Anti-alpha-synuclein therapeutic vaccine

Abstract

The present invention relates to a liposomal vaccine composition comprising a peptide antigen displayed on the surface of a liposome; a peptide comprising a T cell epitope; and an adjuvant, wherein the peptide antigen has the structure: X 1 -X 2 -X 3 -EX 4 -X 5 -PVDPDNEX 6 wherein E is glutamic acid, P is proline, V is valine, D is aspartic acid, and X is a nucleotide sequence consisting essentially of the structure 1 is optionally present and, if present, is G, where G is glycine; X 2 is optional and, if present, is G, where G is as defined above; X 3 is L, K, or S, where L is leucine, K is lysine, and S is serine; X 4 is D, K, or S, where D, K, and S are as defined above; X 5 is M, where M is methionine or methionine sulfoxide; X 6 is A, K, or S, where A is alanine, K and S are as defined above; 3 -EX 4 -X 5 -PVDPDNEX 6 is not LEDMPVDPDNEA and contains 1 to 5 amino acid differences compared to the amino acid sequence GILEDMPVDPDNEA; and the peptide antigen is the dipeptide YE. 6 where Y is tyrosine and E is as defined above.

Description

[Technical field]

[0001] The present invention relates to anti-alpha-synuclein therapeutic vaccines that may be used for the prevention, attenuation, and / or treatment of diseases, disorders, and disorders associated with alpha-synuclein (α-synuclein, A-synuclein, a-synuclein, A-syn, α-syn, aSyn, a-syn) aggregates, including but not limited to Lewy bodies and / or Lewy neurites and / or glial cytoplasmic inclusions, such as Parkinson's disease, multiple system atrophy, Lewy Body dementia (LBD; dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD)), or diffuse Lewy body disease. [Background technology]

[0002] Many degenerative diseases are associated with extracellular or extracellularly deposited amyloid or amyloid-like proteins that contribute to pathogenesis and disease progression. The best-characterized amyloid protein that forms extracellular aggregates is amyloid beta (Aβ). Amyloid-like proteins that primarily form intracellular aggregates include, but are not limited to, alpha-synuclein, tau, and huntingtin (htt).

[0003] aSyn is a 14kD, naturally monomeric protein that is usually associated with the membrane of synaptic vesicles at presynaptic terminals or located in the cytoplasm. Its native function is still poorly understood, but it is thought to be involved in synaptic transmission. During pathogenesis, misfolding and aggregation of aSyn occurs in the central nervous system (CNS) and peripheral nervous system, probably as a result of post-translational modifications, including, inter alia, C-terminal protease cleavage (Dufty 2007, Bassil 2016). Aggregation generates different aSyn species, including oligomers, protofibrils, and fibrils, that have been linked to LB disease pathogenesis. The fibrillar form of aSyn is mostly detected in LBs located in the soma of neurons (Kosaka et al., 1990, Dickson et al, 1989). Aggregates of aSyn have also been detected in astrocytes (Braak 2007).

[0004] Various oligomeric, but not only fibrillar, aSyn species have been detected in diseased human brains. In contrast to fibrillar aSyn, oligomeric aggregates are most often located in neuronal processes and presynaptic terminals, where they can damage synapses and thus oligomeric aSyn has been implicated as a cause of cytotoxicity.

[0005] It has been shown that monomeric aSyn can form different types of aggregates with different appearance, structure, cytotoxicity, and chemical properties under different in vitro conditions. Depending on the monomer conformation and the prevailing permissive conditions, different types of aggregates with different structural properties can result. When seeded, distinct aSyn species (e.g., "fibrils" or "ribbons") recognize their conformation to recipient cells, generating aggregates of the same species in a process called "conformational templating." When injected into rat brains, these types of aggregates display different properties in vivo in terms of inclusion body formation and the generation of behavioral and neurotoxic phenotypes.

[0006] This may be because different types of aSyn aggregates expose different polypeptide chains due to their distinct conformations. These differently exposed surfaces allow different combinations of molecular interactions. Thus, the conformation of a given aSyn species dictates its properties, such as its seeding propensity or preference for specific cell types. Experimental data are beginning to emerge that demonstrate different properties of aSyn species extracted from PD and multiple system atrophy (MSA) material, and analysis of pathological brain material from PD or MSA patients has demonstrated different properties of disseminating aSyn aggregates.

[0007] Diseases involving alpha-synuclein aggregates are generally classified as synucleinopathies (or α-synucleinopathies, alpha-synucleinopathies), including, but not limited to, Parkinson's disease (PD), which includes Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with non-alpha-synuclein mutations, pure autonomic failure, and Lewy body dysphagia), Lewy body dementia (LBD;Dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD), diffuse Lewy body disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2, or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, and Down's syndrome. Synucleinopathies involving aggregates of alpha-synuclein in neurons and glia include, but are not limited to, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration, and olivopontocerebellar atrophy). Other diseases that may have alpha-synuclein immunoreactive lesions include traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and Niemann-Pick disease type C1, frontotemporal dementia with parkinsonism linked to chromosome 17), motor neuron diseases, Huntington's disease, amyotrophic lateral sclerosis (sporadic, familial, and ALS-dementia complex of Guam), neuroaxonal dystrophies, cerebral iron deposition neurodegeneration type 1 (Hallervorden-Spatz syndrome), prion diseases, Creutzfeldt-Jakob disease, ataxia-telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, These disorders include Gerstmann-Sträussler-Scheinker disease, inclusion body myositis, Gaucher disease, Krabbe disease, and other lysosomal storage disorders (including Kufor-Rakeb syndrome and Sanfilippo syndrome) and rapid eye movement (REM) sleep behavior disorder (Jellinger, Mov Disord 2003, 18 Suppl. 6, S2-12; Galvin et al., JAMA Neurology 2001, 58 (2), 186-190; Kovari et al., Acta Neuropathol. 2007, 114(3), 295-8; Saito et al., J Neuropathol Exp Neurol. 2004, 63(4), 323-328;McKee et al., Brain, 2013, 136(Pt 1), 43-64; Puschmann et al., Parkinsonism Relat Disord 2012, 18S1, S24-S27; Usenovic et al., J Neurosci. 2012, 32(12), 4240-4246; Winder-Rhodes et al., Mov Disord. 2012, 27(2), 312-315; Ferman et al., J Int Neuropsychol Soc. 2002, 8(7), 907-914; Smith et al., J Pathol. 2014;232:509-521, Lippa et al., Ann Neurol. 1999 Mar;45(3):353-7; Schmitz et al., Mol Neurobiol. 2018 Aug 22; Charles et al., Neurosci Lett. 2000 Jul 28;289(1):29-32; Wilhelmsen et al., Arch Neurol. 2004 Mar;61(3):398-406; Yamaguchi et al., J Neuropathol Exp Neurol. 2004, 80; th annual meeting, vol.63; Askanas et al., J Neuropathol Exp Neurol. 2000 Jul;59(7):592-8)。

[0008] Parkinson's disease (PD) is a synucleinopathy and the second most common neurodegenerative movement disorder. The prevalence of PD ranges from 100 to 200 per 100,000 in the general population and affects approximately 1% of the population over 60 years of age, with an annual incidence of about 15 per 100,000. It is a chronic progressive disorder defined by motor symptoms (bradycardia, rigidity, rest tremor, and postural instability) and non-motor symptoms (various autonomic dysfunction, sensory abnormalities, and psychiatric abnormalities) that usually precede the motor symptoms. The disease is characterized by severe loss of dopaminergic neurons in the substantia nigra (SN), accompanied by the accumulation of fibrillar proteinaceous inclusions, called Lewy bodies (LB), composed mainly of alpha-synuclein (aSyn). PD, DLB, and other LB diseases show accumulation and redistribution of aSyn in various brain regions and cell populations.

[0009] MSA is another very important synucleinopathic disease. It is a sporadic neurodegenerative disorder characterized by symptoms of L-DOPA-resistant parkinsonism, cerebellar ataxia, and dysautonomia. Patients suffer from multisystem neuronal loss affecting various brain regions, including the striatum, substantia nigra, cerebellum, pons, as well as the inferior olivary nucleus and spinal cord. MSA is characterized by aSyn-positive glial cytoplasmic inclusions (GCIs) and rare neuronal inclusions throughout the central nervous system. These inclusions are associated with substantia nigra degeneration, olivopontocerebellar atrophy, and involvement of autonomic nuclei in the medulla and spinal cord. The importance of GCIs in the pathogenesis of MSA is generally accepted and is highlighted by the recent analysis of a transgenic mouse model that analyzed the effects of aSyn overexpression in oligodendroglia. In tg mice overexpressing human aSyn, both GCI-like aggregates and biochemical markers of MSA were found.

[0010] DLB is the second most common type of neurodegenerative dementia in Western societies after Alzheimer's disease. It represents 4-7% of clinically diagnosed dementias, with an estimated equal number of cases escaping correct clinical diagnosis. Diagnosis of DLB ​​is challenging as the disease is "intermediate" between AD and PD and shows overlapping features of both diseases. Four clinical consensus criteria, two of which must be present to diagnose "probable DLB", are cognitive and attentional fluctuations, recurrent visual hallucinations, REM sleep behavior disorder, and spontaneous parkinsonian motor signs, which occur later in the disease than the other criteria. These may be supported by a variety of additional clinical criteria that may or may not occur, such as syncope and transient unresponsive episodes, apathy, anxiety, depression, psychotic episodes, and neuroleptic responsiveness. Symptoms are not uniform across patients.

[0011] DLB pathology is characterized by proteinaceous inclusions called Lewy bodies (LBs), which are composed mainly of alpha-synuclein (aSyn), which plays a role in the loss of neuronal function and structure. However, in DLB, LBs are diffusely distributed throughout the cortex, whereas in PD, they are found mainly in dopaminergic neurons of the substantia nigra. LBs in DLB are less well-defined, less acidophilic, and less fibrillar than those in PD. In addition, amyloid plaques containing mainly carboxy-terminal extended forms of amyloid beta (Abeta), such as Abeta 1-42, can be found in the brains of DLB ​​patients. Cortical amyloid deposition is associated with a tendency towards temporal lobe hypoperfusion and hippocampal atrophy.

[0012] Potential therapeutics that could modulate the underlying alpha-synuclein-related neurodegeneration are currently under development. (登録商標)Vaccination with PD01 and PD03, the peptides that target alpha-synuclein, has proven effective in reducing aSyn pathology, preserving neuroinflammation, and alleviating behavioral impairments in various animal models of aSyn aggregation disorders (Mandler et al. 2014; WO 2009 / 103105A1, WO 2011 / 020133A1, WO 2017 / 076873A1). These peptides proved to be safe and well-tolerated vaccines capable of inducing target-specific antibodies in humans. Another active immunotherapy targeting alpha-synuclein, UB-312, has been tested in a human alpha-synuclein transgenic mouse model and shown to reduce alpha-synuclein accumulation in the brain and gut. This was accompanied by improved motor performance in treated animals (Nimmo JT et al., 2022 Acta Neuropathol, Jan;143(1):55-73). UB-312 is currently being tested in a Phase 1 / 2 clinical trial that will determine the safety, tolerability, and immunogenicity of UB-312 in healthy participants and patients with PD and MSA (Fleming SM et al., 2022, Neuropharmacology 202).

[0013] Currently, there are no approved anti-alpha synuclein vaccines on the market for preventing and / or treating alpha synuclein-associated diseases. It is therefore desirable to identify new therapeutic vaccine compositions that can prevent and / or treat these diseases. Summary of the Invention

[0014] The inventors have designed a vaccine composition comprising an antigenic peptide that is shown herein to be highly immunogenic and to induce large amounts of aSyn-specific antibodies in the periphery, which is predicted to increase the target binding of the induced antibodies through an oligoclonal antibody response.

[0015] In one general aspect, the present invention relates to a liposome composition comprising an antigenic peptide displayed on the surface of a liposome and an adjuvant, wherein the antigenic peptide has the structure: X1-X2-X3-E-X4-X5-PVDPDNE-X6 having E is glutamic acid, P is proline, V is valine, D is aspartic acid, and N is asparagine; X1 is optional and, if present, is G, where G is glycine; X2 may be present or absent, and if present is I or G, where I is isoleucine and G is as defined above; X3 is L, K, or S, where L is leucine, K is lysine, and S is serine; X4 is D, K, or S, where D, K, and S are as defined above; X5 is M, where M is methionine or methionine sulfoxide; X6 is A, K, or S, where A is alanine, K and S are as defined above. The single letter amino acid codes commonly known in the art are used herein.

[0016] In one embodiment, the present invention relates to a liposomal vaccine composition comprising an antigenic peptide presented on the surface of a liposome and an adjuvant, wherein the antigenic peptide has the structure: X1-X2-LEDMPVDPDNEA having L is leucine, E is glutamic acid, M is methionine or methionine sulfoxide, P is proline, V is valine, D is aspartic acid, N is asparagine, A is alanine; X1 is optional and, if present, is G, where G is glycine; X2 is optional and, if present, is I or G, where I is isoleucine and G is as defined above.

