] Thus, a particularly preferred embodiment of the present invention comprises: (a) β-glucan or mannan (b) at least a B cell epitope polypeptide or a T cell epitope polypeptide, and (c) Carrier protein consisting of or comprising: The three components (a), (b), and (c) are covalently bonded to one another in a conjugate.

[0176] This combination of these three components can be provided in any orientation or order, i.e., (a)-(b)-(c), (a)-(c)-(b), or (b)-(a)-(c), where (b) and / or (c) can be covalently conjugated from N-terminus to C-terminus or C-terminus to N-terminus, or can be conjugated via a functional group in the polypeptide (e.g., via a functional group in a lysine, arginine, aspartic acid, glutamic acid, asparagine, glutamine, serine, threonine, tyrosine, tryptophan, or histidine residue, in particular via the ε-ammonium group of a lysine residue). Of course, β-glucan or mannan can be linked to one or more of each of components (b) and (c), preferably by the methods disclosed herein. Preferably, these components are linked by a linker, in particular by a linker between at least all three components. Preferred linkers are disclosed herein, such as a cysteine ​​residue, or a linker comprising a cysteine ​​or glycine residue, a linker resulting from hydrazide-mediated linkage, a linker resulting from linkage via a heterobifunctional linker such as BMPH, MPBH, EMCH or KMUH, a linker resulting from imidazole-mediated linkage, a linker resulting from reductive amination, a linker resulting from carbodiimide linkage, a -NH-NH2 linker; a peptide linker such as a NRRA linker, a NRRA-C linker, or a NRRA-NH-NH2 linker, a dimeric, trimeric, tetrameric (or longer) peptide group such as CG or CG. In the case of established carrier proteins, particularly CRM, CRM197, and KLH, the preferred order of the at least three components is (a)-(c)-(b), i.e., β-glucan or mannan and at least one B cell epitope polypeptide or one T cell epitope polypeptide are linked to the carrier protein.

[0177] According to another preferred embodiment, the conjugate according to the invention comprises a T cell epitope and no B cell epitope, preferably the conjugate comprises two or more T cell epitopes, in particular two, three, four or five T cell epitopes. This construct is particularly suitable for cancer vaccines. This construct is also particularly suitable for autoantigens, in particular autoantigens associated with autoimmune diseases. The therapeutic effect of the respective conjugate is characterized by a reduction in effector T cells, as shown for example for multiple sclerosis, and an increase in regulatory T cells (T cells), which leads to the suppression of the respective disease, for example an autoimmune disease or an allergic disorder. reg Remarkably, these Treg cells exert a potent bystander immunosuppression, thus ameliorating disease induced by cognate and non-cognate self-antigens.

[0178] Preferred CLECs for use as polysaccharide scaffolds according to the invention are pustulan or other β-(1,6) glucans (including synthetic forms of such glucans); others that may be used are mannans, β-glucan family members, in particular linear β-(1,3) (Saccharomyces cerevisiae β-glucan (e.g. 150 kDa), curdlan) or glucans containing branched β-(1,3) and branched β-(1,6), such as: laminarin (4.5-7 kDa), scleroglucan, schizophyllan, more preferably linear glucans (e.g.: β(1,3): Saccharomyces cerevisiae β-glucan (150 kDa), curdlan (75-80 kDa or more), β-(1,3)+β-(1,4) lichenan (22-250 kDa) β-(1,6) pustulan (20 kDa) Thus, preferred CLECs according to the invention are mannans and β-glucans, including linear and branched β-glucans, characterized by the presence of a β-(1,3)-backbone, a β-(1,3)+β-(1,4)-backbone, and a β(-1,6) backbone, with attached side chains having β-(1,6) residues, more preferably linear β-glucans containing β-(1,3)-, β-(1,3)+β-(1,4) and β-(1,6) chains, more preferably linear β-(1,6) β-glucans, in particular pustulan, fragments thereof, or synthetic variants thereof consisting of polymeric β-(1,6)-glucan saccharides (e.g. 4-mer, 5-mer, 6-mer, 8-mer, 10-mer, 12-mer, 15-mer, 17-mer, or 25-mer).

[0179] Preferably, the minimum length of the CLEC according to the invention is a 6-mer, since smaller polysaccharides would be problematic in the oxidation reaction carried out in the present invention (eventually other linking mechanisms could be used for terminal attachment by addition of such smaller and / or reactive forms). CLECs with 6 or more monomer units (i.e. 6-mers and longer mers) show good dectin binding. Usually, the longer the CLEC, the better the dectin binding. A degree of polymerization (i.e. amount of a single glucose molecule within one glucan entity, DP) of 20-25 (i.e. DP 20-25) clearly confirms good binding and in vivo efficacy (e.g. laminarin is a typical example with DP 20-30).

[0180] The molecular weight of the synthetic CLEC may be smaller, and thus may be for example as low as 1-2 kDa, however preferred molecular weight ranges for glucans and fragments thereof may be 1-250 kDa (e.g. laminarin, lichenan, Saccharomyces cerevisiae β-glucan, pustulan, curdlan, barley glucan, etc.), preferably 4.5-80 kDa (e.g. laminarin, pustulan, curdlan, low molecular weight lichenan, etc.), particularly 4.5-30 kDa (e.g. laminarin, pustulan, low MW lichenan, etc.).

[0181] Mannans are polysaccharides that are linear polymers of the sugar mannose. Plant mannans have β-(1,4) linkages. They are a form of storage polysaccharide. Mannan cell wall polysaccharides found in yeast have an α-(1,6) linked backbone and α-(1,2) and α-(1,3) linked branching. It is serologically similar to the structure found in mammalian glycoproteins.

[0182] To prepare the conjugates according to the invention, the CLECs, especially pustulan, must be activated (e.g., by using mild periodate-mediated oxidation), and the degree of oxidation is important for the immune response. As already disclosed above, a practical oxidation range, especially for pustulan, is about 20-260% oxidation. In many cases, the optimal oxidation range is in the range of low / moderate oxidation (i.e., 20-60% oxidation) to high oxidation (i.e., 140-260% oxidation), i.e., 60-140% oxidation. The optimization of other CLECs can be easily adapted by the skilled person, for example, for lichenan, more than 200% is required to obtain a similar amount of aldehyde groups.

[0183] These ranges may therefore alternatively be defined as reactivity with Schiff's fuchsin reagent, which for example for pustulan can be defined as low / medium oxidation degrees with a molar ratio (sugar monomer:periodic acid) of 0.2-0.6, an optimum range of 0.6-1.4, and a high oxidation degree of 1.4-2.6, respectively.

[0184] In any case, the degree of oxidation should be defined to meet the optimum range of each particular CLEC. Preferably, linear β-glucan, more preferably β-(1,6) β-glucan, in particular pustulan, pustulan fragments, or synthetic variants thereof consisting of polymeric β(1,6)-glucan saccharides (e.g., 4mer, 5mer, 6mer, 8mer, 10mer, 12mer, 15mer, 17mer, or 25mer), is activated by mild periodate oxidation, which results in cleavage of vicinal OH groups, thus generating reactive aldehydes. Mild periodate oxidation refers to the use of sodium periodate (NaIO4), a well-known mild agent to 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 used, aldehydes can be stoichiometrically introduced into a few or many sugar moieties of a given polysaccharide.

[0185] Other exemplary methods for activating carbohydrates are well known in the art and include cyanylation of hydroxyls (e.g., by use of organic cyanylating reagents such as 1-cyano-4-(dimethylamino)-pyridinium tetrafluoroborate (CDAP) or N-cyanotriethylammonium tetrafluoroborate (CTEA)), reductive amination of carbohydrates, or activation and coupling using carboxylic acid reactive chemical groups such as carbodiimides.

[0186] The activated carbohydrate is then reacted with the polypeptide to link it to the activated CLEC, forming a conjugate between the CLEC and the B cell epitope polypeptide or the T cell epitope polypeptide.

[0187] The present invention therefore also relates to a method for producing a conjugate according to the invention, comprising activating a β-glucan or mannan by oxidation and contacting the activated β-glucan or mannan with a B-cell and / or T-cell epitope polypeptide, thereby obtaining a conjugate of β-glucan or mannan with a B-cell and / or T-cell epitope polypeptide.

[0188] Preferably, the β-glucans or mannans are obtained by periodate oxidation at the vicinal hydroxyl groups, by reductive amination, or by cyanylation of the hydroxyl groups.

[0189] According to a preferred embodiment, the β-glucan or mannan is oxidized to a degree of oxidation, defined as the reactivity with Schiff's fuchsin reagent, which corresponds to the degree of oxidation of an equivalent amount of pustulan oxidized with periodic acid in a molar ratio of 0.2 to 2.6, preferably 0.6 to 1.4, in particular 0.7 to 1.

[0190] Preferably, the conjugates are made by hydrazone-based linkages to attach hydrazides to carbonyls (aldehydes), or by using heterobifunctional maleimide-and-hydrazide linkers (e.g., BMPH (N-β-maleimidopropionic acid hydrazide, MPBH (4-[4-N-maleimidophenyl]butyric acid hydrazide), EMCH (N-[ε-maleimidocaproic acid] hydrazide)) or KMUH (N-[κ-maleimidoundecanoic acid] hydrazide)) to attach sulfhydryls (e.g., cysteine) to carbonyls (aldehydes).

[0191] The polypeptide linked to a CLEC according to the invention is or comprises at least one B cell epitope or at least one T cell epitope. Preferably, the polypeptide linked to a CLEC contains a single B cell epitope or T cell epitope (even in embodiments where more than one polypeptide is linked to the CLEC polysaccharide backbone). As also shown in the Examples section, the preferred length of the alpha-synuclein polypeptide is 5-29 amino acid residues long, preferably 5-25 amino acid residues long, more preferably 7-20 amino acid residues long, even more preferably 7-15 amino acid residues long, especially 7-13 amino acid residues long. In this context, it is important to note that these length ranges focus only on the epitope sequence and do not include linkers that include peptide linkers such as cysteine ​​or glycine, or dimeric, trimeric, tetrameric (or longer) peptide groups such as CG or CG, or cleavage sites such as cathepsin cleavage sites, or combinations thereof (e.g. NRRAC). Illustrative examples of epitopes are examined in the Examples section. These results indicate that the platform according to the invention is not limited to any particular polypeptide, and therefore virtually all possible epitopes are eligible for the present invention, including epitopes already known in the art, particularly epitopes already described as being capable of being incorporated into a presentation platform (e.g., together with "classical" carrier molecules or adjuvants).

[0192] It is particularly preferred if the epitope can be linked to the activated β-glucan based on prior art linking methods including hydrazide mediated linkage, linkage via a heterobifunctional linker (e.g. BMPH, MPBH, EMCH, KMUH, etc.), imidazole mediated linkage, reductive amination, carbodiimide linkage, etc. (further additions) The epitopes used include individual peptides, can be contained within a peptide or protein, or can be provided as a peptide-protein conjugate prior to linkage to the CLEC.

[0193] Thus, preferred linking methods used to provide conjugates according to the invention are hydrazide linkages or linkages using thioester formation (e.g. maleimide linkages using BMPH (N-β-maleimidopropionic acid hydrazide), MPBH, EMCH, KMUH, particularly where pustulan is linked to BMPH via hydrazone formation and the polypeptide is linked via a thioester).

[0194] In this embodiment, a polypeptide having two preferred linkers, such as hydrazide polypeptides / epitopes for hydrazone linkage, i.e. N-terminal linkage of peptides: H2N-NH-CO-CH2-CH2-CO-polypeptide-COOH, preferably in combination with succinic acid or an alternative suitable linker, such as other suitable dicarboxylic acids, especially glutaric acid, used as spacer / linker; C-terminal linkage (preferred linkage orientation according to the present invention): NH2-polypeptide-NH-NH2 It is preferred to provide:

[0195] Alternatively, unmodified alpha-synuclein polypeptides / epitopes may be applied in the present invention, e.g. polypeptides containing an (extra) cysteine ​​residue or an alternative source of SH group at either the C-terminus or N-terminus for heterobifunctional linker mediated linkage (especially BMPH, MPBH, EMCH, KMUH): NH2-Cys-Pep-COOH or NH2-Pep-Cys-COOH.

[0196] Preferred B cell polypeptides for use according to the invention are polypeptides having a length of 5 to 19 amino acid residues, preferably 6 to 18 amino acid residues, in particular 7 to 15 amino acid residues. B cell epitopes are preferably short linear polypeptides, glycopolypeptides, lipopolypeptides, other post-translationally modified polypeptides (e.g. phosphorylated, acetylated, nitrated, containing pyroglutamic acid residues, glycosylated, etc.), cyclic polypeptides, etc.

[0197] Preferred T cell polypeptides for use in accordance with the invention have a length of from 8 to 30 amino acid residues, preferably from 13 to 29 amino acid residues, more preferably from 13 to 28 amino acid residues.

[0198] Preferred specificities of the T cell epitopes used in the present invention are short linear peptides suitable or known to be suitable for presentation via MHC I and MHC II (known to the skilled artisan), in particular MHC II epitopes of CD4 effector T cells and CD4 Treg cells, cytotoxic T cells (CD8+) and CD8 T cells, useful for cancer, autoimmune diseases or infectious diseases, with known efficacy in humans or animals. MHCI epitopes for Treg cells; short linear peptides suitable for presentation via MHCI and MHCII appended at the N- or C-terminus (known to those skilled in the art) with lysosomal protease cleavage sites, in particular cathepsin protease family member specific sites, more particularly sites for cysteine ​​cathepsins such as cathepsin B, C, F, H, K, L, O, S, V, X, and W, in particular cathepsin S- or L-, most preferably cathepsin L cleavage sites, with known efficacy in humans or animals, that facilitate efficient endosomal / lysosomal release of the peptide for MHC presentation, in particular MHCII presentation. Cathepsin cleavage sites in various proteins have been identified and are well known in the art. This includes disclosures of sequences or methods for identifying such sequences, for example: Biniossek et al., J.Proteome Res.2011, 10, 12, 5363-5373; Adams-Cioaba et al., Nature Comm.2011, 2:197; Ferrall-Fairbanks PROTEIN SCIENCE 2018 VOL 27:714-724; Kleine-Weber et al., Scientific Reports (2018) 8:1659, https: / / en.wikipedia.org / wiki / Cathepsin_S. In particular, the adaptation of peptide sequences with artificial protease cleavage sites as presented in the present invention is based on the surprising effect of these sequence extensions in eliciting a more efficient immune response after dermal administration of a CLEC vaccine according to the present invention when the antigen is linked to a CLEC. The vaccine according to the present invention is taken up by DCs, after which the peptide antigen is processed in lysosomes and presented on MHC.

[0199] Lysosomes are intracellular membrane-bound organelles characterized by an acidic interior and contain a variety of hydrolases, including lipases, proteases, and glycosidases, involved in cellular catabolism. Among the various enzymes that lysosomes possess, cathepsins are a family of lysosomal proteases with a wide range of functions. All cathepsins are classified into three different protease families: serine proteases (cathepsin A and cathepsin G), aspartic proteases (cathepsin D and cathepsin E), and eleven cysteine ​​cathepsins. In humans, eleven cysteine ​​cathepsins with structures similar to that of papain are known, namely B, C (J, dipeptidyl peptidase I, or DPPI), F, H, K (O2), L, O, S, V (L2), X (P, Y, Z), and W (lymphopain).

[0200] Cathepsins show similarities in their cellular localization and biosynthesis, with some differences in their expression patterns. Among all lysosomal proteases, cathepsins L, B, and D are the most abundant, with their lysosomal concentration equal to 1 mM. Cathepsins B, H, L, C, X, V, and O are ubiquitously expressed, whereas 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.

[0201] In addition to their primary function in lysosomal protein recycling, cathepsins play important roles in a variety of physiological processes. Cathepsin S is the major protease involved in the processing and presentation of MHC II Ag. Cathepsin S-deficient mice show marked variations in the generation and presentation of MHC II-bound li fragments due to a substantial reduction in li degradation in professional APCs, where cathepsin S is abundantly expressed. Furthermore, endocytosis selectively targets exogenous material to cathepsin S in human DCs. Enrichment of MHC II molecules within late endocytic structures has also been consistently observed in splenic DCs from cathepsin S-deficient mice. Recent studies have also suggested that both cathepsin B and cathepsin D are involved, but not essential, in MHC II-mediated Ag presentation. Cathepsin L also plays a role in a wide variety of cellular processes, including antigen processing, tumor invasion and metastasis, bone resorption, and turnover of intracellular and secreted proteins involved in growth regulation. Although generally recognized as a lysosomal protease, cathepsin L is also secreted. This broad-spectrum protease is potent in degrading several extracellular proteins (laminin, fibronectin, collagen I and collagen IV, elastin, and other structural proteins of the basement membrane), as well as serum proteins and cytoplasmic and nuclear proteins.

[0202] As a novel means to enhance the efficacy of T cell epitopes in vaccines, particularly CLEC-based vaccines, N- or C-terminal addition of lysosomal protease cleavage sites is provided as a preferred embodiment of the present invention.

[0203] Such cleavage sites according to the invention can be characterized as follows.

[0204] Cathepsin L-like cleavage site: The intended cathepsin L-like cleavage site is defined based on protease cleavage site sequences known by those skilled in the art, in particular those disclosed in Biniossek et al. (J. Proteome Res. 2011, 10, 5363-5373) and Adams-Cioaba et al. (Nature Comm. 2011, 2:197). The orientation of the site can be N-terminal or C-terminal, preferably C-terminal. A preferred consensus sequence for a C-terminal cathepsin L site consists of the following formula:

[0205] X n -X1-X2-X3-X4-X5-X6-X7-X8 X n : 3 to 27 amino acids derived from an immunogenic peptide X1: any amino acid X2: Any amino acid X3: Any amino acid X4: N / D / A / Q / S / R / G / L; preferably N / D, more preferably N X5: F / R / A / K / T / S / E; preferably F or R, more preferably R X6: F / R / A / K / V / S / Y; preferably F or R, more preferably R X7: any amino acid, preferably A / G / P / F, more preferably A X8: Linker such as cysteine ​​or NHNH2 Most preferred sequence: X n -X1X2X3NRRA-Linker

[0206] Cathepsin S-like cleavage site: The contemplated cathepsin S cleavage sites are based on protease cleavage site sequences known by those of skill in the art, in particular those disclosed in Biniossek et al. (J. Proteome Res. 2011, 10, 5363-5373) and also in https: / / en.wikipedia.org / wiki / Cathepsin_S, and are characterized by the following consensus sequence:

[0207] Xn -X1-X2-X3-X4-X5-X6-X7-X8 X is characterized by X n : 3 to 27 amino acids derived from an immunogenic peptide X1: any amino acid X2: Any amino acid X3: any amino acid, preferably V, L, I, F, W, Y, H, more preferably V X4: any amino acid, preferably V, L, I, F, W, Y, H, more preferably V X5: K, R, E, D, Q, N, preferably K, R, more preferably R X6: Any amino acid X7: any amino acid, preferably A X8:Preferably A X8: Linker such as cysteine ​​or NHNH2 Most preferred sequence: X n -X1X2VVRAA-Linker

[0208] The protein is a carrier protein, in particular a toxic cross-reacting substance (CRM) of diphtheria toxin, in particular the CRM 197 , KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), and outer membrane protein complex (OMPC) of serogroup B Neisseria meningitidis, recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A (rEPA), flagellin, Escherichia coli heat-labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72), virus-like particles, albumin-binding proteins, bovine serum albumin, ovalbumin, synthetic peptide deoxyribonuclease (DR), and erythropoietin (ER) are also included. T cell epitopes contained within a protein suitable for conjugation to CLECs including endrimers, such as multiple antigenic peptides (MAPs), or other commercially available carrier proteins, preferably CRM197 and KLH, most preferably CRM197, wherein the ratio of carrier protein to β-glucan 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, in particular 1 / 0.1 to 1 / 10.

[0209] According to a preferred embodiment of the invention, the CLEC conjugates according to the invention comprise: (a) CLECs bound to individual B-cell epitopes and / or T-cell epitopes of alpha-synuclein, including mixtures of B-cell epitopes or T-cell epitopes, in particular these epitopes linked to pustulan; (b) CLECs bound to a polypeptide-carrier protein conjugate linked to pustulan, preferably a polypeptide-KLH conjugate or a polypeptide-CRM197 conjugate, most preferably a polypeptide-CRM197 conjugate linked to pustulan; (c) CLECs, most preferably CLECs bound to individual B-cell epitopes and T-cell epitopes of alpha-synuclein, linked to pustulan; (d) a polypeptide or protein, such as a carrier protein, autologous (e) CLECs individually ("individually" having the same meaning as (d)) linked to B cell epitopes and T cell epitopes contained within a protein, a foreign protein from a pathogen, an allergen, etc. (where "individually" means that the polypeptide chains are present as separate entities and not as a fusion protein, tandem repeat polypeptide, or peptide-protein conjugate; i.e., separate B cell epitope-containing polypeptides and separate T cell epitope-containing polypeptides); (f) CLECs individually ("individually" having the same meaning as (d)) linked to T cell epitopes represented by linear MHCCI epitopes and linear MHCII epitopes, or contained within a protein, e.g., a carrier protein or a target protein for treating, e.g., a neoplastic disease, or an autoimmune disease.

