Heterogeneous administration of Tau vaccine

Abstract

A method for inducing an immune response against tau protein in subjects with neurodegenerative diseases, disorders, or conditions (e.g., Alzheimer's disease) is described. The method comprises administering a liposome-initiating composition containing a tau peptide (preferably phosphorylated tau peptide) and a conjugate-enhancing composition containing a tau peptide (preferably phosphorylated tau peptide) conjugated to an immunogenic carrier.

Description

Invention Field

[0001] This invention pertains to the medical field. Specifically, it relates to a method for inducing an immune response against tau protein in subjects suffering from neurodegenerative diseases, disorders, or conditions using a liposome-initiating composition containing tau peptide and a conjugate-enhancing composition comprising tau peptide conjugated to an immunogenic carrier.

[0002] background

[0003] Alzheimer's disease (AD) is a progressive, debilitating neurodegenerative disease that is estimated to affect 44 million people worldwide (Alzheimers.net). Currently available clinical treatments for AD aim to slow the progression of clinical symptoms but do not target the underlying pathogenic processes of the disease. Unfortunately, these treatments have only minimal efficacy, and therefore there is an urgent need to develop and test alternative preventative and therapeutic measures.

[0004] The hallmark lesions of Alzheimer's disease are extracellular plaques containing aggregated amyloid-β protein and intracellular "tangles" or hyperphosphorylated tau protein. The molecular events leading to these protein accumulations are poorly characterized. For amyloid, it is hypothesized that aberrant cleavage of amyloid precursor proteins leads to the accumulation of easily aggregated fragments containing amino acids 1-42. For tau, it is hypothesized that dysregulation of kinases, phosphatases, or both leads to aberrant phosphorylation of tau. Once tau becomes hyperphosphorylated, it loses its ability to bind efficiently and stabilize microtubules, instead accumulating in the cytoplasm of affected neurons. Unbound and hyperphosphorylated tau appear to first form oligomers, followed by higher-order aggregates, the presence of which is presumed to negatively impact the function of the neurons in which they form via interference with normal axonal transport.

[0005] In developed countries, individuals diagnosed with Alzheimer's disease or other dementia-related tau conditions are typically treated with cholinesterase inhibitors (such as Aricept®) or memantine (such as Namenda™). These medications, while fairly well-tolerated, have very general efficacy. For example, in approximately 50% of treated individuals, Aricept® delayed symptom progression by 6–12 months. The remaining treatments are nonpharmacological and focused on enabling patients to better manage daily tasks as their cognitive abilities decline.

[0006] Several published studies (Asuni AA et al., J Neurosci. 2007 Aug 22; 27(34):9115-29., Theunis C et al., PLoS One. 2013; 8(8): e72301., Kontsekova E et al., AlzheimersRes Ther. 2014 Aug 1; 6(4):44) have shown that active vaccines containing tau peptides can induce anti-tau immune responses in mice or rats; reduce the accumulation of pathological tau aggregates in the rodent brain; and slow the progression of cognitive decline in animal models of Alzheimer's disease. Active vaccines targeting pathological tau proteins have shown immunogenicity in human patients with Alzheimer's disease (Novak P et al., Lancet Neurology 2017, 16:123-134). WO2010 / 115843 and WO2019 / 084118 describe antigenic phosphopeptides of the major pathological phosphorylated epitopes of tau, a mimic protein, for therapeutic and diagnostic use in tau diseases, including Alzheimer's disease, and related compositions. However, there are currently no approved, effective vaccines on the market to prevent the onset of tau-mediated diseases. There are also no effective drugs on the market that stop or slow the progression of the disease once it begins. Therefore, there is an urgent need to identify new preventative measures (e.g., vaccines) to prevent these diseases. Invention Summary

[0007] Surprisingly, this invention has revealed that heterologous vaccination using liposome compositions and conjugate compositions, each containing a phosphopeptide, increases the epitope coverage of tau phosphopeptide-specific antibodies. Furthermore, heterologous vaccination using liposome-initiated compositions has been found to induce a stronger immune response than heterologous vaccination using conjugate-initiated compositions.

[0008] In one general aspect, the present invention relates to a method for inducing an immune response against tau protein (preferably inducing an antibody against at least one of phosphorylated tau and enriched paired helical filaments (ePHF)) in a subject in need of it (preferably a subject with a neurodegenerative disorder), the method comprising:

[0009] (i) administering to a subject a priming composition comprising an immunologically effective amount of liposomes, said liposomes comprising:

[0010] a. First tau phosphopeptide;

[0011] b. Helper T cell epitopes;

[0012] c. Lipid-modified CpG oligonucleotides; and

[0013] d. Adjuvants containing Toll-like receptor 4 ligands;

[0014] The tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further comprises a pharmaceutically acceptable carrier; and

[0015] (ii) Administering to the subject a first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the structure of formula (I):

[0016]

[0017] Or it may have the structure of formula (II):

[0018]

[0019] in

[0020] x is an integer from 0 to 10 (preferably 2 to 6, most preferably 3);

[0021] n is an integer from 3 to 15 (preferably 3 to 12);

[0022] Tau peptide represents the second tau phosphopeptide; and

[0023] The carrier represents an immunogenic carrier selected from keyhole cyanobacterial hemocyanin (KLH), tetanus toxoid, CRM197, and Neisseria meningitidis (…). N. meningitidis outer membrane protein mixture (OMP) or derivatives thereof; and

[0024] The first enhancement composition further comprises a pharmaceutically acceptable carrier;

[0025] The first tau phosphopeptide and the second tau phosphopeptide each independently comprise an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 5 to SEQ ID NO: 12.

[0026] In some embodiments, the liposomes comprise:

[0027] a. A first tau phosphopeptide having an amino acid sequence selected from SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38;

[0028] b. A helper T cell epitope having an amino acid sequence selected from SEQ ID NO: 39 to SEQ ID NO: 44, preferably the helper T cell epitope consists of an amino acid sequence selected from SEQ ID NO: 13 to SEQ ID NO: 17;

[0029] c. Lipidified CpG oligonucleotide having a nucleotide sequence selected from SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker;

[0030] d. Monophospholipid A (MPLA); and

[0031] The conjugate comprises a second tau phosphopeptide having an amino acid sequence selected from SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38, conjugated to CRM197 via a linker.

[0032] In some embodiments, the conjugate has the following structure:

[0033] ,

[0034] Where n is an integer from 3 to 7.

[0035] According to an embodiment of the present invention, a method for inducing an antibody against at least one of phosphorylated Tau and enriched paired helical filaments (ePHF) in a subject in need comprises:

[0036] (i) administering to a subject a priming composition comprising an immunologically effective amount of liposomes, said liposomes comprising:

[0037] a. A first tau phosphopeptide having an amino acid sequence selected from SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38;

[0038] b. A helper T cell epitope having an amino acid sequence selected from SEQ ID NO: 39 to SEQ ID NO: 44, preferably the helper T cell epitope consists of an amino acid sequence selected from SEQ ID NO: 13 to SEQ ID NO: 17;

[0039] c. Lipidified CpG oligonucleotides having nucleotide sequences selected from SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; and

[0040] d. Monophospholipid A (MPLA);

[0041] The first tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further comprises a pharmaceutically acceptable carrier; and

[0042] (ii) Administering to the subject a first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the following structure:

[0043] ,

[0044] Where n is an integer from 3 to 7, and the first reinforcing composition further comprises a pharmaceutically acceptable carrier.

[0045] According to another embodiment of this application, a method for inducing an antibody against at least one of phosphorylated Tau and enriched paired helical filaments (ePHF) in a subject in need comprises:

[0046] (i) administering to a subject a priming composition comprising an immunologically effective amount of liposomes, said liposomes comprising:

[0047] (1) A first tau phosphopeptide having the amino acid sequence of SEQ ID NO:28;

[0048] (2) Toll-like receptor 4 agonist, which contains monophosphoryl hexa-acyl lipid A, 3-deacylated;

[0049] (3) Helper T cell epitopes containing the amino acid sequence of SEQ ID NO: 39;

[0050] (4) Lipid-modified CpG oligonucleotides comprising the nucleotide sequence of SEQ ID NO:18; and

[0051] (5) At least one lipid selected from 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycerol-3-phospho-3'-rac-glycerol (DMPG), and cholesterol.

[0052] The first tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further comprises a pharmaceutically acceptable carrier; and

[0053] (ii) Administering to the subject a first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the following structure:

[0054] ,

[0055] Where n is an integer from 3 to 7, and the first reinforcing composition further comprises a pharmaceutically acceptable carrier.

[0056] In some embodiments, the method further includes administering the first strengthening composition to the subject at least once after the initial administration of the first strengthening composition.

[0057] In some embodiments, the method further comprises administering a second booster composition to the subject comprising an immunologically effective amount of the liposome and a pharmaceutically acceptable carrier. The second booster composition may be administered before or after the initial administration of the first booster composition.

[0058] In some embodiments, the method further includes administering the second strengthening composition to the subject at least once after the initial administration of the second strengthening composition.

[0059] According to an embodiment of this application, a method for inducing an antibody against at least one of phosphorylated Tau and enriched paired helical filaments (ePHF) in a subject in need comprises:

[0060] (i) administering to a subject a priming composition comprising an immunologically effective amount of liposomes, said liposomes comprising:

[0061] (1) A first tau phosphopeptide having the amino acid sequence of SEQ ID NO:28;

[0062] (2) Toll-like receptor 4 agonist, which contains monophosphoryl hexa-acyl lipid A, 3-deacylated;

[0063] (3) Helper T cell epitopes containing the amino acid sequence of SEQ ID NO: 39;

[0064] (4) Lipid-modified CpG oligonucleotides comprising the nucleotide sequence of SEQ ID NO:18; and

[0065] (5) At least one lipid selected from 1,2-dimyristoyl-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycerol-3-phospho-3'-rac-glycerol (DMPG), and cholesterol.

[0066] The first tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further includes a pharmaceutically acceptable carrier;

[0067] (ii) Administering to the subject a first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the following structure:

[0068] ,

[0069] Where n is an integer from 3 to 7, and the first enhancing composition further comprises a pharmaceutically acceptable carrier; and

[0070] (iii) Administer to the subject a first enhancement composition or a second enhancement composition comprising an immunologically effective amount of the liposome and a pharmaceutically acceptable carrier.

[0071] In some implementations, step (ii) is performed before step (iii).

[0072] In other implementations, step (ii) is performed after step (iii).

[0073] In some implementations, the method includes administering a first booster composition to the subject at least twice to enhance the immune response.

[0074] In some embodiments, the method includes administering a second booster composition to the subject at least once to enhance the immune response.

[0075] In some embodiments, a first reinforcing composition is administered approximately 27-32 days after administration of the initiating composition. Preferably, the method further includes administering the first reinforcing composition approximately 82-87 days after the initial administration of the initiating composition, and optionally further includes administering a second reinforcing composition approximately 167-172 days after administration of the initiating composition.

[0076] In some embodiments, the first reinforcing composition is administered approximately 82-87 days after administration of the initiating composition. Preferably, the method further includes administering a second reinforcing composition approximately 27-32 days after administration of the initiating composition, and optionally further includes administering the first reinforcing composition approximately 167-172 days after administration of the initiating composition.

[0077] In some embodiments, the immunologically effective amount of liposomes contains about 25 nanomoles to about 750 nanomoles of a first tau phosphopeptide per dose, such as about 29.7 nanomoles to about 742.5 nanomoles per dose, preferably about 90 nanomoles to about 715 nanomoles per dose, such as about 89.1 nanomoles to about 712.8 nanomoles per dose, or about 90 nanomoles to about 535 nanomoles per dose, such as about 89.1 nanomoles to about 534.6 nanomoles per dose, or about 90 nanomoles to about 275 nanomoles per dose, such as about 89.1 nanomoles to about 267.3 nanomoles of a first tau phosphopeptide per dose, and the first tau phosphopeptide is present on the surface of the liposomes. In one embodiment, the effective amount of liposomes comprises a first tau phosphopeptide in an amount of about 265 to about 275 nanomoles per dose, such as about 265, about 266, about 267, about 268, about 269, about 270, about 271, about 272, about 273, about 274, or about 275 nanomoles per dose, or any value between the two, such as about 267.3 nanomoles per dose. In another embodiment, the immunologically effective amount of liposomes comprises a first tau phosphopeptide in an amount of about 530 to about 540 nanomoles per dose, such as about 530, about 531, about 532, about 533, about 534, about 535, about 536, about 537, about 538, about 539, or about 540 nanomoles per dose, or any value between the two, such as about 534.6 nanomoles per dose. In another embodiment, the effective amount of liposomes comprises a first tau phosphopeptide in an amount of about 710 to about 720 nanomoles per dose, such as about 710, about 711, about 712, about 713, about 714, about 715, about 716, about 717, about 718, about 719 or about 720 nanomoles per dose, or any value between the two, such as about 712.8 nanomoles per dose.

[0078] In some embodiments, the first tau phosphopeptide consists of an amino acid sequence selected from SEQ ID NO:27 to SEQ ID NO:29 and SEQ ID NO:31 to SEQ ID NO:38, preferably consisting of the amino acid sequence of SEQ ID NO:28. In one embodiment, the immunologically effective amount of liposomes contains a tetrapalmitoylated Tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 28, wherein the tetrapalmitoylated Tau phosphopeptide is present on the surface of the liposomes and is administered in an amount of about 100 µg to about 2500 µg per dose (equivalent to about 29.7 nanomoles to about 742.5 nanomoles per dose), preferably about 300 µg to about 2400 µg per dose (equivalent to about 89.1 nanomoles to about 712.8 nanomoles per dose), such as about 300 µg, about 900 µg, about 1800 µg or about 2400 µg per dose (equivalent to about 89.1 nanomoles, about 267.3 nanomoles, about 534.6 nanomoles or about 712.8 nanomoles per dose).

[0079] In some embodiments, the immunologically effective amount of liposomes comprises a helper T cell epitope in an amount of about 2 nanomoles to about 110 nanomoles per dose, such as about 4.02 nanomoles to about 100.44 nanomoles per dose, or about 4 nanomoles to about 75 nanomoles per dose, such as about 4.02 nanomoles to about 72.32 nanomoles per dose, or about 10 nanomoles to about 105 nanomoles per dose, such as about 12.06 nanomoles to about 100.44 nanomoles per dose, or about 70 nanomoles to about 105 nanomoles per dose, such as about 72.32 nanomoles to about 100.44 nanomoles per dose. In some embodiments, the effective amount of liposomes comprises a helper T cell epitope, preferably composed of the amino acid sequence of SEQ ID NO: 13, in an amount of about 2 nanomoles to about 110 nanomoles, such as about 12.06 nanomoles to about 100.44 nanomoles.

[0080] The present invention also relates to vaccine combinations (e.g., kits) of the liposomes and conjugates of the present invention for inducing an immune response against tau protein in subjects with neurodegenerative disorders, or for treating or preventing neurodegenerative diseases or disorders in subjects in need of them.

[0081] The present invention also relates to the use of the vaccine combination of liposomes and conjugates of the present invention in the manufacture of a medicament for inducing an immune response against tau protein in a subject suffering from a neurodegenerative disorder, or for treating or preventing a neurodegenerative disease or disorder in a subject in need of it.