[0017] In one embodiment, the present invention relates to a liposomal vaccine composition comprising an antigenic peptide presented on the surface of a liposome and an adjuvant, wherein the antigenic peptide comprises, consists essentially of, or consists of the amino acid sequence of GILEDMPVDPDNEA (SEQ ID NO: 1). Thus, the present invention, in one aspect, encompasses the use of the native aSyn peptide (amino acids 111-124, with reference to SEQ ID NO: 28) in a liposomal vaccine composition.

[0018] In one embodiment, the present invention relates to a liposomal vaccine composition comprising an antigenic peptide presented on the surface of a liposome and an adjuvant, wherein the antigenic peptide comprises, consists essentially of, or consists of the amino acid sequence of LEDMPVDPDNEA (SEQ ID NO: 38).Accordingly, the present invention in another aspect encompasses the use of the native aSyn peptide (amino acids 113-124, with reference to SEQ ID NO: 28) in a liposomal vaccine composition.

[0019] The antigenic peptide of the liposomal vaccine composition may comprise, consist essentially of, or consist of the amino acid sequence of GGLEDMPVDPDNEA (SEQ ID NO: 45). Thus, the invention may encompass the use of an aSyn peptide (amino acids 111-124) with one I substituted for G at amino acid position 112 where X2 is G, and thus the aSyn peptide of the liposomal vaccine composition may have the amino acid sequence of GGLEDMPVDPDNEA (SEQ ID NO: 45).

[0020] The antigenic peptide may optionally contain 1 to 2 amino acid differences (i.e., 1, 2 differences) compared to the amino acid sequence GILEDMPVDPDNEA. The differences are generally amino acid substitutions according to the options represented for each position. These differences may be selected from any of the amino acids X1-X2. The absence of X1 and / or X2 is considered a difference. For example, SEQ ID NO: 38 lacks amino acids at positions X1 and X2, i.e., presents two differences compared to SEQ ID NO: 1, and SEQ ID NO: 45 presents an amino acid substitution (I is replaced by G) at position X2, thus presenting one difference compared to the amino acid sequence GILEDMPVDPDNEA.

[0021] In some embodiments, the antigenic peptide of a liposomal vaccine composition comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:38, or SEQ ID NO:45 does not contain the dipeptide YE immediately following the alanine residue (i.e., position X6), where Y is tyrosine and E is glutamic acid.

[0022] Typically, the antigenic peptides included in the liposomal vaccine compositions of the present invention do not contain any further alpha synuclein amino acid residues after the alanine of SEQ ID NO: 1 or SEQ ID NO: 38. In particular, they do not contain the dipeptide YE immediately following the alanine.

[0023] The antigenic peptide of the liposomal vaccine composition may comprise, consist of, or consist essentially of an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:38, or SEQ ID NO:45. However, the antigenic peptide may contain a limited number of additional amino acid residues at the N- and / or C-terminus to allow for insertion of the antigenic peptide into the liposome (in a manner that allows the peptide antigen to be presented on the surface of the liposome). For example, the antigenic peptide may contain additional residues such as lysine residues to facilitate palmitoylation. These residues are typically found at the N- and / or C-terminus of the antigenic peptide sequence. In some embodiments, one to four lysine residues may be added to the N- and / or C-terminus, preferably two lysine residues are added to the N- and C-terminus.

[0024] In this context, the term "consisting essentially of" means that the alpha synuclein-derived peptide antigen comprises 10 to 14 contiguous amino acids beginning at position 111 or 113 of alpha synuclein (SEQ ID NO: 28), but may include a limited number of additional residues, such as two to four lysine residues to facilitate presentation on the surface of a liposome.

[0025] The peptide antigens comprising, consisting of, or consisting essentially of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:38, or SEQ ID NO:45 contained in the liposome composition of the present invention may further comprise at least one chemical modification. Modifications such as amidation, esterification, palmitoylation, formylation, acetylation, and other chemical substitutions may be made to the free C-terminus (or N-terminus) of the peptide or to its side chains. Even if not otherwise specified, such modifications are within the scope of the term "consisting essentially of" as used herein. Palmitoylation is a preferred modification to promote presentation of the peptide antigen on the surface of the liposome, which may be a structural arrangement conductive to the production of neutralizing antibodies.

[0026] Embodiments of the invention are defined below with reference to numbered paragraphs. [Section 1] Peptide antigens displayed on the surface of liposomes; A peptide containing a T cell epitope; and Adjuvants A liposomal vaccine composition comprising the peptide antigen having the structure: X1-X2-LEDMPVDPDNEA comprising, consisting essentially of, or consisting of the structure L is leucine, E is glutamic acid, M is methionine or methionine sulfoxide, P is proline, V is valine, D is aspartic acid, N is asparagine, A is alanine; X1 is optional and, if present, is G, where G is glycine; X2 may be present or absent, and if present is I or G, where I is isoleucine and G is as defined above; The peptide antigen does not contain a dipeptide YE immediately following A, where Y is tyrosine and E is as defined above; Liposomal vaccine compositions. [Section 2] Item 3. The liposome composition according to item 1, wherein X1 and X2 are present. [Section 3] Item 3. The liposome composition according to item 1, wherein X1 and X2 are absent. [Section 4] Item 3. The liposome vaccine composition according to item 1 or 2, wherein X2 is I. [Section 5] The liposome composition according to item 1, wherein the peptide antigen is selected from the group consisting of GILEDMPVDPDNEA (SEQ ID NO: 1), LEDMPVDPDNEA (SEQ ID NO: 38), and GGLEDMPVDPDNEA (SEQ ID NO: 45). [Section 6] The liposome composition of any one of the preceding paragraphs, wherein M is methionine sulfoxide. [Section 7] The liposomal vaccine composition according to any one of the preceding clauses, wherein the peptide antigen further comprises at least one chemical modification. [Section 8] The liposomal vaccine composition according to any one of the preceding clauses, wherein one or both termini of the peptidic antigen further comprise at least one arginine I or glutamic acid I residue. [Section 9] The liposomal vaccine composition according to any one of the preceding clauses, wherein one or both termini of the peptidic antigen further comprise at least three arginine I or glutamic acid I residues. [Section 10] The liposomal vaccine composition of any one of the preceding clauses, wherein the peptide comprising a T cell epitope comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO:35 (PaDre), SEQ ID NO:36 (P2), SEQ ID NO:37 (P30), SEQ ID NO:21 (SAT13), SEQ ID NO:22 (SAT15), SEQ ID NO:23 (SAT17), SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and any combination thereof. [Section 11] The liposomal vaccine composition of any one of the preceding clauses, wherein the T cell epitope-containing peptide comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:29 (SAT42), SEQ ID NO:30 (SAT43), SEQ ID NO:31, (SAT44), SEQ ID NO:32 (SAT47), SEQ ID NO:35 (PaDre), SEQ ID NO:36 (P2) and SEQ ID NO:37 (P30), or a close sequence analog thereof. [Section 12] The liposomal vaccine composition of any one of the preceding clauses, wherein the T cell epitope-containing peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 32 (SAT47) or a close sequence analog thereof. [Section 13] The liposomal vaccine composition according to any one of the preceding clauses, wherein the adjuvant comprises a Toll-like receptor 4 ligand and / or a Toll-like receptor 9 ligand. [Section 14] Item 14. The liposomal vaccine composition of item 13, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA) and / or the Toll-like receptor 9 ligand comprises CpG. [Section 15] The liposomal vaccine composition of any one of the preceding clauses, wherein the adjuvant comprises monophosphoryl lipid A (MPLA). [Section 16] The liposomal vaccine composition according to any one of the preceding clauses, wherein the adjuvant further comprises CpG. [Section 17] The liposomal vaccine composition according to any one of the preceding clauses, wherein the peptide comprising the T cell epitope is encapsulated in a liposome. [Section 18] A peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from GILEDMPVDPDNEA (SEQ ID NO:1), LEDMPVDPDNEA (SEQ ID NO:38), and GGLEDMPVDPDNEA (SEQ ID NO:45); and A T cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 32 (SAT47) or a close sequence analog thereof; and An adjuvant comprising a Toll-like receptor 4 ligand, the Toll-like receptor 4 ligand comprising monophosphoryl lipid A (MPLA). A liposomal vaccine composition comprising: [Section 19] 20. The liposomal vaccine composition of claim 18, wherein the peptide antigen comprises, consists essentially of, or consists of the amino acid sequence GILEDMPVDPDNEA (SEQ ID NO:1). [Section 20] 20. The liposomal vaccine composition of claim 18, wherein the peptide antigen comprises, consists essentially of, or consists of the amino acid sequence LEDMPVDPDNEA (SEQ ID NO: 38). [Section 21] A pharmaceutical composition comprising the liposome vaccine composition according to any one of items 1 to 20, and a pharma- ceutically acceptable carrier, diluent, and / or excipient. [Section 22] A kit comprising the liposome vaccine composition according to any one of Items 1 to 20 or the pharmaceutical composition according to Item 21, and a container. [Section 23] The liposome vaccine composition according to any one of Items 1 to 20, the pharmaceutical composition according to Item 21, or the kit according to Item 22, for use in treating or preventing a disease, disorder, or abnormality associated with alpha synuclein aggregates. [Section 24] Item 24. The liposomal vaccine composition, pharmaceutical composition, or kit according to Item 23, wherein the disease, disorder, or abnormality associated with alpha-synuclein aggregates is a synucleinopathy. [Section 25] A disease, disorder, or abnormality associated with alpha-synuclein aggregates or a synucleinopathic disease is defined as Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with non-alpha-synuclein mutations, pure autonomic failure, and Lewy body dysphagia), Lewy body dementia (LBD; dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD)), diffuse Lewy body disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2, or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, and Down's syndrome, Multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration, and olivopontocerebellar atrophy), traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and Niemann-Pick disease type C1, frontotemporal dementia with parkinsonism linked to chromosome 17), motor neuron diseases, Huntington's disease, amyotrophic lateral sclerosis (sporadic, familial, and Guam ALS-dementia complex), neuroaxonal dystrophies, cerebral iron deposition neurodegeneration type 1 (Hallervorden-Spatz syndrome), prion diseases, Creutzfeldt-Jakob disease, ataxia-telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, 25. The liposomal vaccine composition, pharmaceutical composition, or kit according to item 23 or 24, wherein the liposomal vaccine composition, pharmaceutical composition, or kit is selected from the group consisting of Gerstmann-Straussler-Scheinker disease, inclusion body myositis, Gaucher disease, Krabbe disease, and other lysosomal storage diseases (including Kufuor-Rakeb syndrome and Sanfilippo syndrome), and rapid eye movement (REM) sleep behavior disorder. [Section 26] 26. The liposomal vaccine composition, pharmaceutical composition, or kit according to any one of items 23 to 25, wherein the disease, disorder, or abnormality associated with alpha-synuclein aggregates or synucleinopathies is selected from the group consisting of Lewy bodies and / or Lewy neurites and / or glial cytoplasmic inclusions, such as Parkinson's disease, multiple system atrophy, Lewy body dementia (LBD; dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD)), or diffuse Lewy body disease. [Section 27] Item 27. The liposome vaccine composition, pharmaceutical composition, or kit according to any one of Items 23 to 26, wherein the disease, disorder, or abnormality associated with alpha synuclein aggregates, or the synucleinopathy is multiple system atrophy. [Section 28] Item 27. The liposome vaccine composition, pharmaceutical composition, or kit according to any one of Items 23 to 26, wherein the disease, disorder, or abnormality associated with alpha-synuclein aggregates, or the synucleinopathy is Parkinson's disease. [Section 29] A method for preventing, treating, or alleviating a disease associated with a disease, disorder, or abnormality associated with alpha-synuclein aggregates, comprising administering to a subject the liposome vaccine composition according to any one of items 1 to 20 or the pharmaceutical composition according to item 21. [Section 30] 30. The method of claim 29, wherein the disease, disorder, or abnormality associated with alpha synuclein aggregates is a synucleinopathy. [Section 31] The method according to item 29 or 30, wherein a protective immune response against alpha-synuclein aggregates is induced by administering to a subject the liposome vaccine composition according to any one of items 1 to 20 or the pharmaceutical composition according to item 21. [Brief description of the drawings]

[0027] [Figure 1]Figure 1 is a graph showing IgG titers against SEQ ID NO:2 in mice immunized with SEQ ID NO:2 liposomal vaccine or SEQ ID NO:2 conjugate vaccine. The Y-axis shows anti-SEQ ID NO:2 IgG titers (AU / mL) and the X-axis shows the time of measurement in days post-immunization. The liposomal vaccine-treated group (filled circles) was compared to the conjugate vaccine-treated group (filled squares). [Diagram 2] 2 is a graph showing IgG titers against a-syn protein in mice immunized with SEQ ID NO:2 liposomal vaccine or SEQ ID NO:2 conjugate vaccine. The Y-axis shows anti-a-syn protein IgG titers (AU / mL) and the X-axis shows the time of measurement in days post-immunization. The liposomal vaccine-treated group (filled circles) was compared to the conjugate vaccine-treated group (filled squares). [Diagram 3] 3 is a graph showing IgG titers against a-syn aggregates in mice immunized with SEQ ID NO:2 liposomal vaccine or SEQ ID NO:2 conjugate vaccine. The Y-axis shows anti-a-syn aggregate IgG titers (AU / mL) and the X-axis shows the time of measurement in days post-immunization. The liposomal vaccine-treated group (filled circles) was compared to the conjugate vaccine-treated group (filled squares). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] In one embodiment, the present invention relates to a liposome composition comprising an antigenic peptide and an adjuvant presented on the surface of a liposome, wherein the antigenic peptide has the structure: X1-X2-X3-E-X4-X5-PVDPDNE-X6 having E is glutamic acid, P is proline, V is valine, D is aspartic acid, and N is asparagine; X1 is optional and, if present, is G, where G is glycine; X2 may be present or absent, and if present is I or G, where I is isoleucine and G is as defined above; X3 is L, K, or S, where L is leucine, K is lysine, and S is serine; X4 is D, K, or S, where D, K, and S are as defined above; X5 is M, where M is methionine or methionine sulfoxide; X6 is A, K, or S, where A is alanine, K and S are as defined above.