[0210] Considering these advantageous properties of the conjugates of the invention, the conjugates and vaccines according to the invention are particularly usable for active anti-alpha-synuclein vaccines for treating and preventing synucleinopathies.

[0211] According to a further aspect, the present invention also relates to a pharmaceutical composition comprising a conjugate or a vaccine as defined above and a pharma- ceutically acceptable carrier.

[0212] Preferably, the pharma- ceutically acceptable carrier is a buffer, preferably a phosphate or TRIS-based buffer.

[0213] According to a preferred embodiment of the present invention, the pharmaceutical composition is contained in a needle-based delivery system, preferably a syringe, a mini-needle system, a hollow needle system, a solid microneedle system, or a system with a needle adapter; an ampoule, a needle-free injection system, preferably a jet injector; a patch, a transdermal patch, a microstructured transdermal system, a microneedle array patch (MAP), preferably a solid MAP (S-MAP), a coated MAP (C-MAP), or a dissolved MAP (D-MAP); an electrophoretic system, an iontophoretic system, a laser-based system, in particular an Erbium YAG laser system; or a gene gun system. The conjugate according to the present invention is not limited to any form of preparation, storage, or delivery. Therefore, all traditional and typical forms are compatible with the present invention. Preferably, the composition according to the present invention may contain the conjugate or vaccine contained as a solution or suspension, a deep-frozen solution or a deep-frozen suspension; a lyophilisate, a powder, or a granule.

[0214] The present invention is further illustrated by the following examples and figures, without however being limited thereto. [Brief description of the drawings]

[0215] [Figure 1A] 1A-1F show the in vitro binding activity of CLEC conjugates to ConA and DC receptors (i.e., dectin-1), demonstrating higher binding efficacy to dectin-1 for pustulan (Pus) compared to lichenan (Lich). [Figure 1B] β-glucans derived from oats (oat_BG265, oat_BG391) and barley (barley_BG229) showed limited binding effectiveness compared to pustulan. [Figure 1C]Different glucan types (i.e., pustulan, mannan, and barley glucan (229 kd)) retain high or intermediate receptor binding activity after glucan oxidation as assessed by competitive binding assays. "20% oxidized and 40% oxidized" indicate the oxidation state of the glucan moiety used for conjugate formation. Inhibition (%) indicates inhibition of binding of soluble Dectin-1 receptor (pustulan and barley_BG229) or ConA (mannan) to plate-bound β-glucan or mannan in the presence of the indicated concentrations of test CLEC. [Figure 1D] Pustulan-conjugates retain approximately 50% of the Dectin-1 binding capacity compared to unlinked β-glucan as assessed by competitive binding assays. [Figure 1E] The lichenan-conjugates retain approximately 50% of the Dectin-1 binding capacity compared to unconjugated β-glucan as assessed by competitive binding assays. [Figure 1F] Pustulan-conjugates made via heterobifunctional linkers maintain high Dectin-1 binding efficacy. Data are shown as relative light units (RLU) of luminescence ELISA. Pus70 conjugates 1-3 refer to three different CLEC peptide conjugates (SEQ ID NO:2, SEQ ID NO:10, and SEQ ID NO:16), respectively. Pus 70% and Lich 200% refer to pustulan and lichenan with their respective oxidation states. BMPH Pus refers to activated pustulan. BMPH conjugate 2 refers to the CLEC-SEQ ID NO:10 conjugate.

[0216] [Figure 2A]2A-D show flow cytometric analysis of dendritic cell activation by lipopolysaccharide (LPS) and different pustulan preparations. Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 h with LPS (equal doses were contained in the oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each saccharide (up to 500 μg / mL) starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression and surface expression of CD80 by SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates was measured by flow cytometry. DCs treated with pustulan preparations (=measured) and with an equivalent amount of LPS (=predicted) were analyzed for the expression of activation markers by CytExpert software. [Figure 2B] Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 h with LPS (equal doses contained in oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each sugar (up to 500 μg / mL) starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression and surface expression of CD80 with oxidized pustulan alone was measured by flow cytometry. DCs treated with pustulan preparations (=measured) and DCs treated with an equal amount of LPS (=predicted) were analyzed for expression of activation markers by CytExpert software. [Figure 2C]Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 h with LPS (equal doses contained in oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each sugar (up to 500 μg / mL) starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression and major histocompatibility complex (MHC) class II surface expression by SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates was measured by flow cytometry. DCs treated with pustulan preparations (= measured) and DCs treated with an equal amount of LPS (= predicted) were analyzed for expression of activation markers by CytExpert software. [Figure 2D] Immature bone marrow-derived murine dendritic cells (BMDCs) were generated in vitro using granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF-BMDCs were stimulated for 24 h with LPS (equal doses contained in oxidized pustulan and pustulan conjugate preparations), SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates, or oxidized pustulan alone. Pustulan conjugates and pustulan alone were used at increasing doses of each sugar (up to 500 μg / mL) starting at 62.5 μg / mL. DCs were identified based on CD11c / CD11b expression and major histocompatibility complex (MHC) class II surface expression with oxidized pustulan alone was measured by flow cytometry. DCs treated with pustulan preparations (=measured) and DCs treated with an equal amount of LPS (=predicted) were analyzed for expression of activation markers by CytExpert software.

[0217] [Figure 3A]3A-3C show particle size determination of CLEC conjugates by dynamic light scattering (DLS). Particle size was determined by DLS by measuring random changes in the intensity of light scattered from a suspension or solution. Regularization analysis and corresponding cumulant radius analysis over 24 hours for SEQ ID NO:5 + SEQ ID NO:7 + pustulan (80% oxidation state) conjugate are shown. [Figure 3B] Particle size was determined by measuring random changes in the intensity of light scattered from a suspension or solution by DLS. A regularization analysis and corresponding cumulant radius analysis over a 24 hour period for the SEQ ID NO:6 + CRM + pustulan conjugate is shown. [Figure 3C] Particle size was determined by measuring random changes in the intensity of light scattered from a suspension or solution by DLS. A regularization analysis and corresponding cumulant radius analysis over a 24 hour period for unmodified pustulan is shown.

[0218] [Figure 4A] Figures 4A and 4B show a comparison of immunogenicity of different CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at two-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination and analyzed for anti-peptide responses to mannan-based vaccine, barley-based vaccine, and pustulan-based vaccine (SEQ ID NO:2 + SEQ ID NO:7 + CLEC). [Figure 4B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination and analyzed for anti-peptide responses (SEQ ID NO:3 and SEQ ID NO:11) to the pustulan-based and lichenan-based vaccines (SEQ ID NO:2 + SEQ ID NO:7 + CLEC and SEQ ID NO:10 + SEQ ID NO:7 + CLEC).

[0219] [Diagram 5] Figure 5 shows a comparative immunogenicity analysis of a vaccine consisting of peptide-pustulan conjugates and unconjugated peptide and CLEC. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were taken 2 weeks after the third vaccination and analyzed for anti-peptide responses (SEQ ID NO:3). Vaccines used: SEQ ID NO:2 + SEQ ID NO:7 + CLEC, or a mixture of unconjugated SEQ ID NO:2, SEQ ID NO:7, and CLEC.

[0220] [Figure 6] FIG. 6 shows a comparative analysis of the immunogenicity of pustulan conjugates containing B and T cell epitopes versus conjugates containing only either B or T cell epitopes. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Vaccines used: SEQ ID NO:5 + SEQ ID NO:7 + CLEC, or SEQ ID NO:5 + CLEC, and SEQ ID NO:7 + CLEC. Samples were taken 2 weeks after the third dose and analyzed for anti-peptide responses (SEQ ID NO:6).

[0221] [Figure 7A]Figures 7A-7C show comparative analysis of anti-pustulan antibody responses in mice after repeated immunization with vaccines containing peptide-pustulan conjugates or the individual components unconjugated. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Pre-plasma and t1-t3 represent the immune response detectable before immunization (pre-plasma) or after the first (t1), second (t2), or third (t3) immunization. Samples were taken 2 weeks after the third dose and analyzed for anti-pustulan responses. Analysis of anti-pustulan responses induced by different vaccines. Vaccines used: SEQ ID NO:2 + SEQ ID NO:7 + CLEC or a mixture of SEQ ID NO:2, SEQ ID NO:7, and CLEC unconjugated. [Figure 7B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Pre-plasma and t1-t3 represent the detectable immune response before immunization (pre-plasma) or after the first (t1), second (t2), or third (t3) immunization. Kinetics of immune response. Vaccines used: SEQ ID NO:2 + SEQ ID NO:7 + CLEC, or a mixture of SEQ ID NO:2, SEQ ID NO:7, and CLEC unconjugated. [Figure 7C] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Pre-plasma and t1-t3 represent detectable immune responses before immunization (pre-plasma) or after the first (t1), second (t2), or third (t3) immunization. Inhibition ELISA demonstrating the specificity of the ELISA system. Vaccines used: SEQ ID NO:2 + SEQ ID NO:7 + CLEC, or a mixture of SEQ ID NO:2, SEQ ID NO:7, and CLEC unconjugated.

[0222] [Figure 8A]Figures 8A and 8B show comparative analysis of immune responses elicited by CLEC-based vaccines using different peptide linkage orientations. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the ensuing immune response. Four different CLEC-based prototype vaccine candidates (two different peptides linked to pustulan via the C-terminus or N-terminus) were tested. Samples were collected 2 weeks after the third dose and analyzed for anti-peptide responses. Vaccines used: SEQ ID NO:1 / 2 / 4 / 5 + SEQ ID NO:7 + CLEC. [Figure 8B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Four different CLEC-based prototype vaccine candidates (two different peptides linked to pustulan via the C-terminus or N-terminus) were tested. Samples were collected 2 weeks after the third dose and analyzed for anti-aSyn protein responses. Vaccine used: SEQ ID NO:1 / 2 / 4 / 5 + SEQ ID NO:7 + CLEC.

[0223] [Figure 9A] 9A and 9B show comparative immunogenicity analysis of CLEC-based vaccines using different promiscuous T-helper cell epitopes. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by nine different CLEC-based vaccines (Vaccine 1-9) containing the same B cell epitope and different T helper epitopes (i.e., SEQ ID NO: 7, SEQ ID NO: 22-29) were evaluated against the respective peptide-KLH conjugate (Vaccine 10), respectively. Samples were collected 2 weeks after the third dose and analyzed for anti-peptide responses. [Figure 9B]Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by nine different CLEC-based vaccines (Vaccine 1-9) containing the same B cell epitope and different T helper epitopes (i.e., SEQ ID NO: 7, SEQ ID NO: 22-29) were evaluated against the respective peptide-KLH conjugate (Vaccine 10), respectively. Samples were collected two weeks after the third dose and analyzed for anti-aSyn protein responses.

[0224] [Figure 10A] 10A and 10B show comparative analysis of target-specific and carrier protein-specific immunogenicity induced by CLEC-based and conventional peptide-protein conjugate vaccines using the carrier protein KLH as a source of T-helper cell epitopes. Female BALB / c mice aged 8-12 weeks were given a total of three intradermal or subcutaneous (sc) vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by two peptide-protein conjugate vaccines using KLH as a source of T-helper epitopes in combination with CLEC modification (SEQ ID NO:3+KLH+pustulan and SEQ ID NO:6+KLH+pustulan, respectively) were evaluated against responses induced by conventional peptide-KLH conjugates (i.e., SEQ ID NO:3+KLH and SEQ ID NO:6+KLH) administered sc with Alum / Alhydrogel or id without additional adjuvant. Samples were taken two weeks after the third dose and analyzed for anti-peptide and anti-aSyn protein responses by ELISA. [Figure 10B]Female BALB / c mice aged 8-12 weeks were given a total of three intradermal or subcutaneous (sc) vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by two peptide-protein conjugate vaccines using KLH as a source of T-helper epitopes in combination with CLEC modification (SEQ ID NO:3+KLH+pustulan and SEQ ID NO:6+KLH+pustulan, respectively) were evaluated against responses induced by conventional peptide-KLH conjugates (i.e., SEQ ID NO:3+KLH and SEQ ID NO:6+KLH) administered sc with Alum / Alhydrogel or id without additional adjuvant. Samples were collected 2 weeks after the third dose and analyzed for anti-KLH responses by ELISA.

[0225] [Figure 11A] 11A and 11B show comparative analysis of target-specific and carrier protein-specific immunogenicity induced by CLEC-based and conventional peptide-protein conjugate vaccines using carrier protein CRM197 as a source of T helper cell epitopes. Female BALB / c mice aged 8-12 weeks were given a total of three intradermal or sc vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two different CRM-based vaccine types were used in this study. SEQ ID NO:6+CRM+Pus represents a peptide-CRM conjugate that was subsequently linked to pustulan, and SEQ ID NO:5+CRM+Pus represents a conjugate in which the peptide components and the carrier molecule were individually linked to CLEC. The immune responses induced by both types were evaluated against the respective conventional peptide-CRM conjugates (i.e., SEQ ID NO:6+CRM administered sc with alum / Alhydrogel adjuvant). Samples were taken two weeks after the third dose and analyzed for anti-peptide and anti-aSyn protein responses by ELISA. [Figure 11B]Female BALB / c mice aged 8-12 weeks were given a total of three intradermal or sc vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two different CRM-based vaccine types were used in this study: SEQ ID NO:6+CRM+Pus represents a peptide-CRM conjugate that was subsequently linked to pustulan, and SEQ ID NO:5+CRM+Pus represents a conjugate in which the peptide components and the carrier molecule were individually linked to CLEC. The immune responses induced by both types were evaluated against the respective conventional peptide-CRM conjugates (i.e., SEQ ID NO:6+CRM adjuvanted with alum / Alhydrogel and administered sc). Samples were taken 2 weeks after the third dose and analyzed for anti-CRM responses by ELISA.

[0226] [Figure 12] FIG. 12 shows a comparative analysis of the selectivity of immune responses elicited by CLEC-based vaccines in vivo against two different aSyn forms. Female BALB / c mice aged 8-12 weeks were given a total of three intradermal or sc vaccinations administered at two-week intervals. CLEC-based vaccines (SEQ ID NO:2+SEQ ID NO:7+Pus and SEQ ID NO:5+SEQ ID NO:7+Pus; id administration) and alternative CLEC-based vaccines (SEQ ID NO:3+KLH+Pus and SEQ ID NO:6+CRM+Pus; id administration) were evaluated against the conventional peptide component vaccine (SEQ ID NO:3+KLH+Alum and SEQ ID NO:6+CRM+Alum, sc administration). Samples were taken two weeks after the third dose and subjected to aSyn selectivity assay (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0227] [Figure 13]FIG. 13 shows a comparative avidity analysis of immune responses elicited by CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks were given a total of three intradermal or sc vaccinations administered at two-week intervals. CLEC-based vaccines (SEQ ID NO:2+SEQ ID NO:7+Pus and SEQ ID NO:5+SEQ ID NO:7+Pus, id administered) and alternative CLEC-based vaccines (SEQ ID NO:3+KLH+Pus and SEQ ID NO:6+CRM+Pus, id administered) were evaluated against a conventional peptide component vaccine (SEQ ID NO:3+KLH+Alum and SEQ ID NO:6+CRM+Alum, sc administered). Samples were taken 2 weeks after the second immunization (T2) or 2 weeks after the third immunization (T3) and antibody avidity to aSyn was assessed by an ELISA-based avidity assay.

[0228] [Figure 14] FIG. 14 shows a comparative analysis of the affinity of immune responses elicited by CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks were given a total of three intradermal or sc vaccinations administered at two-week intervals. CLEC-based vaccines (SEQ ID NO:2+SEQ ID NO:7+Pus and SEQ ID NO:5+SEQ ID NO:7+Pus, administered id) and alternative CLEC-based vaccines (SEQ ID NO:3+KLH+Pus and SEQ ID NO:6+CRM+Pus, administered id) were evaluated against a conventional peptide component vaccine (SEQ ID NO:3+KLH+Alum and SEQ ID NO:6+CRM+Alum, administered sc). Samples were taken two weeks after the third dose and antibody equilibrium dissociation constants (Kd) for aSyn were assessed by aSyn displacement ELISA assay.

[0229] [Figure 15A]Figures 15A and 15B show comparative analysis of in vitro functionality of immune responses elicited by CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three intradermal and sc vaccinations administered at 2-week intervals. Samples were taken 2 weeks after the third vaccination and modulation of aSyn aggregation in the presence of aSyn-specific Abs was assessed by ThT fluorescence assay. aSyn was aggregated for 0-72 h in the presence of CLEC vaccine-induced Abs (SEQ ID NO:2 + SEQ ID NO:7 + Pus; administered id), conventional peptide component-induced Abs (SEQ ID NO:3 + KLH + Alum, administered sc), or mouse plasma. Kinetic curves were calculated by normalization of ThT fluorescence at t0, and inhibition of aSyn aggregation (%) was calculated using slope values ​​extracted from linear regression analysis during the exponential growth phase of ThT kinetics. [Figure 15B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal and sc vaccinations administered at 2-week intervals. Samples were taken 2 weeks after the third vaccination and modulation of aSyn aggregation in the presence of aSyn-specific Abs was assessed by ThT fluorescence assay. aSyn or aSyn with preformed fibrils was allowed to aggregate for 0-92 h in the presence of CLEC vaccine-induced Abs (SEQ ID NO:5+SEQ ID NO:7+Pus and SEQ ID NO:6+CRM+Pus, both administered id), conventional peptide component-induced Abs (SEQ ID NO:6+CRM+Alum, administered sc), or mouse plasma. Kinetic curves were calculated by normalization of ThT fluorescence at t0, and inhibition of aSyn aggregation (%) was calculated using slope values ​​extracted from linear regression analysis during the exponential growth phase of ThT kinetics.

[0230] [Figure 16A]16A and 16B show comparative analysis of the effect of immunization routes on immune responses elicited by CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two alternative routes, including subcutaneous (sc) and intramuscular (im), were compared with intradermal (id) administration for the CLEC-based vaccine. Three doses of the CLEC-based vaccine (SEQ ID NO:2 + SEQ ID NO:7 + Pus) were administered per route. Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide responses. [Figure 16B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two alternative routes, including subcutaneous (sc) and intramuscular (im), were compared with intradermal (id) administration for the CLEC-based vaccine. Three doses of the CLEC-based vaccine (SEQ ID NO:2 + SEQ ID NO:7 + Pus) were administered per route. Samples were collected 2 weeks after the third vaccination and analyzed for anti-aSyn protein responses.

[0231] [Figure 17]Figure 17 shows a comparative analysis of target protein specific immunogenicity induced by CLEC-based peptide-CRM197 conjugate vaccines using different peptide-CRM197 / CLEC ratios. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Five different peptide-CRM based vaccines were used in this study, applying different peptide-CRM / pustulan ratios (w / w). All five groups were immunized using SEQ ID NO:6+CRM+Pus conjugate. 1:1, 1:2.5, 1:5, 1:10, and 1:20 represent conjugates with w / w peptide-CRM conjugate / CLEC ratios of 1 / 1, 1 / 2.5, 1 / 5, 1 / 10, and 1 / 20. The induced immune response was assessed using samples taken 2 weeks after the third dose and analyzed for anti-aSyn protein responses by ELISA. Titer determination was based on ODmax / 2 calculation.

[0232] [Figure 18A] Figures 18A and 18B show comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitopes (aa1-8). Female BALB / c mice aged 8-12 weeks were vaccinated (id and sc) for a total of three times, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:12 + SEQ ID NO:7 + pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:13 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for A) anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 18B]Female BALB / c mice aged 8-12 weeks were vaccinated with a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. A CLEC-based vaccine (SEQ ID NO:12 + SEQ ID NO:7 + pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:13 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for A) anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0233] [Figure 19A] Figures 19A and 19B show comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitopes (aa100-108). Female BALB / c mice aged 8-12 weeks were vaccinated (id and sc) for a total of three times, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:16 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:17 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for A) anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 19B]Female BALB / c mice aged 8-12 weeks were vaccinated with a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. A CLEC-based vaccine (SEQ ID NO:16 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:17 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0234] [Figure 20] FIG. 20 shows a comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitopes (aa91-97). Female BALB / c mice aged 8-12 weeks were vaccinated (id and sc) for a total of three doses administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. A CLEC-based vaccine (SEQ ID NO:14 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:15 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected two weeks after the third dose and analyzed for anti-peptide and anti-aSyn protein responses.