[0082] Other aspects, features, and advantages of the invention will become more apparent upon reading the following detailed description and claims of the invention. Brief description of the attached diagram

[0084] The foregoing overview and the detailed description of the following preferred embodiments of this application will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that this application is not limited to the exact embodiments shown in the drawings.

[0085] Figure 1A-1C Displays data from the same source AA ( Figure 1A ) or BB ( Figure 1B ) Vaccination or heterologous AB ( Figure 1C Epitope recognition profiles of antibodies induced by vaccination, as determined by ELISA mapping of epitopes of short octamer overlapping peptides, which cover phosphopeptide SEQ ID NO: 2 and peptide SEQ ID NO: 4;

[0086] Figure 2 The epitope recognition profiles of antibodies induced after heterologous ABB vaccination (day 162) are compared with those induced after heterologous ABB vaccination (day 190) (where A is a liposomal vaccine containing a TLR4 ligand as an adjuvant and a lipotropic CpG oligonucleotide, 1800 μg / dose of tetrapalmitoyl acetate-conjugated phosphorylated Tau peptide of SEQ ID NO:2 (corresponding to SEQ ID NO: 28), and an encapsulated T50 helper T cell epitope, and B is a conjugate vaccine containing 15 μg / dose of CRM197 conjugated to phosphorylated Tau peptide of SEQ ID NO: 2 via linker, and the conjugate vaccine is co-injected with aluminum adjuvant (alum) and CpG oligonucleotide), as determined by epitope mapping ELISA of short octamer overlapping peptides, which overlap phosphopeptides SEQ ID NO: 2 and peptide SEQ ID NO: 4;

[0087] Figure 3 The titers of specific IgGs enriched from the post-mortem brains of Alzheimer's patients (ePHF) isolated at day 190 following heterologous vaccination (ABBA, AABB, or BBAA, where A is a liposomal vaccine containing a TLR4 ligand as an adjuvant and a lipid-modified CpG oligonucleotide, 1800 ug / dose of tetrapalmitoyl acetate-modified phosphorylated Tau peptide of SEQ ID NO: 2, and encapsulated T50 helper T cell epitopes, and B is a conjugate vaccine containing 15 ug / dose of CRM197 conjugated to phosphorylated Tau peptide of SEQ ID NO: 2, and the conjugate vaccine is co-injected with aluminum adjuvant and CpG oligonucleotide) or homologous vaccination (AAAA) are shown, and the fold change compared to AAAA is plotted.

[0088] Figure 4A The titer of specific IgG with the sequence SEQ ID NO: 2(T3.5) in rhesus monkeys at day 106 following homologous (AAA) and heterologous (AAB and ABB) immunization regimens, as measured by ELISA, is shown, where the data are expressed as endpoint titer (EPT). Figure 4B The titer of ePHF-specific IgG in rhesus monkeys at day 106 following homologous (AAA) and heterologous (AAB and ABB) immunization regimens is expressed in arbitrary units (AU) / mL.

[0089] Figure 5 shows the heterogeneous solution ABBA ( Figure 5A ) and AABB ( Figure 5B The quality of the ePHF-induced specific IgG antibody compared with that induced by the homologous AAAA protocol, as measured by binding to ePHF under limited coating conditions, was plotted, and the ePHF-specific IgG titer expressed in AU / mL and measured by MSD on limited coating of ePHF was plotted.

[0090] Figure 6 The titer of specific IgG with the N-terminal biotinylated SEQ ID NO: 2 sequence in rhesus monkey cerebrospinal fluid (CSF) was shown on day 183 after the first immunization following heterologous vaccination (ABBA, AABB, or BBAA, where A is a liposomal vaccine containing a TLR4 ligand as an adjuvant and a lipotropic CpG oligonucleotide, 1800 µg / dose of tetrapalmitoyl acetate-conjugated phosphorylated Tau peptide of SEQ ID NO: 2, and encapsulated T50 helper T cell epitopes, while B is a conjugate vaccine containing 15 µg / dose of CRM197 conjugated to phosphorylated Tau peptide of SEQ ID NO: 2 via linker and co-injected with aluminum adjuvant and CpG oligonucleotide); and the fold change compared to homologous vaccination AAAA was plotted.

[0091] Figure 7 The ratio of the titer of phosphopeptide-specific IgG with the sequence SEQ ID NO: 2 (T3.5) to the titer of T50 peptide-specific IgG in rhesus monkeys at day 190 after the first immunization, following homologous (AAAA) and heterologous (AABB and ABBA) immunizations, as measured by ELISA. Statistical analysis was performed using the Mann-Whitney test (**p<0.001). Invention Details

[0093] The background and throughout this specification reference or describe various publications, articles, and patents, each of which is incorporated herein by reference in its entirety. Discussions of documents, laws, materials, devices, articles, etc., included in this specification are for the purpose of providing background to the invention. Such discussion, with respect to any disclosed or claimed invention, is not an admission that any or all of these matters constitute part of the prior art.

[0094] 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 pertains. Otherwise, certain terms used herein have the meanings set forth in the specification.

[0095] It should be noted that the singular forms “a,” “an,” and “the” used herein and in the appended claims include plural references unless the context clearly indicates otherwise.

[0096] Unless otherwise stated, any numerical value, such as concentrations or concentration ranges described herein, should in all cases be understood to be modified by the term "about". Therefore, numerical values ​​generally include ±10% of the stated value. For example, a concentration of 1 mg / mL includes 0.9 mg / mL to 1.1 mg / mL. Similarly, a concentration range of 1% to 10% (w / v) includes 0.9% (w / v) to 11% (w / v). The use of numerical ranges herein explicitly includes all possible subranges, all individual numerical values ​​within that range, including integers and fractions of such values, unless the context clearly indicates otherwise.

[0097] Unless otherwise specified, the term "at least" preceding a series of elements should be understood to refer to each element in the series. Those skilled in the art will recognize or be able to determine numerous equivalents of the particular embodiments of the invention described herein using no more than conventional experimentation. Such equivalents are contemplated to be covered by the invention.

[0098] As used herein, the terms “comprises,” “comprising,” “includes,” “including,” “has,” “having,” “contains,” or “containing,” or any other variation thereof, shall be understood to mean including the stated integers or groups of integers, but not excluding any other integers or groups of integers, and are intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to those elements, but may include other elements not expressly listed or inherent to such composition, mixture, process, method, article, or apparatus. Further, unless the opposite is explicitly stated, “or” means inclusive or rather than exclusive or. For example, condition 1 or 2 is satisfied by any one of the following: 1 is true (or exists) and 2 is false (or does not exist), 1 is false (or does not exist) and 2 is true (or exists), and both 1 and 2 are true (or exist).

[0099] It should also be understood that, when referring to the size or feature of a component of a preferred invention, the terms “about,” “approximately,” “generally,” “substantially,” and similar terms used herein indicate that the described size / feature is not a strict limit or parameter and does not exclude minor variations that are functionally identical or similar, as would be understood by one of ordinary skill in the art. At a minimum, such references including numerical parameters will include variations that do not alter the least significant digit, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0100] According to embodiments of this application, the effective amount of liposomes comprises Tau phosphopeptide in an amount of about 25 nanomoles to about 750 nanomoles per dose, such as about 29.7 nanomoles to about 742.5 nanomoles per dose, preferably about 90 nanomoles to about 715 nanomoles per dose, such as about 89.1 nanomoles to about 712.8 nanomoles per dose, or about 90 nanomoles to about 535 nanomoles per dose, such as about 89.1 nanomoles to about 534.6 nanomoles per dose, or about 90 nanomoles to about 275 nanomoles per dose, such as about 89.1 nanomoles to about 267.3 nanomoles per dose, comprising an amino acid sequence of one of SEQ ID NO: 1-3 or 5-12. Preferably, the Tau phosphopeptide consists of an amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO: 38. More preferably, the Tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 28. In one embodiment, the effective amount of liposome comprises a Toll-like receptor 4 agonist and a tetrapalmitoylated Tau phosphopeptide consisting of the amino acid sequence SEQ ID NO: 28, in an amount of about 100 µg to about 2500 µg (preferably about 300 µg to about 2400 µg) per dose, which is equivalent to about 29.7 nanomoles to about 742.5 nanomoles (preferably about 89.1 nanomoles to about 712.8 nanomoles).

[0101] According to embodiments of this application, the effective amount of liposomes comprises Tau phosphopeptide, for example, Tau phosphopeptide comprising an amino acid sequence of one of SEQ ID NO:1-3 or 5-12, preferably Tau phosphopeptide comprising an amino acid sequence of one of SEQ ID NO:27 to SEQ ID NO:38 (more preferably SEQ ID NO:28), in an amount of about 100 µg to about 2500 µg, about 300 µg to about 2400 µg, about 300 µg to about 1800 µg, or about 300 µg to about 900 µg per dose, such as about 100 µg, about 150 µg, about 200 µg, about 250 µg, about 300 µg, about 400 µg, about 500 µg, about 600 µg, about 700 µg, about 800 µg, about 900 µg, about 1000 µg, about 1100 µg, about 1200 µg, about 1300 µg per dose. µg, about 1400 µg, about 1500 µg, about 1600 µg, about 1700 µg, about 1800 µg, about 1900 µg, about 2000 µg, about 2100 µg, about 2200 µg, about 2300 µg, about 2400 µg, or about 2500 µg, or any value in between.

[0102] In some embodiments, the effective amount of liposomes comprises a Toll-like receptor 4 agonist in an amount of about 30 µg to about 900 µg per dose, preferably about 100 µg to about 585 µg. In some embodiments, the effective amount of liposomes comprises a Toll-like receptor agonist monophosphorylhexyllipid A, 3-deacylated, in an amount of about 30 µg, about 50 µg, about 100 µg, about 150 µg, about 200 µg, about 250 µg, about 300 µg, about 330 µg, about 360 µg, about 390 µg, about 420 µg, about 450 µg, about 480 µg, about 500 µg, about 520 µg, about 540 µg, about 560 µg, about 580 µg, about 600 µg, about 700 µg, about 800 µg, or about 900 µg per dose.

[0103] In some embodiments, the effective amount of liposomes comprises lipid-modified CpG oligonucleotides in an amount of about 50 µg to about 1250 µg per dose, preferably about 150 µg to about 800 µg. For example, the effective amount of liposomes may comprise lipid-modified CpG oligonucleotides in an amount of about 50 µg, about 100 µg, about 150 µg, about 200 µg, about 250 µg, about 300 µg, about 350 µg, about 400 µg, about 450 µg, about 500 µg, about 550 µg, about 600 µg, about 650 µg, about 700 µg, about 750 µg, about 800 µg, about 850 µg, about 900 µg, about 950 µg, about 1000 µg, about 1050 µg, about 1100 µg, about 1200 µg, or about 1250 µg per dose. In some embodiments, the effective amount of liposomes comprises a CpG oligonucleotide consisting of the nucleotide sequence of SEQ ID NO:18, in an amount of about 50 µg to about 1250 µg per dose, preferably about 150 µg to about 800 µg.

[0104] According to the embodiments of this application, Tau phosphopeptide is present on the surface of liposomes.

[0105] The immunologically effective amount of the conjugates used in this article is defined by the amount of the immunogenic carrier conjugated to the second tau phosphopeptide in the conjugate. For example, 15 µg of conjugate refers to a conjugate composition containing 15 µg of an immunogenic carrier conjugated to the tau phosphopeptide. One or more tau phosphopeptides may be conjugated to an immunogenic carrier.

[0106] According to the embodiments of this application, in view of this disclosure, the effective amount of the conjugate can be determined using methods known in the art (such as clinical trials).

[0107] The Tau phosphopeptide comprises an amino acid sequence of one of SEQ ID NO: 1-3 or 5-12. Preferably, the Tau phosphopeptide present on liposomes consists of an amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38. More preferably, the Tau phosphopeptide present on liposomes consists of an amino acid sequence of SEQ ID NO: 28. Preferably, the Tau phosphopeptide present in the conjugate consists of an amino acid sequence of one of SEQ ID NO: 1-3 or 5-12 (preferably SEQ ID NO: 2).

[0108] The term “tau” or “tau protein” as used in this article, also known as microtubule-associated protein tau, MAPT, neurofibrillary tangles protein, paired helical filament-tau, PHF-tau, MAPTL, and MTBT1, refers to a rich central and peripheral nervous system protein with multiple isoforms. In the human central nervous system (CNS), there are six major tau isoforms ranging in length from 352 to 441 amino acids due to alternative splicing (Hanger et al., Trends Mol Med. 15:112-9, 2009). Examples of tau include, but are not limited to, tau isoforms in the CNS, such as the longest tau isoform (4R2N) with four repeats and two insertions at 441 amino acids, and the shortest (fetal) isoform (3R0N) with three repeats and no insertions at 352 amino acids. Examples of tau also include the “large tau” isoform expressed in the peripheral nervous system, which contains 300 additional residues (exon 4a). Friedhoff et al., Biochimica et Biophysica Acta 1502 (2000) 122-132. Examples of tau include human large tau (a 758-amino acid protein encoded by a 6762-nucleotide mRNA transcript (NM_016835.4)) or its isoforms. The amino acid sequence of the illustrative human large tau is represented by GenBank accession number NP_058519.3. The term “tau” as used herein includes homologs of tau from species other than humans, such as the cynomolgous monkey (Macaca Fascicularis) or the chimpanzee (Pantroglodytes). The term “tau” as used herein includes proteins containing mutations (e.g., point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type tau). The term “tau” also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0109] The term “phosphorylated Tau” as used in this article refers to a tau protein, or a fragment thereof, containing at least one phosphorylated residue.

[0110] The term “enriched paired helical filaments” or “ePHF” used in this article refers to preparations in which tau protein is enriched in paired helical filaments. PHF is a prominent component of neurofibrillary tangles in Alzheimer’s disease.

[0111] As used herein, the term "peptide" or "polypeptide" refers to a polymer composed of amino acid residues, associated naturally occurring structural variants, and synthetic, non-natural analogs linked by peptide bonds. The term refers to a peptide of any size, structure, or function. Generally, a peptide is at least 3 amino acids long. Peptides can be naturally occurring, recombinant, or synthetic, or any combination thereof. Synthetic peptides can be synthesized, for example, using an automated peptide synthesizer. Examples of tau peptides include any peptide of tau protein with a length of about 5 to about 30 amino acids, preferably about 10 to about 25 amino acids, more preferably about 16 to about 21 amino acids. In this disclosure, peptides are listed using standard 3- or 1-letter amino acid abbreviations from N to C-terminus, wherein phosphorylated residues are indicated by "p". Examples of tau peptides useful in this invention include, but are not limited to, tau peptides comprising the amino acid sequence of any one of SEQ ID NO: 1-12, or tau peptides having an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 1-12.

[0112] As used herein, the term "phosphopeptide" or "phosphorylated epitope" refers to a peptide that is phosphorylated at one or more amino acid residues. Examples of tau phosphopeptides include any tau peptide comprising one or more phosphorylated amino acid residues. Examples of tau phosphopeptides useful in this invention include, but are not limited to, tau phosphopeptides comprising the amino acid sequence of any one of SEQ ID NO: 1-3 or 5-12, or tau phosphopeptides having an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity with the amino acid sequence of any one of SEQ ID NO: 1-3 or 5-12.