[0029] The peptides are preferably modified compared to the peptide sequence. Despite the fact that these peptides have sequences different from the native sequence, such peptides can elicit a strong anti-aSyn antibody response, and the induced antibodies show high cross-reactivity with human aSyn. The peptides of the present invention achieved an immune response superior to that induced by the native sequence (i.e. targeting the same native structure). Thus, the present invention provides An antigen peptide displayed on the surface of the liposome, and adjuvants The present invention relates to a liposomal vaccine composition comprising: wherein the antigenic peptide has the structure: X1-X2-X3-E-X4-X5-PVDPDNE-X6 comprising, consisting essentially of, or consisting of the structure E is glutamic acid, P is proline, V is valine, D is aspartic acid, and N is asparagine; X1 is optional and, if present, is G, where G is glycine; X2 may or may not be present, and if present is G, where G is as defined above; X3 is L, K, or S, where L is leucine, K is lysine, and S is serine; X4 is D, K, or S, where D, K, and S are as defined above; X5 is M, where M is methionine or methionine sulfoxide; X6 is A, K, or S, where A is alanine, K and S are as defined above; However, X3-E-X4-X5-PVDPDNE-X6 is not LEDMPVDPDNEA, and the antigenic peptide does not contain the dipeptide YE immediately following X6, where Y is tyrosine and E is as defined above. Thus, the one-letter amino acid code is generally used herein.

[0030] The peptide antigens described herein may contain 1 to 5 amino acid differences (i.e. 1, 2, 3, 4, or 5 differences), preferably 1 to 4 amino acid differences, compared to the amino acid sequence GILEDMPVDPDNEA. The differences are generally amino acid substitutions according to the options represented for each position. However, in some embodiments, X6 is deleted. More preferably, there are 2, 3, 4, or 5 amino acid differences from the amino acid sequence GILEDMPVDPDNEA (SEQ ID NO: 1, which is the wild-type alpha synuclein sequence from amino acids 111 to 124 of SEQ ID NO: 28). These differences may be selected from any of the amino acids X1-X6. In some embodiments, the antigen peptide contains an amino acid difference at one or more positions selected from X1, X2, X3, X4, X5, and X6, compared to the amino acid sequence GILEDMPVDPDNEA. The absence of X1, X2, and / or X6 is considered a difference.

[0031] Methionine is a naturally occurring amino acid. The sulfur-containing amino acids methionine and cysteine ​​are more easily oxidized than other amino acids. Oxidation of the sulfur of methionine results in methionine sulfoxide or methionine sulfone. In some embodiments, the antigenic peptide of the liposome composition of the present invention contains methionine as its oxidized form, methionine sulfoxide. In some embodiments, the liposome composition of the present invention contains an antigenic peptide containing methionine sulfoxide. In some embodiments, X5 is methionine or methionine sulfoxide. In some embodiments, X5 is methionine. In another embodiment, X5 is methionine sulfoxide.

[0032] The liposome composition of the present invention contains antigenic peptides that retain the ability to generate aSyn-specific antibodies when used as an immunogen. Furthermore, the antigenic peptides of the present invention are more immunogenic in terms of generating aSyn-specific antibodies than the corresponding wild-type aSyn peptide (containing the 14-mer GILEDMPVDPDNEA (SEQ ID NO: 1)) as demonstrated in the comparative experiment herein (Example 2). They are also more immunogenic than other aSyn peptides derived from the C-terminal region of aSyn. See Tables 2 and 4 below.

[0033] The antigenic peptides included in the liposomal vaccine composition of the present invention typically do not contain any further alpha synuclein amino acid residues after X6. In particular, they do not contain the dipeptide YE immediately following X6. As described herein, the amino acid Y 125 and E 126 In silico analysis has predicted that peptides containing Tyr 3+ , Tyr 4+ , ​​Tyr 5+ , Tyr 6+ , Tyr 7+ , Tyr 8+ , Tyr 9+ , Tyr 10+ , Tyr 11+ , Tyr 12+ , Tyr 13+ , Tyr 14+ , Tyr 15+ , Tyr 16+ , Tyr 17+ , Tyr 18+ , Tyr 19+ , Tyr 20+ , Tyr 21+ , Tyr 22+ , Tyr 23+ , Tyr 24+ , Tyr 25+ , Tyr 26+ , Tyr 27+ , Tyr 28+ , Tyr 29+ , Tyr 30+ , Tyr 31+ , Tyr 32+ , Tyr 33+ , Tyr 34+ , Tyr 35+ , Tyr 36+ , Tyr 37+ , Tyr 38+ , Tyr 39+ , Tyr 39+ , Tyr 38+ , Tyr 39+ , Tyr 39+ , Tyr 34+ , Tyr 35+ , Tyr 36+ , Tyr 37+ , Tyr 38+ , Tyr 39 ... 125 and Glu 126 There should be no amino acid extension at the C-terminus corresponding to: Accordingly, there should be no Y amino acid residue or YE dipeptide chain at the C-terminus of the peptide, where Y is tyrosine and E is as defined above.

[0034] However, the antigenic peptide included in the liposomal vaccine composition may contain a limited number of additional N-terminal amino acid residues. The antigenic peptide may additionally or alternatively contain a limited number of additional C-terminal amino acid residues to allow for the insertion of the antigenic peptide into the liposome (in a manner that allows the peptide antigen to be presented on the surface of the liposome). For example, the peptide may contain additional residues, such as lysine residues to facilitate palmitoylation. These residues are typically found at the N- and / or C-terminus of the peptide. In some embodiments, there may be one to four lysine residues added to the N- and / or C-terminus, preferably there may be two lysine residues added to the N- or C-terminus, or preferably there may be two lysine residues added to the N- and C-terminus. In this context, a peptide antigen derived from alpha synuclein may contain 10 to 14 consecutive amino acids starting at position 111 or 113 of alpha synuclein (SEQ ID NO: 28), but may contain a limited number of additional residues, such as two to four lysine residues to facilitate presentation on the surface of the liposome.

[0035] In some embodiments of the invention, one or both ends of the peptide antigen may contain additional residues that are not derived from the alpha synuclein protein sequence but are rather designed to modify the properties of the peptide. In some embodiments of the invention, the peptide antigen of the invention further comprises a patch comprising, consisting essentially of, or consisting of at least one amino acid, optionally arginine (R) or glutamic acid (E). In some embodiments of the invention, the peptide antigen of the invention further comprises a patch comprising, consisting essentially of, or consisting of at least three amino acids, optionally arginine (R) or glutamic acid (E). These sequences are referred to as patches. They may consist of sequences such as, for example, a repeating arginine (R) sequence consisting of three (RRR) to eight (RRRRRRRR) arginines or a repeating glutamic acid (E) sequence consisting of three (EEE) to eight (EEEEEEEE) glutamic acids.

[0036] Thus, the antigenic peptides of the present invention are typically 11-22 amino acids in length, preferably 12-14 amino acids in length (i.e. 12, 13, or 14 amino acids in length). It is particularly preferred that the antigenic peptides are 12 or 14 amino acids in length. The antigenic peptides of the liposomal vaccine composition result in an antibody response without a T cell response. That is, the antigenic peptides of the present invention themselves typically do not contain T cell epitopes, particularly cytotoxic T cell epitopes.

[0037] The liposomal vaccine compositions of the present invention containing the antigenic peptides described herein are expected to be highly immunogenic, and the induced antibody responses are expected to have low patient-to-patient variability. Furthermore, the liposomal vaccine compositions of the present invention incorporating palmitoylated peptides may preferentially generate antibodies against alpha-synuclein sequences in a beta-sheet conformation, resulting in enhanced affinity for pathological and / or aggregated aSyn species. Periodic peptide arrangements, such as in liposomal formulations, are known to very effectively cross-link immunoglobulin antigen receptors on B cells, resulting in strong stimulation for antibody production (Pihlgren et al., 2013).

[0038] In one embodiment, a liposome composition is provided comprising an antigenic peptide displayed on the surface of a liposome, wherein the antigenic peptide has the structure: X1-X2-X3-E-X4-X5-PVDPDNE-X6 having E is glutamic acid, P is proline, V is valine, D is aspartic acid, and N is asparagine; X1 is optional and, if present, is G, where G is glycine; X2 may be present or absent, and if present is I or G, where I is isoleucine and G is as defined above; X3 is L, K, or S, where L is leucine, K is lysine, and S is serine; X4 is D, K, or S, where D, K, and S are as defined above; X5 is M, where M is methionine or methionine sulfoxide; X6 is A, K, or S, where A is alanine, K and S are as defined above.

[0039] In one embodiment, a liposome composition is provided comprising an antigenic peptide displayed on the surface of a liposome, wherein the antigenic peptide has the structure: X1-X2-X3-E-X4-X5-PVDPDNE-X6 having E is glutamic acid, P is proline, V is valine, D is aspartic acid, and N is asparagine; X1 is optional and, if present, is G, where G is glycine; X2 may or may not be present, and if present is G, where G is as defined above; X3 is L, K, or S, where L is leucine, K is lysine, and S is serine; X4 is D, K, or S, where D, K, and S are as defined above; X5 is M, where M is methionine or methionine sulfoxide; X6 is A, K, or S, where A is alanine, K and S are as defined above; However, X3-E-X4-MPVDPDNE-X6 is not LEDMPVDPDNEA.

[0040] In some embodiments, X1 and X2 are absent. In certain embodiments, the present invention provides a compound having the structure: X3-E-X4-X5-PVDPDNE-X6 The present invention relates to a liposome composition comprising an antigenic peptide having the formula: E is glutamic acid, P is proline, V is valine, D is aspartic acid, and N is asparagine; X3 is L, K, or S, where L is leucine, K is lysine, and S is serine; X4 is D, K, or S, where D, K, and S are as defined above; X5 is M, where M is methionine or methionine sulfoxide; X6 is A, K, or S, where A is alanine, K and S are as defined above; However, X3-E-X4-X5-PVDPDNE-X6 is not LEDMPVDPDNEA.

[0041] In some embodiments, X1 is present and is G. In some embodiments, X2 is present and is G. In some embodiments, when X1 is present, X2 is also present and is G. In some embodiments, X3 is L, K, or S. In some embodiments, X4 is D, S, or K. In some embodiments, X5 is methionine or methionine sulfoxide, preferably methionine sulfoxide. In some embodiments, X6 is A, K, or S.

[0042] In some embodiments, X1, X2 are present and each is G, and X3 is K. In some embodiments, X1, X2 are present and each is G, X3 is K, and X6 is A. In a preferred embodiment, X1, X2 are present and each is G, X3 is K, X4 is S, and X6 is A. In a preferred embodiment, X1, X2 are present and each is G, X3 is K, X4 is D, and X6 is A.

[0043] In some embodiments, X3 is L or K. In some embodiments, X4 is D or S. In some embodiments, X5 is M. In some embodiments, X6 is A or S.

[0044] In some embodiments, X1 and X2 are absent. In some embodiments, X1, X2 are absent, and X3 is L. In a preferred embodiment, X1, X2 are absent, X3 is L, and X6 is S. In another preferred embodiment, X1, X2 are absent, X3 is L, and X4 is D.

[0045] The peptide antigen contained in the liposome composition of the present invention may further include at least one chemical modification. Modifications such as amidation, esterification, palmitoylation, formylation, acetylation, and other chemical substitutions of the free C-terminus (or N-terminus) of the peptide or its side chains are clearly included within the scope of the present invention. Even if not otherwise specified, such modifications are within the scope of the term "consisting essentially of" as used herein. Palmitoylation is a preferred modification to promote presentation of peptide antigens on the surface of liposomes, which may be a structural arrangement that can induce the production of neutralizing antibodies.