[0235] [Figure 21A]Figures 21A and 21B show comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitope (aa130-140). Female BALB / c mice aged 8-12 weeks were vaccinated (id and sc) for a total of three times, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The CLEC-based vaccine (SEQ ID NO:20 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:21 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for anti-peptide and anti-aSyn protein responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 21B] Female BALB / c mice aged 8-12 weeks were vaccinated with a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. A CLEC-based vaccine (SEQ ID NO:20 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:21 conjugated to KLH and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0236] [Figure 22A]Figures 22A and 22B show comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitope (aa115-122). Female BALB / c mice aged 8-12 weeks were vaccinated (id and sc) for a total of three times, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. CLEC-based vaccines (SEQ ID NO:51 + SEQ ID NO:7 and pustulan, id) were evaluated against conventional peptide component conjugate-based vaccines (SEQ ID NO:52 conjugated to CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for anti-peptide and anti-aSyn fibril responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 22B] Female BALB / c mice aged 8-12 weeks were vaccinated with a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. CLEC-based vaccines (SEQ ID NO:51 + SEQ ID NO:7 and pustulan, id) were evaluated against conventional peptide component conjugate-based vaccines (SEQ ID NO:52 conjugated to CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0237] [Figure 23A]Figures 23A and 23B show comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitope (aa115-124). Female BALB / c mice aged 8-12 weeks were vaccinated (id and sc) for a total of three times, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. CLEC-based vaccines (SEQ ID NO:67 + SEQ ID NO:7 and pustulan, id) were evaluated against conventional peptide component conjugate-based vaccines (SEQ ID NO:68 conjugated to CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for anti-peptide and anti-aSyn fibril responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 23B] Female BALB / c mice aged 8-12 weeks were vaccinated with a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. A CLEC-based vaccine (SEQ ID NO:67 + SEQ ID NO:7 and pustulan, id) was evaluated against a conventional peptide component conjugate-based vaccine (SEQ ID NO:68 conjugated to CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0238] [Figure 24A]Figures 24A and 24B show comparative analysis of immune responses elicited by CLEC vaccines containing aSyn-derived B cell epitope (aa107-113). Female BALB / c mice aged 8-12 weeks were vaccinated (id and sc) for a total of three times, administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. CLEC-based vaccines (SEQ ID NO:73 + SEQ ID NO:7 and pustulan, id) were evaluated against conventional peptide component conjugate-based vaccines (SEQ ID NO:74 conjugated to CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for anti-peptide and anti-aSyn fibril responses. Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition. [Figure 24B] Female BALB / c mice aged 8-12 weeks were vaccinated with a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. CLEC-based vaccines (SEQ ID NO:73 + SEQ ID NO:7 and pustulan, id) were evaluated against conventional peptide component conjugate-based vaccines (SEQ ID NO:74 conjugated to CRM and Alhydrogel (Alum), sc). Samples were collected 2 weeks after the third dose and analyzed for aSyn selectivity (inhibition ELISA). Black line: monomeric aSyn used for inhibition; dashed line: fibrillar aSyn used for inhibition.

[0239] [Figure 25A]25A-D show comparative in vitro functionality analysis of immune responses elicited by CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination and ThT kinetic measurements (i.e., fibrillar fraction of aSyn) were assessed in the presence of CLEC vaccine-induced Abs (SEQ ID NO:67 + SEQ ID NO:7 and pustulan, id) or conventional peptide component-induced Abs (SEQ ID NO:68 bound to CRM and Alhydrogel, sc). [Figure 25B] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination to assess ThT kinetic measurements (i.e., fibrillar fraction of aSyn) in the presence of A-C) CLEC vaccine-induced Abs (SEQ ID NO:71 + SEQ ID NO:7 and pustulan, id) or conventional peptide component-induced Abs (SEQ ID NO:72 bound to CRM and Alhydrogel, sc). [Figure 25C] Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination to assess ThT kinetic measurements (i.e., fibrillar fraction of aSyn) in the presence of A-C) CLEC vaccine-induced Abs (SEQ ID NO:73 + SEQ ID NO:7 and pustulan, id) or conventional peptide component-induced Abs (SEQ ID NO:74 bound to CRM and Alhydrogel, sc). [Figure 25D]Female BALB / c mice aged 8-12 weeks received a total of three vaccinations (id and sc) administered 2 weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination and ThT kinetic measurements (i.e., fibrillar fraction of aSyn) were assessed in the presence of aSyn-specific monoclonal Ab LB09 or naive mouse plasma.

[0240] [Figure 26A] Figures 33A-26D show mouse DC receptor (i.e. Dectin-1) binding activity by CRM197-CLEC conjugates in vitro. Comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Pus refers to unmodified pustulan, pus oxi refers to activated pustulan. CRM-pus conjugate 1 refers to SEQ ID NO:6+CRM197+pustulan conjugate, CRM conjugate 1 refers to CRM197+SEQ ID NO:6 conjugate without β-glucan modification. Negative control refers to sample without inhibitor. [Figure 26B] Figure 1 shows a comparative analysis of Dectin-1 binding capacity as determined by ELISA. SEQ ID NOs: 52 / 66 / 68 / 70 / 72 refer to CRM197-pustulan conjugates with the indicated B cell epitopes. [Figure 26C] Figure 2 shows a comparative analysis of Dectin-1 binding ability as determined by ELISA. Lich oxi refers to activated Lichenan, CRM-Lich conjugate 1 refers to SEQ ID NO:6 + CRM197 + Lichenan conjugate. [Figure 26D] Figure 2 shows a comparative analysis of Dectin-1 binding capacity as determined by ELISA. Lam oxi refers to activated laminarin, CRM-Lam conjugate 1 refers to SEQ ID NO:6 + CRM197 + laminarin conjugate.

[0241] [Figure 27]Figure 27 shows the human DC receptor (i.e. Dectin-1) binding activity of CRM197-CLEC conjugates in vitro. Comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Lich conjugate refers to SEQ ID NO:6 + CRM197 + Lichenan conjugate, Pus conjugate refers to SEQ ID NO:6 + CRM197 + Pustulan conjugate, and Lam conjugate refers to SEQ ID NO:6 + CRM197 + Laminarin conjugate. Negative control refers to sample without inhibitor.

[0242] [Figure 28A] Figures 28A and 28B show a comparison of immunogenicity of different CRM-pustulan based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination and analyzed for anti-peptide responses. [Figure 28B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered two weeks apart. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third vaccination and analyzed for anti-aggregatory aSyn fiber responses.

[0243] [Figure 29] FIG. 29 shows a comparative analysis of the selectivity of immune responses elicited by peptide+CRM+pustulan-based vaccines in vivo against aSyn fibers. Female BALB / c mice aged 8-12 weeks received a total of three intradermal or sc vaccinations administered at 2-week intervals. The CRM-pustulan-based vaccine was evaluated against the conventional CRM vaccine. Samples were taken 2 weeks after the third dose and subjected to an aSyn selectivity assay (inhibition ELISA). IC50 values ​​of antibodies inhibited with increasing doses of aSyn fibers are shown.

[0244] [Diagram 30]30 shows the avidity of antibodies induced by peptide+CRM197+pustulan vaccine, the stability of aSyn-antibody complexes induced by peptide+CRM197+pustulan vaccine or peptide+CRM197 vaccine after challenge with different concentrations of the chaotropic agent sodium thiocyanate (NaSCN), and the determined avidity index.

[0245] [Figure 31A] Figures 31A and 31B show a comparison of immunogenicity of different CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were taken 2 weeks after the third vaccination and analyzed for anti-SEQ ID NO:6 peptide responses induced by peptide+carrier+glucan-based vaccine or CLEC-unmodified vaccine adjuvanted with alum; dose: 20 μg peptide equivalents / injection; pustulan indicates SEQ ID NO:6+CRM+pustulan, lichenan indicates SEQ ID NO:6+CRM+lichenan, laminarin indicates SEQ ID NO:6+CRM+laminarin, and sc+alum indicates CLEC-unmodified vaccine SEQ ID NO:6+CRM adjuvanted with alum. [Figure 31B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third vaccination and analyzed for anti-aSyn fiber responses induced by peptide+carrier+glucan-based vaccine or CLEC-unmodified vaccine adjuvanted with alum; dose: 20 μg peptide equivalents / injection; pustulan indicates SEQ ID NO:6+CRM+pustulan, lichenan indicates SEQ ID NO:6+CRM+lichenan, laminarin indicates SEQ ID NO:6+CRM+laminarin, and sc+alum indicates CLEC-unmodified vaccine adjuvanted with alum, SEQ ID NO:6+CRM.

[0246] [Figure 32A] Figure 32A shows the in vitro binding activity of peptide-CLEC conjugates to mouse DC receptors (i.e., Dectin-1). Comparative analysis of Dectin-1 binding ability as determined by ELISA is shown. Lich conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + Lichenan conjugate, Pus conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + Pustulan conjugate, and Lam conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + Laminarin conjugate. Negative control refers to a sample without inhibitor. [Figure 32B] Figure 32B shows human DC receptor (i.e. Dectin-1) binding activity by peptide-CLEC conjugates in vitro. Comparative analysis of Dectin-1 binding ability as determined by ELISA. Lich conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + Lichenan conjugate, Pus conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + Pustulan conjugate, and Lam conjugate refers to SEQ ID NO:5 + SEQ ID NO:7 + Laminarin conjugate. Negative control refers to sample without inhibitor.

[0247] [Figure 33A] Figures 33A and 33B show a comparison of immunogenicity of different CLEC-based vaccines. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at two-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Two weeks after the third vaccination, samples were collected and analyzed for anti-peptide responses (SEQ ID NO:6, shown as peptide) and anti-aSyn responses (shown as protein) induced by peptide-glucan-based vaccines (i.e., SEQ ID NO:5 + SEQ ID NO:7 + CLEC, dose: 5 μg / injection; Lichenan indicates SEQ ID NO:5 + SEQ ID NO:7 + Lichenan; Laminarin indicates SEQ ID NO:5 + SEQ ID NO:7 + Laminarin; Pustulan indicates SEQ ID NO:5 + SEQ ID NO:7 + Pustulan). [Figure 33B]Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected two weeks after the third dose and analyzed for anti-peptide responses (SEQ ID NO:6, shown as peptide) and anti-aSyn responses (shown as protein) induced by the peptide-glucan-based vaccine (i.e., SEQ ID NO:5 + SEQ ID NO:7 + CLEC, dose: 20 μg / injection; Lichenan indicates SEQ ID NO:5 + SEQ ID NO:7 + Lichenan; Laminarin indicates SEQ ID NO:5 + SEQ ID NO:7 + Laminarin; Pustulan indicates SEQ ID NO:5 + SEQ ID NO:7 + Pustulan).

[0248] [Figure 34A] Figures 34A-H show that SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine-induced antibodies inhibit aSyn aggregation in a PFF model in vivo. C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized 4 times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLEC as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Plasma titers of antibodies specific to the peptide used for immunization collected 2 weeks after the third immunization on day 126. Error bars represent the mean ± SEM of n = 5-9 animals per group. Statistical differences were assessed by unpaired t-test; **p<0.01; *p<0.05. [Figure 34B]C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized four times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLEC as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Comparison of CSF and plasma titers of antibodies specific to the B cell peptide of the vaccine on day 126. Error bars represent the mean ± SEM of n=5-9 animals per group. Statistical differences were assessed by unpaired t-test; **p<0.01; *p<0.05. [Figure 34C] C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized four times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLECs as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Analysis of phosphor-S129 aSyn positive aggregates across all brain regions in SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccinated and CLEC-treated mice. Error bars indicate mean ± SEM of n = 5-9 animals per group. Statistical differences were assessed by unpaired t-test; **p<0.01; *p<0.05. [Fig. 34D] C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized four times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLEC as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Correlation between antibody responses and levels of synucleinopathy in vaccine recipients (r=-0.9391; CI (95%) -0.9961 to -0.3318, p=0.0179, and R2=0.882). [Figure 34E]C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized four times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLEC as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Representative pSer129 aSyn staining in the injected brain hemisphere at the level of the substantia nigra. Vehicle-treated mice after PFF injection. [Fig. 34F] C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized four times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLEC as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Representative pSer129 aSyn staining in the injected brain hemisphere at the level of the substantia nigra. Vaccine-treated mice after PFF injection. [Figure 34G] C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized four times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLEC as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Representative pSer129 aSyn staining in the injected brain hemisphere at the level of the striatum. Vehicle-treated mice after PFF injection. [Fig. 34H] C57BL / 6 mice stereotaxically injected with recombinant aSyn PFFs into the right substantia nigra were immunized four times with SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine (vaccine) or unconjugated CLEC as control (vehicle) starting on the day of PFF inoculation. Plasma was collected after the third immunization. Brain, plasma, and CSF were collected 126 days after PFF inoculation. Representative pSer129 aSyn staining in the injected brain hemisphere at the level of the striatum. Vaccine-treated mice after PFF injection.

[0249] [Diagram 35] Figure 35 shows the analysis of carrier-specific immunogenicity of peptide+CLEC and peptide+CRM+CLEC conjugates. Female BALB / c mice aged 8-12 weeks received a total of three intradermal / sc vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune response elicited by SEQ ID NO:6+CRM197+pustulan was evaluated against the respective peptide+CRM197 conjugates adjuvanted with alum. Samples were collected 2 weeks after the third dose and analyzed for in vivo induced anti-CRM responses.

[0250] [Figure 36A] Figures 36A and 36B show analysis of CLEC-specific immunogenicity of peptide + CRM + CLEC conjugates. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by the different CLEC-based vaccines were evaluated. Samples were taken 2 weeks after the third administration and analyzed for anti-pustulan responses in vivo; Samples: SEQ ID NO:6 + CRM197 + pustulan, SEQ ID NO:6 + CRM197 + lichenan; SEQ ID NO:6 + CRM197 + laminarin; Pre-serum: Samples obtained before immunization; Positive control: Samples from animals immunized with non-oxidized pustulan alone. [Figure 36B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. The immune responses elicited by 14 different CLEC-based vaccines were evaluated. Samples were collected 2 weeks after the third dose and analyzed for in vivo anti-pustulan responses; samples: SEQ ID NO:6 + CRM197 + pustulan; pustulan conjugated at the indicated conjugate / pustulan ratio (w / w); pre-serum: samples obtained before immunization; positive control: samples from animals immunized with non-oxidized pustulan alone.

[0251] [Figure 37A] Figures 37A and 37B show analysis of immunogenicity of vaccines consisting of peptide + carrier + glucan conjugate and peptide + carrier conjugate with unconjugated glucan. Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were taken at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were taken 2 weeks after the third vaccination and analyzed for anti-SEQ ID NO:6 peptide responses. Vaccines used: SEQ ID NO:6 + CRM197 + pustulan, SEQ ID NO:6 + CRM197 mixed with non-oxidized pustulan, and SEQ ID NO:6 + CRM197 unadjuvanted with CLEC unmodified. [Figure 37B] Female BALB / c mice aged 8-12 weeks received a total of three intradermal vaccinations administered at 2-week intervals. Blood samples were collected at baseline and after each vaccination to characterize the kinetics of the subsequent immune response. Samples were collected 2 weeks after the third dose and analyzed for anti-aSyn monomer responses. Vaccines used: SEQ ID NO:6-CRM197-pustulan: SEQ ID NO:6+CRM197+pustulan, SEQ ID NO:6-CRM197+Pus (unconjugated): unadjuvanted SEQ ID NO:6+CRM197 mixed with non-oxidized pustulan, and SEQ ID NO:6-CRM197: CLEC-unmodified, unadjuvanted SEQ ID NO:6+CRM197. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0252] Working Example: Materials and Methods 1) Oxidation of CLEC / glucan backbone To form vaccine conjugates, polysaccharides, especially CLEC / β-glucans, need to be chemically modified to generate reactive groups that can be used to link proteins / peptides. Two commonly used methods for polysaccharide activation are periodate oxidation at vicinal hydroxyls and cyanylation of hydroxyls. Additional methods of polysaccharide activation are possible and well known in the art. The example shown in this Example section relies on mild periodate oxidation.

[0253] CLECs and β-glucans (eg, β-glucans from mannan, lichenan, pustulan, or barley) are oxidized using periodate oxidation in aqueous solution or DMSO, depending on their solubility.

[0254] The degree of oxidation is predetermined based on adding periodate in molar ratios (periodate:sugar subunits; 100% = 1 Mol of periodate per Mol of sugar monomer) ranging from 1:5 (i.e., 20% oxidation) to 2.6:1 (260% oxidation degree).

[0255] Briefly, sodium periodate is added in a molar ratio of 1:5 to 2.6:1 (periodate:sugar subunits corresponding to oxidation degrees of 20% and 260%) to open the furanose ring between the vicinal diols of β-glucan, leaving two aldehyde groups as substrates for the subsequent ligation reaction. 10% (v / v) 2-propanol is added as a radical scavenger. The reaction is incubated for 4 h at room temperature on an orbital shaker (1000 rpm) in the dark. The oxidized glucan is then dialyzed three times against water using Slide-A-Lyzer™ (Thermo Scientific) cassettes or Pur-A-Lyzer™ (Sigma Aldrich) cassettes with a 20 kDa cutoff to remove sodium (per)iodate and low molecular weight glucan impurities. The dialyzed glucan can be directly subjected to peptide conjugation reaction or stored at -20°C or lyophilized and stored at 4°C for further use.

[0256] 2) Conjugate formation of WISIT vaccine 2a. Via hydrazone formation The polypeptide contains a hydrazide group at the N-terminus or C-terminus for aldehyde linkage. If the orientation of linkage to the aldehyde group of the glucan moiety via the N-terminus of the selected peptide is intended, the peptide is designed to contain a suitable linker / spacer, such as succinic acid. Alternatively, intact proteins (e.g., CRM197) have also been used for glucan linkage.

[0257] A typical example of such a peptide is the N-terminal linkage of the peptide: H2N-NH-CO-CH2-CH2-CO-polypeptide-COOH; C-terminal linkage: NH2-polypeptide-NH-NH2.

[0258] For conjugation, the activated glucan solution (i.e., oxidized pustulan) is stirred with hydrazide-modified peptides or intact proteins (e.g., CRM197) dissolved in a conjugation buffer (using either sodium acetate buffer at pH 5.4 or DMEDA at neutral pH (6.8), depending on the isoelectric point of the peptide). Free hydrazide groups in the peptide react with aldehyde groups to form hydrazone bonds, forming the final conjugates. In the case of proteins, conjugation to activated glucans is based on the reaction of amino groups of existing lysine residues with reactive aldehydes on the glucan moiety in the presence of sodium cyanoborohydride.

[0259] The conjugates are then reduced by adding sodium borohydride in borate buffer (pH 8.5). This step reduces the hydrazine bond to a stable secondary amine and converts the unreacted aldehyde groups in the sugar backbone to primary alcohols. The carbohydrate concentration in the conjugates was estimated using the anthrone method, and the peptide concentration was estimated by UV spectroscopy or determined by amino acid analysis.

[0260] 2b. Linkage using heterobifunctional linkers The second conjugation technique applied relies on heterobifunctional linkers (e.g.: BMPH (N-β-maleimidopropionic acid hydrazide), MPBH (4-[4-N-maleimidophenyl]butyric acid hydrazide), EMCH (N-[ε-maleimidocaproic acid] hydrazide), or KMUH (N-[κ-maleimidoundecanoic acid] hydrazide) short chain maleimide- and -hydrazide crosslinkers) for conjugating sulfhydryls (cysteines) to carbonyls (aldehydes).

[0261] The polypeptide contains a cysteine ​​(Cys) at the N-terminus or C-terminus for maleimide linkage. Typical examples of such peptides are N-terminal linkage of peptide: Cys-peptide-COOH; C-terminal linkage: NH2-peptide-Cys-COOH.