[0113] The tau peptide of this invention can be synthesized via solid-phase peptide synthesis or via a recombinant expression system. Automated peptide synthesizers are commercially available from many vendors, such as Applied Biosystems (Foster City, Calif.). Recombinant expression systems may include bacteria (such as E. coli), yeast, insect cells, or mammalian cells. The procedure for recombinant expression is described by Sambrook et al., Molecular Cloning: A Laboratory Manual (CSHP Press, NY 2nd Edition, 1989).

[0114] Tau is a human "self" protein. This means that, in principle, all lymphocytes carrying tau-specific receptors should have been deleted during development (central tolerance) or become unresponsive due to peripheral tolerance mechanisms. This issue has proven to be a significant obstacle to developing vaccines targeting self or "altered self" proteins (such as tumor antigens).

[0115] The production of high-quality antibodies against antigens (autogenous or infectious) requires not only the action of antibody-producing B lymphocytes but also the action of CD4+ T "helper" lymphocytes. CD4+ T cells provide crucial survival and maturation signals for B lymphocytes, while CD4+ T cell-deficient animals suffer from profound immunosuppression. CD4+ T cells are also affected by tolerance mechanisms, and another obstacle to producing strong anti-autogenous (e.g., anti-tau) antibody responses is that tau-reactive CD4+ T cells are likely rare to non-existent in human / animal libraries.

[0116] While not wishing to be bound by theory, but believed, but in no way limiting the scope of the invention, this problem is circumvented by the vaccine compositions of the invention.

[0117] In one embodiment, liposomes containing tau peptides are generated, which also contain T-cell epitopes capable of binding most or all of the HLA DR (human leukocyte antigen-associated D) molecules. The T-cell epitopes then activate CD4+ T cells and provide necessary maturation and survival signals to tau-specific B cells. In another embodiment, conjugates of tau peptides with carrier proteins are generated, which produce a strong helper T-cell response. In this embodiment, “non-connected recognition” is used, where carrier-specific T cells provide survival and maturation signals to self-reactive B cells. Thus, tau-specific B cells receive key signals to trigger affinity maturation, immunoglobulin class switching, and the establishment of a long-term memory pool. Tau liposomes and tau conjugates can be used to generate high-quality antibodies against tau antigens in homologous or heterologous immunization protocols, where the liposomes or conjugates are used to initiate and / or enhance the response.

[0118] Liposomes

[0119] In this application, liposomes are used in the initiating composition and optionally in the enhancing composition. Liposomes useful to the methods of the present invention comprise:

[0120] Tau peptide, preferably tau phosphopeptide; and

[0121] Helper T cell epitopes

[0122] Tau peptide is present on the surface of liposomes.

[0123] The liposomes of the embodiments of the present invention are also referred to herein as "modified liposomes", "modified liposomal vaccines" or "liposomal vaccines of the embodiments of the present invention" or "Tau liposomes" or "second-generation liposomes" or "optimized liposomal vaccines".

[0124] The term "liposome" as used in this article generally refers to lipid vesicles made of materials with high lipid content, such as phospholipids and cholesterol. The lipids in these vesicles are typically organized in the form of a lipid bilayer. The lipid bilayer usually encapsulates a certain volume, which is dispersed between multiple onion-shaped shells within the lipid bilayer, forming multilayered lipid vesicles (MLVs), or contained within an amorphous central lumen. Lipid vesicles with an amorphous central lumen are monolayered lipid vesicles, i.e., lipid vesicles with a single peripheral bilayer surrounding the lumen. Large monolayered vesicles (LUVs) typically have a diameter of 100 nm to several micrometers, such as 100-200 nm or larger, while small monolayered lipid vesicles (SUVs) typically have a diameter less than 100 nm, such as 20-100 nm, generally 15-30 mm.

[0125] According to a specific implementation, liposomes contain one or more tau peptides. The tau peptides in the liposomes may be the same or different, depending on the specific implementation.

[0126] In view of this disclosure, any suitable tau peptide known to those skilled in the art can be used in this invention. According to a particular embodiment, one or more tau peptides comprise an amino acid sequence of one of SEQ ID NO: 1-12. In other embodiments, one or more tau peptides comprise an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity with an amino acid sequence of one of SEQ ID NO: 1-12, wherein none of the amino acid residues are phosphorylated, or one or more amino acid residues are phosphorylated.

[0127] According to a specific embodiment, one or more tau peptides are tau phosphopeptides. According to a specific embodiment, the one or more tau phosphopeptides comprise an amino acid sequence of one of SEQ ID NO: 1-3 or 5-12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity with an amino acid sequence of one of SEQ ID NO: 1-3 or 5-12, wherein one or more of the indicated amino acid residues are phosphorylated. Preferably, the tau phosphopeptide comprises an amino acid sequence of one of SEQ ID NO: 1-3. The tau peptide may have an amidated C-terminus.

[0128] According to embodiments of this application, tau peptides are present on the surface of liposomes. In view of this disclosure, methods known in the art can be used to make tau peptides (preferably tau phosphopeptides) present on the surface of liposomes. See, for example, the relevant disclosures in U.S. Patent Nos. 8,647,631 and 9,687,447, the contents of which are incorporated herein by reference. According to specific embodiments, the one or more tau peptides (including phosphopeptides) further comprise one or more modifications, such as palmitoylation or dodecyl modification, to allow the tau peptide to be present on the surface of the liposomes. Additional amino acid residues, such as Lys, Cys, or sometimes Ser or Thr, may be added to the tau peptide to promote modification. The position of the lipid anchor has been reported to induce different conformations of the peptide sequence (Hickman et al., J. Biol. Chem. vol. 286, NO. 16, pp. 13966-13976, April 22, 2011). While not wishing to be bound by theory, it is believed that the addition of hydrophobic moieties at both ends can increase the pathological β-sheet conformation of the tau peptide. Therefore, the one or more tau peptides further include hydrophobic portions at both ends. The modified tau peptide may have an amidated C-terminus. Preferably, the tau peptide present on the liposome surface consists of an amino acid sequence of one of SEQ ID NO:27 to SEQ ID NO:38. More preferably, the peptide is a tau phosphopeptide having an amino acid sequence selected from SEQ ID NO:27 to SEQ ID NO:29 or SEQ ID NO:31 to SEQ ID NO:38.

[0129] As used in this article, the term "helper T cell epitope" refers to a polypeptide containing an epitope that can be recognized by helper T cells. Examples of helper T cell epitopes include, but are not limited to, tetanus toxoid (e.g., P2 and P30 epitopes, also referred to as T2 and T30, respectively), hepatitis B surface antigen, cholera toxoid B, toxoid, diphtheria toxoid, measles virus F protein, Chlamydia trachomatis major outer membrane protein, Plasmodium falciparum cyclosporine T, Plasmodium falciparum CS antigen, Schistosoma mansoni triphosphate isomerase, Bordetella pertussis, Clostridium tetani, Pertusaria trachythallina, Escherichia coli TraT, and influenza virus hemagglutinin (HA).

[0130] In view of this disclosure, any suitable helper T cell epitope known to those skilled in the art can be used in this invention. According to a particular embodiment, the helper T cell epitope comprises at least one amino acid sequence selected from SEQ ID NO:23 to SEQ ID NO:26. Preferably, the helper T cell epitope comprises two or more amino acid sequences of SEQ ID NO:23 to SEQ ID NO:26 fused together via a linker (e.g., a peptide linker containing one or more amino acids such as Val(V), Ala(A), Arg(R), Gly(G), Ser(S), Lys(K)). The length of the linker can vary, preferably 1-5 amino acids. Preferably, the helper T cell epitope comprises three or more amino acid sequences of SEQ ID NO:23 to SEQ ID NO:26 fused together via one or more linkers selected from VVR, GS, RR, RK. The helper T cell epitope may have an amidated C-terminus.

[0131] According to embodiments of this application, helper T cell epitopes can be incorporated into the surface of liposomes, for example, by being anchored by a covalently bound hydrophobic portion, wherein the hydrophobic portion is an alkyl group, fatty acid, triglyceride, diglyceride, steroid, sphingolipid, glycolipid, or phospholipid, particularly alkyl or fatty acids, especially those having a carbon skeleton of at least 3 carbon atoms, particularly at least 4 carbon atoms, particularly at least 6 carbon atoms, particularly at least 8 carbon atoms, particularly at least 12 carbon atoms, particularly at least 16 carbon atoms. In one embodiment of the invention, the hydrophobic portion is palmitic acid. Alternatively, the helper T cell epitope can be encapsulated in liposomes. According to a specific embodiment, the helper T cell epitope is encapsulated in liposomes.

[0132] In view of this disclosure, helper T cell epitopes can be modified at desired locations in liposomes using methods known in the art. According to specific embodiments, helper T cell epitopes useful to the present invention comprise an amino acid sequence of one of SEQ ID NO:39 to SEQ ID NO:44. Preferably, the helper T cell epitope consists of an amino acid sequence selected from SEQ ID NO:13 to SEQ ID NO:17.

[0133] According to a specific implementation scheme, the liposomes contain tau peptide and helper T cell epitopes in a weight ratio of 1:1, 2:1, 3:1, 4:1, 5:1 or 6:1.

[0134] In embodiments, the liposomes further comprise at least one adjuvant containing a Toll-like receptor ligand. Therefore, in another general aspect, the present invention relates to liposomes comprising:

[0135] Tau peptides, preferably tau phosphopeptides;

[0136] Helper T cell epitopes; and

[0137] At least one of the following:

[0138] Toll-like receptor 9 ligand, and

[0139] Toll-like receptor 4 ligand.

[0140] As used in this article, the term "toll-like receptor" or "TLR" refers to a class of pattern recognition receptor (PRR) proteins that play a crucial role in the innate immune response. TLRs recognize pathogen-associated molecular patterns (PAMPs) from microbial pathogens (such as bacteria, fungi, parasites, and viruses) that are distinguishable from host molecules. TLRs are transmembrane proteins that generally function as dimers and are expressed by cells involved in the innate immune response, including antigen-presenting dendritic cells and phagocytic macrophages. There are at least 10 human TLR family members, TLR1 through TLR10, and at least 12 murine TLR family members, TLR1 through TLR9 and TLR11 through TLR13, distinguished by the types of antigens they recognize. For example, TLR4 recognizes lipopolysaccharide (LPS), a component found in many Gram-negative bacteria, as well as viral proteins, polysaccharides, and endogenous proteins such as low-density lipoprotein, β-defensins, and heat shock proteins; while TLR9 is a nucleotide-sensing TLR that is activated by unmethylated cytosine-phosphate-guanine (CpG) single- or double-stranded dinucleotides, abundant in prokaryotic genomes but scarce in vertebrate genomes. TLR activation leads to a series of signaling events resulting in the production of type I interferon (IFN), inflammatory cytokines, and chemokines, as well as the induction of an immune response. Ultimately, this inflammation also activates the adaptive immune system, which subsequently leads to the clearance of invading pathogens and infected cells.

[0141] As used herein, the term "ligand" refers to a molecule that forms a complex with a biomolecule (e.g., a receptor) to serve a biological purpose. According to a specific implementation, a toll-like receptor ligand is a toll-like receptor agonist.

[0142] As used herein, the term "agonist" refers to a molecule that binds to one or more TLRs and induces a receptor-mediated response. For example, agonists can induce, stimulate, increase, activate, promote, enhance, or upregulate the activity of a receptor. This activity is referred to as "agonistic activity." For example, TLR4 or TLR9 agonists can activate or increase cell signaling by binding to a receptor. Agonists include, but are not limited to, nucleic acids, small molecules, proteins, carbohydrates, lipids, or any other molecules that bind to or interact with a receptor. Agonists can mimic the activity of natural receptor ligands. Agonists may be identical to these natural receptor ligands in sequence, conformation, charge, or other characteristics, making them recognizable by the receptor. This recognition can lead to physiological and / or biochemical changes within the cell, causing the cell to respond to the presence of the agonist in the same manner as the natural receptor ligand. According to a particular embodiment, a toll-like receptor agonist is at least one of a toll-like receptor 4 agonist and a toll-like receptor 9 agonist.

[0143] As used herein, the term "toll-like receptor 4 agonist" refers to any compound that acts as a TLR4 agonist. In view of this disclosure, any suitable toll-like receptor 4 agonist known to those skilled in the art may be used in this invention. Examples of toll-like receptor 4 ligands useful to this invention include TLR4 agonists, including but not limited to monophospholipid A (MPLA). As used herein, the terms "monophospholipid A" or "MPLA" refer to a modified form of lipid A, which is the biologically active portion of Gram-negative bacterial lipopolysaccharide (LPS) endotoxin. MPLA is less toxic than LPS while maintaining immunostimulatory activity. 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-deacylated-4'-monophospholipid A, monophospholipid hexa-acyl lipid A, 3-deacylated, monophospholipid 3-deacylated lipid A, and their structurally related variants. The MPLA useful in this invention can be obtained using methods known in the art or from commercial sources, such as 3D-(6-acyl)PHAD®, PHAD®, PHAD®-504, 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA), or MPL™ from various commercial sources. According to a particular embodiment, the toll-like receptor 4 agonist is an MPLA.

[0144] As used herein, the term "toll-like receptor 9 agonist" refers to any compound that acts as a TLR9 agonist. In view of this disclosure, any suitable toll-like receptor 9 agonist known to those skilled in the art may be used in this invention. Examples of toll-like receptor 9 ligands useful to this invention include TLR9 agonists, including but not limited to CpG oligonucleotides.

[0145] As used herein, the terms “CpG oligonucleotide,” “CpG oligodeoxynucleotide,” or “CpG ODN” refer to an oligonucleotide containing at least one CpG motif. The terms “oligonucleotide,” “oligodeoxynucleotide,” or “ODN” as used herein refer to a polynucleotide composed of multiple linked nucleotide units. Such oligonucleotides may be obtained from existing nucleic acid sources or may be produced synthetically. The term “CpG motif” as used herein refers to a nucleotide sequence containing an unmethylated cytosine-phosphate-guanine (CpG) dinucleotide (i.e., cytosine (C) followed by guanine (G)) linked by a phosphate ester bond or phosphodiester backbone or other internucleotide bonds.

[0146] According to a specific implementation scheme, CpG oligonucleotides are lipid-modified, that is, conjugated (covalently linked) to the lipid moiety.

[0147] As used herein, "lipid moieties" refers to the portion containing a lipophilic structure. Lipid moieties, such as alkyl groups, fatty acids, triglycerides, diglycerides, steroids, sphingolipids, glycolipids, or phospholipids, particularly sterols (such as cholesterol) or fatty acids, can significantly enhance plasma protein binding and thus increase the circulating half-life of hydrophilic molecules when linked to highly hydrophilic molecules (such as nucleic acids). Furthermore, binding to certain plasma proteins (such as lipoproteins) has been shown to increase uptake in specific tissues expressing the corresponding lipoprotein receptors (e.g., LDL receptor, HDL receptor, or scavenger receptor SR-B1). In particular, lipid moieties conjugated to phosphopeptides and / or CpG oligonucleotides allow said peptides and / or oligonucleotides to be anchored to the membrane of liposomes via a hydrophobic portion.

[0148] According to a specific implementation, and in view of this disclosure, CpG oligonucleotides may contain any suitable internucleotide bonds.