[0046] The peptides of the invention (such as the "(antigenic) peptide(s) (of the invention)", "X1 to X6") may be provided as compositions suitable for the intended use for the prevention and / or treatment of synucleinopathies, in particular pharmaceutical compositions, preferably in combination with a pharma- ceutically acceptable carrier. Such pharmaceutical compositions may be administered to a patient in need thereof in an amount effective to achieve a prophylactic and / or therapeutic effect, as described in more detail herein.

[0047] In a particular embodiment, the antigenic peptide of the present invention is selected from the group consisting of GILEDMPVDPDNEA (SEQ ID NO: 1), GGKESMPVDPDNEA (SEQ ID NO: 2), GGKEDMPVDPDNEA (SEQ ID NO: 3), LESMPVDPDNEA (SEQ ID NO: 4), LEKMPVDPDNEA (SEQ ID NO: 5), GGSESMPVDPDNEA (SEQ ID NO: 6), GGSESMPVDPDNES (SEQ ID NO: 7), KEDMPVDPDNEA (SEQ ID NO: 8), SESMPVDPDNEA (SEQ ID NO: 9), LESMPVDPDNEA (SEQ ID NO: 10), GGKESMPVDPDNES (SEQ ID NO: 11). ), KESMPVDPDNEA (SEQ ID NO: 12), KESMPVDPDNES (SEQ ID NO: 13), SEKMPVDPDNEA (SEQ ID NO: 14), LEKMPVDPDNES (SEQ ID NO: 15), GGSESMPVDPDNEK (SEQ ID NO: 16), LEKMPVDPDNEK (SEQ ID NO: 17), GGKEKMPVDPDNEA (SEQ ID NO: 18), GGKESMPVDPDNEK (SEQ ID NO: 19), GGKEKMPVDPDNEK (SEQ ID NO: 20), LEDMPVDPDNEA (SEQ ID NO: 38) and GGLEDMPVDPDNEA (SEQ ID NO: 45).

[0048] Thus, in certain embodiments, the antigenic peptides of the present invention are GGKESMPVDPDNEA (SEQ ID NO: 2), GGKEDMPVDPDNEA (SEQ ID NO: 3), LESMPVDPDNEA (SEQ ID NO: 4), LEKMPVDPDNEA (SEQ ID NO: 5), GGSESMPVDPDNEA (SEQ ID NO: 6), GGSESMPVDPDNEA (SEQ ID NO: 7), KEDMPVDPDNEA (SEQ ID NO: 8), SESMPVDPDNEA (SEQ ID NO: 9), LESMPVDPDNEA (SEQ ID NO: 10), GGKESMP VDPDNES (SEQ ID NO:11), KESMPVDPDNEA (SEQ ID NO:12), KESMPVDPDNES (SEQ ID NO:13), SEKMPVDPDNEA (SEQ ID NO:14), LEKMPVDPDNES (SEQ ID NO:15), GGSESMPVDPDNEK (SEQ ID NO:16), LEKMPVDPDNEK (SEQ ID NO:17), GGKEKMPVDPDNEA (SEQ ID NO:18), GGKESMPVDPDNEK (SEQ ID NO:19) and GGKEKMPVDPDNEK (SEQ ID NO:20).

[0049] Preferably, the antigenic peptide of the present invention is selected from the group consisting of GGKESMPVDPDNEA (SEQ ID NO: 2), GGKEDMPVDPDNEA (SEQ ID NO: 3), LESMPVDPDNES (SEQ ID NO: 4), LEKMPVDPDNEA (SEQ ID NO: 5) and KESMPVDPDNEA (SEQ ID NO: 12).

[0050] More preferably, the antigenic peptide of the present invention is selected from the group consisting of GGKESMPVDPDNEA (SEQ ID NO: 2), GGKEDMPVDPDNEA (SEQ ID NO: 3), and LESMPVDPDNES (SEQ ID NO: 4). In one embodiment, the antigenic peptide of the present invention is GGKESMPVDPDNEA (SEQ ID NO: 2). In one embodiment, the antigenic peptide of the present invention is KESMPVDPDNEA (SEQ ID NO: 12).

[0051] According to these embodiments, the antigenic peptide preferably has up to 1, 2, 3, 4, or 5 mutations compared to the native GILEDMPVDPDNEA sequence. In some embodiments, the antigenic peptide has no mutations compared to the native GILEDMPVDPDNEA sequence. The mutations are amino acid substitutions according to the options represented for each position X1 to X6.

[0052] The peptide antigen derived from alpha synuclein is presented on the surface of the liposome composition. This is typically by insertion into the outer surface of the liposome. Insertion into the outer surface of the liposome may be facilitated through linking the peptide antigen derived from alpha synuclein to a moiety that is inserted into the outer surface of the liposome. The liposome may be any liposome suitable for presenting a peptide antigen derived from alpha synuclein on its surface and may encapsulate a peptide that contains a T cell epitope, particularly a peptide unrelated to aSyn that contains a universal helper T cell epitope. Typically, this moiety includes a hydrophobic moiety to ensure insertion into the lipid bilayer membrane of the liposome. This moiety may be any suitable moiety, but is preferably a fatty acid. The fatty acid may include a palmitoyl residue.

[0053] In one embodiment of the present invention, the liposome composition comprises an antigenic peptide as described herein linked to one or two palmitoyl residues at the N- or C-terminal region of the peptide. That is, the antigenic peptide is mono- or di-palmitoylated at one or, optionally, both of the terminal regions of the peptide. This may be facilitated by incorporating lysine residues at the N- and C-terminal regions of the peptide antigen derived from alpha synuclein. The lysine residues are palmitoylated.

[0054] In another embodiment of the present invention, the liposome composition comprises an antigenic peptide as described herein linked to at least one palmitoyl residue at the N- and C-terminal regions of the antigenic peptide. That is, the antigenic peptide is dipalmitoylated. This may be facilitated by incorporating at least one lysine residue at the N- and C-terminal regions of the peptide antigen derived from alpha synuclein. The lysine residue is palmitoylated.

[0055] In another embodiment of the present invention, the liposome composition comprises an antigen peptide linked to two palmitoyl residues at the N- and C-terminal regions of the peptide. That is, the antigen peptide is tetrapalmitoylated. This may be facilitated by incorporating two lysine residues at the N- and C-terminal regions of the peptide antigen derived from alpha synuclein. The lysine residues are palmitoylated.

[0056] The liposomal vaccine composition of the present invention comprises a peptide comprising a T cell epitope, in particular a helper T cell epitope. Preferably, the peptide comprising a T cell epitope comprises at least two T cell epitopes. The peptide comprising a T cell epitope may be at least partially encapsulated in a liposome, and / or may be presented on the surface of the liposome, and / or may be at least partially incorporated into the lipid bilayer of the liposome. The peptide comprising a T cell epitope is preferably encapsulated. This is typically achieved by forming the liposome in a solution containing the peptide comprising a T cell epitope. It should be noted that during the encapsulation process, a certain percentage of the peptide comprising a T cell epitope may be adsorbed on the outer surface of the liposome, and a certain percentage of the peptide comprising a T cell epitope may be incorporated into the lipid bilayer of the liposome. That is, when referring to a T cell epitope being encapsulated, it is intended to mean that at least some of the peptide comprising a T cell epitope is encapsulated.

[0057] The percentage of the total peptide containing the T cell epitope associated with the liposome may be at least 20%, 25%, 30%, 35%, 40%, 50%, 60%, 70%, 80% or 90% (the remainder is adsorbed to the surface or incorporated into the surface of the lipid bilayer). The exact degree of encapsulation will depend on the properties of the peptide containing the T cell epitope.

[0058] In some embodiments of the invention, the liposomal vaccine composition comprises at least one or at least two (2) T cell epitopes. The T cell epitopes can be at least two (2) linear T cell epitopes or a conjugate of multiple T cell epitopes linked via a spacer or not. In some embodiments of the invention, the T cell epitopes comprise multiple T cell epitopes linked via a spacer comprising at least one amino acid, preferably at least three amino acids, more preferably an amino acid sequence comprising a VVR. The conjugate comprises at least two, three, or four different T cell epitopes encapsulated in the liposome. In some embodiments, the T cell epitopes are derived from diphtheria toxin, tetanus toxin, Epstein-Barr virus, influenza hemagglutinin, and / or keyhole limpet hemocyanin. Thus, specific preferred combinations of T cell epitopes are selected from the following: a) Diphtheria toxin and tetanus toxin T cell epitope combination b) A combination of Epstein-Barr virus and tetanus toxin T cell epitopes c) a combination of Epstein-Barr virus, tetanus toxin, and keyhole limpet hemocyanin T cell epitopes; or d) A combination of influenza hemagglutinin, diphtheria toxin, tetanus toxin, and Epstein-Barr virus T cell epitopes.

[0059] The above combinations are preferably included in the order listed, but may be included in other orders, for example if there are three T cell epitopes, A, B, and C, they may be included in any order: ABC, ACB, BAC, BCA, CAB, or CBA.

[0060] Such peptides are preferably included in the vaccine composition of the present invention. Thus, peptides useful in the present invention comprise, consist essentially of, or consist of an amino acid sequence selected from SEQ ID NO:21 (SAT13), SEQ ID NO:22 (SAT15), SEQ ID NO:23 (SAT17), SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and / or any combination thereof. In a preferred embodiment, the T cell epitope comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and / or any combination thereof. In some embodiments, the T cell epitope comprises, consists essentially of, or consists of SEQ ID NO:24. In some embodiments, the T cell epitope comprises, consists essentially of, or consists of SEQ ID NO:25. In some embodiments, the T cell epitope comprises, consists essentially of, or consists of SEQ ID NO:26. In some embodiments, the T cell epitope comprises, consists essentially of, or consists of SEQ ID NO:27. In other embodiments, the T cell epitope comprises at least two amino acid sequences selected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. In another embodiment, the T cell epitope comprises at least three amino acid sequences selected from SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. In a most preferred embodiment, the T cell epitope comprises SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27. Combinations of these peptides may also be appropriately truncated to a length of 10 to 20 amino acids and included in the vaccine composition of the present invention. The combined peptides are preferably linked by one or more linkers as defined herein.

[0061] In some embodiments, the T cell epitopes of the invention may contain one or more modifications, such as, but not limited to, amino acid substitutions and post-translational modifications, In a more preferred embodiment, the T cell epitopes of the invention contain one to five amino acid modifications.

[0062] The Pan DR epitope (PADRE or PaDre) peptide (having the sequence AKFVAAWTLKAAA (SEQ ID NO: 35)) is known. See, for example, Alexander et al. (1994) and de Guercio et al (1997). The PADRE peptide has been shown to be a peptide that helps antibody responses and provides helper T cell activity in vivo. These properties suggest that constructs containing the PADRE peptide may be as effective as large multivalent antigens in generating an immune response.

[0063] The P2 (having the amino acid sequence QYIKANSKFIGITEL (SEQ ID NO:36)) and P30 peptides (having the amino acid sequence FNNFTVSFWLRVPKVSASHLE (SEQ ID NO:37)) are derived from tetanus toxin. See, e.g., Panina-Bordignon et al (1989) and Boeckler et al., (1999).

[0064] In some embodiments, a liposomal vaccine composition of the invention comprises a universal T cell epitope comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:29 (SAT42), SEQ ID NO:32 (SAT47), or close sequence analogs thereof as defined herein. In some embodiments, a liposomal vaccine composition of the invention comprises a universal T cell epitope comprising, consisting essentially of, or consisting of SEQ ID NO:32 (SAT47), or close sequence analogs thereof as defined herein.

[0065] In some embodiments, a liposomal vaccine composition of the invention comprises a peptide having a universal T cell epitope comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or SEQ ID NO: 33 (SAT58). In some embodiments, a liposomal vaccine composition of the invention comprises a peptide having a universal T cell epitope comprising, consisting essentially of, or consisting of SEQ ID NO: 33 (SAT58).

[0066] Preferably, the combination of T cell epitopes is provided as a peptide comprising the amino acid sequence of SEQ ID NO:29 (SAT42), SEQ ID NO:30 (SAT43), SEQ ID NO:31 (SAT44), SEQ ID NO:32 (SAT47), SEQ ID NO:35 (PaDre), SEQ ID NO:36 (P2), SEQ ID NO:37 (P30), SEQ ID NO:21 (SAT13), SEQ ID NO:22 (SAT15), SEQ ID NO:23 (SAT17), SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, or a variant having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, preferably 99% sequence identity thereto (which may be referred to as a close sequence analogue), or a variant comprising one to five amino acid substitutions (provided that the resulting peptide retains the T cell epitope).

[0067] In one embodiment, the combination of T cell epitopes is provided as a peptide comprising the amino acid sequence of SEQ ID NO: 32 (SAT47), or a variant having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, preferably 99% sequence identity thereto (which may be referred to as a close sequence analog), or a variant containing one to five amino acid substitutions (but wherein the resulting peptide retains the T cell epitope).

[0068] In one embodiment, the combination of T cell epitopes is provided as a peptide comprising the amino acid sequence of SEQ ID NO: 33 (SAT58), or a variant having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, preferably 99% sequence identity thereto (which may be referred to as a close sequence analog), or a variant containing one to five amino acid substitutions (but wherein the resulting peptide retains the T cell epitope).