[0262] For conjugation, the activated glucan solution (i.e., oxidized pustulan) is reacted with BMPH (BMPH:pustulan ratios used are 1:1 ratio (w / w) to 2:1 ratio) overnight and then dialyzed three times in PBS. The BMPH-activated glucan is then mixed with the Cys-modified polypeptide dissolved in conjugation buffer (e.g., phosphate-buffered saline, PBS). The maleimide group reacts with the sulfhydryl group from the peptide to form a stable thioether bond, which together with the hydrazone formed between the linker and the reactive aldehyde results in a stable conjugate. The carbohydrate concentration in the conjugate was estimated using the anthrone method, and the polypeptide concentration was determined by amino acid analysis or Ellman's assay using Ellman's reagent (5,5'-dithio-bis-(2-nitrobenzoic acid), DTNB). DTNB reacts with sulfhydryl groups to produce a colored product, providing a reliable method for measuring reduced cysteine ​​and other free sulfhydryls in solution by spectrophotometry (λmax = 412 nm; ε = 14,150 / M cm).

[0263] 2c) Polypeptide KLH / CRM Conjugate Formation Polypeptides (containing N- or C-terminal Cys residues, see above) were coupled to carriers CRM-197 (e.g., EcoCRM, Fina Biosolutions) or KLH (Sigma Aldrich) by using the heterobifunctional crosslinkers GMBS or SMCC (Thermo Fisher). Briefly, CRM-197 / KLH was activated by mixing with excess GMBS or SMCC at room temperature (following the manufacturer's protocol), followed by removal of excess GMBS by desalting column centrifugation. Excess peptide was then added to the activated carrier for coupling (buffer: 200 mM Na phosphate (pH = 6.8)) and then dialyzed three times against PBS. Coupling efficiency / peptide content was assessed using the Ellman's assay (Ellman's reagent: 5,5'-dithio-bis-(2-nitrobenzoic acid) used to quantify free sulfhydryl groups in solution). The polypeptide CRM-197 / KLH conjugate was further formulated with alum (Alhydrogel® adjuvant 2%) and administered subcutaneously to animals. When the CRM-197 / KLH vaccine was compared to other vaccines according to the invention, the same amount of conjugate polypeptide was injected per mouse.

[0264] 2d) Gluconoconjugate formation using polypeptides, KLH / CRM197, and glucan The polypeptide-KLH conjugates and polypeptide-CRM197 conjugates prepared as described in 2c) were also linked to activated glucan at different polypeptide-KLH and polypeptide-CRM197 to glucan ratios (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 polypeptide conjugate formation, dithiothreitol (DTT) is used to reduce the Pep-KLH conjugate or Pep-CRM conjugate. The reduced carrier conjugate is linked to the activated glucan in the presence of an excess of the heterobifunctional linker BMPH. Linkage is achieved between the sulfhydryl residue of the reduced KLH conjugate or CRM197 conjugate, which forms a stable thioether bond, and between the aldehyde group in the glycan and the hydrazide group of BMPH via the maleimide group of BMPH. After 2 hours at room temperature, the resulting hydrazone is reduced to a stable secondary amine by overnight incubation with sodium cyanoborohydride. The gluconeoconjugate is then dialyzed three times against PBS or water using a Slide-A-Lyzer™ (Thermo Scientific) or Pur-A-Lyzer™ (Sigma Aldrich) cassette to remove low molecular weight impurities (see also Example 23).

[0265] 3) Determining the biological activity of CLEC conjugates in vitro The biological activity of mannan and glucan conjugates in vitro was analyzed by ELISA using soluble mouse Fc-Dectin-1a receptor (InvivoGen) or ConA as described in Korotchenko et al., 2020. Briefly, ELISA plates are coated with a reference glucan (CLR agonist, CLEC), e.g., pustulan, lichenan, or mannan, and reacted with fluorescently labeled ConA (for mannan) or soluble mouse Fc-Dectin-1a receptor (for pustulan and other β-D-glucans), which can be detected by an HRP-labeled secondary antibody. Oxidized carbohydrate and gluconeo conjugates are tested in a competitive ELISA (increasing concentrations of CLEC or conjugate are added to the soluble receptor used in the assay to reduce receptor binding to the coated CLEC) to demonstrate their functionality. IC 50 The value is used to determine biological activity (ie, binding effectiveness to the soluble receptor compared to the unoxidized, unconjugated ligand).

[0266] 4) Activation analysis using bone marrow-derived dendritic cells Bone marrow-derived dendritic cells (BMDCs) were harvested from mouse femurs and mouse tibias and incubated with 20ng / mL mouse GM-CSF (Immunotools) as described (with minor modifications) in Korotchenko et al., 2020. The effects of the various conjugates and a positive control (=LPS) on the expression of CD80 and MHCII were examined using CD11c + MHCII + CD11b int Evaluation was performed by FACS analysis on GM-CSF-derived DCs (GM-DCs).

[0267] 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 g for 15 min (Merck Millipore, Ultrafree-MC-VV Durapore PVDF). All sample wells were sealed with silica oil to prevent evaporation, and data were collected continuously for approximately 24 h. All measurements were performed using a WYATT DynaPro PlateReader-II at 25 °C in 1536-well plates (1536W SensoPlate, Greiner Bio-One). Samples were measured in triplicate. All measurements were filtered for a baseline value of 1.00 ± 0.005, so that only curves that returned values ​​between 0.995 and 1.005 were considered for further analysis (e.g., cumulant radius and regularization analysis). Analysis of the samples was performed according to https: / / www.wyatt.com / library / application-notes / by-technique / dls.html and according to the DYNAMICS User Guide (M1406 Rev.C, version 7.6.0), Technical Notes TN2004 and TN2005 (all on www.wyatt.com).

[0268] 6) Animal testing Female BALB / c mice, n=5 mice per group, were immunized with either the different CLEC conjugates (id, im, sc), peptide-CRM-197 / KLH conjugates (id), or peptide-CRM-197 / KLH conjugates adsorbed to alum (sc), and the respective controls (e.g., unconjugated CLEC, a mixture of CLEC and peptide, etc.). Animals were vaccinated three times at biweekly intervals, and blood samples were routinely taken 1 day before each vaccination and 2 weeks after the last dose, unless otherwise indicated.

[0269] 7) Quantification of vaccine-induced antibodies in mouse plasma using ELISA Whole blood was collected from mice using heparin as anticoagulant and plasma was obtained by centrifugation. Plasma samples were stored at -80°C. To detect anti-target specific antibodies, ELISA plates (Nunc Maxisorb) were coated with peptide-BSA conjugates or recombinant proteins / fragments (usual concentration 1 μg / ml) using 50 mM sodium carbonate buffer overnight at 4°C. All anti-polypeptide ELISAs used in the examples provided are performed using Pep-BSA conjugates (e.g. SEQ ID NO: 3 with a C-terminal C for linkage with maleimide-activated BSA (SEQ ID NO: DQPVLPD); nomenclature: Pep1c (DQPVLPD-C, SEQ ID NO: 3) used as bait for anti-Pep1 specific responses elicited by Pep1b (SEQ ID NO: 2; DQPVLPD-(NH-NH2))-containing conjugate vaccines and Pep1c-containing conjugate vaccines). Plates were blocked with 1% bovine serum albumin (BSA) and plasma samples were serially diluted onto the plates. Detection of target-specific antibodies was performed using biotinylated anti-mouse IgG (Southern Biotech) and subsequent color development reaction using streptavidin-POD (Roche) and TMB. EC 50 Values ​​were calculated according to nonlinear regression analysis (four-parameter logistic fit function) using GraphPad Prism software (GraphPad Prism www.graphpad.com / scientific-software / prism / ).

[0270] [Table 3]

[0271] 8) Characterization of the binding preferences of aSyn-specific antibodies by inhibition ELISA ELISA plates (Nunc Maxisorb) were coated with either aSyn monomer (Abcam) or aSyn fibrils (Abcam) and blocked with 1% bovine serum albumin (BSA). Control antibodies and plasma samples were incubated with serially diluted aSyn monomer or aSyn fibrils in low-binding ELISA plates. The pre-incubated antibody / plasma samples were then added to the monomer / fibril-coated plates and detection of binding was performed using biotinylated anti-mouse IgG (Southern Biotech) and subsequent color development reaction using streptavidin-POD (Roche) and TMB. The logIC50 values ​​were calculated as the concentration of monomeric or fibrillar aSyn required to quench half of the ELISA signal and were used as an estimate of Ab selectivity for the investigated antigen. The logIC 50 Values ​​were calculated according to nonlinear regression analysis (four-parameter logistic fit function) using GraphPad Prism software (GraphPad Prism www.graphpad.com / scientific-software / prism / ).

[0272] 9) Quantification of aSyn aggregation Protein aggregation assays in an automated format were performed in a reaction volume of 0.1 ml in black flat-bottom 96-well plates with continuous orbital shaking in a GENIOS microplate reader (Tecan, Austria). Kinetics were monitored by top readings of fluorescence intensity every 20 min using a 450 nm excitation filter and a 505 nm emission filter. Fibril formation in the absence and presence of antibodies (antibody / protein molar ratio of 6 × 10 -5 From 3×10 -3 The incubation time (which varied from 10 to 30 min) was started by shaking aSyn solution at a concentration of 0.3 mg / ml (20.8 μM) 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).

[0273] Furthermore, fibril formation in the absence and presence of antibodies was initiated by the presence of preformed fibrils. Briefly, aSyn preformed fibrils (1 μM) were aggregated for 0–24 h in the presence of activated aSyn monomer (10 μM) and 10 μM ThT in 100 μl of PBS.

[0274] For data analysis, the average of the negative control samples, i.e., background fluorescence of ThT, was calculated and divided from each sample at a given time point, e.g., in Microsoft Excel. To compare different conditions / inhibitors in the aggregation assay, each sample was normalized to a fluorescence reading determined at the beginning of the assay and set to 1 (t0=1).

[0275] To evaluate the kinetic curves, the Michaelis-Menten kinetic model was applied: Km (substrate concentration resulting in half-maximal velocity) and Vmax (maximal velocity) values ​​for each condition were calculated according to enzyme kinetic analysis (Michaelis-Menten) using GraphPad Prism software.

[0276] To compare different conditions / inhibitors in the aggregation assay, slope values ​​in the exponential growth phase of ThT kinetics were calculated using GraphPad Prism software according to linear regression.

[0277] 10) Determination of affinity and avidity To determine Ab avidity, a modification of the standard ELISA assay was used in which replicate wells containing antigen-bound antibody were exposed to increasing concentrations of the chaotropic thiocyanate ion. Resistance to thiocyanate elution was used as a measure of avidity, and an index representing 50% of effective antibody binding (Avidity Index) was used to compare different sera. Briefly, plasma was diluted 1 / 500 in PBS and dispensed onto coated and blocked ELISA plates (Nunc Maxisorb). After 1 h incubation, sodium thiocyanate (NaSCN, SIGMA; in PBS) was added to the samples at concentrations between 0.25 and 3 M. Plates were incubated for 15 min at room temperature before washing, detection, and subsequent color development using streptavidin-POD (Roche) and TMB. Absorbance readings in the absence of NaSCN were assumed to represent effective total binding of specific antibody (100% bound), and subsequent absorbance readings in the presence of increasing concentrations of NaSCN were converted to the appropriate percentage of total bound antibody. Data were fitted to a graph of (% bound) versus (log) concentration of NaSCN, and an avidity index was estimated by linear regression analysis, representing the concentration of NaSCN required to reduce the initial optical density by 50%. Data were rejected if the correlation coefficient of the line fit was less than 0.88.

[0278] k for aSyn fibers D To determine the binding affinity (k value), the k of the complex formed by the Ab and its competing ligand is D A displacement ELISA was used, which allows for the simple determination of free antibody values. Briefly, equal concentrations of Ab were incubated with increasing concentrations of free aSyn fibers prior to measurement of free antibody titers on plates with immobilized aSyn fibers. Relative binding of Ab is expressed as a percentage of the maximum binding observed in the assay for each sample; a competitive reaction with aSyn fibers (5 μg / ml) was defined to represent 0% binding (nonspecific binding), and a reaction without competition was interpreted as showing 100% (maximum) binding in the displacement curve.

[0279] Analysis of competitive binding curves was performed according to a one-site model using computer-assisted curve fitting software from GraphPad.

[0280] 11) Induction of synucleinopathy in mice To induce synucleinopathy, 9-week-old male C57BL / 6 mice were stereotactically injected with preformed polymorph fibrils (PFFs; i.e., preformed sonicated tau-polymorph aSyn fibrils 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 PFF 1B solution (concentration: 2.5 mg / ml) into the area just above the right substantia nigra (coordinates from bregma: -2.9 AP, ±1.3 L and -4.5 DV) at a flow rate of 0.4 μL / min [Sci.Adv.2020, 6, eabc4364, doi:10.1126 / sciadv.abc4364; DOI:10.1126 / sciadv.abc4364] and the needle was left in place for 5 min before being slowly withdrawn from the brain.

[0281] Starting on the same day as inoculation, animals were immunized id three times every two weeks (i.e., weeks 0, 2, and 4) with either the CLEC-based vaccine (n=5) or unconjugated CLEC as a control (n=10), followed by a boost immunization at week 10. At the end of the study (day 126), cerebrospinal fluid (CSF) was collected by cisternal puncture and brains were carefully removed and fixed in paraformaldehyde (PFA; 4%). Serial coronal sections of whole brains were taken at 50 μm intervals (rostral anterior cortex to striatum to medulla oblongata - i.e., bregma -6.72 mm) using a cryostat and processed for immunohistochemistry.

[0282] 12) Immunohistochemistry (IHC) IHC staining for phosphor-S129 aSyn (pS129 aSyn) on coronal serial sections was performed as previously described [Sci.Adv.2020, 6, eabc4364, doi:10.1126 / sciadv.abc4364; DOI:10.1126 / sciadv.abc4364]. Monoclonal rabbit anti-pS129 aSyn antibody EP1536Y (ab51253, Abcam) was used, followed by incubation with labeled multimer-HRP anti-rabbit (Dako EnVision+TM kit, K4011). Visualization of pS129 aSyn staining was performed with DAB (K3468) from Dako, and sections were counterstained with Nissl stain. The actual number of pS129 aSyn aggregates per structure (cerebral cortex, striatum, thalamus, substantia nigra, and brainstem) and the total number of pS129 aSyn aggregates were assessed using whole-section acquisition with Panoramic Scan II (3DHISTECH, Hungary) and further processed with the specially developed QuPath algorithm.

[0283] Example 1: Determination of biological activity of CLEC conjugates in vitro PAMPs such as CLECs are recognized by PRRs present in APCs. Binding of CLECs to their cognate PRRs (e.g., Dectin-1 in the case of β-glucan) is necessary to control adaptive immunity at various levels, for example by inducing downstream carbohydrate-specific signaling, as well as cell activation, cell maturation and migration of cells to draining lymph nodes, or through crosstalk with other PRRs. Therefore, to provide the novel vaccine platform technology proposed in this application, it is important that the CLECs used retain their PRR-binding capacity that demonstrates the biological activity of the selected CLECs and CLEC-based conjugates.

[0284] Along these lines, binding to Dectin-1 was assessed by ELISA to ensure that 1) the structure of CLECs is not destroyed during mild periodate oxidation, and 2) the polysaccharides remain biologically active after conjugation. First, several different CLECs were oxidized by mild periodate oxidation to generate the reactive sugar backbone of the proposed vaccine. These CLECs include mannan, pustulan (20 kDa), lichenan (245 kDa), barley β-glucan (229 kDa), oat β-glucan (295 kDa), and oat β-glucan (391 kDa). Vaccine conjugates were then generated by hydrazone conjugation using different B cell epitope peptides (SEQ ID NO:2, SEQ ID NO:10, SEQ ID NO:16), and SEQ ID NO:7 as the T helper epitope peptide, all of which contain a C-terminal hydrazide linker for conjugation. Additionally, a peptide-pustulan conjugate made by linking SEQ ID NO: 10 via the heterobifunctional linker BMPH was also used.

[0285] The non-oxidized and oxidized CLECs, as well as the novel CLEC-based conjugates, were then evaluated for biological activity using a competitive ELISA system based on competitive binding of soluble mouse Fc-Dectin-1a receptor (InvivoGen) or ConA, as described in Korotchenko et al. 2020.

[0286] result: Different CLECs tested show different efficiencies of PRR binding. In a series of ELISA experiments, the ligands of dectin-1, pustulan, lichenan, barley β-glucan, and oat β-glucan, were evaluated for their binding efficiencies to dectin-1. Subsequent experiments revealed that pustulan, a medium molecular weight (20 kDa) linear β-(1,6)-linked β-D-glucan, surprisingly exerts a significantly higher binding efficiency (about three-fold) to dectin-1 than the larger high molecular weight linear β-(1,3)β-(1,4)-β-D-glucan, lichenan (about 245 kDa) (see Figure 1).

[0287] This difference was even more pronounced when pustulan was compared with other linear β-(1,3)β-(1,4)-β-D-glucans from oats and barley (barley β-glucan (229 kDa), oat β-glucan: 265 kd and 391 kd), which showed very limited binding effectiveness compared to pustulan (e.g., about 30-fold lower for barley β-glucan (229 kDa)).

[0288] Mild periodate oxidation of selected CLECs results in a decrease in Dectin-1 binding. Oxidation of mannan reduced its binding ability to the lectin ConA to an extent similar to that described for oxidized pustulan-Dectin-1 binding after periodate oxidation. Similarly, oxidation of glucan results in a similar proportional decrease in PRR binding (see Figure 1A).

[0289] Importantly, conjugation also resulted in a decrease in the PRR-binding ability of the peptide-CLEC conjugates compared to unconjugated CLEC, as shown for mannan-containing conjugates as well as for conjugates of pustulan, lichenan, or the various barley and oat β-glucans tested (see FIG. 1B).

[0290] Experiments revealed that pustulan, a linear β-(1,6)-linked β-D-glucan, exerted the highest binding efficacy, regardless of oxidation or conjugate formation, despite its smaller size and absence of β-(1,3) glycosidic bonds (Note: β-(1,3)-containing glucans have been described as the ligand of choice for Dectin-1). For example, pustulan-containing conjugates retained approximately three-fold higher binding than lichenan-based constructs.

[0291] Regarding IC50 values, the binding results according to Figure 1 show the binding of the various constructs to the soluble mouse Fc-Dectin-1a receptor. The IC50 values ​​obtained are as follows (Figure 1): Oat beta-glucan 265: 860μg / ml Oat beta-glucan 391: 820μg / ml Barley β-glucan 229: 145μg / ml Lichenan (Figure 1E): 13 μg / ml Lichenan 200% conjugate (Figure 1E): 27 μg / ml (i.e., about half of unconjugated Lichenan) Pustulan: 3.5 μg / ml (FIG. 1B) and 5 μg / ml (FIG. 1D) (at least 30-fold stronger binding than barley β-glucan 229 at 145 μg / ml) Pustulan conjugates (Figure 1D): 11 μg / ml, 14 μg / ml, and 15 μg / ml (i.e., about half of the unconjugated pustulan) Pustulan BMPH conjugate (FIG. 1F): 80 μg / ml (peptide was linked to pustulan using a heterobifunctional linker).

[0292] Figures 1A and 1B further demonstrate that conjugation of the peptides via hydrazone formation or heterobifunctional linkers is equally suitable for WISIT conjugation, with both types of conjugates retaining high Dectin-1 binding efficacy.

[0293] Example 2: Determination of DC activation following pustulan exposure in vitro An important function of the proposed vaccines is their ability to activate DCs after PRR binding and internalization. To demonstrate that CLEC-based conjugates not only bind PRRs but also exert biological functions in their target cells, i.e., DCs, DC activation experiments were performed.

[0294] First, mouse bone marrow cells were incubated with mGM-CSF to generate BMDCs according to published protocols. These GM-CSF DCs were then exposed to either the peptidoglycan conjugate P SEQ ID NO:2 + SEQ ID NO:7 + pustulan, or an equivalent amount of oxidized but unconjugated saccharide. In both cases, the conjugate / saccharide was titrated from 500 μg to 62.5 μg / mL of each saccharide. For comparison, the strong activator LPS was used as a control, starting at a concentration of 2 ng / ml. Importantly, the pustulan preparation used for oxidation and conjugation also contains small amounts of LPS. Therefore, an equivalent dose of LPS was used to normalize the effects. DCs were then assessed for expression of markers for DC activation and DC maturation using FACS analysis, including CD80 and MHCII.

[0295] result: GM-CSF DC stimulated in vitro with SEQ ID NO:2-SEQ ID NO:7-pustulan conjugates demonstrated a significant increase in expression of CD80 and MHCII (see FIG. 2). The levels were significantly higher than the effect observed with an equivalent dose of LPS contained within the conjugate preparation. In contrast, an equivalent amount of oxidized but unconjugated sugar resulted in a slight decrease in CD80 expression, as expected from the LPS levels in the preparation, and a significantly less pronounced induction of MHCII compared to the pustulan conjugate.