[0149] As used herein, the term "nucleotide internucleotide bond" refers to the chemical bond that links two nucleotides together via the sugar of the nucleotide, consisting of a phosphorus atom and a charged or neutral group between adjacent nucleosides. Examples of nucleotide internucleotide bonds include phosphodiester (po), thiophosphate (ps), dithiophosphate (ps2), methylphosphonate (mp), and methylthiophosphate (rp). Thiophosphates, dithiophosphates, methylphosphonates, and methylthiophosphates are stable nucleotide internucleotide bonds, while phosphodiester is a naturally occurring nucleotide internucleotide bond. Oligonucleotide thiophosphates are generally synthesized as a random racemic mixture of Rp and Sp thiophosphate bonds.

[0150] In view of this disclosure, any suitable CpG oligonucleotide known to those skilled in the art may be used in this invention. Examples of such CpG oligonucleotides include, but are not limited to, CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336.

[0151] In view of this disclosure, CpG oligonucleotides can be lipidated using methods known in the art. In some embodiments, the 3' end of the CpG oligonucleotide is covalently linked to a cholesterol molecule via a phosphate ester bond (optionally via a PEG linker). Other lipophilic moieties may also be covalently linked to the 3' end of the CpG oligonucleotide. For example, the CpG oligonucleotide may be (optionally via a PEG linker) covalently linked to a lipid anchor of the same length as the phospholipid in the liposome: one palmitic acid chain (using Pal-OH or the like, activated for coupling) or two palmitic acid chains (e.g., using 1,2-dispalmitoyl-sn-glycerol-3-phosphate ethanolamine-N-(succinyl) or the like, activated for coupling). See, for example, the relevant disclosure in U.S. Patent No. 7,741,297, the contents of which are incorporated herein by reference. The length of the PEG may vary, for example, from 1 to 5 PEG units.

[0152] Other linkers can also be used to covalently link CpG oligonucleotides to lipophilic moieties (such as cholesterol molecules), examples of which include, but are not limited to, alkyl spacer groups having 3 to 12 carbons. Short linkers such as aminodiols, which are chemically compatible with the oligonucleotide, are required. In some embodiments, no linker is used for covalent bonding. See, for example, Ries et al., “Convenientsynthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes, Org. Biomol. Chem., 2015, 13, 9673,” the relevant disclosure of which is incorporated herein by reference.

[0153] According to a specific embodiment, the lipidated CpG oligonucleotide useful to the present invention comprises a nucleotide sequence selected from SEQ ID NO:18 to SEQ ID NO:22, wherein the nucleotide sequence comprises one or more phosphate thioester nucleotide internucleotide bonds, and the nucleotide sequence is covalently linked to at least one cholesterol molecule via a linker. Any suitable linker can be used to covalently link the CpG oligonucleotide to a cholesterol molecule. Preferably, the linker comprises polyethylene glycol (PEG).

[0154] According to a specific implementation scheme, liposomes comprise:

[0155] tau phosphopeptide;

[0156] Helper T cell epitopes;

[0157] Lipid-modified CpG oligonucleotides; and

[0158] Toll-like receptor 4 ligand;

[0159] Tau phosphopeptide is present on the surface of liposomes, and helper T cell epitopes are encapsulated within liposomes.

[0160] According to a specific implementation scheme, liposomes comprise:

[0161] tau peptide having an amino acid sequence selected from SEQ ID NO:27 to SEQ ID NO:29 or SEQ ID NO:31 to SEQ ID NO:38;

[0162] Helper T cell epitopes having an amino acid sequence selected from SEQ ID NO:39 to SEQ ID NO:44, preferably the helper T cell epitope is composed of an amino acid sequence selected from SEQ ID NO:13 to SEQ ID NO:17;

[0163] Lipidified CpG oligonucleotides having nucleotide sequences selected from SEQ ID NO:18 to SEQ ID NO:22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; and

[0164] Monophospholipid A (MPLA).

[0165] According to a specific implementation, the liposome further comprises one or more lipids selected from the following: 1,2-dimyristic-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristic-sn-glycerol-3-phospho-3'-rac-glycerol (DMPG), and cholesterol.

[0166] According to specific embodiments, the liposomes further comprise a buffer. In view of this disclosure, any suitable buffer known to those skilled in the art may be used in this invention. In one embodiment, the liposomes comprise a phosphate-buffered saline solution. According to specific embodiments, the buffer comprises histidine and sucrose.

[0167] According to a specific implementation scheme, the liposomes contain DMPC, DMPG, cholesterol, tau phosphopeptide, and helper T cell epitopes in a molar ratio of 9:1:7:0.07:0.04.

[0168] In view of this disclosure, the liposomes of the present invention can be prepared using methods known in the art.

[0169] The exemplary liposomes of this application comprise a tau tetrapalmitoyl phosphopeptide (pTau peptide T3, SEQ ID NO:28) present on the surface of the liposome via two palmitic acids at each end of the tau peptide; a TLR-9 ligand containing lipid-modified CpG (adjuvant CpG7909-Chol) covalently linked to the liposome membrane; a TLR-4 ligand (adjuvant 3D-(6-acyl)PHAD®) incorporated into the membrane; and an encapsulated helper T cell epitope (PAN-DR binding agent T50).

[0170] Conjugate

[0171] The conjugates are used in the enhanced compositions of this application. The conjugates used in the methods of this invention comprise conjugates of tau peptides (preferably tau phosphopeptides) and immunogenic carriers conjugated thereto.

[0172] Depending on specific aspects, the conjugate has the following structure:

[0173] ,

[0174] Or the structure of formula (II):

[0175]

[0176] in

[0177] x is an integer from 0 to 10;

[0178] n is an integer from 2 to 15 (preferably 3 to 11);

[0179] The vector represents an immunogenic vector; and

[0180] Tau peptide stands for tau phosphopeptide.

[0181] According to a specific implementation scheme, x is an integer from 1 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3. According to a specific implementation scheme, x is 3.

[0182] Depending on the specific implementation scheme, n can be 2 to 15, 3 to 11, 3 to 9, 3 to 8, or 3 to 7.

[0183] According to a specific embodiment, the conjugate comprises one or more tau peptides. According to a specific embodiment, the tau peptides of the conjugate may be the same or different.

[0184] According to specific embodiments, and in view of this disclosure, any suitable tau peptide may be used in this invention. According to specific embodiments, one or more tau peptides comprise an amino acid sequence of one of SEQ ID NO:1-12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity with an amino acid sequence of one of SEQ ID NO:1-12, wherein none of the amino acid residues are phosphorylated.

[0185] According to a specific embodiment, one or more tau peptides are tau phosphopeptides. According to a specific embodiment, the one or more tau phosphopeptides comprise an amino acid sequence of one of SEQ ID NO: 1-3 or 5-12, or an amino acid sequence having at least 75%, 80%, 85%, 90%, or 95% identity with an amino acid sequence of one of SEQ ID NO: 1-3 or 5-12, wherein one or more of the indicated amino acid residues are phosphorylated.

[0186] According to a specific implementation scheme, the tau phosphopeptide consists of an amino acid sequence of one of SEQ ID NO:1-3.

[0187] As used herein, the term "immunogenic carrier" refers to an immunogenic substance conjugable to tau peptide. The immunogenic portion conjugated to tau peptide can induce an immune response and generate antibodies that specifically bind to tau peptide. The immunogenic portion is the active portion, which includes proteins, polypeptides, glycoproteins, complex polysaccharides, particles, nucleic acids, polynucleotides, etc., that are recognized as foreign substances and thereby elicit an immune response in the host. In view of this disclosure, any suitable immunogenic carrier known to those skilled in the art can be used in this invention. According to a particular embodiment, the immunogenic carrier is keyhole cyanobacterium hemocyanin (KLH), tetanus toxoid, CRM197 (a non-toxic form of diphtheria toxin), a mixture of outer membrane proteins (OMP) of Neisseria meningitidis, or a derivative thereof. According to a particular embodiment, the immunogenic carrier is KLH or CRM197.

[0188] According to specific embodiments, the tau peptide is conjugated to a carrier via a linker. As used herein, the term "linker" refers to the attachment of an immunogenic carrier to the chemical moiety of the tau peptide. In view of this disclosure, any suitable linker known to those skilled in the art may be used in this invention. Linkers may be, for example, monovalent bonds, substituted or unsubstituted alkyl groups, substituted or unsubstituted heteroalkyl moieties, polyethylene glycol (PEG) linkers, peptide linkers, sugar-based linkers, or cleavable linkers (such as disulfide bonds or protease cleavage sites) or amino acids or combinations thereof. Examples of linkers may include polyethylene glycol (PEG), succinimide 3-(bromoacetamido)propionate (SBAP), m-maleimide benzoyl-N-hydroxysuccinimide (MBS), or one or more amino acids (such as Cys, Lys, or sometimes Ser or Thr) or combinations thereof.

[0189] According to a specific implementation, the connector comprises (C2H4O)x-cysteine-acetaminopropionamide or m-maleimide benzoyl-N-hydroxysuccinimide ester-cysteine-(C2H4O)x, where x is an integer from 0 to 10, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0190] According to a specific implementation scheme, the vector is covalently linked to the N-terminus of the tau peptide via a linker.

[0191] According to other specific implementation schemes, the vector is covalently linked to the C-terminus of the tau peptide via a linker.

[0192] According to a specific implementation scheme, the conjugate has the following structure:

[0193] ,

[0194] Where n is an integer from 2 to 15 (preferably 3 to 11, more preferably 3 to 7).

[0195] In view of this disclosure, the conjugates of the present invention can be prepared by methods known in the art. For example, the above conjugates can be prepared by using succinimide 3-(bromoacetamido)propionate (SBAP):

[0196]

[0197] It is formed by reacting with the amino group of CRM197 to form an amide bond. This CRM197 precursor can then be reacted with a tau peptide (e.g., the phosphorylated tau peptide of SEQ ID NO:2) to form a tau phosphopeptide conjugate, wherein the tau peptide is conjugated at its N-terminus or at its C-terminus with a PEG-cysteine ​​linker having a free nucleophilic thiol group.

[0198] An exemplary conjugate according to an embodiment of this application comprises a plurality of tau phosphopeptides (pTau peptide T3.76) covalently linked to the carrier protein CRM197.

[0199] Pharmaceutical Composition

[0200] Liposomes and conjugates are administered to subjects in pharmaceutical compositions. The pharmaceutical composition is a composition containing a therapeutically effective amount of liposomes or a composition containing a therapeutically effective amount of the conjugates of the present invention, each liposome and conjugate being accompanied by a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include excipients and / or carriers known in the art (see Remington's Pharmaceutical Science (15th edition), Mack Publishing Company, Easton, Pa., 1980). Preferred formulations of the pharmaceutical composition depend on the intended administration method and therapeutic application. The composition may contain a pharmaceutically acceptable nontoxic carrier or diluent, defined as a medium commonly used to formulate pharmaceutical compositions for administration to animals or humans. Diluents are selected so as not to affect the bioactivity of the composition. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextran solution, and Hank's solution. Additionally, the pharmaceutical composition or formulation may contain other carriers, adjuvants, or nontoxic, non-therapeutic, non-immunogenic stabilizers, etc. It should be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application, such as intramuscular, subcutaneous, oral, intradermal, transdermal, intramucosal (e.g., intestinal), intranasal, or intraperitoneal routes. Preferably, the pharmaceutically acceptable carrier included in the pharmaceutical composition is suitable for intramuscular administration.

[0201] Pharmaceutical compositions can be formulated into vaccines (also known as "immunogenic compositions"), such as initiating compositions or booster compositions, using methods known in the art.

[0202] The pharmaceutical composition may contain a mixture of tau peptides of the same immunogenicity. Alternatively, the pharmaceutical composition may contain a mixture of tau peptides of different immunogenicities according to the present invention.

[0203] Another issue associated with vaccines targeting neuronal diseases is the potential for exceptionally high antibody titers to ensure efficacy. This is because the vaccine's target antigens are located in the brain. The brain is separated from the circulation by a specialized cellular structure called the blood-brain barrier (BBB). The BBB restricts substances from entering the brain from the circulation. This prevents toxins, microorganisms, and other pathogens from entering the central nervous system. The BBB also has the potentially less desirable effect of preventing immune mediators, such as antibodies, from effectively entering the interstitial tissue and cerebrospinal fluid surrounding the brain.

[0204] Approximately 0.1% of antibodies present in the systemic circulation cross the BBB and enter the brain. This means that the systemic titer induced by a vaccine targeting CNS antigens must be at least 1000 times the minimum effective titer in the brain.

[0205] Therefore, according to specific embodiments, the pharmaceutical compositions of the present invention further comprise one or more suitable adjuvants. Thus, the tau peptide of the present invention, present in liposomes or conjugates, can be administered in combination with a suitable adjuvant to obtain a desired immune response in a subject. The suitable adjuvant can be administered before, after, or simultaneously with the administration of the liposomes or conjugates of the present invention. Preferred adjuvants enhance the intrinsic response to an immunogen without causing conformational changes in the qualitative form of the immunogen that affect the response. Examples of adjuvants are aluminum salts (aluminum adjuvants), such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate. Other examples of adjuvants include CpGs, such as CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336. This adjuvant may be used with or without other specific immunostimulants, such as MPLAs (3-de-O-acylated monophosphoryl lipid A (MPL™), monophosphoryl hexa-acyl lipid A, 3-deacylated compounds (3D-(6-acyl)PHAD®, PHAD™, PHAD®-504, 3D-PHAD®) lipid A), polymeric or monomeric amino acids (such as polyglutamic acid or polylysine). This adjuvant may be used with or without other specific immunostimulants, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycerol-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosamine-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoyloxypropionamide (DTP-DPP) Theramide™) or other bacterial cell wall components. Oil-in-water emulsions include MF59 (see WO 90 / 14837), containing 5% squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally containing various amounts of MTP-PE), formulated into submicron particles using a microfluidizer; SAF, containing 10% squalene, 0.4% Tween 80, 5% Pronic block polymer L121, and thr-MDP, microfluidized into a submicron emulsion or vortexed to produce an emulsion with a larger particle size; and the Ribi™ adjuvant system (RAS) (Ribi ImmunoChem, Hamilton, Mont.) 0.2% Tween 80 and one or more bacterial cell wall components selected from monophosphoryl lipid A (MPL™), trehalose dimethicone ester (TDM), and cell wall skeleton (CWS) (preferably MPL™+CWS (Detox™)).Other adjuvants include complete Freund's adjuvant (CFA) and cytokines such as interleukins (IL-1, IL-2, and IL-12), macrophage colony-stimulating factor (M-CSF), and tumor necrosis factor (TNF).

[0206] As used herein, the term "therapeutic effective dose" refers to the amount of an active ingredient or component that elicits the desired biological or medical response in a subject. For the purposes described herein, the therapeutic effective dose can be determined empirically and in a conventional manner. For example, in vitro assays may optionally be used to help determine the optimal dose range. Those skilled in the art can determine the selection of a specific effective dose based on considerations of several factors (e.g., clinical trials), including the disease to be treated or prevented, the symptoms involved, the patient's weight, the patient's immune status, and other factors known to the technician. The precise dose used in the formulation also depends on the route of administration and the severity of the disease, and should be determined based on the judgment of the practitioner and the condition of each patient. The effective dose can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[0207] In some embodiments, a “therapeutic effective amount” is an “immunologic effective amount,” meaning an amount of composition sufficient to induce a desired immune effect or immune response in a subject in need of it. In one embodiment, an immunogenic effective amount means an amount sufficient to induce an immune response in a subject in need of it. In another embodiment, an immunogenic effective amount means an amount sufficient to produce immunity (e.g., to provide a therapeutic effect against a neurodegenerative disease, disorder, or condition) in a subject in need of it. The immunogenic effective amount can vary depending on various factors such as the subject’s physical condition, age, weight, health, etc. In view of this disclosure, those skilled in the art can readily determine the immunogenic effective amount.