[0069] Alignment of variants is typically based on comparison with the full-length sequence of the original linear peptide. Various alignment algorithms are available and routinely used, such as CLUSTALW and GAP. The composition of these peptides is described in more detail with reference to Table 1 below. Such peptides are included in the liposomal vaccine composition of the present invention. Preferably, the liposomal vaccine composition of the present invention comprises the amino acid sequence of SEQ ID NO:29 (SAT42), SEQ ID NO:32 (SAT47), or its close sequence analogues as defined herein.

[0070] More preferably, the liposomal vaccine composition comprises a peptide comprising the amino acid sequence of SEQ ID NO: 32 (SAT47) or a close sequence analog thereof. For example, the liposomal vaccine composition may comprise a peptide comprising the amino acid sequence of SEQ ID NO: 33 (SAT58). [Table 1]

[0071] In some embodiments of the invention, the T cell epitope may be encapsulated or displayed on the surface of the liposome. Depending on the nature of the T cell epitope, it may bind or associate to some degree with the liposome membrane, allowing some level to be surface displayed and / or some level to be within the liposome membrane.

[0072] In some embodiments, the liposome has a negative surface charge; the liposome is anionic.Preferably, the liposome comprises phospholipids, more preferably, the phospholipid comprises dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG).The liposome may further comprise cholesterol.In some embodiments, the molar ratio of these three components is 9:1:7.

[0073] Thus, the most preferred constructs comprise antigenic peptides reconstituted into liposomes, and therefore these compositions of the invention may be generally referred to herein as "liposomal vaccine compositions of the invention."

[0074] The composition of the present invention typically comprises at least one adjuvant. In some embodiments of the present invention, the composition of the present invention comprises two adjuvants. The purpose of the adjuvant is to improve or stimulate an immune response in a subject. Preferably, at least one adjuvant is part of the carrier (rather than being encapsulated in the carrier). That is, at least one adjuvant may form part of a liposome or may form part of a lipid bilayer membrane. The adjuvant may be a TLR agonist. Thus, the adjuvant may be a lipid-based adjuvant. The adjuvant may be presented, at least in part, on the surface of the liposome, which may be a result of the adjuvant forming part of the lipid bilayer membrane. The adjuvant may comprise a TLR4 agonist and / or a TLR9 agonist.

[0075] Examples of TLR4 ligands useful in the present invention include TLR4 agonists, including but not limited to monophosphoryl lipid A (MPLA). As used herein, the term "monophosphoryl lipid A" or "MPLA" refers to a modified form of lipid A, the biologically active portion of the lipopolysaccharide (LPS) endotoxin of gram-negative bacteria. MPLA maintains immunostimulatory activity but is less toxic than LPS. As a vaccine adjuvant, MPLA stimulates both cellular and humoral responses to vaccine antigens. Examples of MPLA include, but are not limited to, 3-O-desacyl-4'-monophosphoryl lipid A, monophosphoryl hexaacyl lipid A, 3-deacyl, monophosphoryl 3-deacyl lipid A, and their structurally related variants. MPLA useful in the present invention can be obtained using methods known in the art or can be purchased from Avanti Polar Lipids (Alabaster, Alabama, USA) as 3D-(6-acyl) PHAD. (登録商標) , P.H.A.D. (登録商標) , P.H.A.D. (登録商標) -504, 3D-PHAD (登録商標) or MPL from a variety of commercial sources. (商標) According to a particular embodiment, the TLR4 agonist is MPLA.

[0076] Examples of TLR9 ligands useful in the present invention include TLR9 agonists, including but not limited to CpG oligonucleotides.

[0077] Other adjuvants that may be used according to the present invention include aluminum hydroxide (alum) and / or CpG, among others. Any suitable CpG known to those skilled in the art may be used in the present invention in view of the present disclosure. Examples of such CpG oligonucleotides include, but are not limited to, CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, or CpG2336. CpG may be lipidated using methods known in the art in view of the present disclosure. In some embodiments, the 3' or 5' end of the CpG oligonucleotide is covalently linked to a cholesterol molecule through a phosphate bond, optionally via a PEG linker. Thus, a preferred adjuvant is CpG-chol (cholesterol). In some embodiments of the present invention, the CpG comprises CpG2006 (also known as CpG7909), which is defined by the following nucleotide sequence (SEQ ID NO: 34): 5'-tcgtcgttttgtcgttttgtcgtt-3' (SEQ ID NO: 34) Here the base is phosphorothioate (ps).

[0078] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; b A peptide containing a T cell epitope; and C. Adjuvant Includes.

[0079] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; b A peptide containing a T cell epitope; and C. Adjuvant wherein the adjuvant comprises at least one TLR ligand.

[0080] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; b A peptide containing a T cell epitope; and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0081] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; b. a peptide comprising a T cell epitope, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, or any combination thereof; and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0082] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, and SEQ ID NO:20; b. a T cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO:32 (SAT47) or a close sequence analog of SEQ ID NO:32; and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0083] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; b. a peptide comprising a T-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:24 and / or SEQ ID NO:25 and / or SEQ ID NO:27; and c. An adjuvant comprising a Toll-like receptor 4 ligand, preferably monophosphoryl lipid A (MPLA), and / or a Toll-like receptor 9 ligand, preferably CpG. Includes.

[0084] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:24, and SEQ ID NO:25, and SEQ ID NO:27; and c. An adjuvant comprising a Toll-like receptor 4 ligand, preferably monophosphoryl lipid A (MPLA), and / or a Toll-like receptor 9 ligand, preferably CpG. Includes.

[0085] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; b. a peptide comprising a T-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:46 and / or SEQ ID NO:25 and / or SEQ ID NO:27; and c. An adjuvant comprising a Toll-like receptor 4 ligand, preferably monophosphoryl lipid A (MPLA), and / or a Toll-like receptor 9 ligand, preferably CpG. Includes.

[0086] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:46, and SEQ ID NO:25, and SEQ ID NO:27; and c. An adjuvant comprising a Toll-like receptor 4 ligand, preferably monophosphoryl lipid A (MPLA), and / or a Toll-like receptor 9 ligand, preferably CpG. Includes.

[0087] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:12; b. a peptide comprising a T-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:46 and / or SEQ ID NO:25 and / or SEQ ID NO:27; and c. An adjuvant comprising a Toll-like receptor 4 ligand, preferably monophosphoryl lipid A (MPLA), and / or a Toll-like receptor 9 ligand, preferably CpG. Includes.

[0088] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:12; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:46, and SEQ ID NO:25, and SEQ ID NO:27; and c. An adjuvant comprising a Toll-like receptor 4 ligand, preferably monophosphoryl lipid A (MPLA), and / or a Toll-like receptor 9 ligand, preferably CpG. Includes.

[0089] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; b A peptide containing a T cell epitope; and C. Adjuvant Includes.

[0090] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:12; b A peptide containing a T cell epitope; and C. Adjuvant Includes.

[0091] In a further embodiment of the invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; b. a peptide comprising a T cell epitope, comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27, or any combination thereof; and C. Adjuvant Includes.

[0092] In a further embodiment of the invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:12; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:24, and SEQ ID NO:25, and SEQ ID NO:26, and SEQ ID NO:27, or any combination thereof; and C. Adjuvant Includes.

[0093] In a further embodiment of the invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; and C. Adjuvant Includes.

[0094] In a further embodiment of the invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:12; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:46 and SEQ ID NO:25 and SEQ ID NO:26 and SEQ ID NO:27; and C. Adjuvant Includes.

[0095] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2; b. a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:32 (SAT47) or a close sequence analog of SEQ ID NO:32; and C. Adjuvant Includes.

[0096] In some embodiments, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2; b. a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33 (SAT58); and C. Adjuvant Includes.

[0097] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:12; b. a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:32 (SAT47) or a close sequence analog of SEQ ID NO:32; and C. Adjuvant Includes.

[0098] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:12; b. a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33 (SAT58); and C. Adjuvant Includes.

[0099] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2; b. a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33 (SAT58); and c. An adjuvant comprising a Toll-like receptor 4 ligand and / or a Toll-like receptor 9 ligand Optionally, the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA) and the Toll-like receptor 9 ligand comprises CpG.

[0100] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or a close sequence analogue of SEQ ID NO: 32; c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0101] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:12; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or a close sequence analogue of SEQ ID NO: 32; c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0102] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; b) a peptide containing a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 32 (SAT47) or a close sequence analog thereof; and c) an adjuvant comprising a Toll-like receptor 4 ligand, the Toll-like receptor 4 ligand comprising monophosphoryl lipid A (MPLA); Includes.

[0103] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2; b) a peptide containing a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 32 (SAT47) or a close sequence analog thereof; and c) an adjuvant comprising a Toll-like receptor 4 ligand and / or a Toll-like receptor 9 ligand, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA) and the Toll-like receptor 9 ligand comprises CpG; Includes.

[0104] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or a close sequence analogue of SEQ ID NO: 32; c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0105] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 33 (SAT58); c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0106] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; and b) a peptide containing a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33 (SAT58); and c) an adjuvant comprising a Toll-like receptor 4 ligand, the Toll-like receptor 4 ligand comprising monophosphoryl lipid A (MPLA); Includes.

[0107] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:3; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or a close sequence analogue of SEQ ID NO: 32; c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0108] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:4; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or a close sequence analogue of SEQ ID NO: 32; c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0109] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:12; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or a close sequence analogue of SEQ ID NO: 32; c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0110] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a peptide comprising a T cell epitope; and c) Adjuvants wherein the peptide antigen comprises one or two palmitoyl residues at the N- and / or C-terminal regions of the peptide.

[0111] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:12; b) a peptide comprising a T cell epitope; and c) Adjuvants wherein the peptide antigen comprises one or two palmitoyl residues at the N- and / or C-terminal regions of the peptide.

[0112] In some embodiments of the present invention, the liposomal vaccine composition d) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; e) a peptide comprising a T cell epitope; and f) Adjuvants wherein the peptide antigen comprises two palmitoyl residues at the N- or C-terminal region of the peptide.

[0113] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a peptide comprising a T cell epitope; and c) Adjuvants wherein the peptide antigen comprises two palmitoyl residues at the N- and C-terminal regions of the peptide.

[0114] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:12; b) a peptide comprising a T cell epitope; and c) Adjuvants wherein the peptide antigen comprises one or two palmitoyl residues at the N-terminal region of the peptide.

[0115] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:2; b) a peptide comprising a T cell epitope; and c) Adjuvants wherein the peptide antigen comprises one or two palmitoyl residues at the N-terminal region of the peptide.

[0116] In some embodiments of the present invention, the liposomal vaccine composition a) a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of SEQ ID NO:12; b) a peptide comprising a T cell epitope; and c) Adjuvants wherein the peptide antigen comprises one or two palmitoyl residues at the N-terminal region of the peptide.

[0117] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b A peptide containing a T cell epitope; and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0118] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b. a peptide comprising a T-cell epitope comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27, or any combination thereof; and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0119] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequences of SEQ ID NO:24 and SEQ ID NO:25 and SEQ ID NO:26 and SEQ ID NO:27; and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0120] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b. a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:46, SEQ ID NO:25, SEQ ID NO:26, or SEQ ID NO:27; C. Adjuvant Includes.

[0121] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b. a peptide comprising a T cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:46 and SEQ ID NO:25 and SEQ ID NO:26 and SEQ ID NO:27; C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0122] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b. a T cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO:32 (SAT47) or a close sequence analog of SEQ ID NO:32; and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0123] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b. a T cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 33 (SAT58); and C. Adjuvant wherein the adjuvant comprises at least one TLR4 ligand and / or TLR9 ligand.

[0124] In some embodiments of the present invention, the liposomal vaccine composition a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b. a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33 (SAT58); and c. An adjuvant comprising a Toll-like receptor 4 ligand and / or a Toll-like receptor 9 ligand Optionally, the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA) and the Toll-like receptor 9 ligand comprises CpG.

[0125] In some embodiments of the present invention, the liposomal vaccine composition a) peptide antigens presented on the surface of liposomes comprising, consisting essentially of, or consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b) a T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of SEQ ID NO: 32 (SAT47) or a close sequence analogue of SEQ ID NO: 32; c) TLR4 ligands including monophosphoryl lipid A (MPLA); and d) CpG-containing TLR9 ligands Includes.

[0126] In some embodiments of the present invention, the liposomal vaccine composition a) peptide antigens presented on the surface of liposomes comprising, consisting essentially of, or consisting of SEQ ID NO:1, SEQ ID NO:38, and SEQ ID NO:45; b) a peptide comprising a T cell epitope; and c) Adjuvants wherein the peptide antigen comprises one or two palmitoyl residues at the N- and / or C-terminal regions of the peptide.

[0127] The liposomal vaccine composition of the present invention may be administered to a subject once to generate a protective immune response. However, in some embodiments, the vaccine composition of the present invention is administered multiple times to the same subject. That is, a so-called prime-boost regimen may be employed in accordance with the present invention. The administration of the vaccine is typically separated by an intervening period of at least one week, and often about 1 to 12 months.