[0296] In summary, upregulation of MHC-II indicates DC activation. Furthermore, CD80 is upregulated more than would be expected by the same amount of LPS, strongly indicating that pustulan conjugates contribute significantly to DC maturation and activation (beyond the effect explained by LPS exposure alone). Thus, Examples 1 and 2 clearly demonstrate the biological activity of the pustulan vaccine.

[0297] Example 3: Particle size determination by DLS Separate experiments were carried out analyzing the particle size / hydrodynamic radius of the different glucan conjugates.

[0298] For DLS analysis, different peptide-glucan conjugates and peptide-carrier-glucan conjugates were analyzed and compared with unconjugated pustulan, respectively. All analyses were performed in triplicate using a WYATT DynaPro PlateReader-II. The results obtained show a particle size distribution with a maximum in the low nm spectrum for all conjugates tested.

[0299] Conjugates tested: [Table 4]

[0300] result: This analysis shows a mean primary particle hydrodynamic radius (HDR) of about 5 nm for the peptide-pustulan conjugate SEQ ID NO:2 + SEQ ID NO:7 + pustulan used in this assay. A minor second peak detectable at about 60 nm indicates very few aggregates present in the preparation (see FIG. 3A). However, the majority of the conjugate preparation appears to be present as a monomeric form. This prevalence of monomeric conjugates rather than cross-linked or aggregated conjugates is also strongly supported by the fact that monomeric pustulan (about 20 kDa) is detectable at approximately 5 nm (as shown in the control sample, see also FIG. 3C), which also supports the prevalence of monomeric pustulan conjugates (giving an HDR of about 5 nm for monomeric pustulan). As shown by cumulant radius analysis over 24 hours, the conjugates are stable for their HDR and do not tend to aggregate, also supporting the prevalence of monomeric conjugates.

[0301] To characterize the vaccine based on peptide-carrier-glucan conjugates, we analyzed SEQ ID NO:6 + CRM197 conjugates further conjugated to pustulan. Again, DLS analysis revealed an average HDR of 11 nm and a second minor peak at about 75 nm, again indicating the presence of a small number of aggregates (see Figure 3B). As CRM197 is about 60 kDa in size, the slight increase to 11 nm most likely reflects an increase in the MW of the resulting conjugates. No significant aggregation or cross-linking of the CRM conjugates could be detected, and cumulant radius analysis over 24 hours also indicates that the conjugates are stable for their HDR and not prone to aggregation. Again, DLS analysis of this alternative type of CLEC-based vaccine supports the prevalence of monomeric conjugates.

[0302] The control sample (i.e., non-oxidized pustulan) showed a much larger HDR averaging approximately 600 nm, as well as two additional small peaks at 5 nm and 46 nm, respectively (see Figure 3C). Pustulan monomer has an HDR of approximately 5 nm, which fits well with the assumed MW of 20 kD, and larger aggregates can be easily detected, with the majority of the glucan present as large, high MW particles. Importantly, cumulant radius analysis over 24 h also shows that, in contrast to pustulan conjugates, unconjugated pustulan tends to aggregate strongly over time, resulting in the prevalent formation of large particles, in agreement with various literature reports.

[0303] Exemplary graphs for these two conjugates and the non-oxidized pustulan control are shown in FIG.

[0304] The results obtained in this example further demonstrate the so far unique features of CLEC-based conjugates compared to examples well known in the art (e.g., Wang et al., 2019, Jin et al., 2018) that show small (i.e., 5-11 nm), generally monomeric sugar-based nanoparticles with HDRs below 150 nm, the size of which is generally considered the preferred size for immunotherapeutically active conjugate vaccines. This is mainly due to the PRR binding and activation properties of larger particles, including whole glucan particles as well. Larger particles (>150 nm up to 2-4 μm) are known to interact more efficiently with their receptors and can initiate DC signaling, DC activation, DC maturation, and DC migration to draining lymph nodes, whereas small, even soluble, PRR ligands are believed to be able to bind to their receptors but block subsequent DC activation (Goodridge et al., 2011). However, these data, together with the data described in Examples 1, 2, and 3, as well as in the other Examples provided below, demonstrate for the first time that small soluble peptide-based gluconeoconjugates based on monomeric β-glucans, such as the linear β(1,6)-β-D-glucan pustulan, as a scaffold, effectively bind to PRR (Dectin-1), activate respective APCs (exemplified by GM-CSF DCs), and exhibit extremely high biological activity and immunogenicity specifically in the skin, significantly exceeding the efficacy of classical conjugate vaccines.

[0305] Example 4: In vivo comparison of different CLEC-based vaccines CLEC-based vaccines that were able to bind to their DC receptors (e.g. Dectin-1 or ConA) were tested for their ability to induce robust and specific immune responses after repeated dosing in n=5 Balb / c mice per group. Routine experiments were performed applying B cell epitope peptides with a net peptide content of 5 μg per dose.

[0306] In the first set of experiments, three different CLECs were compared in which the a-synuclein-derived peptide SEQ ID NO:2 or the amyloid β42 (Aβ42)-derived peptide SEQ ID NO:10 and the promiscuous T-helper cell epitope SEQ ID NO:7 were linked via a C-terminal hydrazide linker to oxidized pustulan (20% oxidized), mannan (20% oxidized), or barley β-glucan (229 kDa, 20% oxidized).

[0307] Vaccines used: [Table 5]

[0308] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (route: id) and the subsequent immune response to the injected peptides (i.e., SEQ ID NO: 2 and SEQ ID NO: 10, respectively) was analyzed using mouse plasma collected two weeks after the third immunization.

[0309] result: As shown in FIG. 4A, all three CLEC vaccines (SEQ ID NO:2 + SEQ ID NO:7 + mannan, SEQ ID NO:2 + SEQ ID NO:7 + pustulan (linear β(1,6) β-glucan), and barley SEQ ID NO:2 + SEQ ID NO:7 + β-glucan (229 kDa)) were able to induce detectable immune responses. Interestingly, immunization with the barley high molecular weight β-glucan-based vaccine induced only a very low anti-peptide response (OD max / 2 In contrast, the pustulan-based conjugate was able to induce a significantly higher response with a mean titer of about 11,000. The mannan-based conjugate showed about 7-fold lower immunogenicity compared to the pustulan-based conjugate in this experiment, since it reached a mean titer of about 1500 after immunization.

[0310] FIG. 4B shows results from a second set of experiments comparing the immunogenicity of two different variants of glucan-based conjugates using either the a-synuclein-derived peptide SEQ ID NO:2 or the amyloid β42 (Aβ42)-derived peptide SEQ ID NO:10 as the B cell epitope and the T cell epitope SEQ ID NO:7. The first variant also relies on pustulan as a CLEC for conjugate formation, while the second variant was made using the linear β-(1,3)β-(1,4)-β-D glucan lichenan (approximately 245 kDa). As shown in FIG. 4B, both variants were able to induce high titer immune responses against the injected peptides (i.e., SEQ ID NO:2 / 3 (SEQ ID NO:3=SEQ ID NO:2 compatible with BSA conjugate formation) and SEQ ID NO:10 / 11 (SEQ ID NO:11=SEQ ID NO:10 compatible with BSA conjugate formation)). However, the peptide lichenan conjugates showed significantly lower immunogenicity than the peptide pustulan conjugates in these experiments (4-8 times higher anti-peptide titers at 5 μg dose), which is also consistent with the lower Dectin-1 binding capacity shown in Example 1. This demonstrates that in vitro Dectin-1 binding efficacy can be directly related to the in vivo immunogenicity and biological activity of the vaccine. This identifies pustulan or a fragment thereof (i.e. linear β(1,6)-β-D glucan) as the most effective glucan variant proposed in this application. The vaccine is also functional with different peptides, demonstrating the potential of this vaccine type platform.

[0311] Example 5: In vivo comparison of peptide pustulan conjugates with unconjugated peptide vaccines To assess whether conjugation of CLEC to peptide immunogens is necessary for the induction of good immunogenicity of the vaccine according to the invention, a series of experiments was initiated to compare the two conjugates (SEQ ID NO:2 + SEQ ID NO:7 + pustulan or SEQ ID NO:2 + SEQ ID NO:7 + mannan) with a vaccine preparation containing all components as a mixture without conjugation (i.e. SEQ ID NO:2 and SEQ ID NO:7 + either non-oxidized pustulan or non-oxidized mannan, respectively). Again, n=5 female Balb / c mice were immunized id three times at biweekly intervals and the subsequent immune response to the injected peptide (i.e. SEQ ID NO:3) was analyzed using mouse plasma collected two weeks after the third immunization.

[0312] Vaccines used: [Table 6]

[0313] result: Figure 5 shows a comparison of anti-peptide (SEQ ID NO:3) specific immune responses detectable after three immunizations. In this experiment, SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugate (20% oxidized) was able to induce a four-fold higher immune response than reported for the mixture of unconjugated peptides SEQ ID NO:2, SEQ ID NO:7, and non-oxidized pustulan (i.e., 1 / 12000 vs. 1 / 3000). Similarly, SEQ ID NO:2 + SEQ ID NO:7 + mannan conjugate (20% oxidized) was also more efficient in inducing peptide-specific immune responses than administration of the mixture of the components (1 / 7000 vs. 1 / 4000; 1.75-fold increase). These data indicate that conjugation of peptide immunogens to activated CLECs is necessary to induce strong and sustainable immune responses in vivo.

[0314] Example 6: In vivo comparison of SEQ ID NO:5 + SEQ ID NO:7 + pustulan and SEQ ID NO:2 + pustulan or SEQ ID NO:7 + pustulan conjugates To assess whether a CLEC-based vaccine requires B and T cell epitopes to induce a sustainable anti-B cell epitope-specific immune response in vivo, a series of experiments was initiated comparing three conjugates: SEQ ID NO:5 + SEQ ID NO:7 + pustulan, SEQ ID NO:5 + pustulan, and SEQ ID NO:7 + pustulan. n=5 female Balb / c mice were immunized id three times at biweekly intervals and the subsequent immune response to the injected peptide (i.e., SEQ ID NO:6) was analyzed using mouse plasma collected two weeks after the third immunization.

[0315] [Table 7]

[0316] result: As shown in Figure 6, SEQ ID NO:5 + SEQ ID NO:7 + pustulan conjugates (80% oxidized) are able to induce high and highly specific immune responses against the injected peptide moiety (i.e., the a-synuclein-derived peptide SEQ ID NO:6) reaching an average titer of 1 / 36000 in these experiments. Peptide-pustulan conjugates containing either SEQ ID NO:5 or SEQ ID NO:7 alone linked to pustulan via a hydrazone linkage were able to induce a 12-fold lower immune response (1 / 3000) in the case of SEQ ID NO:5 + pustulan, or were unable to induce a SEQ ID NO:6-specific immune response (titer <1 / 100, below the detection limit, for SEQ ID NO:7 + pustulan conjugates) after three biweekly immunizations (route: id).

[0317] These data indicate that conjugation of peptide immunogens to activated CLECs is necessary to induce strong and sustainable immune responses in vivo. However, it also demonstrates that pustulan conjugation to individual short B cell epitopes in the absence of T cell epitopes (e.g., SEQ ID NO: 5 alone) allows the induction of T cell-independent B cell responses in vivo, albeit with significantly lower efficacy than reported for CLEC conjugates containing T and B cell epitopes.

[0318] Example 7: In vivo analysis of anti-pustulan / glucan immune responses following peptide-pustulan immunization The analysis of anti-CLEC antibodies is important on two levels for the novelty and efficacy of the proposed CLEC vaccine according to the invention.

[0319] 1) β-glucan is a major constituent of the cell walls of various fungi, lichens, and plants that confers to the cell walls their normal strength against intracellular osmotic pressure. Therefore, β-glucan is also considered to be a common microbial pathogen-associated molecular pattern (PAMP) and the primary target of high-titer circulating natural Abs in healthy human subjects. PAMPs are common and relatively invariant molecular structures shared by many pathogens that are potent 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)-glucan and β-(1,6)-glucan can be found in normal human serum, and β-(1,6)-glucan appears to be a much more potent antigen than the β-(1-3) variant. Moreover, the β-(1→6)-β-glucan moiety has been identified as one of the typical microbial PAMPs, acting as a recognition and attack center for immunological malignancy monitoring and defense against microbial invasion. Pustulan, the preferred glucan backbone for the CLEC conjugate according to the present invention, is composed of linear β-(1-6)-β-glucan moieties, and several research groups have reported that anti-pustulan immune responses can be detected in plasma from naive non-pustulan immunized human subjects. Therefore, it is important to investigate the potential of CLEC-based vaccines against anti-pustulan immune reactivity activation. Anti-β-glucan antibodies could specifically interact with peptide-pustulan in vivo, forming antigen-antibody complexes, thereby resulting in rapid elimination by preventing the induction of efficient immune responses.Alternatively, induction / boosting of anti-pustulan antibody responses after immunization could also promote immunogenicity as potential cross-presentation of CLEC conjugates by anti-pustulan specific IgG antibodies and uptake into APCs could also increase the efficacy of the administered vaccine.

[0320] No formal studies have been published investigating the presence of anti-pustulan antibodies in naive mice. However, Ishibashi et al. and Harada et al. were able to demonstrate the presence of β-glucan IgG against soluble scleroglucan / β-glucan (i.e., 1,3 / 1,6-β-glucan) in the serum of naive DBA / 2 mice.

[0321] 2) As previously reported (e.g. Torosantucci et al., Bromuro et al., Donadei et al., Liao et al.), CLEC-protein conjugates, e.g. CRM197 linked to laminarin, curdlan, or synthetic β(1,3) β-D glucan acted as potent immunogens inducing not only high anti-CRM197 titers but also high anti-glucan titers combined with protection from anti-fungal infection. Thus, previous attempts using such conjugates were directed towards using CLEC as true disease / fungal infection specific immunogens, instead of using CLEC as a carrier and immunologically inert scaffold, as proposed in this application.

[0322] Along these lines, an extensive analysis of plasma samples from naive and peptide-CLEC conjugate-immunized Balb / c mice (n=5 / group) for the presence of anti-pustulan antibodies, respectively, before and after repeated immunizations, was initiated.

[0323] Vaccines used: [Table 8]

[0324] result: Therefore, samples from peptide-pustulan (SEQ ID NO:2 + SEQ ID NO:7 + pustulan (20%)) and peptide-mannan (SEQ ID NO:2 + SEQ ID NO:7 + mannan (20%)) immunized animals were analyzed (all vaccines: 4 μg aSyn targeting peptide / dose). For control purposes, animals receiving vaccines consisting of unconjugated peptide and non-oxidized CLEC were also used (i.e. SEQ ID NO:2 + SEQ ID NO:7 + non-oxidized pustulan, SEQ ID NO:2 + SEQ ID NO:7 + non-oxidized mannan). As shown in Figure 7A, the analyzed Balb / c animals showed a low level of pre-existing immune response against pustulan / β(1,6)-β-D glucan. Both CLEC vaccines tested (SEQ ID NO:2 + SEQ ID NO:7 + pustulan (20%) and SEQ ID NO:2 + SEQ ID NO:7 + mannan (20%)) failed to induce a strong de novo immune response against the glucan backbone in vivo. In contrast, repeated administration of unconjugated, non-oxidized pustulan present in the control group (receiving a mixture of all three components) resulted in the induction of a strong anti-glucan immune response by boosting antibody levels against pustulan by 18.5-fold (compared to pre-immune plasma). Mannan-containing conjugates or mixtures failed to induce anti-pustulan titers, demonstrating the specificity of the detected anti-glucan response. Kinetic analysis of anti-pustulan antibody titers showed a steady increase over time in animals immunized with unconjugated, non-oxidized pustulan, with a strong increase after the third immunization (see Figure 7B). Competitive ELISA using increasing amounts of native pustulan also demonstrated the specificity of the antibody response detectable in groups receiving the mixture of components (Figure 7C).

[0325] In summary, these analyses were able to demonstrate that, despite the presence of low levels of autoreactivity (IgG) against pustulan in naive Balb / c mice, immunization with the various CLEC conjugates induces no / very low vaccination-dependent increase in anti-pustulan immune reactivity. Thus, the CLECs 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 therefore constitutes a surprising and original novel feature of the carbohydrate scaffolds (e.g. β-glucans or mannans, in particular the pustulan scaffolds) according to the invention.

[0326] Furthermore, since the injected peptide responses for both experiments revealed high anti-peptide titers, it appears that pre-existing anti-pustulan responses do not interfere with immune responses to the peptide components of the WISIT vaccine.

[0327] Example 8: Analysis of the immunogenicity of glucan conjugates with peptide immunogens linked to the N- or C-terminus To evaluate whether the orientation of the linker used in the linkage interferes with the immunogenicity of the vaccine, four different vaccine candidates were generated. In this experiment, the a-synuclein derived peptides SEQ ID NO:1 / 2 and SEQ ID NO:4 / 5 were linked to oxidized pustulan (80%) via an N-terminal hydrazide linker or a C-terminal hydrazide linker. Additionally, each of the four vaccines had the promiscuous T helper cell epitope SEQ ID NO:7 linked to the CLEC scaffold via a C-terminal hydrazide linker.

[0328] Vaccines used: [Table 9]

[0329] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (route: id) and the subsequent immune response against the injected peptides (i.e., SEQ ID NO:3 and SEQ ID NO:6) and the target protein, i.e., recombinant a-synuclein, was analyzed using mouse plasma collected two weeks after the third immunization.

[0330] result: As shown in FIG. 8, all four CLEC vaccines using N- or C-terminally linked B cell epitopes were able to induce strong and highly specific immune responses against both the injected peptide moiety (FIG. 8A) and the target protein a-synuclein (FIG. 8B). Interestingly, the linkage orientation affected immunogenicity differently. For example, the N-terminally linked SEQ ID NO:1+SEQ ID NO:7+CLEC vaccine induced 7-fold lower anti-injected peptide responses and 10-fold lower immune responses against rec.aSyn compared to the C-terminally linked SEQ ID NO:2+SEQ ID NO:7+CLEC vaccine, respectively. In contrast, the C-terminally linked SEQ ID NO:5+SEQ ID NO:7+CLEC vaccine induced approximately 4-fold lower injected peptide responses but comparable anti-aSyn responses compared to the N-terminally linked SEQ ID NO:4+SEQ ID NO:7+CLEC vaccine, respectively. Thus, the linkage orientation can be changed based on peptide-specific properties without affecting the generation of high titer immune responses.

[0331] However, as shown in Figure 8, altering the linkage orientation of the immunogenic peptide can significantly increase the specificity of the subsequent response to the target protein and can therefore be used to generate novel and unprecedented immune responses.

[0332] For example, vaccination with SEQ ID NO:1 resulted in a 4.5-fold higher response to the peptide compared to the target protein, which compares with a 3.3-fold higher anti-peptide response compared to the protein induced by vaccination with SEQ ID NO:2.

[0333] In contrast, the vaccine of sequence number 4 induced a 1.7-fold higher response to the peptide compared to the protein, and the vaccine of sequence number 5 was able to reverse this ratio, resulting in a 2.5-fold higher protein-specific response compared to the detectable injected peptide response.

[0334] In summary, these data clearly demonstrate that vaccines using either linkage orientation are biologically active and suitable for this application, and also show that linkage orientations can be used to select particularly preferred and unprecedentedly active vaccines depending on the peptide and target to be addressed.

[0335] Example 9: Analysis of immunogenicity of CLEC conjugates using different T helper cell epitopes In this example, the immunogenicity of a CLEC-based vaccine containing a non-natural pan-DR epitope (PADRE, SEQ ID NO: 7, containing an artificial cathepsin cleavage site) was compared with other known T helper cell epitopes. For this purpose, several promiscuous epitopes were selected that were either adapted using a novel artificially contained cathepsin L cleavage site or left unchanged for efficient endosomal / lysosomal release after receptor-mediated uptake in APCs / DCs. The selected epitopes include:

[0336] [Table 10]

[0337] To evaluate whether peptide vaccines carrying these T helper cell epitope peptides could initiate high immune responses after repeated immunization and induce better immune responses than conventional conjugate vaccines, ten different vaccine candidates were tested.

[0338] In this experiment, the aSyn-derived peptide SEQ ID NO:2 was used as a peptide-CLEC vaccine (i.e., SEQ ID NO:2 combined with different T helper cell epitopes linked to oxidized pustulan (80%) via a C-terminal hydrazide linker) or a conventional peptide conjugate was made using SEQ ID NO:3, which contains a C-terminal cysteine ​​for linkage to GMBS-activated KLH.