[0208] In one embodiment, the immunogenic composition is a triggering composition for initiating an immune response, which is administered prior to the administration of a strengthening composition. According to an embodiment of the invention, the triggering composition comprises an immunologically effective amount of the liposomes described herein. According to an embodiment of the invention, the immunogenic composition is a strengthening composition for enhancing an immune response, which is administered after the administration of a strengthening composition. According to an embodiment of the invention, the strengthening composition comprises an immunologically effective amount of the conjugates described herein. According to embodiments of this application, an immunogenic composition comprising an immunologically effective amount of the liposomes described herein can be used as both a triggering composition for initiating an immune response and a strengthening composition for enhancing an immune response. The triggering composition can be used in combination with one or more strengthening compositions. The strengthening composition can be administered more than once.

[0209] As used herein, the term "combination" refers to the use of more than one therapy in the context of administering two or more treatments to a subject. The use of the term "combination" does not limit the order in which the treatments are administered to the subject. For example, a first therapy (e.g., the liposome composition described herein) may be administered to the subject before (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks or longer) the second therapy (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 ​​hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks or longer).

[0210] The pharmaceutical compositions of the present invention can be formulated according to methods well known in the art. In view of this disclosure, the optimal ratio of each component in the composition can be determined by techniques well known to those skilled in the art.

[0211] In some embodiments, the pharmaceutical composition comprises the liposomes described herein and a buffer containing one or more amino acids (e.g., histidine or glycine) and / or one or more carbohydrates (e.g., glucose or sucrose).

[0212] In other embodiments, the pharmaceutical composition comprises the conjugates described herein and a buffer comprising one or more amino acids (e.g., histidine or glycine), one or more carbohydrates (e.g., glucose or sucrose) and / or a surfactant (e.g., polysorbate 80, polysorbate 20, etc.).

[0213] How to use

[0214] The present invention provides a method for inducing and enhancing an immune response against tau protein in subjects with neurodegenerative disorders using liposomal vaccines containing tau peptides and conjugate vaccines containing tau peptides conjugated to an immunogenic carrier.

[0215] In a general sense, methods for inducing an immune response against tau protein in subjects with neurodegenerative disorders include:

[0216] A priming composition comprising an immunologically effective amount of liposomes is administered to the subject, said liposomes comprising:

[0217] First tau phosphopeptide;

[0218] Helper T cell epitopes;

[0219] Lipid-modified CpG oligonucleotides; and

[0220] Adjuvants containing Toll-like receptor 4 ligands;

[0221] Tau phosphopeptide is present on the surface of liposomes.

[0222] Furthermore, the initiating composition further comprises a pharmaceutically acceptable carrier; and

[0223] The subject was given a first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the structure of formula (I):

[0224]

[0225] Or it may have the structure of formula (II):

[0226]

[0227] in

[0228] x is an integer from 0 to 10 (preferably 2 to 6, most preferably 3);

[0229] n is an integer from 3 to 15 (preferably 3 to 12);

[0230] The carrier represents an immunogenic carrier selected from keyhole cyanobacterial hemocyanin (KLH), tetanus toxoid, CRM197, and an outer membrane protein mixture (OMP) of Neisseria meningitidis, or derivatives thereof; and

[0231] Tau peptide represents the second tau phosphopeptide, and

[0232] The first enhancing composition further comprises a pharmaceutically acceptable carrier.

[0233] The first tau phosphopeptide and the second tau phosphopeptide each independently have an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 5 to SEQ ID NO: 12.

[0234] In some embodiments, an immunologically effective amount of the conjugate is administered together with one or more adjuvants (such as those described herein). In one embodiment, an immunologically effective amount of the conjugate is administered together with one or more aluminum salts (aluminum adjuvants) (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate) and / or one or more CpGs (e.g., CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336).

[0235] In some embodiments, the first tau phosphopeptide and the second tau phosphopeptide are the same. In other embodiments, the first tau phosphopeptide and the second tau phosphopeptide are different, preferably sharing at least one common epitope.

[0236] In some embodiments, the method further comprises administering a second enhancement composition to the subject containing an immunogenically effective amount of the liposomes.

[0237] Depending on the specific aspect, the immune response is an induced antibody against phosphorylated Tau protein (preferably ePHF).

[0238] In some embodiments, a first booster composition is administered approximately 27-32 days (e.g., approximately 27, 28, 29, 30, 21, or 32 days) after administration of the initiating composition. In some embodiments, the first booster composition is administered again approximately 82-87 days (e.g., approximately 82, 83, 84, 85, 86, or 87 days) after administration of the initiating composition. In some embodiments, a second booster composition comprising an immunogenically effective amount of the liposome is administered approximately 167-172 days (e.g., approximately 167, 168, 169, 170, 171, or 172 days) after administration of the initiating composition.

[0239] In some embodiments, the first reinforcing composition is administered about 82-87 days after administration of the initiating composition (e.g., about 82, 83, 84, 85, 86, or 87 days). In some embodiments, the second reinforcing composition is administered about 27-32 days after administration of the initiating composition (e.g., about 27, 28, 29, 30, 21, or 32 days). In some embodiments, the first reinforcing composition is administered again about 167-172 days after administration of the initiating composition (e.g., about 167, 168, 169, 170, 171, or 172 days).

[0240] Those skilled in the art will be able to modify the exact timing, frequency of administration, dosage, etc., of initiating and enhancing the composition based on the teachings of this article and clinical experience.

[0241] Any of the initiating and enhancing compositions described herein can be used in methods for inducing an immune response against tau protein in subjects with neurodegenerative disorders. Embodiments of initiating compositions, enhancing compositions, liposomes, and / or conjugates, etc., that can be used in the methods of the present invention are discussed in detail in the illustrative examples above and below.

[0242] As used herein, the terms “induction” and “stimulation” and their variations refer to any measurable increase in cell activity. Induction of an immune response may include, for example, the activation, proliferation, or maturation of immune cell populations, increased cytokine production, and / or an increase in another indicator of immune function. In some embodiments, induction of an immune response may include increased B cell proliferation, production of antigen-specific antibodies, increased proliferation of antigen-specific T cells, improved antigen presentation by dendritic cells, and / or increased expression of certain cytokines, chemokines, and co-stimulatory markers.

[0243] The ability to induce or stimulate an anti-tau immune response in animal or human organisms after administration can be assessed in vitro or in vivo using a variety of assays standard in the art. For a general description of techniques that can be used to assess the occurrence and activation of an immune response, see, for example, Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & Sons Inc, National Institute of Health). Measurements of cellular immunity can be performed by methods readily known in the art, such as by measuring the cytokine profile secreted by activated effector cells (including those derived from CD4+ and CD8+ T cells) (e.g., quantifying IL-4 or IFNγ-producing cells via ELISPOT); by determining the activation status of immune effector cells (e.g., T cell proliferation assays via classical [3H]thymidine uptake); and by measuring antigen-specific T lymphocytes in sensitized subjects (e.g., peptide-specific cleavage in cytotoxicity assays).

[0244] The ability to stimulate cellular and / or humoral responses can be determined by testing for the presence of antibodies against the immunogenic tau peptide administered with the pharmaceutical composition in biological samples from the subject (e.g., blood, plasma, serum, PBMCs, urine, saliva, feces, CSF, or lymph) (see, for example, Harlow, 1989, Antibodies, Cold Spring Harbor Press). For instance, the titer of antibodies generated in response to administration of the immunogenic composition can be measured by enzyme-linked immunosorbent assay (ELISA), Meso scale Discovery (MSD), dot blot, SDS-PAGE gel, ELISPOT, or antibody-dependent phagocytosis (ADCP) assay.

[0245] The present invention provides a method for treating or preventing neurodegenerative diseases or disorders in subjects who require them using liposomal vaccines containing tau peptides and conjugate vaccines containing tau peptides conjugated to an immunogenic carrier.

[0246] Those skilled in the art will be able to modify the strengthening composition, the exact timing of initiation and strengthening of the composition, its frequency of administration, its dosage, etc., based on the teachings of this document and clinical experience.

[0247] Any initiating and enhancing compositions described herein can be used in methods for treating or preventing neurodegenerative diseases or disorders in subjects in need of them. Embodiments of initiating compositions, enhancing compositions, liposomes, and / or conjugates, etc., that can be used in the methods of the present invention are discussed in detail in the illustrative examples above and below.

[0248] As used herein, the term "subject" refers to an animal. Depending on the specific implementation, a subject is a mammal, including non-primates (e.g., camels, donkeys, zebras, cattle, pigs, horses, goats, sheep, cats, dogs, rats, rabbits, guinea pigs, or mice) or primates (e.g., monkeys, chimpanzees, or humans). Depending on the specific implementation, a subject is a human.

[0249] As used herein, the terms “treat,” “treating,” and “treatment” are intended to refer to an improvement or reversal of at least one measurable physical parameter associated with a neurodegenerative disease, disorder, or condition, which is not necessarily identifiable in the subject but may be identifiable in the subject. The terms “treat,” “treating,” and “treatment” can also refer to causing the remission of a disease, disorder, or condition, preventing its progression, or at least slowing its progression. In one particular implementation, “treat,” “treating,” and “treatment” refers to the reduction of one or more symptoms associated with a neurodegenerative disease, disorder, or condition, preventing its development or onset, or reducing its duration. In one particular implementation, “treat,” “treating,” and “treatment” refers to the prevention of relapse of a disease, disorder, or condition. In one particular implementation, “treat,” “treating,” and “treatment” refers to an increase in the survival of a subject with a disease, disorder, or condition. In a particular implementation, “treat,” “treating,” and “treatment” refer to the elimination of a subject’s disease, disorder, or ailment.

[0250] According to a specific implementation plan, a therapeutically effective dose refers to a dose sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing or improving the severity of the disease, disorder, or condition to be treated, or related symptoms; (ii) reducing the duration of the disease, disorder, or condition to be treated, or related symptoms; (iii) preventing the progression of the disease, disorder, or condition to be treated, or related symptoms; (iv) causing the resolution of the disease, disorder, or condition to be treated, or related symptoms; (v) preventing the development or onset of the disease, disorder, or condition to be treated, or related symptoms; (v (i) to prevent the recurrence of a disease, disorder, or condition to be treated, or related symptoms; (vii) to reduce hospitalization of subjects with a disease, disorder, or condition to be treated, or related symptoms; (viii) to reduce the length of hospitalization of subjects with a disease, disorder, or condition to be treated, or related symptoms; (ix) to increase the survival of subjects with a disease, disorder, or condition to be treated, or related symptoms; (xi) to suppress or reduce a disease, disorder, or condition to be treated, or related symptoms in subjects; and / or (xii) to enhance or improve the preventive or therapeutic effect of another therapy.

[0251] In view of this disclosure, the term “neurodegenerative disease, disorder, or condition” as used herein includes any neurodegenerative disease, disorder, or condition known to those skilled in the art. Examples of neurodegenerative diseases, disorders, or conditions include neurodegenerative diseases or disorders caused by or associated with neurofibrillary damage, such as tau-related diseases, disorders, or conditions known as tau disease. According to a specific implementation plan, neurodegenerative diseases, disorders, or conditions include any disease or disorder showing the coexistence of tau and amyloid pathology, including but not limited to Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jacob disease, boxer's dementia, Down's syndrome, Gerstmann-Sträussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis (ALS), Guam Parkinsonism-dementia complex, non-Guamanian motor neuron disease with neurofibrillary tangles, argyrophilic granular dementia, corticobasal degeneration, Lewy body dementia, and ALS. Sclerosis), diffuse neurofibrillary tangles with calcification, frontotemporal dementia, preferably frontotemporal dementia associated with chromosome 17 with Parkinson's syndrome (FTDP-17), frontotemporal lobar dementia, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease (type C), Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangles-only dementia, postencephalitic Parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), cerebrovascular angiopathy, or Lewy body dementia (LBD). Depending on the specific implementation plan, the neurodegenerative disease, disorder, or condition is Alzheimer's disease or another type of tau disease.

[0252] The clinical progression of Alzheimer's disease can be divided into several stages, characterized by a progressive pattern of cognitive and functional impairment. These stages can be defined using grading scales known in the art, including, for example, the NIA-AA Research Framework. See, for example, Dubois et al. Alzheimer's & Dementia 12 (2016) 292-323, Dubois et al., Lancet Neurol 2014; 13: 614–29, Jack et al. Alzheimer's & Dementia 14 (2018) 535-562, all of which are incorporated herein by reference in their entirety.

[0253] According to the preferred implementation scheme, the neurodegenerative disease, disorder, or condition is early Alzheimer's disease, mild cognitive impairment (MCI) caused by Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.

[0254] In some implementations, the subjects requiring treatment have positive amyloid protein in their brains but have not yet shown significant cognitive impairment. Amyloid protein deposition in the brain can be detected using methods known in the art, such as PET scans, immunoprecipitation mass spectrometry, or other methods.

[0255] The present invention also provides a method for promoting the clearance of tau aggregates from the brain of a subject, the method comprising administering to the subject a pharmaceutical composition of the embodiments of the present invention, wherein the clearance of tau aggregates is effectively promoted from the subject's brain. According to a particular embodiment, the tau aggregates are neurofibrillary tangles or pathological tau precursors thereof.

[0256] The present invention also provides a method for slowing the progression of tau-pathology-related behavioral phenotypes in subjects, the method comprising administering a pharmaceutical composition of the present invention to a subject, provided that the progression of tau-pathology-related behavioral phenotypes is effectively slowed in the subject.

[0257] In a preferred embodiment of the invention, administration of tau peptide via the pharmaceutical composition of the present invention induces an active immune response in a subject against tau peptide and against pathological forms of tau, thereby promoting the clearance of associated tau aggregates, slowing the progression of tau-pathology-related behaviors, and / or treating underlying tau disease. According to this aspect of the invention, the immune response involves the development of a beneficial humoral (antibody-mediated) response against tau peptide and a cellular (mediated by antigen-specific T cells or their secretory products) response against T cell epitopes or immunogenic carriers.

[0258] The tau-pathology-related behavioral phenotypes used in this article are not limited to include cognitive impairment, early personality changes and disinhibition, apathy, lack of will, mutism, apraxia, continuous movements, stereotyped movements / behaviors, hyperorality, disorganization, inability to plan or organize tasks in sequence, selfishness / apathy, antisocial traits, lack of empathy, halting, grammatical impairment with frequent incoherence but relatively preserved comprehension, impaired comprehension and difficulty in word search, slow progressive gait instability, backward gait, stiffness, frequent falls, non-levodopa responsive axial stiffness, supranuclear gaze palsy, square wave jerks, bradykinesia, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory loss, and tremor.