[0128] The timing of administration (e.g., injections) can vary widely, from once a day to once a year to once every ten years. A typical regimen consists of an immunization followed by booster administrations (e.g., injections) at regular time intervals, such as every 4-6 weeks. However, for reasons of convenience and compliance, less regular booster administrations, such as annual boosts, may be preferred.

[0129] One or more immunizations may be provided. A typical regimen consists of an immunization followed by a booster injection at time intervals such as 4-6 week intervals. Another regimen may consist of an immunization followed by a booster injection 1, 2, 6, 9, and / or 12 months later.

[0130] Alternatively, booster injections may be administered at irregular intervals depending on monitoring of the immune response (eg, when antibody levels fall below a threshold determined by a physician or skilled artisan).

[0131] The vaccine composition of the present invention is a novel and powerful therapeutic option for the prevention and treatment of diseases, disorders, or disorders associated with alpha-synuclein aggregates, including but not limited to Lewy bodies and / or Lewy neurites and / or glial cytoplasmic inclusions, such as Parkinson's disease, multiple system atrophy, Lewy body dementia (LBD; dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD)), or diffuse Lewy body disease. In some embodiments, the same vaccine composition is administered at each stage (homologous vaccination regimen). Homologous vaccination refers to an immunization regimen that uses the same vaccine for both prime (first immunization) and boost (second or subsequent immunization).

[0132] The vaccine composition of the present invention may be administered to a subject by any suitable route of administration. As will be appreciated by those skilled in the art, the vaccine composition may be administered topically, orally, rectally, nasally, or parenterally (intravenously, intradermally, subcutaneously, or intramuscularly). In addition, the vaccine composition may be incorporated into a sustained release matrix, such as a biodegradable polymer, which is implanted near or in close proximity to where delivery is desired. In a preferred embodiment, the vaccine composition is administered intramuscularly or subcutaneously.

[0133] The vaccine compositions of the invention are administered to a subject to treat, prevent, induce a protective immune response against, or attenuate symptoms associated with a disease, disorder, or condition associated with alpha synuclein aggregates.

[0134] Thus, the vaccine composition may have both prophylactic and therapeutic uses.The subject is a mammal, typically a human.

[0135] In certain embodiments of the invention, the disease, disorder, or condition associated with alpha synuclein aggregates is a synucleinopathy.

[0136] In other embodiments of the invention, the disease, disorder, or condition associated with alpha synuclein aggregates or synucleinopathies is selected from the group consisting of Parkinson's disease (sporadic, familial with alpha synuclein mutations, familial with non-alpha synuclein mutations, pure autonomic failure, and Lewy body dysphagia), Lewy body dementia (LBD; dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD)), diffuse Lewy body disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2, or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, and Down's syndrome, multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration, and olivopontocerebellar atrophy), traumatic brain injury, chronic The disease may be selected from the group consisting of traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and Niemann-Pick disease type C1, frontotemporal dementia with parkinsonism linked to chromosome 17), motor neuron diseases, Huntington's disease, amyotrophic lateral sclerosis (sporadic, familial, and Guam ALS-dementia complex), neuroaxonal dystrophies, cerebral iron deposition neurodegeneration type 1 (Hallervorden-Spatz syndrome), prion diseases, Creutzfeldt-Jakob disease, ataxia-telangiectasia, Meige syndrome, subacute sclerosing panencephalitis, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, Gaucher disease, Krabbe disease and other lysosomal storage diseases (including Kufour-Rakeb syndrome and Sanfilippo syndrome), and rapid eye movement (REM) sleep behavior disorder.

[0137] In a preferred embodiment, the disease, disorder, or abnormality associated with alpha-synuclein aggregates or synucleinopathies is selected from the group consisting of Lewy bodies and / or Lewy neurites and / or glial cytoplasmic inclusions, such as Parkinson's disease, multiple system atrophy, Lewy body dementia (LBD; Dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease type dementia (PDD)), or diffuse Lewy body disease. In an embodiment, the disease, disorder, or abnormality associated with alpha-synuclein aggregates or synucleinopathies is Parkinson's disease or multiple system atrophy, more preferably multiple system atrophy.

[0138] Thus, the present invention provides a method for treating, preventing, inducing a protective immune response against, or attenuating a condition associated with alpha-synuclein aggregates in a subject, said method comprising administering to the subject a vaccine composition of the present invention.

[0139] Such methods may also be expressed in the form of medical uses of the vaccine compositions of the invention.Accordingly, the invention also provides the vaccine compositions of the invention for use in treating, preventing, inducing a protective immune response against, or attenuating a symptom associated with an alpha-synuclein aggregate-associated disease, disorder, or condition in a subject.

[0140] Thus, the present invention further comprises administering to a subject a liposomal vaccine composition or pharmaceutical composition of the present invention to induce an immune response against alpha synuclein aggregates. Some embodiments of the present invention further comprise administering to a subject a liposomal vaccine composition or pharmaceutical composition of the present invention to induce an immune response against alpha synuclein protein. In some embodiments, administering to a subject a liposomal vaccine composition or pharmaceutical composition of the present invention induces a protective immune response against alpha synuclein aggregates. In some embodiments, administering to a subject a liposomal vaccine composition or pharmaceutical composition of the present invention induces an immune response against alpha synuclein protein, preferably human alpha synuclein protein.

[0141] Similarly, the invention provides the use of a vaccine composition of the invention in the manufacture of a medicament for use in treating, preventing, inducing a protective immune response against, or attenuating a symptom associated with an alpha-synuclein aggregate-associated disease, disorder, or abnormality in a subject.

[0142] It is an object of the present invention to provide a medicament in the form of a vaccine for use in the prevention and treatment of synucleinopathies. It is a further object to provide a vaccine peptide suitable for use in humans.

[0143] It is one object of the present invention to provide a liposomal composition in the form of a vaccine for use in the prevention and treatment of synucleinopathies. It is a further object to provide an antigenic peptide suitable for use in humans.

[0144] All embodiments herein, however expressed, apply to such methods or medical uses. Administering the vaccine composition of the present invention to a subject results in the production of IgG antibodies, typically polyclonal, that bind to pathological and / or aggregated forms of alpha synuclein. As already explained, these pathological and / or aggregated forms of alpha synuclein include multimers. Thus, the antibodies produced may be referred to as "alpha synuclein multimer-specific" antibodies.

[0145] The present invention provides liposomal vaccine compositions containing antigenic peptides in a therapeutically effective amount. The term "therapeutically effective amount" refers to an amount of an antigenic / immunogenic composition that elicits an immune response when administered to a human or animal. An effective amount can be readily determined by one of skill in the art following routine procedures.

[0146] In absolute terms, it is preferred to use an amount of antigenic peptide in a dose of at least 10 μg, for example at least 50 μg. In this regard, it is important to note that the "μg peptide" referred to in the present invention refers to the amount of antigenic peptide in a dose and does not include other components of the liposomal vaccine composition.

[0147] The present invention further provides a kit containing the vaccine composition of the present invention. Thus, a kit is provided for treating, preventing, inducing a protective immune response against, or attenuating symptoms associated with a disease, disorder, or disorder associated with alpha-synuclein aggregates in a subject, comprising the liposomal vaccine composition of the present invention described herein. Such a kit may be provided with appropriate instructions for use. The instructions for use may describe the administration schedule of the composition. Thus, the kit may include multiple (separate) doses of the vaccine composition of the present invention. The instructions for use may further describe the storage conditions of the composition, especially during the period between administrations of doses of the vaccine composition. These kits may be applied to all of the related methods of the present invention disclosed herein.

[0148] A method for producing the liposomal vaccine composition of the present invention may rely on crossflow injection, as exemplified herein. Such a method may comprise the following steps: a) dissolving liposome-forming lipids (and adjuvants, if lipid-based) in solution; b) dissolving a peptide comprising a (universal) T-cell epitope in solution c) mixing the solutions of steps a and b using a cross-flow injection module to form intermediate liposomes encapsulating peptides containing universal T-cell epitopes. d) Extruding intermediate liposomes through a membrane to reduce their size and polydispersity e) mixing a solution containing a peptide antigen as described herein with the solution of step d using a cross-flow injection module, thereby inserting the peptide antigen into the lipid bilayer of the liposome.

[0149] Such methods are exemplified herein, the details of which may be applied to these aspects of the invention. In general terms, the methods use cross-flow injection to encapsulate peptides containing universal T-cell epitopes and insert alpha-synuclein-derived peptide antigens into the lipid bilayer of liposomes.

[0150] definition By "T cell epitope" is meant an epitope specific for T cells present in the majority of the human population. These generally originate from antigens to which humans are normally exposed during their lifetime. Examples include antigens incorporated into routinely administered vaccines. Specific examples of T cell epitopes include, but are not limited to, tetanus, influenza and diphtheria (including their non-toxic variants such as CRM197), keyhole limpet hemocyanin (KLH), Epstein-Barr virus (EBV) and PaDre (Pan HLA DR binding epitope). The ability of a T cell epitope to activate a T cell is the result of at least two complementary properties: i) the affinity of binding to the HLA groove (meaning the strength of binding) and ii) the ability to bind promiscuously to different HLA haplotypes (meaning the ability to cover a highly diverse human population in terms of differences in expression of HLA molecules). A T cell epitope may bind to the majority of MHC class II alleles present in the human population. Therefore, the T cell epitope contained in the vaccine composition of the present invention may be capable of stimulating CD4 T cell response.Thus, the T cell epitope contained in the vaccine composition of the present invention may be capable of stimulating helper T cell response that enhances the production of alpha-synuclein-related antibodies by B cells.Herein, these may be referred to as "universal" T cells, a term commonly used in the art.

[0151] Thus, a T cell epitope is herein understood as an epitope capable of stimulating helper T cells or a T cell helper response.

[0152] A "close sequence analog" of the invention may contain one or more modifications of the sequences described herein, such as, but not limited to, amino acid substitutions, post-translational modifications, extensions (addition of additional amino acids), shortening of length (removal of some amino acids), etc. In a preferred embodiment, a close sequence analog of a T cell epitope of the invention contains one to five amino acid modifications, preferably one, two, or three modifications.

[0153] The term "Toll-like receptor 4 agonist" or "TLR4" refers to any compound that acts as an agonist of TLR4. Any suitable TLR4 agonist known or discovered by those skilled in the art can be used in the present invention. Examples of TLR4 ligands useful in the present invention include TLR4 agonists, including but not limited to monophosphoryl lipid A.

[0154] The term "Toll-like receptor agonist" or "TLR9" refers to any compound that acts as an agonist of TLR9. Any suitable TLR9 agonist known or discovered by those skilled in the art can be used in the present invention. Examples of TLR9 ligands useful in the present invention include TLR9 agonists, including but not limited to CpG oligonucleotides.

[0155] Pharmaceutically acceptable carriers, diluents, adjuvants and excipients are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences 15, the disclosure of which is incorporated herein by reference. th. or 18 th. Ed.(Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990);Remington: the Science and Practice of Pharmacy 19 th Ed.(Lippincott, Williams & Wilkins, 1995);Handbook of Pharmaceutical Excipients 3 rd Ed.(Arthur H. Kibbe, ed.;Amer. Pharmaceutical Assoc, 1999);Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12 thEd.(Walter Lund ed.;Pharmaceutical Press, London, 1994);The United States Pharmacopeia: The National Formulary (United States Pharmacopeial Convention);Fiedler's "Lexikon der Hilfstoffe" 4 th Ed., Edition Cantor Verlag Aulendorf 2002;"The Handbook of Pharmaceutical Excipients", 4 th Ed., American Pharmaceuticals Association, 2003; and Goodman and Gilman's: the Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992).

[0156] Carriers, diluents, adjuvants, and pharmaceutical excipients can be selected having regard to the intended route of administration and standard pharmaceutical practice. It must be observed that these compounds are not harmful to the recipient. See Remington's Pharmaceutical Sciences 15, the disclosure of which is incorporated herein by reference. th. or 18 th. Ed.(Alfonso R. Gennaro, ed.; Mack Publishing Company, Easton, PA, 1990);Remington: the Science and Practice of Pharmacy 19 th Ed.(Lippincott, Williams & Wilkins, 1995);Handbook of Pharmaceutical Excipients 3 rdEd.(Arthur H. Kibbe, ed.;Amer. Pharmaceutical Assoc, 1999);Pharmaceutical Codex: Principles and Practice of Pharmaceutics 12 th Ed.(Walter Lund ed.;Pharmaceutical Press, London, 1994);The United States Pharmacopeia: The National Formulary (United States Pharmacopeial Convention);Fiedler's "Lexikon der Hilfstoffe" 5 th Ed., Edition Cantor Verlag Aulendorf 2002;"The Handbook of Pharmaceutical Excipients", 4 th Ed., American Pharmaceuticals Association, 2003; and Goodman and Gilman's: the Pharmacological Basis of Therapeutics (Louis S. Goodman and Lee E. Limbird, eds.; McGraw Hill, 1992).