[0339] Vaccines used: [Table 11]

[0340] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines (adjuvanted with Alhydrogel)) and the subsequent immune response against the injected peptide (i.e., SEQ ID NO:3) and target protein, i.e., recombinant human a-synuclein, was analyzed using mouse plasma collected 2 weeks after the third immunization.

[0341] result: As shown in FIG. 9, all nine CLEC vaccines using different T helper cell epitopes and KLH conjugates were able to induce strong and specific immune responses against both the injected peptide moiety (SEQ ID NO:3, FIG. 9A) and the target protein, recombinant a-synuclein (FIG. 9B).

[0342] All T helper epitopes were able to induce anti-peptide titers similar to or better than the conventional SEQ ID NO:3+KLH conjugate. Vaccine 1 (containing SEQ ID NO:2 and SEQ ID NO:7 linked to pustulan) was able to induce, for example, a 60% higher response compared to the KLH control, whereas vaccine 8 (containing SEQ ID NO:28, a well-known T helper epitope particularly suitable for administration in Balb / c animals, SEQ ID NO:2, and pustulan) was able to induce a 5.5-fold higher response than the control. Even the promiscuous T helper epitope SEQ ID NO:24 (a weak T helper epitope for Balb / c animals derived from diphtheria toxin, disclosed in WO 2019 / 21355 A1) was able to induce a sustainable immune response, albeit weaker than the KLH control.

[0343] Similarly, all T helper epitopes were able to induce anti-protein titers similar to or better than the conventional SEQ ID NO:3-KLH conjugate. Importantly, vaccine 1 (containing SEQ ID NO:2 and SEQ ID NO:7 linked to pustulan) was able to induce, for example, a 2.5-fold higher response compared to the KLH control, and vaccine 8 (containing SEQ ID NO:28, a well-known T helper epitope particularly suitable for administration in Balb / c animals, SEQ ID NO:2, and pustulan) was able to induce a 3-fold higher response than the control, again supporting the fact that CLEC-based vaccines according to the invention are able to induce superior anti-target responses.

[0344] This example also shows that the introduction of an additional cathepsin L cleavage site into a well-described T helper epitope results in the induction of an efficient immune response compared to conventional vaccines and to CLEC-based vaccines lacking this artificial sequence.

[0345] For example, SEQ ID NO:25, a modified variant of SEQ ID NO:24, a weak T-helper epitope containing a cleavage site, was able to induce a 7.5-fold higher anti-peptide response and a 3.6-fold higher anti-protein response compared to the unmodified peptide (Vaccine 5 vs. Vaccine 4). Furthermore, this change also resulted in a 40% increase in anti-protein titer compared to the KLH control. SEQ ID NO:27, a cathepsin L cleavage site modified variant of SEQ ID NO:26 (epitope derived from measles virus fusion protein, disclosed in WO 2019 / 21355 A1), was also able to significantly increase titer with a 1.8-fold increase in anti-peptide titer and a 3.2-fold increase in anti-protein titer compared to the SEQ ID NO:26-CLEC vaccine (i.e., Vaccine 7 vs. Vaccine 6). Vaccine 7 also induced a 2.2-fold higher anti-peptide response and a 1.6-fold higher anti-protein response as the KLH control. The SEQ ID NO:7 based CLEC vaccine also induced superior anti-protein titers (20% increase) compared to unmodified variants (e.g., SEQ ID NO:22), and both peptides produced an approximate doubling of anti-SEQ ID NO:2 peptide titers and anti-aSyn titers, respectively, compared to the KLH control.

[0346] The addition of a cathepsin cleavage site results in the formation of peptide variants with an additional N (e.g., at the C-terminus, which is released upon cleavage). For example, SEQ ID NO: 22, PADRE is released as AKFVAAWTLKAAA, and SEQ ID NO: 7, modified PADRE is released as AKFVAAWTLKAAA-N. This N may also adversely affect further processing and MHCII presentation, thus reducing the effectiveness of the respective peptide. This phenomenon can be seen in the example of SEQ ID NO: 28 and SEQ ID NO: 29, which are very strong OVA-derived epitopes. While the unmodified peptide induces a very high immune response, the modified variant peptide 17 induces a 75% reduced anti-peptide titer and a 98% reduced anti-protein titer compared to the unmodified variant.

[0347] Example 10: Analysis of immunogenicity of CLEC conjugates using carrier protein as T helper cell epitope: KLH In this example, the immunogenicity of a CLEC-based conjugate vaccine containing the well-known carrier protein KLH was compared with that of a conventional KLH vaccine. For this purpose, two aSyn-derived epitopes (SEQ ID NO: 3 and SEQ ID NO: 6) linked to GMBS-activated KLH were selected. The Pep-KLH conjugates were then linked to the reactive aldehydes of oxidized pustulan using a BPMH crosslinker to form a CLEC-based conjugate vaccine with KLH as a source of T-helper cell epitopes to induce a sustainable immune response.

[0348] Vaccines used: [Table 12]

[0349] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 20 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines and unadjuvanted KLH-based vaccines, sc for Alhydrogel-adjuvanted KLH-based vaccines) and the subsequent immune response against the injected peptides (i.e., SEQ ID NO:3 and SEQ ID NO:6) and the target protein, i.e., recombinant human a-synuclein, was analyzed using mouse plasma collected 2 weeks after the third immunization.

[0350] result: As shown in FIG. 10A, all six vaccines that used KLH as a source of T-helper epitopes were able to induce strong and specific immune responses against both the injected peptide moieties (SEQ ID NO:3 and SEQ ID NO:6) and the target protein, recombinant a-synuclein.

[0351] The CLEC modification of the KLH conjugates results in a significantly better immune response with the use of both peptides SEQ ID NO:3 and SEQ ID NO:6, respectively. SEQ ID NO:3+KLH+pustulan was able to induce a 2.3-fold higher anti-peptide response compared to SEQ ID NO:3+KLH adjuvanted with Alhydrogel, and a 14-fold higher response compared to that obtained after id administration of unadjuvanted SEQ ID NO:3+KLH. Similarly, the anti-protein titers were increased 8.5-fold (compared to SEQ ID NO:3+KLH adjuvanted with Alhydrogel) and 17-fold compared to the unadjuvanted material. SEQ ID NO:6+KLH+pustulan was also 2-fold (injected peptide) to 4.6-fold (α-synuclein) more effective than adjuvanted SEQ ID NO:6+KLH, and 8.7-fold (injected peptide) and 11-fold (α-synuclein) more immunogenic than the unadjuvanted SEQ ID NO:6+KLH vaccine, respectively.

[0352] In addition to the general increase in immunogenicity of the CLEC-modified vaccine, the results also show that the CLEC-modification according to the invention leads to a significant increase in the relative amount of induced antibodies binding to the target molecule, i.e., protein, thereby significantly increasing the target specificity of the subsequent immune response.Thus, the relative amount of antibodies detecting alpha-synuclein (i.e., the ratio of total anti-injected peptide titers compared to anti-alpha-synuclein specific titers) is 3.7 times higher for SEQ ID NO:3+KLH+pustulan-induced responses compared to adjuvanted SEQ ID NO:3+KLH, and 2.2 times higher for SEQ ID NO:6+KLH+pustulan compared to adjuvanted conjugates.

[0353] In a second set of experiments, the same vaccines used (total vaccines: 5 μg aSyn targeting peptide / dose; route: id for CLEC-based vaccines, sc for Alhydrogel-adjuvanted KLH-based vaccines) were compared for their ability to induce anti-carrier specific antibody responses. As expected, the conventional SEQ ID NO:3+KLH-based vaccine and SEQ ID NO:6+KLH-based vaccine were able to induce high anti-KLH titers (SEQ ID NO:3+KLH: 1 / 2100 and SEQ ID NO:6+KLH: 1 / 7700), whereas the CLEC-based SEQ ID NO:3+KLH+pustulan vaccine and SEQ ID NO:6+KLH+pustulan vaccine were essentially unable to induce sustainable anti-carrier antibodies. The titers obtained were close to the limit of detection, 150-fold lower for SEQ ID NO:3 + KLH + pustulan and less than 100-fold lower for SEQ ID NO:6 + KLH + pustulan, respectively, thus creating a novel and previously undescribed optimization strategy for peptide-conjugate vaccines to increase target-specific titers while reducing undesirable anti-carrier responses.

[0354] Example 11: Analysis of immunogenicity of CLEC conjugates using carrier proteins as T helper 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 conventional CRM197 vaccine. For this purpose, the α-synuclein-derived epitope SEQ ID NO:6 was linked to maleimide-activated CRM197. The SEQ ID NO:6+CRM197 conjugate was then linked to activated pustulan using the heterobifunctional linker BPMH to form a CLEC-based conjugate vaccine with CRM197 as a source of T-helper cell epitopes to induce a sustainable immune response. Alternatively, SEQ ID NO:5-(NH-NH2; SEQ ID NO:5) and CRM197 were independently linked to activated pustulan. This was done by reaction of the hydrazide at the C-terminus of SEQ ID NO:5 and via the lysine present in CRM197 to the reactive aldehyde on activated pustulan.

[0355] Vaccines used: [Table 13]

[0356] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 20 μg α-synuclein-targeted peptide / dose; route: id for CLEC-based vaccines, sc for CRM197-based vaccines adjuvanted with Alhydrogel), and the subsequent immune response against the injected peptide (i.e., SEQ ID NO: 6) and the target proteins, i.e., recombinant human a-synuclein and α-synuclein fibrils, was analyzed using mouse plasma collected two weeks after the third immunization.

[0357] result: As shown in FIG. 11A, all three vaccines that used CRM197 as a source of T-helper epitopes were able to induce strong and specific immune responses against both the injected peptide moiety (SEQ ID NO: 6) and the target protein, recombinant α-synuclein.

[0358] Also, CLEC modification of CRM197 conjugates resulted in significantly better immune responses: SEQ ID NO:6 + CRM197 + pustulan was able to induce a 28-fold higher anti-peptide response compared to SEQ ID NO:6 + CRM197 adjuvanted with Alhydrogel. Similarly, the anti-protein titer against recombinant aSyn was also increased by 15-fold (compared to SEQ ID NO:6 + CRM197 adjuvanted with Alhydrogel), and the titer against α-synuclein fibrils, the aggregated form of α-synuclein, was increased by 11-fold.

[0359] Vaccines made by independently linking SEQ ID NO:5 and CRM197 to pustulan also induced 1.7-fold higher injected peptide titers compared to SEQ ID NO:6+CRM197 adjuvanted with conventional Alhydrogel. Reactivity to recombinant aSyn was also increased by 6.6-fold, and the antifibrotic response was increased by 4.25-fold, respectively.

[0360] Comparison of anti-carrier specific antibody responses revealed that the conventional SEQ ID NO:6 + CRM197 based vaccine was able to induce high anti-CRM197 titers (1 / 6600), whereas the CLEC based SEQ ID NO:6 + CRM197 + pustulan vaccine was essentially unable to induce sustainable anti-carrier antibodies. The titers obtained were close to the detection limit, less than 1 / 100 for SEQ ID NO:6 + CRM197 + pustulan, respectively.

[0361] Thus, the experiments show that CLEC modification of conventional peptide-protein conjugates significantly impairs the generation of anti-carrier responses and leads to a significant enhancement of the target specificity of the subsequent immune response, providing a novel and unprecedented strategy to optimize current prior art conjugate vaccines based on carrier proteins such as KLH, CRM197, etc.

[0362] Independent ligation of CRM197 and SEQ ID NO:6 to pustulan resulted in sustainable responses to B cell epitopes present on CRM197, but at a lower rate (approximately 400-fold lower titres) than that detectable with conventional CLEC-unmodified conjugates, demonstrating that the CLEC scaffold according to the invention is also suitable for presenting B cell epitopes from CLEC-linked immunogenic proteins for use as vaccines.

[0363] Example 12: Analysis of the selectivity of immune responses elicited by CLEC-based vaccines in vivo Aggregation of the presynaptic protein aSyn has been implicated as a major pathological cause in synucleinopathies such as Parkinson's disease, whereas monomeric non-aggregated aSyn has important neuronal functions. Therefore, reducing / eliminating aggregated aSyn without affecting the available pool of present non-aggregated molecules may be important for the treatment of synucleinopathies, for example by active or passive immunotherapy.

[0364] To further characterize the immune responses elicited by CLEC-based vaccines containing aSyn-targeting peptides SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:6 compared to conventional peptide-carrier vaccines (i.e., SEQ ID NO:3 + KLH and SEQ ID NO:6 + CRM197), a series of experiments were performed to analyze the selectivity of the subsequent immune responses elicited against two different forms of the presynaptic protein aSyn: non-aggregated, predominantly monomeric aSyn and aggregated aSyn fibrils.

[0365] Vaccines used: [Table 14]

[0366] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 20 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines, sc for Alhydrogel-adjuvanted KLH-based vaccines and CRM197-based vaccines), and the subsequent immune response against the target proteins, i.e., recombinant human α-synuclein and aSyn fibrils, was analyzed using mouse plasma collected 2 weeks after the third immunization. Plasma samples were subjected to an aSyn-specific inhibition ELISA and IC50 values ​​were determined.

[0367] result: In summary, all CLEC-based conjugates used in this study demonstrate superior immunogenicity and a-synuclein aggregate-specific targeting selectivity compared to conventional peptide-conjugate vaccines (i.e., SEQ ID NO:3+KLH and SEQ ID NO:6+CRM, see FIG. 12).

[0368] Conventional peptide conjugate vaccines can induce antibody responses with slightly increased selectivity for aSyn aggregates (i.e., fibrils) compared to monomeric / recombinant aSyn. SEQ ID NO:3+KLH adjuvanted with Alhydrogel initiated an immune response with 9-fold higher selectivity for aSyn aggregates compared to recombinant aSyn. SEQ ID NO:6+CRM197 adjuvanted with Alhydrogel induced a less selective immune response reaching 3.5-fold selective binding for aggregates compared to predominantly monomeric recombinant aSyn.

[0369] In contrast, the antibodies induced by the CLEC-based peptide conjugate vaccine were characterized by several-fold more selective binding compared to the KLH conjugate vaccine or the CRM197 conjugate vaccine. Plasma induced by SEQ ID NO:2 + SEQ ID NO:7 + pustulan and SEQ ID NO:5 + SEQ ID NO:7 + pustulan showed approximately 97-fold higher aggregate selectivity (i.e., 14-fold higher than the comparative vaccine SEQ ID NO:3 + KLH, Alhydrogel) and 50-fold higher aggregate selectivity (i.e., 14-fold higher than the comparative vaccine SEQ ID NO:6 + CRM, Alhydrogel). SEQ ID NO:3 + KLH + pustulan and SEQ ID NO:6 + CRM197 + pustulan were similarly selective, reaching 40-fold (i.e., 5-fold higher than SEQ ID NO:3 + KLH) and 50-fold (i.e., 14-fold higher than SEQ ID NO:6 + CRM) higher selectivity against aSyn aggregates, respectively.

[0370] Thus, the experiments demonstrate that CLEC modification of peptide and peptide-protein conjugates results in strongly enhanced target specificity of the subsequent immune response, providing a novel and unprecedented strategy for optimizing current prior art conjugate vaccines.

[0371] Example 13: Avidity and affinity analysis of immune responses elicited by CLEC-based vaccines To further characterize the immune responses elicited by CLEC-based vaccines containing aSyn-targeting peptides SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:5, and SEQ ID NO:6 compared to conventional peptide-carrier vaccines (i.e., SEQ ID NO:3 + KLH and SEQ ID NO:6 + CRM197), a series of experiments were performed to analyze the binding activity and affinity of antibodies elicited against aSyn.

[0372] Vaccines used: [Table 15]

[0373] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 20 μg aSyn-targeting peptide / dose; route: id for CLEC-based vaccines, sc for Alhydrogel-adjuvanted KLH-based vaccines and CRM197-based vaccines), and the subsequent immune response to the target proteins, i.e., recombinant human aSyn and aSyn fibrils, was analyzed using mouse plasma collected 2 weeks after each immunization. To determine the avidity of induced Abs to recombinant aSyn, a modification of a standard ELISA assay was used in which replicate wells containing antigen-bound antibody were exposed to increasing concentrations of chaotropic thiocyanate ions. Resistance to thiocyanate elution was used as a measure of avidity, and plasma samples were compared (both between treatment groups and between time points) using an index representing 50% of effective antibody binding (avidity index).

[0374] In addition, the k of antibodies against aSyn fibers 2 weeks after the last immunization D Values ​​(antibody affinity to aSyn fibrils) were also determined based on aSyn competitive ELISA.

[0375] result: As shown in FIG. 13, the conventional SEQ ID NO:3+KLH conjugate (adjuvanted with Alhydrogel) showed very limited avidity maturation for aSyn binding when comparing immune samples obtained 2 weeks after the second immunization (T2) or 2 weeks after the third immunization (Avidity Maturation (AM, IC for T2 samples). 50 Values ​​and IC for T3 samples 50 Compare values: 1.1). In contrast, CLEC-based vaccines such as SEQ ID NO:2 + SEQ ID NO:7 + pustulan were able to induce a strong maturation of anti-aSyn responses as shown by an AI of 2.2, which is associated with a strong increase in the binding activity of T3 samples to a-synuclein. Samples obtained from SEQ ID NO:3 + KLH + pustulan immunized animals also showed significantly higher binding activity and slightly increased maturation compared to SEQ ID NO:3 + KLH alone.

[0376] Similarly, the avidity of the immune response elicited against the aSyn protein was also significantly higher for SEQ ID NO:5 + SEQ ID NO:7 + pustulan vaccine-induced antibodies and SEQ ID NO:6 + CRM197 + pustulan vaccine-induced antibodies compared to the SEQ ID NO:6 + CRM197 benchmark vaccine (analyzed at T3; i.e., 3-3.8-fold higher chaotropic salt levels were required to reduce binding), and affinity maturation was also increased compared to T2 and T3 values, respectively. SEQ ID NO:6 + CRM197 did not result in increased avidity for aSyn compared to T2 and T3, whereas the two CLEC-based vaccines resulted in a strong increase in aSyn-specific binding compared to T2 and T3.

[0377] aSyn fiber k for immune responses elicited by CLEC-based vaccines and conventional benchmark vaccines DExperiments quantifying the affinity of antibodies induced by the CLEC-based vaccine for aSyn revealed a highly significant increase in the overall affinity of the antibodies (see FIG. 14) induced by the CLEC-based vaccine. The SEQ ID NO:2 + SEQ ID NO:7 + pustulan conjugates and SEQ ID NO:3 + KLH + pustulan conjugates showed 6-9 times higher affinity (i.e., Kd: k of 1 mM) than the benchmark vaccine SEQ ID NO:3 + KLH adjuvanted with Alhydrogel. D The SEQ ID NO:5 + SEQ ID NO:7 + pustulan conjugate and the SEQ ID NO:6 + CRM + pustulan conjugate showed 12-15 fold better Kd values ​​(i.e., Kd: k of 750 nM) compared to the benchmark control SEQ ID NO:6 + CRM197 adjuvanted with Alhydrogel. D (50 nM and 60 nM compared to

[0378] Thus, the experiments demonstrate that CLEC modifications of peptide and peptide-protein conjugates result in strongly enhanced target specificity and affinity of the subsequent immune response, providing a novel and unprecedented strategy for optimizing current prior art conjugate vaccines.

[0379] Example 14: Analysis of the in vitro functionality of immune responses elicited by CLEC-based vaccines To analyze whether the aSyn-specific antibodies elicited by the CLEC-based vaccine (containing the aSyn-targeting peptides SEQ ID NO:2 / 3 and SEQ ID NO:5 / 6) were biologically active, a series of experiments were performed to analyze the ability of the antibodies to inhibit aSyn aggregation in vitro.

[0380] Vaccines used: [Table 16]

[0381] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 20 μg aSyn-targeting peptide / dose; route: id for CLEC-based vaccines, sc for Alhydrogel-adjuvanted KLH-based vaccines and CRM197-based vaccines). Samples of mouse plasma taken two weeks after each immunization and respective control samples (e.g., non-aSyn-binding antibodies or pre-immune plasma obtained before immunization) were analyzed for in vitro agglutination inhibition capacity.

[0382] result: As shown in Figure 15A, control antibodies or plasma taken from animals before immunization did not significantly affect the aggregation kinetics of aSyn, confirming the specificity of the assay. Abs induced by the classical SEQ ID NO:3 + KLH conjugate (with Alhydrogel adjuvant) were able to significantly reduce aSyn aggregation, as indicated by a 40% reduced slope value (aSyn monomer alone: ​​100%; KLH.60%). Abs induced by SEQ ID NO:2 + SEQ ID NO:7 + pustulan vaccine strongly inhibited aSyn aggregation, as indicated by an 85% reduced slope value (aSyn monomer alone: ​​100%; CLEC: 15%) in this assay, demonstrating a significantly higher inhibitory capacity compared to Abs induced by classical vaccines.