[0259] In implementing the method of the present invention, it is preferred to select subjects suffering from or at risk of Alzheimer's disease or other tau diseases, subjects with tau aggregates in their brains, or subjects exhibiting tangles-related behavioral phenotypes before administering the immunogenic peptides or antibodies of the present invention. Suitable subjects for treatment include individuals at risk of the disease but asymptomatic, as well as patients currently exhibiting symptoms. In the case of Alzheimer's disease, almost anyone is at risk of developing it. Therefore, this method can be administered prophylactically to the general population without any risk assessment of the subjects. This method is particularly useful for individuals with a known genetic risk of Alzheimer's disease. Such individuals include those with relatives who have experienced the disease, and those whose risk has been determined by analyzing genetic or biochemical markers.

[0260] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, 30 years). However, treatment is typically not initiated until the patient reaches 40, 50, 60, or 70 years of age. The treatment generally requires multiple doses over a period of time. Treatment can be monitored by measuring the response to the therapeutic agent over time, either by measuring antibodies or activated T or B cells. If the response decreases, a booster dose is indicated.

[0261] In prophylactic use, a pharmaceutical composition containing tau peptides is given to patients susceptible to Alzheimer's disease or other tau diseases, or otherwise at risk, in an amount sufficient to eliminate or reduce the risk of the disease, lessen its severity, or delay its onset. These diseases include biochemical, histological, and / or behavioral symptoms, complications, and intermediate pathological phenotypes that occur during disease development. In therapeutic use, a pharmaceutical composition containing tau peptides is given to patients suspected of having or already having the disease in an amount sufficient to cure or at least partially suppress disease symptoms (biochemical, histological, and / or behavioral, including complications and intermediate pathological phenotypes in disease development).

[0262] The effective dosage of the pharmaceutical compositions of the present invention for the prevention and / or treatment of neurodegenerative diseases, disorders, or conditions varies depending on many different factors, including the administration pattern, target site, patient's physiological state, other drugs administered, and whether the treatment is preventative or therapeutic. The amount of the pharmaceutical composition depends on whether an adjuvant is also administered; a higher dosage is required in the absence of an adjuvant. In the method according to the invention, the subject is administered the initiating composition at least once and the enhancing composition at least once. Regardless of the number of enhancing compositions used, the antigens in the respective initiating and enhancing compositions need not be identical, but should share antigenic determinants or be substantially similar to each other.

[0263] Those skilled in the art will readily recognize that the regimen for initiating and boosting administration can be adjusted based on the immune response measured after administration. For example, the boosting composition is typically administered weeks or months after administration of the initiating composition, such as about 2 weeks, or about 3 weeks, or about 4 weeks, or about 8 weeks, or about 12 weeks, or about 16 weeks, or about 20 weeks, or about 24 weeks, or about 26 weeks, or about 28 weeks, or about 30 weeks, or about 32 weeks, or about 36 weeks, or about 1 to 2 years after administration of the initiating composition.

[0264] Pharmaceutical compositions may be administered via parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intradermal, intranasal, or intramuscular routes for prophylactic and / or therapeutic purposes. The most typical route of administration for immunogenic substances is subcutaneous or intramuscular injection. This latter type of injection is most commonly performed in the muscle of the arm or leg.

[0265] If desired, the composition may be present in a kit, package, or dispenser, which may contain one or more unit dosage forms containing the active ingredient. The kit may include, for example, metal or plastic foil, such as blister packaging. Instructions may be provided with the kit, package, or dispenser.

[0266] According to a specific embodiment, the kit contains a pharmaceutical composition comprising liposomes according to an embodiment of the invention and a pharmaceutical composition comprising conjugates according to an embodiment of the invention.

[0267] Implementation Plan

[0268] The present invention also provides the following non-limiting embodiments.

[0269] Implementation scheme 1 is a method for inducing an immune response against tau protein (preferably inducing antibodies against tau phosphopeptide and / or ePHF) in subjects with neurodegenerative disorders, the method comprising:

[0270] A priming composition comprising an immunologically effective amount of liposomes is administered to the subject, said liposomes comprising:

[0271] a. First tau peptide; and

[0272] b. Helper T cell epitopes;

[0273] Tau peptide is present on the surface of liposomes.

[0274] and pharmaceutically acceptable carriers; and

[0275] The subject was given a first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the structure of formula (I):

[0276]

[0277] Or it may have the structure of formula (II):

[0278]

[0279] in

[0280] x is an integer from 0 to 10 (preferably 2 to 6, most preferably 3); and

[0281] n is an integer from 3 to 15 (preferably 3 to 12);

[0282] The carrier represents an immunogenic carrier; preferably, the immunogenic carrier is selected from keyhole cyanobacterial hemocyanin (KLH), tetanus toxoid, CRM197 and an outer membrane protein mixture (OMP) of Neisseria meningitidis, or derivatives thereof; and

[0283] Tau peptide represents the second tau phosphopeptide;

[0284] Furthermore, the first enhancing composition further comprises a pharmaceutically acceptable carrier.

[0285] Implementation scheme 2 is the method of implementation scheme 1, wherein the tau peptide of the liposome is a tau phosphopeptide.

[0286] Implementation scheme 3 is the method of implementation scheme 1 or 2, wherein the liposomes further comprise a toll-like receptor ligand.

[0287] Implementation scheme 4 is the method of implementation scheme 3, wherein the toll-like receptor ligand includes at least one of toll-like receptor 4 ligand and toll-like receptor 9 ligand.

[0288] Implementation scheme 5 is the method of implementation scheme 3 or 4, wherein the toll-like receptor ligand is the toll-like receptor 4 ligand.

[0289] Implementation scheme 6 is the method of implementation scheme 5, wherein the toll-like receptor 4 ligand comprises monophospholipid A (MPLA).

[0290] Implementation scheme 7 is the method of implementation scheme 3 or 4, wherein the toll-like receptor ligand is the toll-like receptor 9 ligand.

[0291] Implementation scheme 8 is the method of implementation scheme 7, wherein the toll-like receptor 9 ligand contains lipid-modified CpG oligonucleotides.

[0292] Implementation Scheme 9 is the method of Implementation Scheme 1, wherein the initiating composition comprises an immunologically effective amount of liposomes, which includes:

[0293] a. tau peptide;

[0294] b. Helper T cell epitopes; and

[0295] c. at least one of the following

[0296] i. Toll-like receptor 9 ligand, and

[0297] ii. Toll-like receptor 4 ligand.

[0298] Implementation scheme 10 is the method of implementation scheme 9, wherein the tau peptide is a tau phosphopeptide.

[0299] Implementation scheme 11 is the method of implementation scheme 9 or 10, wherein the toll-like receptor 9 ligand is a lipid-modified CpG oligonucleotide.

[0300] Implementation scheme 12 is a method of any one of implementation schemes 9 to 11, wherein the liposomes contain a Toll-like receptor 4 ligand and a Toll-like receptor 9 ligand.

[0301] Implementation scheme 13 is the method of implementation scheme 12, wherein the toll-like receptor 4 ligand comprises monophospholipid A (MPLA).

[0302] Implementation Scheme 14 is the method of Implementation Scheme 1, wherein the initiating composition comprises an immunologically effective amount of liposomes, which includes:

[0303] tau phosphopeptide;

[0304] Helper T cell epitopes;

[0305] Lipid-modified CpG oligonucleotides; and

[0306] Adjuvants containing Toll-like receptor 4 ligands;

[0307] Tau phosphopeptide is present on the surface of liposomes.

[0308] Implementation scheme 15 is the method of implementation scheme 14, wherein the toll-like receptor 4 ligand comprises monophospholipid A (MPLA).

[0309] Implementation scheme 16 is a method of any one of implementation schemes 1 to 15, wherein helper T cell epitopes are encapsulated in liposomes.

[0310] Implementation scheme 16a is a method of any one of implementation schemes 1 to 15, wherein helper T cell epitopes are incorporated into the membrane of liposomes.

[0311] Implementation scheme 16b is a method of any one of implementation schemes 1 to 15, wherein helper T cell epitopes are present on the surface of liposomes.

[0312] Embodiment 17 is a method according to Embodiments 1 to 16b, wherein the initiating composition comprises an immunologically effective amount of the liposomal composition, which comprises:

[0313] tau phosphopeptide;

[0314] Helper T cell epitopes;

[0315] Lipid-modified CpG oligonucleotides; and

[0316] Monophospholipid A (MPLA);

[0317] Tau phosphopeptide is present on the surface of liposomes, and

[0318] T cell epitopes are encapsulated in liposomes.

[0319] Implementation scheme 17a is the method of implementation scheme 17, wherein MPLA is 3-O-deacylated-4'-monophosphoryl lipid A, preferably MPL™.

[0320] Implementation scheme 17b is the method of implementation scheme 17, wherein MPLA is monophosphoryl hexa-acyl lipid A, 3-deacylated, preferably 3D-(6-acyl) PHAD®.

[0321] Implementation scheme 17c is the method of implementation scheme 17, wherein MPLA is monophosphoryl 3-deacylated lipid A, preferably 3D-PHAD®.

[0322] Embodiment 18 is a method of any one of Embodiments 1 to 17c, wherein the liposome composition initiating the composition further comprises one or more lipids selected from: 1,2-dimyristic-sn-glycerol-3-phosphocholine (DMPC), 1,2-dimyristic-sn-glycerol-3-phospho-3'-rac-glycerol (DMPG) and cholesterol.

[0323] Implementation scheme 19 is a method of any one of implementation schemes 1 to 18, wherein the tau peptide of the liposome has an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:12 or has at least 85%, 90% or 95% identity with an amino acid sequence selected from SEQ ID NO:1 to SEQ ID NO:12.

[0324] Implementation Scheme 19-1 is the method of Implementation Scheme 19, wherein the tau peptide of the liposome is a phosphopeptide containing an amino acid sequence selected from SEQ ID NO: 1-3 and 5-12.

[0325] Implementation scheme 19-2 is the method of implementation scheme 19-1, wherein the tau phosphopeptide of the liposome contains the amino acid sequence of SEQ ID NO:1.

[0326] Implementation scheme 19-3 is the method of implementation scheme 19-1, wherein the tau phosphopeptide of the liposome contains the amino acid sequence of SEQ ID NO:2.

[0327] Implementation scheme 19-4 is the method of implementation scheme 19-1, wherein the tau phosphopeptide of the liposome contains the amino acid sequence of SEQ ID NO:3.

[0328] Implementation scheme 19a is a method of any one of implementation schemes 19, 19-1, 19-2, 19-3 and 19-4, wherein the amino acid sequence further comprises one or more modifications to allow the tau peptide to be present on the surface of the liposome.

[0329] Embodiment 19b is the method of Embodiment 19a, wherein the one or more modifications comprise at least one of palmitoylation and dodecyl modification.

[0330] Implementation scheme 19c is a method of implementation scheme 19a or 19b, wherein the tau peptide of the liposome is modified at its N-terminus by one or more of the modifications described above.

[0331] Implementation scheme 19d is a method of any one of implementation schemes 19a to 19c, wherein the tau peptide of the liposome is modified at its C-terminus by one or more of the modifications described above.

[0332] Implementation scheme 19e is the method of implementation scheme 19d, wherein the tau peptide of the liposome is palmitoylated at both its N-terminus and C-terminus.

[0333] Implementation scheme 19f is a method of any of implementation schemes 19a to 19e, wherein the tau peptide of the liposome further comprises one or more additional amino acids to promote the one or more modifications.

[0334] Implementation scheme 19g is the method of implementation scheme 19f, wherein one or more additional amino acids are selected from Lys, Cys, Ser and Thr.

[0335] Implementation scheme 19h is a method of any one of implementation schemes 19 to 19g, wherein the tau peptide of the liposome is amidated at its C-terminus.

[0336] Implementation scheme 19i is the method of any one of implementation schemes 19 to 19h, wherein the tau peptide of the liposome consists of an amino acid sequence selected from SEQ ID NO:27 to SEQ ID NO:38.

[0337] Implementation scheme 19j is the method of any one of implementation schemes 19 to 19i, wherein the tau peptide of the liposome consists of the amino acid sequence of SEQ ID NO:27.

[0338] Implementation scheme 19k is a method of any one of implementation schemes 19 to 19i, wherein the tau peptide of the liposome consists of the amino acid sequence of SEQ ID NO:28.

[0339] Implementation scheme 19l is the method of any one of implementation schemes 19 to 19i, wherein the tau peptide of the liposome consists of the amino acid sequence of SEQ ID NO:29.

[0340] Implementation scheme 20 is a method of any one of implementation schemes 1 to 19, wherein the helper T cell epitope of the liposome contains at least one amino acid sequence selected from the following: SEQ ID NO:23 to SEQ ID NO:26.

[0341] Implementation scheme 20a is the method of implementation scheme 20, wherein the helper T cell epitope comprises at least two amino acid sequences selected from the following: SEQ ID NO:23 to SEQ ID NO:26.

[0342] Implementation scheme 20b is the method of implementation scheme 20, wherein the helper T cell epitope comprises at least three amino acid sequences selected from the following: SEQ ID NO:23 to SEQ ID NO:26.

[0343] Implementation scheme 20c is the method of implementation scheme 20, wherein the helper T cell epitope comprises four or fewer amino acid sequences: SEQ ID NO:23 to SEQ ID NO:26.

[0344] Implementation scheme 20d is the method of any one of implementation schemes 20a to 20c, wherein two or more amino acid sequences selected from SEQ ID NO:23 to SEQ ID NO:26 are covalently linked by a linker.

[0345] Implementation scheme 20e is the method of implementation scheme 20d, wherein the linker comprises one or more amino acids selected from Val (V), Ala (A), Arg (R), Gly (G), Ser (S), Lys (K).

[0346] Implementation scheme 20f is the method of implementation scheme 20e, wherein the linker comprises an amino acid sequence selected from VVR, GS, RR and RK.

[0347] Implementation scheme 20g is a method of any one of implementation schemes 20 to 20f, wherein the helper T cell epitope is amidated at its C-terminus.

[0348] Implementation scheme 20h is a method of any one of implementation schemes 20 to 20g, wherein the helper T cell epitope is modified to insert into the membrane of the liposome, be present on the surface of the liposome, or be encapsulated in the liposome (depending on the intended location of the helper T cell epitope).

[0349] Implementation scheme 20i is a method of any one of implementation schemes 20 to 20h, wherein the helper T cell epitope consists of an amino acid sequence selected from SEQ ID NO:13 to SEQ ID NO:17.

[0350] Implementation scheme 20j is a method of any one of implementation schemes 1 to 20i, wherein the liposomes contain tau peptide and helper T cell epitopes in a weight ratio of 6:1.

[0351] Implementation scheme 20k is a method of any one of implementation schemes 1 to 20i, wherein the liposomes contain tau peptide and helper T cell epitopes in a weight ratio of 5:1.

[0352] Implementation scheme 201 is a method of any one of implementation schemes 1 to 20i, wherein the liposomes contain tau peptide and helper T cell epitopes in a weight ratio of 4:1.

[0353] Implementation scheme 20m is a method of any one of implementation schemes 1 to 20i, wherein the liposomes contain tau peptide and helper T cell epitopes in a weight ratio of 3:1.

[0354] Implementation scheme 20n is a method of any one of implementation schemes 1 to 20i, wherein the liposomes contain tau peptide and helper T cell epitopes in a weight ratio of 2:1.

[0355] Implementation scheme 20o is a method of any one of implementation schemes 1 to 20i, wherein the liposomes contain tau peptide and helper T cell epitopes in a weight ratio of 1:1.

[0356] Implementation scheme 21 is a method of any one of implementation schemes 1 to 20, wherein the lipid-modified CpG oligonucleotide comprises a nucleotide sequence selected from SEQ ID NO:18 to SEQ ID NO:22.