[0157] As used herein, the term "liposomal vaccine composition" may be used interchangeably with "liposomal immunogenic composition." The "close sequence analogs" of the present invention may include one or more modifications of the sequences described herein, such as, but not limited to, amino acid substitutions, post-translational modifications, elongations (addition of additional amino acids), shortening of length (removal of some amino acids).

[0158] When an amino acid is referred to herein, it is typically a naturally occurring amino acid, however, reference to an amino acid also encompasses unnatural amino acids (e.g., beta amino acids, gamma amino acids, D-amino acids) and, in the context of the overall peptide, combinations of natural and unnatural amino acids.

[0159] The native sequence of human alpha-synuclein is MDVFMKGLSKAKEGVVAAAEKTKQGVAEAAGKTKEGVLYVGSKTKEGVVHGVATVAEKTKEQVTNVGGAVVTGVTAVAQKTVEGAGSIAAATGFVKKDQLGKNEEGAPQEGILEDMPVDPDNEAYEMPSEEGYQDYEPEA (sequence number 28).

[0160] An "immune response" involves the production of anti-aSyn antibodies, preferably antibodies that specifically bind to alpha synuclein. The production of such antibodies can be tested by any suitable method, such as an immunoassay, in particular by ELISA. EXAMPLES

[0161] The invention will be further understood with reference to the following non-limiting examples.

[0162] Example 1. Vaccine synthesis and formulation The vaccine is produced as described below by a three-step approach: preparation of intermediate liposomes, followed by incorporation of CpG-Chol to generate fully adjuvanted liposomes, and final insertion of the antigenic peptide.

[0163] Intermediate Liposomes: First, lipids (DMPG, DMPC, cholesterol and monophosphoryl hexaacyl lipid A, 3-deacyl, first adjuvant) are dissolved in ethanol at 60°C. After complete dissolution, the lipid / ethanol solution is filtered through a 0.2 μm pore size filter into an injection system preheated to 60°C. In a separate container, a peptide containing a T cell epitope (e.g. SAT47, or a close sequence analogue) is solubilized with 10 mM histidine, 250 mM sucrose. This solution is filtered through a 0.2 μm pore size filter and heated to 40°C. Next, the lipid / ethanol solution and the peptide containing a T cell epitope (SAT47 or a close sequence analogue) are mixed using a cross-flow injection module to form intermediate liposomes. The intermediate liposomes are subsequently subjected to active cooling and then size reduction using repeated extrusion cycles. Finally, ultrafiltration / diafiltration (UDF) is performed to remove the ethanol. The intermediate liposomes are filtered through a 0.2 μm pore size filter and stored at 4° C. until use.

[0164] CpG-Chol integration : The intermediate liposomes are diluted to a lipid concentration of 1 mg / mL and warmed to 60°C. The second adjuvant, CpG-Chol, is added dropwise to the liposomes. The liposome dispersion is incubated for an additional 30 minutes at 60°C with stirring. The fully adjuvanted liposomes are purified with UDF, then filtered through a 0.45 μm pore size filter, then a 0.2 μm pore size filter, and stored at 4°C.

[0165] Insertion of antigenic peptides derived from alpha-synuclein (Asyn): The fully adjuvanted liposomes are diluted to the desired lipid concentration. At the same time, palmitoylated antigen peptides (such as peptides having the amino acid sequences of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) are dissolved to a final concentration of 0.1 to 10 mg / mL of peptide. Finally, the liposome and peptide solutions are mixed using a cross-flow injection module, and the liposome dispersion is incubated with stirring at a temperature of 30°C to 60°C. A UDF step is performed to exchange the buffer into the formulation system. The result is concentrated to its final volume and filtered through a 0.45 μm pore size filter, followed by a 0.2 μm pore size filter. The bulk formulation is stored at 4°C.

[0166] Example 2: Immunogenicity studies in mice Immunogenicity testing of vaccines presenting different alpha synuclein-derived antigenic peptides conjugated via cysteine ​​to CRM197, including the native alpha synuclein sequence and its derivatives (VARIOTOPES), was performed in BALB / c mice. Mice received a total of three subcutaneous (sc) immunizations (10 μg peptide net per injection) on days 0, 4 and 28. For this purpose, 200 μl of individual vaccines were administered into the flank of the mice using an insulin 20 syringe with a G30 gauge needle. Blood was collected 2 weeks after the last injection to measure alpha synuclein-specific IgG titers by ELISA. For this purpose, 96-well plates (Nunc-Maxisorp) were coated with recombinant human alpha synuclein (1 μg / ml) and titers were calculated as EC50 values ​​by nonlinear regression analysis (four-parameter logistic fit function) in PRISM® 5.04 (GraphPad Inc, San Diego, CA).

[0167] The immunogenicity of vaccines containing serine-exchanged peptides from the alpha-synuclein 113-124 native sequence was evaluated in BALB / c mice and compared with the immunogenicity of vaccines containing the native sequence for a direct comparison (Table 2).

[0168] Table 2 : Treatment groups and the peptide sequences contained in the individual vaccines are indicated. Group 1 was immunized with a vaccine containing the native sequence for direct comparison. [Table 2]

[0169] Vaccines containing peptides with serine exchanges at either positions 1 and 3 (group 3) or positions 3 and 12 (group 2) induced alpha-synuclein-specific IgG responses 4- to 6-fold higher than vaccines containing the native peptide sequence (group 1). Vaccines containing peptides with serine exchanges at position 4 (groups 4, 6, and 7) did not show a significant increase in immunogenicity compared to vaccines containing the native sequence (Table 3). Vaccines containing peptides with serine exchanges at positions 1 and 12 (group 5) showed less than a 2-fold increase in immunogenicity compared to vaccines containing the native sequence (Table 3).

[0170] Table 3 : Potencies against alpha-synuclein were assessed in single mice, values ​​represent median values ​​compared to those obtained with the native sequence. [Table 3]

[0171] Since vaccines containing peptides with amino acid exchanges at positions 1, 3, and 12 to serine were shown to induce high alpha-synuclein-specific IgG titers, further immunogenicity studies were performed using vaccines containing peptides with serine or lysine exchanges at positions 1, 3, and 12. Some of the tested peptides were extended at their N-terminus by adding two glycine residues (Table 4). Furthermore, for direct comparison, these vaccines were compared to a vaccine containing the previously selected antigenic peptide sequence DQPVLPD (SEQ ID NO: 43).

[0172] Table 4: Treatment groups and the peptide sequences contained in the individual vaccines are indicated. Group 1 was immunized with a vaccine containing the peptide sequence LESMPVDPDNES (Group 2, Table 2) to link this experiment with the previous one. [Table 4]

[0173] All newly designed vaccines induced higher alpha-synuclein-specific IgG titers (up to 3-fold in group 6) compared to the already highly immunogenic vaccine containing the antigenic peptide LESMPVDPDNES. Furthermore, all vaccines in groups 1 to 7 listed in Table 4 elicited substantially higher alpha-synuclein-specific titers than the vaccine containing the antigenic peptide DQPVLPD (SEQ ID NO: 43) (Table 5).

[0174] Table 5 : Titers against alpha-synuclein were assessed in single mice, values ​​represent median values ​​compared to those obtained with a vaccine containing the antigenic peptide LESMPVDPDNES. [Table 5]

[0175] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes related to the present invention.

[0176] Example 3: Immunogenicity study of SEQ ID NO:2 liposomal vaccine and SEQ ID NO:2 conjugate vaccine in mice The immunogenicity of a liposomal vaccine containing SEQ ID NO:2 as an antigenic peptide (referred to as "SEQ ID NO:2 liposomal vaccine") and a protein conjugate vaccine containing SEQ ID NO:2 as an antigenic peptide linked to the protein carrier CRM197 (referred to as "SEQ ID NO:2 conjugate vaccine") was evaluated in C57BL / 6J female mice.

[0177] a) Synthesis and formulation of SEQ ID NO:2 liposomal vaccine The vaccine is produced according to Example 1 in a three step approach as follows.

[0178] Intermediate liposomes: First, lipids (DMPG, DMPC, cholesterol and monophosphoryl hexaacyl lipid A, (3D-(6-acyl)PHAD (商標) (Avanti Polar Lipids, USA), the first adjuvant) was dissolved in ethanol at 60°C. After complete dissolution, the lipid / ethanol solution was filtered through a 0.2 μm pore size filter into an injection system preheated to 60°C. In a separate vessel, a peptide containing the T cell epitope SAT58 was dispersed in ethanol with the aid of sonication at room temperature and solubilized by dilution with 10 mM histidine, 250 mM sucrose. The SAT58 solution was filtered through a 0.2 μm pore size filter and heated to 40°C. Next, the lipid / ethanol solution and the SAT58 solution were mixed using a cross-flow injection module to form intermediate liposomes. The intermediate liposomes were subsequently subjected to active cooling followed by size reduction using repeated extrusion cycles. Finally, ultrafiltration / diafiltration (UDF) was performed to remove the ethanol. The intermediate SAT58 liposomes were filtered through a 0.2 μm pore size filter and stored at 4°C until use.

[0179] CpG-Chol synthesis: intermediate liposomes were diluted with 20 mM histidine, 145 mM NaCl to a lipid concentration of 1 mg / mL and warmed to 60°C. The second adjuvant, CpG-cholesterol, was added dropwise to the liposomes. The liposome dispersion was incubated for an additional 30 minutes at 60°C with stirring. The fully adjuvanted liposomes were purified with UDF, then filtered through a 0.45 μm pore size filter, then a 0.2 μm pore size filter, and stored at 4°C.

[0180] Insertion of alpha synuclein (Asyn) derived antigenic peptide: The fully adjuvanted liposomes were diluted with 20 mM histidine, 145 mM NaCl to a concentration of 1 mg / mL of total lipid content. At the same time, the N-terminally dipalmitoylated SEQ ID NO:2 peptide was dissolved in 20 mM histidine, 145 mM NaCl at 60°C to a final concentration of 1 mg / mL and the solution was filtered through a 0.2 μm pore size filter. Finally, the liposome and peptide solutions were mixed using a cross-flow module and the liposome dispersion was incubated with stirring at a temperature of 60°C for 30 minutes. A UDF step was performed to exchange the buffer into the final formulation system, i.e. 10 mM histidine, 250 mM sucrose buffer. The resultant was concentrated to its final volume and filtered through a 0.45 μm pore size filter and then through a 0.2 μm pore size filter. The bulk formulation was stored at 4°C.

[0181] b) SEQ ID NO:2 Conjugate Vaccine Synthesis and Formulation The SEQ ID NO:2 conjugate vaccine is the conjugation of the antigen peptide SEQ ID NO:2 to the carrier protein CRM197. The antigen peptide is modified to contain a cysteine ​​residue. The antigen peptide is conjugated to the carrier protein CRM197 via a cysteine ​​residue. The conjugation is a directed procedure that uses the side chain amino group of the lysine residue of CRM197 and the free thiol group of the amino (N)-terminal cysteine ​​of the peptide. For activation of CRM197, an aqueous CRM197 solution is adjusted to 10 mM phosphate buffered saline (PBS) and then gently shaken with the bifunctional linker 4-maleimidobutyric acid N-hydroxysuccinimide ester (GMBS). The excess of unreacted GMBS is then removed by either dialysis or ultrafiltration. The resulting activated CRM197 solution is then incubated with the antigen peptide (SEQ ID NO:2) dissolved in phosphate buffer. The free thiol group of the cysteine ​​in the peptide reacts with the maleimide group to form the final product, antigen peptide (SEQ ID NO:2)-CRM197.

[0182] c) Study design A total of 20 C57BL / 6J female mice, approximately 10 weeks of age at the time of the first immunization, were assigned to two groups (10 in group 1 and 10 in group 2) as shown in Table 6. The two groups were immunized three times by subcutaneous (sc) injection into the dorsal region on days 1, 15, and 29 with either the SEQ ID NO:2 conjugate vaccine (group 1) or the SEQ ID NO:2 liposomal vaccine (group 2).

[0183] Table 6 Study design [Table 6] sc: subcutaneous

[0184] Dose selection of SEQ ID NO:2 liposomal vaccine and SEQ ID NO:2 conjugate vaccine for this study in C57BL / 6J mice is based on peak antibody levels in the mice.

[0185] d) Immunogenicity studies in plasma Immunogenicity against SEQ ID NO:2 peptide, a-syn full-length human protein and a-syn aggregates was assessed in plasma samples collected on day 1 (before the first dose) and one week after each immunization (days 8, 22, and 36). a-syn aggregates were prepared according to the protocol described by Kumar et al. 2020.