[0383] Antibodies induced by SEQ ID NO:5 + SEQ ID NO:7 + pustulan based vaccine and SEQ ID NO:6 + CRM + pustulan based vaccine show 86-92% inhibition of the formation of aggregates starting with rec.aSyn (low content aggregates) and 67-82% inhibition of the formation of aggregates starting with preformed fibrils (= true aggregates) compared to 68% and 57% for the benchmark vaccine SEQ ID NO:6 + CRM, Alhydrogel induced antibodies (see Figure 15B).

[0384] Example 15: Analysis of the effect of immunization route on immune responses elicited by CLEC-based vaccines A series of immunizations was performed to compare id administration with alternative routes including subcutaneous (sc) and intramuscular (im).

[0385] Vaccines used: [Table 17]

[0386] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 1 μg, 5 μg, and 20 μg of aSyn-targeted peptide / dose), and the subsequent immune responses against the injected peptides and target proteins, i.e., recombinant human a-synuclein and aSyn fibrils, were analyzed using mouse plasma collected 2 weeks after the third immunization.

[0387] result: Tables 1 and 2 and FIG. 16 show that SEQ ID NO:2 + SEQ ID NO:7 + pustulan vaccine administered via im or sc route was able to induce high immune responses against both the injected peptide (FIG. 16A) and anti-aSyn responses (FIG. 16B). The maximum titers reached were significantly lower than those after id administration at all doses tested. The sc administration showed a similar dose-response behavior as id, whereas im showed no significant difference between 5 μg and 20 μg, indicating that saturation was reached at these doses / administration volumes. Similar results were obtained for reactivity against monomeric and aggregated aSyn, respectively. These results demonstrate the high selectivity of the CLEC scaffold presented in the invention for application to the skin, as opposed to other routes / tissues.

[0388] [Table 18] Table: Anti-SEQ ID NO:2 / 3 induced antibody responses after WISIT vaccination via different routes

[0389] [Table 19] Table: Anti-aSyn-induced antibody responses following WISIT vaccination via different routes

[0390] Example 16: Analysis of immunogenicity of CLEC conjugates using carrier proteins as T helper cell epitopes: different conjugate / CLEC ratios In this example, the immunogenicity of CLEC-based conjugate vaccines containing the well-known carrier protein CRM197 using different peptide+CRM / CLEC ratios was compared. For this purpose, the aSyn-derived epitope SEQ ID NO:6 was linked to maleimide-activated CRM197. The SEQ ID NO:6+CRM197 conjugate was then linked to activated pustulan at different w / w ratios using the heterobifunctional linker BPMH to form a CLEC-based conjugate vaccine with CRM197 as a source of T-helper cell epitopes to induce a sustainable immune response.

[0391] Vaccines used: [Table 20]

[0392] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines) and the subsequent immune response against the injected peptide (i.e., SEQ ID NO: 6) and target proteins, i.e., recombinant human a-synuclein and aSyn fibrils, was analyzed using mouse plasma collected 2 weeks after the third immunization.

[0393] result: As shown in FIG. 17, all five vaccines that used CRM197 as a source of T-helper epitopes were able to induce strong and specific immune responses against both the injected peptide moiety (SEQ ID NO: 6) and the target protein, recombinant a-synuclein.

[0394] The CLEC modification of CRM197 conjugates resulted in highly efficient immune responses at all w / w conjugate / CLEC ratios tested. SEQ ID NO:6+CRM197+pustulan (w / w 1 / 10) resulted in the highest anti-aSyn specific immune response compared to the other variants tested. Thus, SEQ ID NO:6+CRM197 conjugates with medium / high conjugate / CLEC ratios are particularly suitable for inducing optimal immune responses (e.g., 1 / 5, 1 / 10, and 1 / 20).

[0395] Thus, the experiments demonstrate that CLEC modification of conventional peptide-protein conjugates results in strong target specificity of the subsequent immune response and offers a novel and unprecedented strategy to optimize current prior art conjugate vaccines based on carrier proteins such as KLH, CRM197, etc.

[0396] Example 17: Analysis of immunogenicity of CLEC conjugates and peptide conjugates using carrier proteins as T helper cell epitopes - aSyn N-terminus (aa1-10) In this example, it was evaluated whether a CLEC-based conjugate vaccine according to the invention could induce a superior immune response against aSyn aggregates compared to the respective peptide conjugates that used prior art carrier proteins as a source of T cell epitopes.

[0397] We therefore initiated a series of experiments comparing two conjugates, both of which contained epitopes suggested to be suitable aSyn targeting epitopes, which allowed us to demonstrate the selectivity of the immune responses elicited against the injected peptide and aSyn protein, and the subsequent immune responses elicited against two different forms of the presynaptic protein aSyn: non-aggregated, predominantly monomeric aSyn and aggregated aSyn fibrils.

[0398] For example, Weihofen et al. (2019, aa1-10 as an epitope for sipanemab) and WO2016 / 062720 (aa1-8 as an epitope in VLP-based immunotherapeutics) suggest the N-terminal aSyn sequence derived from position aa1-10 as a potentially suitable epitope for aSyn-targeted immunotherapy. Therefore, to evaluate whether CLEC modification indeed results in a superior immune response, we compared a CLEC-based vaccine containing aSyn sequence aa1-8 (SEQ ID NO:12 + SEQ ID NO:7 + pustulan) with the respective conventional peptide-KLH vaccine (SEQ ID NO:13 + KLH adjuvanted with alum).

[0399] Vaccines used: [Table 21]

[0400] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines adjuvanted with Alhydrogel) and the subsequent immune response against the injected peptides and target proteins by ELISA and EC50 values ​​was determined. Furthermore, to assess the selectivity of the immune response, plasma samples were subjected to an aSyn-specific inhibition ELISA and expressed as a percentage of maximum binding.

[0401] result: The aSyn N-terminus-targeted CLEC-based conjugate vaccine (SEQ ID NO:12 + SEQ ID NO:7 + Pus) used in this experiment demonstrates superior immunogenicity against the aSyn protein compared to conventional peptide-conjugate vaccines (i.e., SEQ ID NO:13 + KLH, see FIG. 18A). The CLEC-based vaccine induces a 1.8-fold increase in anti-aSyn titers and a concomitant 3-fold increase in the ratio of anti-peptide to anti-protein responses compared to the control group. This strongly supports the teaching of the present invention that CLEC modifications result in superior immune responses compared to similar conventional vaccines.

[0402] Furthermore, conventional peptide-KLH conjugate vaccines induce antibody responses with greatly increased selectivity (about 10-fold) for aSyn monomers compared to aggregates (i.e., fibrils, see FIG. 18B). In contrast to this finding, and very surprisingly, CLEC-based conjugates result in a completely different selectivity: SEQ ID NO:12 + SEQ ID NO:7 + pustulan induce antibodies with about 10-fold higher selectivity for aSyn aggregates compared to recombinant aSyn, thereby completely changing the profile of induced antibodies (see FIG. 18B).

[0403] Thus, the experiments show that conventional peptide vaccines applying aSyn aa1-8 are poorly suited to initiate efficient and selective immune responses in vivo, suggesting that this epitope is not suitable for aggregate-selective immunotherapy. Importantly, the results also demonstrate that CLEC modification of peptide conjugates results in strongly enhanced target specificity of the subsequent immune response and altered selectivity towards aggregates, thus providing a novel and unprecedented conjugate vaccine targeting aSyn.

[0404] Example 18: Analysis of immunogenicity of CLEC conjugates and peptide conjugates using carrier proteins as T helper cell epitopes - aSyn aa100-108 Here, we initiated a series of experiments to compare two conjugates that both contain epitopes suggested to be suitable as aSyn-targeting epitopes by analyzing the selectivity of the immune responses elicited against the injected peptide and aSyn protein, and the subsequent immune responses elicited against two different forms of the presynaptic protein aSyn: non-aggregated, predominantly monomeric aSyn and aggregated aSyn fibrils.

[0405] For example, WO 2011 / 020133 and WO2016 / 062720 suggest aSyn sequences derived from positions aa100-108 / 109 (either as native sequence or mimotope, i.e. 100-108) as potentially suitable epitopes for aSyn-targeted immunotherapy. Therefore, to assess whether CLEC modification indeed results in superior immune responses using this epitope region, we compared a CLEC-based vaccine containing aSyn aa100-108 (SEQ ID NO:16+SEQ ID NO:7+pustulan) with the respective conventional peptide-KLH vaccine (SEQ ID NO:17+KLH adjuvanted with alum).

[0406] Vaccines used: [Table 22]

[0407] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines adjuvanted with Alhydrogel) and the subsequent immune response against the injected peptides and target proteins by ELISA and EC50 values ​​was determined. Furthermore, to assess the selectivity of the immune response, plasma samples were subjected to an aSyn-specific inhibition ELISA and expressed as a percentage of maximum binding.

[0408] result: The aSyn-targeted CLEC-based conjugate vaccine used in this experiment (SEQ ID NO:16 + SEQ ID NO:7 + Pus) demonstrates an overall very low anti-aSyn protein response, even lower than a conventional peptide-conjugate vaccine (i.e., SEQ ID NO:17 + KLH, see FIG. 19A). The conventional vaccine induces an immune response characterized by a 2.1-fold increase in anti-aSyn titers, but at the same time a 2-fold decrease in the ratio of anti-peptide / anti-protein titers, compared to the CLEC-based vaccine. The latter finding supports the teaching of the present invention that CLEC modifications result in superior anti-target protein responses, even in the case of overall lower immunogenicity compared to similar conventional vaccines.

[0409] Moreover, both conventional peptide conjugate and CLEC-based vaccines are less favorable for inducing aggregate-selective immune responses (see FIG. 19B). Thus, the presented experiments show that CLEC-based and conventional peptide vaccines targeting the region aa100-108 are less suitable for initiating efficient and selective immune responses in vivo, suggesting that this epitope may not be the optimal choice for aggregate-selective immunotherapy according to the present invention.

[0410] Example 19: Analysis of immunogenicity of CLEC conjugates and peptide conjugates using carrier proteins as T helper cell epitopes - aSyn aa91-100 In this example, we began a series of experiments comparing two conjugates that both contain epitopes suggested to be suitable aSyn targeting epitopes by analyzing the immune responses elicited against the injected peptide and aSyn protein.

[0411] For example, US 2014 / 0377271 A1 suggests that epitope aa91-99 acts as a self-epitope in PD patients and therefore constitutes a potentially suitable epitope for aSyn-targeted immunotherapy. Therefore, to evaluate whether CLEC modification actually applies this epitope to result in a superior immune response, we compared a CLEC-based vaccine containing aSyn aa91-100 (SEQ ID NO:14+SEQ ID NO:7+pustulan) with the respective conventional peptide-KLH vaccine (SEQ ID NO:15+KLH with alum adjuvant).

[0412] Vaccines used: [Table 23]

[0413] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines adjuvanted with Alhydrogel) and the subsequent immune response to the injected peptides and the target protein aSyn by ELISA and EC50 values ​​was determined.

[0414] result: Surprisingly, both vaccines, although inducing relevant anti-peptide titers, were less successful in inducing detectable anti-aSyn protein titers (see FIG. 20). Thus, the presented experiments show that CLEC-based vaccines and traditional peptide vaccines targeting the region aa91-100 are less suitable for mounting efficient and selective immune responses in vivo, suggesting that this epitope may not be the optimal choice for aggregate-selective immunotherapy according to the present invention.

[0415] Example 20: Analysis of immunogenicity of CLEC conjugates and peptide conjugates using carrier proteins as T helper cell epitopes - aSyn C-terminal region aa131-140 In this example, we began a series of experiments comparing two conjugates that both contain epitopes suggested to be suitable aSyn targeting epitopes by analyzing the immune responses elicited against the injected peptide and aSyn protein, and further evaluated the selectivity of the subsequent immune responses elicited against the two different forms of the presynaptic protein aSyn.

[0416] For example, US 2015 / 0232524 and WO2016 / 062720 suggest C-terminal aSyn sequences derived from positions aa126-140 and aa131-140 as potentially suitable epitopes for aSyn-targeted immunotherapy. Therefore, to evaluate whether CLEC modification indeed results in superior immune responses, we compared a CLEC-based vaccine containing aSyn aa131-140 (SEQ ID NO:20+SEQ ID NO:7+pustulan) with the respective conventional peptide-KLH vaccines (SEQ ID NO:21+KLH adjuvanted with alum).

[0417] Vaccines used: [Table 24]

[0418] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines adjuvanted with Alhydrogel) and the subsequent immune response against the injected peptides and target proteins by ELISA and EC50 values ​​was determined. Furthermore, to assess the selectivity of the immune response, plasma samples were subjected to an aSyn-specific inhibition ELISA and expressed as a percentage of maximum binding.

[0419] result: The aSyn-targeted CLEC-based conjugate vaccine used in this experiment (SEQ ID NO:20 + SEQ ID NO:7 + Pus) demonstrates an overall lower anti-aSyn protein response compared to a conventional peptide-conjugate vaccine (i.e., SEQ ID NO:21 + KLH, see FIG. 21A). The conventional vaccine induces an immune response characterized by a 1.8-fold increase in anti-aSyn titers, but a 45% decrease in the ratio of anti-peptide / anti-protein titers, compared to the CLEC-based vaccine. The latter finding supports the teaching of the present invention that CLEC modifications result in superior anti-target protein responses, even in the case of overall lower immunogenicity compared to similar conventional vaccines.

[0420] Furthermore, conventional peptide conjugates are less suitable for inducing aggregate-selective immune responses (see FIG. 21B). In contrast, CLEC-based vaccines induce antibodies with approximately 10-fold increased selectivity for monomeric aSyn at the expense of aggregated aSyn (see FIG. 21B). Thus, the presented experiments indicate that CLEC-based vaccines and conventional peptide vaccines targeting the region aa131-140 are less suitable for initiating efficient and selective immune responses against aggregated aSyn in vivo, suggesting that this epitope may not be the optimal choice for aggregate-selective immunotherapy according to the present invention.

[0421] Example 21: Analysis of immunogenicity of CLEC conjugates and peptide conjugates using carrier proteins as T helper cell epitopes - aSyn C-terminal region aa103-135 In this example, it was evaluated whether a CLEC-based conjugate vaccine according to the present invention could induce a superior immune response against aSyn compared to the respective peptide conjugates that used prior art carrier proteins as a source of T cell epitopes.

[0422] We therefore initiated a series of experiments comparing several conjugates derived from epitope regions suggested to be suitable as aSyn targeting epitopes. These experiments allowed us to demonstrate the selectivity of the immune responses elicited against the injected peptides and aSyn protein, and the subsequent immune responses elicited against two different forms of the presynaptic protein aSyn: non-aggregated, predominantly monomeric aSyn and aggregated aSyn fibrils.

[0423] Several studies have suggested that the C-terminal aSyn sequence derived from positions aa103-135 is a potentially suitable epitope for aSyn-targeted immunotherapy as a source of peptides containing either self-epitopes, the original sequence or its mimotopes. Therefore, to evaluate whether CLEC modification indeed results in superior immune responses using this region within aSyn, we compared several CLEC-based vaccines (using peptides within region 107-126) with the respective conventional peptide-CRM vaccines (adjuvanted with alum).

[0424] Vaccines used: [Table 25]

[0425] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines, sc for KLH-based vaccines adjuvanted with Alhydrogel) and the subsequent immune response against the injected peptides and target proteins by ELISA and EC50 values ​​was determined. Furthermore, to assess the selectivity of the immune response, plasma samples were subjected to an aSyn-specific inhibition ELISA and expressed as a percentage of maximum binding.

[0426] result: [Table 26] Table 1: Immune responses elicited by vaccines covering aa107-126

[0427] Both CLEC-based vaccines containing 5-mer and 6-mer peptides and CRM-based vaccines were less suitable for inducing high anti-aSyn fiber titers in this experiment. The CLEC-based conjugate vaccines (7-12-mer peptides) targeting the aSyn C-terminus used in this experiment (see Table 1 and Figures 22A, 23A, and 24A) all demonstrate superior immunogenicity against aSyn fiber compared to conventional peptide conjugate vaccines (see Table 1, up to 4-fold increase). This strongly supports the teaching of the present invention that CLEC modifications result in superior immune responses compared to similar conventional vaccines using epitopes derived from 103-135, especially 107-126.

[0428] Analysis of the selectivity of aggregated aSyn further supports this teaching. As shown in Figures 22B and 23B, CLEC vaccines containing an epitope derived from sequence aa115-126 are surprisingly effective at eliciting highly aggregate-selective immune responses. As shown in Figure 22B, the CLEC-based vaccine SEQ ID NO:51+SEQ ID NO:7+Pus containing an 8mer aSyn-targeting epitope induces antibodies with 10-fold higher selectivity for aSyn aggregates, whereas the respective conventional vaccine (SEQ ID NO:52+CRM+Alum) fails to induce aggregate-selective antibodies. Similarly, the CLEC-based vaccine SEQ ID NO:67+SEQ ID NO:7+Pus containing a 10mer aSyn-targeting epitope induces approximately 10-fold higher selectivity for aSyn aggregates compared to monomers, whereas the respective conventional vaccine (SEQ ID NO:68+CRM+Alum) induces antibodies that are approximately 3-fold more selective for monomers compared to aggregates (see Figure 23B).

[0429] Selectivity analysis for vaccines containing epitopes aa107-114 (SEQ ID NO:73 + SEQ ID NO:7 + Pus and SEQ ID NO:74 + CRM + Alum, see FIG. 24B) surprisingly showed that despite the presence of high anti-aSyn fiber titers (i.e., excellent immunogenicity) induced by the CLEC-based vaccine, neither the CLEC vaccine nor the conventional vaccine was able to induce aggregate-selective antibodies, indicating that only highly selective peptide sequences within aa103-135 are suitable as immunotherapeutic agents specifically targeting aggregated aSyn.

[0430] It should also be noted that, as shown previously (see Figures 18-20), epitopes derived from aa91-100, aa100-108, and aa131-140 are all poorly suited as potential immunotherapeutic regions for specifically targeting aggregated aSyn.

[0431] Example 22: Analysis of the in vitro functionality of immune responses elicited by CLEC-based vaccines To analyze whether the aSyn-specific antibodies elicited by the CLEC-based vaccine (containing an aSyn-targeting peptide derived from the epitope region aa103-135) were biologically active, a series of experiments were performed analyzing the ability of the antibodies to inhibit aSyn aggregation in vitro.

[0432] Vaccines used: [Table 27]

[0433] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 20 μg aSyn-targeting peptide / dose; route: id for CLEC-based vaccines, sc for CRM197-based vaccines adjuvanted with Alhydrogel). Samples of mouse plasma taken two weeks after each immunization and respective control samples (e.g., aSyn-binding antibody LB509, epitope aa115-122, or pre-immune plasma obtained before immunization) were analyzed for in vitro agglutination inhibition capacity.

[0434] result: As shown in Figure 25D, plasma collected from animals prior to immunization did not significantly affect the aggregation kinetics of aSyn, confirming the specificity of the assay.

[0435] SEQ ID NO:67 + SEQ ID NO:7 + Pustulan vaccine (containing a 10-mer aSyn-derived peptide) induced Abs greatly inhibited aSyn aggregation as shown by a 40% decrease in aggregation over time in this assay, while the respective CRM conjugate vaccines showed only minimal effects showing significantly higher inhibitory potency compared to classical vaccine induced Abs (Figure 25A). Similar results could be achieved analyzing antibodies induced by SEQ ID NO:71 + SEQ ID NO:7 + Pustulan (containing a 12-mer aSyn-derived peptide), which were able to reduce aggregation even more (70%-80% inhibition), exceeding the inhibitory potency of antibodies induced by a conventional CRM vaccine (SEQ ID NO:72 + CRM + Alhydrogel) by 2-2.5 fold. Figure 25C shows that antibodies induced by a CLEC-based vaccine based on epitope aa107-114 (containing an 8-mer aSyn-derived peptide) and a conventional vaccine, in contrast, were unable to inhibit aSyn aggregation.

[0436] As shown in Figure 25D, the aSyn-specific antibody LB509 is unable to inhibit aSyn aggregation, and conversely, a slight increase in aggregation can be detected in this assay.