[0357] Implementation scheme 21a is the method of implementation scheme 21, wherein the CpG oligonucleotide has one or more phosphate thioester nucleotide inter-bonds.

[0358] Implementation scheme 21b is the method of implementation scheme 21a, wherein the CpG oligonucleotide has nucleotide internucleotide bonds that are all thiophosphates.

[0359] Implementation scheme 21c is a method of any one of implementation schemes 21 to 21b, wherein the lipotropic CpG oligonucleotide comprises a CpG oligonucleotide covalently linked to at least one lipophilic group via a linker.

[0360] Implementation scheme 21d is the method of implementation scheme 21c, wherein the connector comprises (C2H4O)n, where n is an integer from 0 to 10.

[0361] Implementation scheme 21e is the method of implementation scheme 21c, wherein the connector comprises an alkyl spacer group having 3 to 12 carbons.

[0362] Implementation scheme 21f is a method of any one of implementation schemes 21 to 21e, wherein at least one lipophilic group is cholesterol.

[0363] Implementation scheme 21g is a method of any one of implementation schemes 21 to 21f, wherein the lipid-modified CpG oligonucleotide comprises a nucleotide sequence of SEQ ID NO:18 or SEQ ID NO:19 covalently linked to a cholesterol molecule via a linker comprising (C2H4O)n, wherein n is an integer from 3 to 5.

[0364] Implementation scheme 22 is a method for inducing an immune response against tau protein in subjects with neurodegenerative disorders, the method comprising:

[0365] A priming composition comprising an immunologically effective amount of liposomes is administered to the subject, said liposomes comprising:

[0366] The first tau phosphopeptide has an amino acid sequence selected from SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38;

[0367] Helper T cell epitopes having an amino acid sequence selected from SEQ ID NO:39 to SEQ ID NO:44, preferably the helper T cell epitope is composed of an amino acid sequence selected from SEQ ID NO:13 to SEQ ID NO:17;

[0368] Lipidified CpG oligonucleotides having nucleotide sequences selected from SEQ ID NO:18 to SEQ ID NO:22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; and

[0369] Monophospholipid A (MPLA),

[0370] and pharmaceutically acceptable carriers; and

[0371] The subject was given a first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the structure of formula (I):

[0372]

[0373] Or it may have the structure of formula (II):

[0374]

[0375] in

[0376] x is an integer from 0 to 10 (preferably 2 to 6, most preferably 3); and

[0377] n is an integer from 3 to 15 (preferably 3 to 12);

[0378] The vector represents an immunogenic vector;

[0379] Tau peptide represents the second tau phosphopeptide;

[0380] And pharmaceutically acceptable carriers.

[0381] Implementation scheme 22a is the method of implementation scheme 22, wherein the liposome comprises:

[0382] a. A first tau phosphopeptide, which consists of the amino acid sequence of SEQ ID NO:27, SEQ ID NO:28 or SEQ ID NO:29;

[0383] b. Helper T cell epitopes, consisting of the amino acid sequence of SEQ ID NO:13.

[0384] c. Lipid-modified CpG oligonucleotides, comprising a nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19 covalently linked to cholesterol via a linker containing (C2H4O)n, wherein n is an integer from 3 to 7; and

[0385] d. Monophospholipid A (MPLA).

[0386] Implementation scheme 22b is the method of implementation scheme 22 or 22a, wherein MPLA is 3-O-deacylated-4′-monophosphoryl lipid A, preferably MPL™.

[0387] Implementation scheme 22c is the method of implementation scheme 22 or 22a, wherein MPLA is monophosphoryl hexa-acyl lipid A, 3-deacylated, preferably 3D-(6-acyl) PHAD®.

[0388] Implementation scheme 22d is the method of implementation scheme 22 or 22a, wherein MPLA is monophosphoryl 3-deacylated lipid A, preferably 3D-PHAD®.

[0389] Implementation scheme 23 is a method of any one of implementation schemes 22 to 22d, wherein helper T cell epitopes are encapsulated in liposomes.

[0390] Implementation scheme 24 is a method of any one of implementation schemes 1 to 23, where x is an integer from 2 to 6.

[0391] Implementation scheme 25 is any one of the methods in implementation schemes 1 to 24, where x is 3.

[0392] Implementation scheme 26 is any one of the methods in implementation schemes 1 to 25, where n is 3 to 7.

[0393] Implementation scheme 27 is a method of any one of implementation schemes 1 to 26, wherein the carrier is an immunogenic carrier selected from keyhole cyanobacterial cyanobacterial (KLH), tetanus toxoid, CRM197 and a mixture of outer membrane proteins of Neisseria meningitidis (OMP) or derivatives thereof.

[0394] Implementation scheme 28 is the method of any one of implementation schemes 1 to 27, wherein the second tau phosphopeptide of the conjugate consists of an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 5 to SEQ ID NO: 12.

[0395] Implementation scheme 28a is the method of implementation scheme 28, wherein the first tau phosphopeptide of the liposome and the second tau phosphopeptide of the conjugate are the same.

[0396] Implementation scheme 28b is the method of implementation scheme 28, wherein the first tau phosphopeptide of the liposome and the second tau phosphopeptide of the conjugate are different.

[0397] Implementation scheme 28c is the method of implementation scheme 28b, wherein the liposome's first tau phosphopeptide and the conjugate's second tau phosphopeptide share at least one common epitope.

[0398] Implementation scheme 29 is the method of any one of implementation schemes 28-28c, wherein the second tau phosphopeptide consists of the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0399] Implementation scheme 30 is any one of the methods in implementation schemes 1 to 29, wherein the carrier is CRM197.

[0400] Implementation scheme 31 is a method of any one of implementation schemes 1-30, wherein the conjugate has the following structure:

[0401] ,

[0402] Where n is between 3 and 7.

[0403] Implementation scheme 32 is a conjugate of any one of implementation schemes 1-29, wherein the conjugate has the following structure:

[0404]

[0405] in

[0406] Tau peptide is composed of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3;

[0407] x is an integer from 0 to 10;

[0408] n is an integer from 2 to 15;

[0409] Tau peptide represents tau phosphopeptide; and

[0410] KLH stands for keyhole hemocyanin.

[0411] Implementation scheme 33 is a method of any one of implementation schemes 1 to 32, further comprising administering a subject a second enhancement composition comprising an immunogenically effective amount of the liposome.

[0412] Embodiment 33a is the method of Embodiment 33, wherein the initiating composition, the first reinforcing composition and / or the second reinforcing composition further comprise an adjuvant.

[0413] Implementation scheme 33b is the method of implementation scheme 33a, wherein the adjuvant comprises at least one of TLR-4 ligand and TLR-9 ligand.

[0414] Embodiment 34 is the method of any one of Embodiments 1 to 33b, wherein the first reinforcing composition is given about 27-32 days (preferably about 29 days) after the initial administration of the initiating composition.

[0415] Embodiment 35 is the method of Embodiment 34, further comprising administering a first reinforcing composition after the initial administration of the initiating composition for about 82-87 days (preferably about 85 days).

[0416] Embodiment 36 is the method of Embodiment 35, further comprising administering a second reinforcing composition after the initial administration of the initiating composition for about 167-172 days (preferably about 169 days).

[0417] Embodiment 37 is the method of any one of Embodiments 1 to 33b, wherein the first reinforcing composition is given about 82-87 days (preferably about 85 days) after the initial administration of the initiating composition.

[0418] Embodiment 38 is the method of Embodiment 37, further comprising administering a second reinforcing composition about 27-32 days (preferably about 29 days) after the initial administration of the initiating composition.

[0419] Embodiment 39 is the method of Embodiment 35, further comprising administering a first reinforcing composition after the initial administration of the initiating composition for about 167-172 days (preferably about 169 days).

[0420] Implementation scheme 40 is a method for treating or preventing neurodegenerative diseases or disorders in subjects who require it, comprising:

[0421] A priming composition comprising an immunologically effective amount of liposomes is administered to the subject, said liposomes comprising:

[0422] The first tau phosphopeptide has an amino acid sequence selected from SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38;

[0423] Helper T cell epitopes having an amino acid sequence selected from SEQ ID NO: 39 to SEQ ID NO: 44, preferably the helper T cell epitope consists of an amino acid sequence selected from SEQ ID NO: 13 to SEQ ID NO: 17;

[0424] Lipidified CpG oligonucleotides having nucleotide sequences selected from SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; and

[0425] Monophospholipid A (MPLA);

[0426] Tau phosphopeptide is present on the surface of liposomes.

[0427] and pharmaceutically acceptable carriers; and

[0428] The subject was given a first enhancement composition comprising an immunologically effective amount of a conjugate, said conjugate comprising a tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the following structure:

[0429] ,

[0430] Where n is an integer from 3 to 7,

[0431] And pharmaceutically acceptable carriers.

[0432] Implementation scheme 41 is the method of implementation scheme 40, wherein the first reinforcing composition is given about 27-32 days (preferably about 29 days) after the initial administration of the initiating composition.

[0433] Implementation scheme 42 is the method of implementation scheme 41, further comprising administering a first reinforcing composition after the initial administration of the initiating composition for about 82-87 days (preferably about 85 days).

[0434] Embodiment 43 is the method of Embodiment 42, further comprising administering a second enhancement composition containing an immunogenically effective amount of the liposomes after the initial administration of the initiating composition for about 167-172 days (preferably about 169 days).

[0435] Implementation scheme 44 is the method of implementation scheme 40, wherein the first reinforcing composition is given about 82-87 days (preferably about 85 days) after the initial administration of the initiating composition.

[0436] Embodiment 45 is the method of Embodiment 44, further comprising administering a second enhancement composition containing an immunogenically effective amount of the liposomes about 27-32 days (preferably about 29 days) after the initial administration of the initiating composition.

[0437] Embodiment 46 is the method of Embodiment 45, further comprising administering a first reinforcing composition after the initial administration of the initiating composition for about 167-172 days (preferably about 169 days).

[0438] Implementation scheme 46a is a method of any one of implementation schemes 1 to 46, wherein an immunologically effective amount of the conjugate is administered together with one or more adjuvants.

[0439] Implementation scheme 46b is the method of implementation scheme 46a, wherein the adjuvant comprises an aluminum salt, such as aluminum hydroxide, aluminum phosphate and aluminum sulfate.

[0440] Implementation scheme 46c is a method of implementation scheme 46a or 46b, wherein the adjuvant comprises CpG, such as CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826 or CpG2336.

[0441] Implementation scheme 46d is the method of implementation scheme 46c, wherein an immunologically effective amount of the conjugate is given together with an aluminum salt (e.g., aluminum hydroxide, aluminum phosphate, and aluminum sulfate) and CpG (e.g., CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336).

[0442] Implementation scheme 46e is the method of implementation scheme 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum phosphate and CpG1018.

[0443] Implementation scheme 46f is the method of implementation scheme 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum hydroxide and CpG7909.

[0444] Implementation scheme 46g is the method of implementation scheme 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum sulfate and CpG2395.

[0445] Implementation scheme 46h is the method of implementation scheme 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum hydroxide and CpG1826.

[0446] Implementation scheme 47 is a method of any one of implementation schemes 1 to 46h, wherein the neurodegenerative disease or disorder is caused by or related to the formation of neurofibrillary damage.

[0447] Implementation scheme 48 is a method of any one of implementation schemes 1 to 47, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, boxer's dementia, Down syndrome, Göstadmann disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis (ALS), Guam Parkinson's-dementia syndrome, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic granular dementia, corticobasal degeneration, Lewy body dementia, amyotrophic lateral sclerosis, and so on. Diffuse neurofibrillary tangles with calcification, frontotemporal dementia (preferably frontotemporal dementia associated with chromosome 17 with Parkinson's syndrome (FTDP-17)), frontotemporal dementia, Hastings-Schwarzman disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangles-only dementia, post-encephalitis Parkinson's syndrome, myotonic dystrophy, chronic traumatic encephalopathy (CTE), cerebrovascular disease, or Lewy body dementia (LBD).

[0448] Implementation scheme 49 is any one of the methods in implementation schemes 1 to 48, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down syndrome, progressive supranuclear palsy (PSP), frontotemporal dementia with Parkinson's syndrome associated with chromosome 17 (FTDP-17), Pick's disease and PART (primary age-related tau disease), corticobasal degeneration, Lewy body dementia, amyotrophic lateral sclerosis, myotonic dystrophy, chronic traumatic encephalopathy (CTE), cerebrovascular disease, or Lewy body dementia (LBD).

[0449] Implementation scheme 50 is the method of any one of implementation schemes 1 to 49, wherein the neurodegenerative disease or disorder is Alzheimer's disease, progressive supranuclear palsy (PSP), frontotemporal dementia associated with chromosome 17 with Parkinson's syndrome (FTDP-17), or Pick's disease and PART (primary age-related tau disease).

[0450] Implementation scheme 51 is any one of the methods in implementation schemes 1 to 50, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down syndrome, frontotemporal dementia with Parkinson's syndrome associated with chromosome 17 (FTDP-17), corticobasal degeneration, Lewy body dementia, amyotrophic lateral sclerosis, myotonic dystrophy, chronic traumatic encephalopathy (CTE), cerebrovascular disease, or Lewy body dementia (LBD).

[0451] Implementation scheme 51a is any one of the methods in implementation schemes 1 to 51, wherein the neurodegenerative disease or disorder is early Alzheimer's disease, mild cognitive impairment (MCI) caused by Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.

[0452] Embodiment 52 is a combination (such as a kit) that includes the initiating composition and the first reinforcing composition used in any of embodiments 1-51a.

[0453] Implementation scheme 53 is the method of any one of implementation schemes 1 to 51a, or a combination of implementation scheme 52, wherein the immunologically effective amount of liposomes contains about 25 nanomoles to about 750 nanomoles per dose, preferably about 90 nanomoles to about 715 nanomoles per dose, or about 90 nanomoles to about 535 nanomoles per dose, for example, about 25 nanomoles, about 30 nanomoles, about 35 nanomoles, about 40 nanomoles, about 45 nanomoles, about 50 nanomoles, about 55 nanomoles, about 60 nanomoles, about 65 nanomoles, about 70 nanomoles, about 75 nanomoles, about 80 nanomoles, about 85 nanomoles, about 90 nanomoles, about 95 nanomoles, about 100 nanomoles, about 125 nanomoles per dose. The first tau phosphopeptide in nanomoles, approximately 150 nanomoles, approximately 175 nanomoles, approximately 200 nanomoles, approximately 225 nanomoles, approximately 250 nanomoles, approximately 275 nanomoles, approximately 300 nanomoles, approximately 325 nanomoles, approximately 350 nanomoles, approximately 375 nanomoles, approximately 400 nanomoles, approximately 425 nanomoles, approximately 450 nanomoles, approximately 475 nanomoles, approximately 500 nanomoles, approximately 525 nanomoles, approximately 550 nanomoles, approximately 575 nanomoles, approximately 600 nanomoles, approximately 625 nanomoles, approximately 650 nanomoles, approximately 675 nanomoles, approximately 700 nanomoles, approximately 725 nanomoles, approximately 750 nanomoles, or any value between the two.