[0186] Anti-SEQ ID NO:2, anti-a-syn protein, or anti-a-syn aggregate IgG titers were analyzed by enzyme-linked immunosorbent assay (ELISA) at each time point. Briefly, SEQ ID NO:2 peptide or a-syn protein or a-syn aggregates conjugated with BSA were immobilized overnight on 96-well microtiter plates. After washing and blocking, the plates were incubated with plasma samples for 2 hours at 37°C to allow antibodies present in the plasma to bind to the peptide or protein. After incubation, the plates were washed to remove non-reactive plasma components. Antibody / antigen complexes were detected via an anti-mouse IgG secondary antibody conjugated with alkaline phosphatase. pNPP (p-nitrophenyl phosphate) substrate was added to the wells, and the optical density was read at 405 nM on an ELISA plate reader. Anti-SEQ ID NO:2 peptide, anti-a-syn protein, or anti-a-syn aggregate IgG titers were back-calculated against a standard curve with eight two-fold serial dilutions using an unweighted four-parameter logistic regression model with Gen5 software (BioTek, Switzerland). Results are expressed in AU / mL.

[0187] Data are expressed over time as geometric mean ± 95% confidence interval (CI), n = 10 / group (n = 9 for group 2 on days 22 and 36). Two-way ANOVA with Fisher's uncorrected LSD multiple comparison test was used for statistical analysis. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001.

[0188] The results (Figures 1, 2, 3) show that animals immunized with SEQ ID NO:2 liposomal vaccine developed strong anti-SEQ ID NO:2 peptide, anti-a-syn protein, and anti-a-syn aggregate responses after one immunization, whereas mice immunized with SEQ ID NO:2 conjugate vaccine required two immunizations. At all time points, SEQ ID NO:2 liposomal vaccine induced statistically significantly higher titers than SEQ ID NO:2 conjugate vaccine for all measures tested.

[0189] In conclusion, these results confirm the ability of SEQ ID NO:2 liposomal vaccine to induce strong immune response against alpha synuclein protein and aggregated alpha synuclein in vivo.Furthermore, these results show that SEQ ID NO:2 liposomal vaccine produces surprisingly improved immune response against anti-SEQ ID NO:2 peptide, alpha synuclein protein and aggregated alpha synuclein in vivo compared with SEQ ID NO:2 conjugate vaccine.

[0190] The present invention should not be limited in scope to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the above description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Moreover, all aspects and embodiments of the present invention described herein are broadly applicable and can be combined, where appropriate, with any other non-inconsistent embodiments, including embodiments taken from other aspects of the invention (including those taken alone).

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Claims

1. a. a peptide antigen displayed on the surface of a liposome; b. a peptide comprising a T cell epitope; and c. Adjuvant 1. A liposomal vaccine composition comprising: X １ -X ２ -X ３ -E-X ４ -X ５ -P-V-D-P-D-N-E-X ６ comprising, consisting essentially of, or consisting of; E is glutamic acid, P is proline, V is valine, D is aspartic acid, N is asparagine; X １ is optionally present and, if present, is G, wherein G is glycine; X ２ is optionally present and, if present, is G, where G is as defined above; X ３ is L, K, or S, where L is leucine, K is lysine, and S is serine; X ４ is D, K, or S, where D, K, and S are as defined above; X ５ is M, where M is methionine or methionine sulfoxide; X ６ is A, K, or S, where A is alanine, K and S are as defined above; However, X ３ -E-X ４ -X ５ -PV-D-P-D-NE-X ６ is not L-E-D-M-P-V-D-P-D-N-E-A and contains one to five amino acid differences compared to the amino acid sequence G-I-L-E-D-M-P-V-D-P-D-N-E-A; and The peptide antigen is the dipeptide Y-E. ６ wherein Y is tyrosine and E is as defined above; Liposomal vaccine compositions.

2. X １ and X ２ The liposome composition of claim 1 , wherein

3. X １ and X ２ The liposome composition of claim 1 , wherein:

4. X ５ The liposome composition of claim 1 , wherein is methionine sulfoxide.

5. X ６ The liposome composition of claim 1 , wherein is A or S.

6. X ３ The liposome composition of claim 1 , wherein is L or K.

7. X ４ The liposome composition of claim 1 , wherein is D or S.

8. The peptide antigens are GGKESMPVDPDNEA (SEQ ID NO: 2), GGKEDMMPVDPDNEA (SEQ ID NO: 3), LESMPVDPDNEA (SEQ ID NO: 4), LEKMPVDPDNEA (SEQ ID NO: 5), GGSESMPVDPDNEA (SEQ ID NO: 6), GGSESMPVDPDNEA (SEQ ID NO: 7), KEDMMPVDPDNEA (SEQ ID NO: 8), SESMPVDPDNEA (SEQ ID NO: 9), LESMPVDPDNEA (SEQ ID NO: 10), GGKESMPVDPDNEA (SEQ ID NO: 11), KESM 2. The liposome composition of claim 1, wherein the liposome composition is selected from the group consisting of PVDPDNEA (SEQ ID NO: 12), KESMPVDPDNES (SEQ ID NO: 13), SEKMPVDPDNEA (SEQ ID NO: 14), LEKMPVDPDNES (SEQ ID NO: 15), GGSESMPVDPDNEK (SEQ ID NO: 16), LEKMPVDPDNEK (SEQ ID NO: 17), GGKEKMPVDPDNEA (SEQ ID NO: 18), GGKESMPVDPDNEK (SEQ ID NO: 19), and GGKEKMPVDPDNEK (SEQ ID NO: 20).

9. 2. The liposomal vaccine composition of claim 1, wherein the peptide antigen is selected from the group consisting of GGKESMPVDPDNEA (SEQ ID NO: 2), GGKEDMMPVDPDNEA (SEQ ID NO: 3), LESMPVDPDNES (SEQ ID NO: 4), and KESMPVDPDNEA (SEQ ID NO: 12).

10. 2. The liposomal vaccine composition of claim 1, wherein the peptide antigen consists of GGKESMPVDPDNEA (SEQ ID NO: 2) or KESMPVDPDNEA (SEQ ID NO: 12).

11. The liposomal vaccine composition of claim 1 , wherein the peptide antigen further comprises at least one chemical modification.

12. 2. The liposomal vaccine composition of claim 1, wherein the peptide comprising a T cell epitope comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO:35 (PaDre), SEQ ID NO:36 (P2), SEQ ID NO:37 (P30), SEQ ID NO:21 (SAT13), SEQ ID NO:22 (SAT15), SEQ ID NO:23 (SAT17), SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and / or any combination thereof.

13. 2. The liposomal vaccine composition of claim 1, wherein the peptide comprising the T cell epitope comprises, consists essentially of, or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:29 (SAT42), SEQ ID NO:30 (SAT43), SEQ ID NO:31 (SAT44), SEQ ID NO:32 (SAT47), SEQ ID NO:35 (PaDre), SEQ ID NO:36 (P2) and SEQ ID NO:37 (P30), or a close sequence analog thereof.

14. 2. The liposomal vaccine composition of claim 1, wherein the T cell epitope-containing peptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 33 (SAT58).

15. The liposomal vaccine composition of claim 1 , wherein the adjuvant comprises a Toll-like receptor 4 ligand and / or a Toll-like receptor 9 ligand.

16. 16. The liposomal vaccine composition of claim 15, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA) and / or the Toll-like receptor 9 ligand comprises CpG.

17. The liposomal vaccine composition of claim 1, wherein the peptide containing the T cell epitope is encapsulated in a liposome.

18. A peptide antigen presented on the surface of a liposome, comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; b. A peptide containing a T-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 46, and / or SEQ ID NO: 25, and / or SEQ ID NO: 27; and c. An adjuvant comprising a Toll-like receptor 4 ligand, preferably monophosphoryl lipid A (MPLA), and / or a Toll-like receptor 9 ligand, preferably CpG. A liposomal vaccine composition comprising:

19. 19. The liposomal vaccine of claim 18, wherein the peptide antigen comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

12.

20. a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:12; and b. A T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33 (SAT58); and c. An adjuvant comprising a Toll-like receptor 4 ligand, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA). A liposomal vaccine composition comprising:

21. 21. The liposomal vaccine composition of claim 20, wherein the peptide antigen comprises, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:

12.

22. 21. The liposomal vaccine composition of claim 20, wherein the peptide antigen comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

12.

23. a. a peptide antigen presented on the surface of a liposome comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:2; b. A T-cell epitope-containing peptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 33 (SAT58); and c. An adjuvant comprising a Toll-like receptor 4 ligand and / or a Toll-like receptor 9 ligand, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA) and the Toll-like receptor 9 ligand comprises CpG. A liposomal vaccine composition comprising:

24. A pharmaceutical composition comprising the liposomal vaccine composition of any one of claims 1 to 23, and a pharmaceutically acceptable carrier, diluent and / or excipient.

25. A kit comprising a liposome vaccine composition according to any one of claims 1 to 23 or a pharmaceutical composition containing the liposome vaccine composition according to any one of claims 1 to 23, and a container.

26. A liposomal vaccine composition according to any one of claims 1 to 23, or a pharmaceutical composition comprising the liposomal vaccine composition according to any one of claims 1 to 23, or a kit comprising the liposomal vaccine composition according to any one of claims 1 to 23 or the pharmaceutical composition comprising the liposomal vaccine composition according to any one of claims 1 to 23, for use in the treatment or prevention of a disease, disorder, or abnormality associated with alpha synuclein aggregates.

27. 27. The liposomal vaccine composition, pharmaceutical composition, or kit of claim 26, wherein the disease, disorder, or abnormality associated with alpha-synuclein aggregates is a synucleinopathy.

28. a disease, disorder, or abnormality associated with alpha-synuclein aggregates or synucleinopathies, including Parkinson's disease (sporadic, familial with alpha-synuclein mutations, familial with non-alpha-synuclein mutations, pure autonomic failure, and Lewy body dysphagia), Lewy body dementia (LBD; dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease dementia (PDD)), diffuse Lewy body disease (DLBD), sporadic Alzheimer's disease, familial Alzheimer's disease with APP mutations, familial Alzheimer's disease with PS-1, PS-2, or other mutations, familial British dementia, Lewy body variant of Alzheimer's disease, and Down's syndrome; Multiple system atrophy (Shy-Drager syndrome, striatonigral degeneration, and olivopontocerebellar atrophy), traumatic brain injury, chronic traumatic encephalopathy, dementia pugilistica, tauopathies (Pick's disease, frontotemporal dementia, progressive supranuclear palsy, corticobasal degeneration and Niemann-Pick disease type C1, frontotemporal dementia with parkinsonism linked to chromosome 17), motor neuron disease, Huntington's disease, amyotrophic lateral sclerosis (sporadic, familial, and Guam ALS-dementia complex), neuroaxonal dystrophies, cerebral iron deposition neurodegeneration type 1 (Hallervorden-Spatz syndrome), prion diseases, Creutzfeldt-Jakob disease, ataxia-telangiectasia, Meige's syndrome, subacute sclerosing panencephalitis, 27. The liposomal vaccine composition, pharmaceutical composition, or kit of claim 26, wherein the liposomal vaccine composition, pharmaceutical composition, or kit is selected from the group consisting of Gerstmann-Straussler-Scheinker disease, inclusion body myositis, Gaucher disease, Krabbe disease and other lysosomal storage diseases (including Kufuor-Rakeb syndrome and Sanfilippo syndrome), and rapid eye movement (REM) sleep behavior disorder.

29. 27. The liposomal vaccine composition, pharmaceutical composition, or kit of claim 26, wherein the disease, disorder, or abnormality associated with alpha-synuclein aggregates or synucleinopathies is selected from the group consisting of Lewy bodies and / or Lewy neurites and / or glial cytoplasmic inclusions, such as Parkinson's disease, multiple system atrophy, dementia with Lewy bodies (LBD; dementia with Lewy bodies (DLB) ("pure" Lewy body dementia), Parkinson's disease type dementia (PDD)), or diffuse Lewy body disease.

30. 27. The liposomal vaccine composition, pharmaceutical composition, or kit of claim 26, wherein the disease, disorder, or abnormality associated with alpha-synuclein aggregates or synucleinopathy is multiple system atrophy or Parkinson's disease.

31. A method for preventing, treating, and alleviating a disease associated with a disease, disorder, or abnormality associated with alpha-synuclein aggregates, comprising administering to a subject a liposomal vaccine composition described in any one of claims 1 to 23 or a pharmaceutical composition containing the liposomal vaccine composition described in any one of claims 1 to 23.

32. 32. The method of claim 31, wherein the disease, disorder, or condition associated with alpha synuclein aggregates is a synucleinopathy.

33. The method described in claim 31, wherein an immune response against alpha-synuclein aggregates is induced by administering to a subject a liposomal vaccine composition described in any one of claims 1 to 23 or a pharmaceutical composition containing the liposomal vaccine composition described in any one of claims 1 to 23.

34. The method described in claim 31, wherein a protective immune response against alpha-synuclein aggregates is induced by administering to a subject a liposomal vaccine composition described in any one of claims 1 to 23 or a pharmaceutical composition comprising the liposomal vaccine composition described in any one of claims 1 to 23.

35. The method described in claim 31, wherein an immune response to alpha synuclein protein, preferably human alpha synuclein protein, is induced by administering to a subject a liposomal vaccine composition described in any one of claims 1 to 23 or a pharmaceutical composition containing the liposomal vaccine composition described in any one of claims 1 to 23.

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