[0437] This is a highly surprising effect in view of the teachings of the present invention (see Example 14 and Figure 15, which also analyze epitopes derived from the aSyn sequence aa115-126, and in particular from aa115-121, as well as Table 1 and Figures 22-25, which describe a vaccine covering epitopes 115-126 as monoclonal LB509 (epitope aa:115-122), known to bind different forms of aSyn (Jakes et al. Neurosci.Lett.1999 Jul 2;269(1):13-6) and sharing the same epitope as the biologically effective vaccine according to the present invention (see Figure 25D). The biologically superior effect obtained with the CLEC-based vaccine is indeed surprising and indicates that highly selected peptide sequences contained within aa115-126 are preferred within the region aa103-135 as immunotherapeutic agents specifically targeting aggregated aSyn.

[0438] Example 23: Determination of the biological activity of peptide+CRM197+CLEC conjugates in vitro against the mouse Dectin-1 receptor In a series of ELISA experiments, conjugates containing the dectin-1 ligands pustulan, lichenan, and laminarin were evaluated for their binding efficacy to mouse dectin-1. The biological activity of peptide + CRM197 + CLEC conjugates is represented by their PRR binding ability. Along these lines, binding to mouse dectin-1 was evaluated to ensure that the CLEC (pustulan, lichenan, laminarin) structures remain biologically active after conjugation. Then, non-oxidized and oxidized pustulan, lichenan, and laminarin, as well as CRM conjugate vaccines, and peptide + CRM197 + CLEC-based novel conjugates were evaluated for biological activity using a competitive ELISA system based on competitive binding of soluble mouse Fc-Dectin-1a receptor (InvivoGen) as described in Korotchenko et al. 2020.

[0439] result: Subsequent experiments revealed that pustulan, a medium molecular weight (20 kDa) linear β-(1,6)-linked β-D-glucan, and laminarin, a linear β(1-3) glucan with β(1-6) linkages, exerted significantly higher binding efficacy (approximately 10-fold) for mouse dectin-1 than lichenan (approximately 245 kDa), a larger high molecular weight linear β-(1,3)β-(1,4)-β-D-glucan (see Figure 26).

[0440] As shown in Figure 26A, the dectin-1 ligand pustulan, oxidized pustulan, SEQ ID NO:6 + CRM conjugate (CRM-conjugate 1), and SEQ ID NO:6 + CRM + pustulan conjugate (CRM-Pus conjugate 1) were evaluated for their binding efficacy to mouse dectin-1 by ELISA analysis. Subsequent experiments revealed that the peptide + CRM197 + pustulan conjugate exhibited binding efficacy to mouse dectin-1 similar to that of oxidized pustulan. In contrast, the conventional CRM-conjugate 1 did not exhibit specific mouse dectin-1 binding. High binding efficacy to mouse dectin-1 was also exhibited by five novel CRM + pustulan conjugates (SEQ ID NO:52 / 66 / 68 / 70 / 72) (Figure 26B). Subsequent experiments revealed that peptide-CRM197-pustulan conjugates bearing different B cell epitopes, ranging from a 7-mer B cell epitope (SEQ ID NO:6+CRM+Pus; Figure 26A) to a 12-mer B cell epitope (SEQ ID NO:71+CRM+Pus; Figure 1B), exhibited binding efficacy to mouse dectin-1 similar to oxidized pustulan. As shown in Figure 26C, high molecular weight (approximately 22-245 kDa) linear β-(1,3)β-(1,4)-β-D-glucan lichenan exerts lower binding efficacy than linear β-(1,6)-linked β-D-glucan pustulan-based constructs, regardless of oxidation or conjugate formation. For example, pustulan-containing CRM197-peptide conjugates retain approximately 10-fold higher binding than lichenan-based constructs. High binding efficacy for mouse dectin-1 was also shown by laminarin, a linear β(1-3)-glucan with β(1-6)-linkages (Figure 26D). Subsequent experiments revealed that peptide + CRM197 + laminarin conjugates showed similar binding efficacy for mouse dectin-1, independent of oxidation or conjugate formation, as did the pustulan-based constructs.

[0441] Experiments revealed that the peptide+CRM197+CLEC conjugate demonstrated biological activity on dendritic cells via binding to Dectin-1 in a mouse system.

[0442] Example 24: Determination of the biological activity of peptide+CRM197+CLEC conjugates in vitro against the human Dectin-1 receptor In a series of ELISA experiments, the dectin-1 ligands pustulan, lichenan, and laminarin were evaluated for their binding efficacy to human dectin-1. The biological activity of peptide+CRM197+CLEC conjugates is represented by their PRR binding ability. Along these lines, to ensure that the CLEC (pustulan, lichenan, laminarin) structures remain biologically active after conjugation, binding to human dectin-1 was evaluated by a competitive ELISA system based on competitive binding of soluble human Fc-dectin-1a receptor (InvivoGen).

[0443] result: As shown in FIG. 27, SEQ ID NO:6+CRM conjugates linked to either lichenan (Lich conjugate), pustulan (Pus conjugate), or laminarin (Lam conjugate) were evaluated for their binding efficacy to human dectin-1 by ELISA analysis.

[0444] Subsequent experiments revealed that the peptide+CRM197+pustulan vaccine exerted significantly higher binding efficacy (approximately 30-fold) to human dectin-1 than the vaccine conjugated to lichenan (see Figure 27). In contrast, the peptide+CRM197+laminarin vaccine showed weak binding to human dectin-1.

[0445] Example 25: In vivo comparison of different peptides + CRM197 + pustulan-based vaccines Novel CRM197-pustulan-based vaccines bearing different B cell epitopes ranging from 8mer to 11mer, which were able to bind to their DC receptors (e.g., Dectin-1), were tested for their ability to induce robust and specific immune responses after repeated dosing in Balb / c mice, n=5 per group. Routine experiments were performed applying B cell epitope peptides with a net peptide content of 5 μg per dose.

[0446] In this experiment, aSyn-derived peptides SEQ ID NO:52+CRM197 conjugate and SEQ ID NO:66 / 68 / 70+CRM conjugate were linked to oxidized pustulan. Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (route: id) with either the β-glucan modified peptide-CRM conjugate or the unmodified peptide-CRM conjugate, and the subsequent immune response against the injected peptides (i.e., SEQ ID NO:52 / 66 / 68 / 70, respectively) and aggregated aSyn fibers was analyzed using mouse plasma collected 2 weeks after the third immunization.

[0447] Vaccines used: [Table 28]

[0448] result: As shown in FIG. 28A, all four CRM+pustulan-based vaccines (SEQ ID NOs: 52 / 66 / 68 / 70 / 72) were able to induce significantly higher responses against both the injected peptide moiety (e.g., SEQ ID NOs: 52 / 66 / 68 / 70) and aggregated aSyn fibers when compared to the unmodified peptide+CRM-based vaccine adjuvanted with Alhydrogel.

[0449] The peptide+CRM+pustulan-based conjugates were able to induce 2-5 times higher titers against the respective peptides (maximum titer 1 / 190,000) and 3-13 times higher titers against aSyn fibers (maximum titer 1 / 29,000) compared to the unmodified peptide+CRM-based vaccine.

[0450] Example 26: Analysis of the selectivity of immune responses elicited by peptide + CRM + pustulan-based vaccines in vivo To further characterize the immune response elicited by the peptide+CRM197+pustulan-based vaccine containing distinct B cell epitopes compared to the conventional peptide+CRM197 vaccine, we performed a series of experiments analyzing the selectivity of the subsequent immune response elicited against aggregated aSyn fibers.

[0451] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for 4 peptide+CRM197+CLEC-based vaccine (SEQ ID NO:52 / SEQ ID NO:66 / 68 / 70+CRM197+pus), sc for 4 peptide-CRM197-based vaccine adjuvanted with Alhydrogel (SEQ ID NO:52 / SEQ ID NO:66 / 68 / 70-CRM197)) and subsequent immune responses against the target proteins, i.e., recombinant human α-synuclein and aSyn fibrils, were analyzed using mouse plasma collected 2 weeks after the third immunization. Plasma samples were subjected to aSyn-specific inhibition ELISA and IC50 values ​​were determined.

[0452] Vaccines used: [Table 29]

[0453] result: In brief, all CLEC-based conjugates used in this study demonstrate superior aSyn aggregate-specific targeting selectivity as determined by much lower IC50 values ​​against aSyn fibers compared to the conventional peptide-CRM197 conjugate vaccine (see Figure 29).

[0454] All four conventional peptide-CRM197 conjugate vaccines tested in this study induced antibodies that demonstrated very weak selectivity for aSyn fibers, as indicated by very high IC50 values ​​ranging from 400 to 1.700 ng / ml.

[0455] In contrast, all antibodies induced by the novel peptide + CRM197 + pustulan-based conjugate vaccine were characterized by much lower IC50 values ​​on aSyn fibers, ranging from 3.5 to 15 ng / ml.

[0456] Thus, the experiments demonstrate that CLEC modification of CRM197 conjugates results in greatly enhanced target specificity of the subsequent immune response, regardless of the epitope used, providing a novel and unprecedented strategy to optimize current prior art conjugate vaccines.

[0457] Example 27: Analysis of the avidity of immune responses elicited by peptide + CRM197 + pustulan-based vaccines To further characterize the immune response elicited by the peptide-CRM197-pustulan-based vaccine, which contains distinct B cell epitopes compared to the conventional peptide+CRM197 vaccine, we performed a series of experiments analyzing the binding activity of antibodies elicited against aSyn fibers.

[0458] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (all vaccines: 5 μg aSyn-targeted peptide / dose; route: id for CLEC-based vaccines (SEQ ID NO: 52 / 66 / 68 / 70 + CRM197 + pustulan), sc for CRM197-based vaccines adjuvanted with Alhydrogel (SEQ ID NO: 52 / 66 / 68 / 70 + CRM197)) and the subsequent immune response to the target protein, i.e., aSyn fibers, was analyzed using mouse plasma collected 2 weeks after each immunization. To determine the avidity of induced Abs to aSyn fibers, a modification of a standard ELISA assay was used in which replicate wells containing antigen-bound antibody were exposed to increasing concentrations of chaotropic thiocyanate ions. Resistance to thiocyanate elution was used as a measure of avidity, and plasma samples were compared using an index representing 50% of effective antibody binding (avidity index).

[0459] Vaccines used: [Table 30]

[0460] result: As shown in Figure 30, all conventional peptide-CRM197 conjugate (with Alhydrogel adjuvant) induced antibodies tested showed limited binding strength to aSyn fibers as demonstrated by very low avidity indexes ranging from 0.25 to 0.85. In contrast, all novel peptide + CRM197 + pustulan-based vaccine induced antibodies showed significantly higher binding strength to aSyn fibers with AIs ranging from 0.5 to 2.2.

[0461] Thus, the experiments show that CLEC modification of peptide-CRM197 conjugates results in greatly enhanced target-specific immune responses (titers) regardless of the epitope used, as well as greatly enhanced target specificity and affinity of the induced antibody responses, providing a novel and unprecedented strategy to optimize current prior art protein conjugate vaccines containing CRM197.

[0462] Example 28: In vivo comparison of different peptide + CRM197 + CLEC based vaccines SEQ ID NO:6+CRM197 conjugates, aSyn-derived peptides linked to either pustulan, lichenan, or laminarin, were tested for their ability to induce robust and specific immune responses after repeated dosing in Balb / c mice, n=5 per group. Routine experiments were performed applying B cell epitope peptides at 5 μg net peptide content per dose. Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (route: id) and the subsequent immune response against the injected peptide (i.e., SEQ ID NO:6) and aggregated aSyn fibers was analyzed using mouse plasma collected 2 weeks after the third immunization.

[0463] Vaccines used: [Table 31]

[0464] result: The tested vaccine was able to induce significant immune responses against the injected peptide (eg, SEQ ID NO:6) and aggregated aSyn fibers after repeated immunization in mice.

[0465] The peptide-CRM-pustulan-based conjugates induce higher titers against each peptide and higher titers against aSyn fibers compared to conventional peptide-CRM-based vaccines and to peptide-CRM-based vaccines conjugated to laminarin or lichenan (see Figure 31).

[0466] Specifically, SEQ ID NO:6 + CRM197 + pustulan induced 1.6-fold higher titers compared to SEQ ID NO:6 + CRM197 + lichenan and 12-fold higher titers compared to SEQ ID NO:6 + CRM197 + laminarin against the injected peptide SEQ ID NO:6, and SEQ ID NO:6 + CRM197 + lichenan was able to induce 7.5-fold higher titers compared to SEQ ID NO:6 + CRM197 + laminarin, respectively.

[0467] Similarly, SEQ ID NO:6 + CRM197 + pustulan induces 3.1-fold higher potency against aSyn aggregates (fibrils) compared to SEQ ID NO:6 + CRM197 + lichenan, 7.6-fold higher potency compared to SEQ ID NO:6 + CRM197 + laminarin, and 6-fold higher potency compared to CLEC-unmodified SEQ ID NO:6 + CRM197 with alum adjuvant. SEQ ID NO:6 + CRM197 + lichenan was able to induce 2.4-fold higher potency compared to SEQ ID NO:6 + CRM197 + laminarin and 2-fold higher potency compared to CLEC-unmodified SEQ ID NO:6 + CRM197 with alum adjuvant, respectively.

[0468] CLEC modification of peptide-CRM197 conjugates offers a novel and unprecedented strategy to optimize current prior art protein conjugate vaccines containing CRM197.

[0469] Example 29: Determination of the biological activity of peptide+CLEC-conjugates in vitro against mouse Dectin-1 receptor and human Dectin-1 receptor In a series of ELISA experiments, the dectin-1 ligands pustulan, lichenan, and laminarin were evaluated for their binding efficacy to mouse dectin-1 and human dectin-1. The biological activity of peptide-CLEC conjugates is represented by their PRR-binding ability. Along these lines, to ensure that the CLEC (pustulan, lichenan, laminarin) structures remain biologically active after conjugation, binding to mouse dectin-1 and human dectin-1 was evaluated by a competitive ELISA system based on competitive binding of soluble mouse Fc-dectin-1a receptor and soluble human Fc-dectin-1a receptor (InvivoGen).

[0470] result: As shown in Figure 32, SEQ ID NO:5 + SEQ ID NO:7 + CLEC conjugates linked to either lichenan (Lich conjugate), pustulan (Pus conjugate), or laminarin (Lam conjugate) were evaluated for their binding efficacy against mouse dectin-1 and human dectin-1 by ELISA analysis.

[0471] Subsequent experiments revealed that the peptide-pustulan vaccine exerted significantly higher binding efficacy to mouse dectin-1 and human dectin-1 than the vaccine conjugated to lichenan (see Figure 32A+B). In contrast, the peptide-laminarin vaccine showed very high binding to mouse dectin-1 (Figure 32A), but only weak binding to human dectin-1 (Figure 32B).

[0472] Example 30: In vivo comparison of different peptide+CLEC-based vaccines CLEC-based vaccines that were able to bind mouse Dectin-1 and / or human Dectin-1 were tested for their ability to induce robust and specific immune responses after repeated dosing in Balb / c mice, n=5 per group. Routine experiments were performed applying B cell epitope peptides at 5 μg and 20 μg net peptide content per dose.

[0473] In this experiment, the a-synuclein derived peptide SEQ ID NO:5 and the promiscuous T helper cell epitope SEQ ID NO:7 were linked to oxidized pustulan, oxidized lichenan, or oxidized laminarin via a C-terminal hydrazide linker.

[0474] Vaccines used: [Table 32]

[0475] Animals (female Balb / c mice) were vaccinated three times at biweekly intervals (dose: (A) 5 μg and (B) 20 μg; route: id) and the subsequent immune response to the injected peptide (i.e., SEQ ID NO: 6) was analyzed using mouse plasma collected two weeks after the third immunization.

[0476] result: As shown in FIG. 33, all three CLEC vaccines (SEQ ID NO:5 + SEQ ID NO:7 + pustulan, SEQ ID NO:5 + SEQ ID NO:7 + lichenan, and SEQ ID NO:5 + SEQ ID NO:7 + laminarin) were able to induce detectable immune responses. Interestingly, immunization with the laminarin-based vaccine induced only very low anti-peptide and anti-aSyn responses. In contrast, the pustulan-based conjugate was able to induce significantly higher responses. The lichenan-based conjugate showed lower immunogenicity compared to the pustulan-based conjugate in this experiment, but was able to induce higher titers compared to the laminarin-based conjugate.

[0477] This demonstrates that Dectin-1 binding efficacy in vitro, and particularly against human Dectin-1, can be directly related to the in vivo immunogenicity and biological activity of the vaccine, identifying pustulan or a fragment thereof (i.e., linear β(1,6)-β-D glucan) as the most effective glucan variant proposed in this application.

[0478] Example 31: Analysis of the in vivo functionality of immu...

Claims

1. consisting of or comprising at least β-glucan or mannan, and at least a B-cell or T-cell epitope polypeptide; the β-glucan or mannan is covalently conjugated to the B cell and / or T cell epitope polypeptide to form a conjugate of the β-glucan or mannan and the B cell and / or T cell epitope polypeptide; The conjugate, wherein the B cell and / or T cell epitope polypeptide is an alpha-synuclein polypeptide.

2. the β-glucan is a dectin-1 binding β-glucan, preferably pustulan, lichenan, laminarin, curdlan, β-glucan peptide (BGP), schizophyllan, scleroglucan, whole glucan particle (WGP), zymosan, or lentinan, more preferably pustulan, laminarin, lichenan, lentinan, schizophyllan, or scleroglucan, in particular the β-glucan is pustulan; and / or the β-glucan is a strong dectin-1 binding β-glucan, preferably an IC50 value of less than 10 mg / ml as determined by competitive ELISA, and / or the conjugate according to claim 1, which is a β-glucan that binds to soluble mouse Fc-Dectin-1a receptor with an IC50 value of less than 1 mg / ml, more preferably less than 500 μg / ml, and particularly less than 200 μg / ml, as determined by competitive ELISA.

3. 3. The conjugate according to claim 1 or claim 2, wherein the β-glucan is a predominantly linear β-(1,6)-glucan having a ratio of (1,6)-linked to non-β-(1,6)-linked monosaccharide moieties of at least 1:1, preferably at least 2:1, more preferably at least 5:1, in particular at least 10:

1.

4. The conjugate of any one of claims 1 to 3, wherein the alpha-synuclein polypeptide comprises at least one B-cell epitope.

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

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

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

8. The conjugate is preferably a carrier protein, preferably a non-toxic cross-reacting substance (CRM) of diphtheria toxin (especially CRM １９７ ), KLH, diphtheria toxoid (DT), tetanus toxoid (TT), Haemophilus influenzae protein D (HipD), and serogroup B meningococcal outer membrane protein complex (OMPC), recombinant non-toxic form of Pseudomonas aeruginosa exotoxin A (rEPA), flagellin, Escherichia coli heat labile enterotoxin (LT), cholera toxin (CT), mutant toxins (e.g., LTK63 and LTR72), virus-like particles, albumin binding proteins, bovine serum albumin, ovalbumin, synthetic peptide dendrimers, such as multiple antigenic peptides (MAPs), The conjugate according to any one of claims 1 to 7.

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

10. A T cell epitope, preferably a T cell epitope comprising the amino acid sequence AKFVAAWTLKAAA, optionally linked to a linker, preferably AKFVAAWTLKAAANRRA-(NH-NH2), AKFVAAWTLKAAAN-C, AKFVAAWTLKAAA-C, AKFVAAWTLKAAANRRA-C; or aKXVAAWTLKAAAaZC, aKXVAAWTLKAAAaZCNRRA, aKXVAAWTLKAAAa, aKXVAAWTLKAAAaNRRA, aA 9. The conjugate of any one of claims 1 to 8, comprising a T cell epitope comprising a variant thereof selected from (X)AAAKTAAAAa, aA(X)AAATLKAAa, aA(X)VAAATLKAAa, aA(X)IAAATLKAAa, aK(X)VAAWTLKAAa, and aKFVAAWTLKAAa, wherein X is L-cyclohexylalanine, Z is aminocaproic acid, and a is an aliphatic amino acid residue selected from alanine, glycine, valine, isoleucine, and leucine.

11. The conjugate according to any one of claims 1 to 10, wherein the ratio of β-glucan or mannan to B-cell and / or T-cell epitope polypeptide in the conjugate is between 10:1 (w / w) and 0.1:1 (w / w), preferably between 8:1 (w / w) and 2:1 (w / w), in particular 4:1 (w / w).

12. A conjugate according to any one of claims 1 to 11 for use as an active anti-alpha-synuclein vaccine for the treatment and prevention of synucleinopathies, preferably Parkinson's disease (PD), Dementia with Lewy Bodies (DLB), Multiple System Atrophy (MSA), Parkinson's Disease Dementia (PDD), Neuroaxonal Dystrophy, Amygdala-Restricted Alzheimer's Disease with Lewy Bodies (AD / ALB).

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

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

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

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