[0454] Embodiment 53a is the method of any one of embodiments 1 to 51a, or a combination of embodiments 52, wherein the immunologically effective amount of liposomes contains about 25 nanomoles to about 750 nanomoles per dose, for example about 29.7 nanomoles to about 742.5 nanomoles per dose, preferably about 90 nanomoles to about 715 nanomoles per dose, for example about 89.1 nanomoles to about 712.8 nanomoles per dose, or about 90 nanomoles to about 535 nanomoles per dose, for example about 89.1 nanomoles to about 534.6 nanomoles per dose, or about 90 nanomoles to about 268 nanomoles per dose, for example about 89.1 nanomoles to about 267.3 nanomoles per dose, of the first tau phosphopeptide.

[0455] Implementation scheme 53b is a method or combination of implementation scheme 53 or 53a, wherein the immunologically effective amount of liposomes contains about 100 µg to about 2500 µg per dose, preferably about 300 µg to about 2400 µg, for example about 100 µg, about 150 µg, about 200 µg, about 250 µg, about 300 µg, about 400 µg, about 500 µg, about 600 µg, about 700 µg, about 800 µg, about 900 µg, about 1000 µg, about 1100 µg, about 1200 µg, about 1300 µg, about 1400 µg, about 1500 µg, about 1600 µg, about 1700 µg, about 1800 µg, about 1900 µg, about 2000 µg, about 2100 µg, about 2200 µg, about 2300 µg, etc. The first tau phosphopeptide, in µg, about 2400 µg, about 2500 µg, or any value in between.

[0456] Implementation scheme 53c is a method or combination of any of implementation schemes 53 to 53b, wherein the first tau phosphopeptide consists of an amino acid sequence of one of SEQ ID NO:27 to SEQ ID NO:38.

[0457] Implementation scheme 53d is a method or combination of implementation scheme 53c, wherein the first tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 28.

[0458] Implementation scheme 54 is any one of the methods or combinations of implementation schemes 53 to 53d, wherein the second tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 1-3 or 5-12.

[0459] Implementation scheme 54a is a method or combination of implementation scheme 54, wherein the second tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 1, 2 or 3. Example

[0460] Example 1

[0461] Preparation of liposome vaccines

[0462] Preparation of a control liposome vaccine (ethanol injection technique)

[0463] The control liposome vaccine was produced via ethanol (EtOH) injection followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, Netherlands), and MPLA (Avanti Polar Lipids, AL, USA) were dissolved at 60°C in a molar ratio of 9:1:7:0.05 in a 20:1 (v / v) mixture of EtOH and tert-butanol (t-BuOH). The lipid / ethanol solution was diluted at 60°C in phosphate-buffered saline (PBS) pH 7.4 to maintain a 10% EtOH concentration, resulting in the formation of multilayer liposome vesicles (MLVs). The MLVs were then subjected to five sequential extrusions using an Emulsiflex-C5 (Avestin, Canada) filter through three consecutive polycarbonate filters (Whatman) with 0.08 μm pore sizes. The resulting liposomes were diluted in PBS pH 7.4 and heated to 60°C to obtain a liposome solution, followed by the addition of tau peptide.

[0464] The tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO:2 (Bachem AG, Switzerland) (corresponding to SEQ ID NO:28, referred to herein as the active pharmaceutical ingredient (API)) was dissolved at a concentration of 1 mg / mL in PBS pH 11.4 containing 2.0% octyl-β-D-glucopyranoside (Sigma-Aldrich, USA). The peptide solution was injected into the liposome solution at 60 °C and stirred at 60 °C for 30 min. The solution was concentrated to the target final volume by ultrafiltration, with 10 buffer exchanges with PBS pH 7.4 during perfiltration. The resulting liposomes, with the API present on the liposome surface, were then aseptically filtered through two consecutive 0.2 μm polycarbonate syringe filters, and the final product was stored at 5 °C.

[0465] Preparation of liposome Z and Z+ vaccines

[0466] The liposomal Z+ vaccine with a final API concentration of 1200 µg / ml and a final T50 concentration of 1200 µg / ml was produced by ethanol injection followed by extrusion, while the liposomal Z vaccine with a final API concentration of 400 µg / ml and a final T50 concentration of 100 µg / ml was produced by thin lipid membrane technology followed by homogenization and extrusion.

[0467] Liposome Z vaccine prepared using thin-film lipid membrane technology

[0468] The liposome Z vaccine was produced using a thin lipid membrane technology followed by homogenization and extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, Netherlands), and a monophosphoryl hexa-acyl lipid A 3-deacylated compound (3D-(6-acyl)PHAD®) (Avanti Polar Lipids, AL, USA) were dissolved in EtOH at a molar ratio of 9:1:7:0.05 at 60°C. Ethanol was then evaporated using a vacuum rotary evaporator to obtain the thin lipid membrane.

[0469] The lipid membrane was rehydrated with PBS pH 7.4 containing 0.15 mg / mL T50 peptide (Peptides & Elephants, Germany) and 5% DMSO (both from Sigma-Aldrich). The sample was gently stirred for 15 minutes and then further vortexed vigorously to dissolve the thin lipid membrane. The resulting multilayer vesicles were subjected to 10 freeze-thaw cycles (liquid N2 and a 37°C water bath), homogenized, and then sequentially extruded through a polycarbonate membrane (Whatman, UK) with a pore size of 0.08 μm. Both homogenization and extrusion steps were performed in an EmulsiFlex-C5 (Avestin, Canada). The extruded liposomes with encapsulated T50 peptide were concentrated by ultrafiltration and the buffer was exchanged with PBS pH 7.4 by perfiltration. The resulting liposomes were diluted in PBS pH 7.4 and heated to 60°C to obtain a liposome solution, followed by the addition of tau peptide and adjuvant.

[0470] CpG2006-cholesterol (CpG2006-Chol) (Microsynth, Switzerland) is a DNA oligonucleotide in which all internucleotide bonds are phosphate thioesters. Its 5' end is modified with a cholesterol molecule via a PEG spacer group through a phosphate ester bond. CpG2006-cholesterol (CpG2006-Chol) (Microsynth, Switzerland) was dissolved at 1 mg / mL in PBS pH 7.4 and injected into a liposome solution, then incubated for 15 minutes before API insertion.

[0471] API (Bachem AG, Switzerland) was dissolved at a concentration of 1 mg / mL in PBS pH 11.4 containing 2.0% octyl-β-D-glucopyranoside (Sigma-Aldrich, USA). The peptide solution was infused into the liposome solution at 60 °C and stirred at 60 °C for 30 min. Concentration was achieved by ultrafiltration to obtain the target values ​​(400 μg / mL API and 100 μg / mL T50 for liposomes Z), with 10 buffer exchanges with PBS pH 7.4 during perfiltration. The resulting liposomes with API present on the liposome surface were then aseptically filtered through a 0.2 μm polycarbonate syringe filter, and the final product was stored at 5 °C.

[0472] Liposome Z+ vaccine prepared by ethanol injection

[0473] The liposome Z+ vaccine is produced using an ethanol-based injection process. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, Netherlands), and 3D-(6-acyl)PHAD® (Avanti Polar Lipids, AL, USA) are dissolved in EtOH at 60°C in a molar ratio of approximately 9:1:7:0.04. T50 peptide (Bachem AG, Switzerland) is dissolved in 10 mM His / 270 mM sucrose (pH 5.8–6.0). Then, the liposome ethanol solution is injected into the T50 peptide-containing solution and gently stirred for 15 minutes to form multilayer vesicles (MLVs). The MLVs are homogenized (6 times for liposome Z+) and then sequentially extruded through a 0.08 μm pore size polycarbonate membrane (Whatman, UK) (5 times for liposome Z+). For liposomes Z+, both homogenization and extrusion steps were performed in an EmulsiFlex-C5 (Avestin, Canada). The extruded liposomes were concentrated by ultrafiltration and the buffer was exchanged with 20 mM His / 145 mM NaCl pH 7.4 by percolation. The resulting liposomes containing the encapsulated T50 peptide were diluted in 20 mM His / 145 mM NaCl pH 7.4 and heated to 60°C to obtain a liposome solution, followed by the addition of API and adjuvant.

[0474] CpG2006-Chol (Microsynth, Switzerland for liposome Z+) was dissolved at 1 mg / mL in 20 mM His / 145 mM NaCl pH 7.4 and injected into the liposome solution, then incubated for 15 minutes before API insertion.

[0475] API (Bachem AG, Switzerland) was dissolved at a concentration of 1 mg / mL in carbonate buffer (pH 10.2) containing 1% octyl-β-D-glucopyranoside (Sigma-Aldrich, USA). The peptide solution was infused into the liposome Z+ solution at 60 °C and stirred at 60 °C for 30 min. The peptide solution was then mixed into the liposome Z+ solution at 60 °C using T-Line mixing and stirred at 60 °C for 30 min. Concentration was achieved by ultrafiltration to obtain the target values ​​(1200 µg / mL API and 1200 µg / mL T50 for liposome Z+), with 10 buffer exchanges of 10 mM His / 270 mM sucrose at pH 6.5 during perfiltration. The resulting Z+ liposomes with API present on the liposome surface were then aseptically filtered through a 0.2 μm polycarbonate syringe / capsule filter, and the final product was stored at 5 °C.

[0476] Example 2

[0477] Preparation of conjugate vaccines

[0478] Peptides and adjuvants

[0479] The phosphorylated tau peptide (SEQ ID NO: 2) used in this study was synthesized by adding phosphorylated residues during synthesis (Pepscan, NL). The conjugate containing the phosphorylated tau peptide having the amino acid sequence of SEQ ID NO: 2, covalently linked to a CRM vector via a linker, is referred to herein as conjugate X.

[0480] The vaccine peptide was conjugated to the carrier protein CRM197 via a polyethylene glycol (PEG)-cysteine-acetaminopropionamide linker. A phosphorylated tau peptide having the amino acid sequence SEQ ID NO: 2 was synthesized by adding a phosphorylated residue and a PEG3 spacer during synthesis (Polypeptide Laboratories SAS). Conjugate X was prepared by conjugating the carrier protein CRM197 to a cysteine ​​residue at the N-terminus of the peptide via a 3-(bromoacetamino)propionate succinimide ester (SBAP) linker. SBAP was attached to the primary amine (-NH2) of the CRM197 protein via NHS ester reaction chemistry. Excess SBAP linkers were removed using ultrafiltration and diafiltration (UF / DF). The CRM197-SBAP intermediate was conjugated to the phosphorylated tau peptide, and the conjugation reaction was terminated by adding excess L-cysteine ​​to quench the reaction once complete. The crude CRM197-peptide conjugate was purified using a Capto QImpRes (GE Healthcare) column and eluted using salt isocratic elution. The purified CRM197-peptide was then reconstituted into a buffer containing Tris and sucrose (e.g., 20 mM Tris, 250 mM sucrose), pH 8.1, using UF / DF. A CRM197-tau peptide drug substance (DS) stock solution was prepared by adding polysorbate 80 (PS80) stock buffer (e.g., 10% PS80 stock buffer) to achieve a final concentration of 0.01% PS80. The solution was thoroughly mixed before filtration. Prior to injection, the stock solution was diluted with PBS and CpG / Alum to a first concentration of 0.8 mg / mL CRM197-tau peptide, and subsequently further diluted with PBS and CpG / Alum adjuvant to a final concentration of 30 μg / mL CRM197-tau peptide for injection. For Examples 3 through 7, the CRM197-tau peptide stock solution was maintained at a concentration of 3.1 mg / mL in 10 mM PBS (pH 7.3) and further diluted in PBS to achieve the desired working concentration. CpG oligonucleotides, aluminum adjuvant, and PBS were then added to achieve a final concentration of 30 ug / mL based on CRM197-tau peptide, and the final formulation was thoroughly mixed prior to injection.

[0481] One concern with using live vaccines targeting CNS antigens is that nonspecific or off-target inflammation could cause unwanted neuropathological changes. To investigate this, whole brains from mice immunized with the conjugate composition were collected and stained to visualize perivascular or other cellular infiltrations. None of the immunized animals showed any signs of neuroinflammation, cellular infiltration, or other unwanted neuropathological changes (data not shown). This suggests that vaccine-induced antibodies, and the innate immune response to vaccination, do not cause neuropathological changes in mice.

[0482] The following examples illustrate different aspects of the immune response induced by different vaccine regimens.

[0483] Example 3

[0484] Heterologous vaccination in rhesus monkeys increases epitope coverage of Tau phosphopeptide-specific antibodies.

[0485] All animal experiments were approved and conducted in accordance with local regulations governing animal testing. Rhesus macaques (Macaca mulatta) were obtained from Kunming Biomed International Ltd, Yunnan Yinmore Bio-Tech Co. LTD, and Yunnan LaboratoryPrimates Inc., China. Animals were 2 to 5 years old at the start of immunization, with a minimum weight of 2.5 kg. Detailed clinical examinations were performed before treatment and weekly thereafter. Furthermore, the macaques were observed twice daily, and clinical signs were recorded.

[0486] Liposome vaccines, such as liposome Z or liposome Z+ (both containing tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2, 3D-(6-acyl)PHAD®, lipidated CpG oligonucleotide CpG2006, and T-cell peptide T50), are referred to as “A” throughout this application, while conjugate vaccines, such as conjugate X (phosphorylated tau peptide of SEQ ID NO: 2 linked to CRM197), are referred to as “B” throughout this application.

[0487] Rhesus monkeys (n=3 males and 3 females per group) were subcutaneously immunized on days 1 and 29 using the following: i) 1800 μg of tetrapalmitoyl acetate-treated phosphorylated tau peptide of SEQ ID NO: 2 / dose liposome Z vaccine (AA regimen); ii) 15 μg / dose conjugate X vaccine (BB regimen); or iii) 1800 μg of tetrapalmitoyl acetate-treated phosphorylated tau peptide of SEQ ID NO: 2 / dose liposome Z vaccine on day 1, followed by 15 μg / dose conjugate X vaccine on day 29 (AB regimen). Epitope recognition profiles of the antibodies were determined three weeks after the second immunization (day 50) using an N-terminal biotinylated octamer peptide library by epitope mapping ELISA, which was shifted one amino acid and covered the complete sequence of the phosphorylated tau peptide of SEQ ID NO: 2 and the sequence of SEQ ID NO: 4 (VYKSPVVSGDTSPRHL, the non-phosphorylated peptide of SEQ ID NO: 2).

[0488] Figures 1A to 1C Monkeys immunized with the liposomal vaccine (liposomal Z) produced IgG antibodies (AA regimen) that primarily bind to the N-terminal portion of the peptide. Figure 1A However, monkeys immunized with the conjugate vaccine (conjugate X) produced IgG antibodies that primarily bind to the C-terminal portion of the peptide (BB regimen). Figure 1B Although liposomal vaccines and conjugate vaccines contain the same phosphorylated Tau peptide as SEQ ID NO: 2, the heterologous protocol induces antibody binding to both the N- and C-terminal portions of the peptide (AB protocol). Figure 1C This increases the epitope coverage of the induced antibodies. Furthermore, IgG antibodies induced in monkeys immunized with a liposomal vaccine (liposome Z) did not bind to the non-phosphorylated library, while IgG antibodies induced in monkeys immunized with a conjugate vaccine (conjugate X) did recognize the non-phosphorylated octamer peptide. IgG antibodies induced in monkeys immunized with the heterologous protocol (AB) showed an intermediate degree of binding to the non-phosphorylated library.

[0489] Example 4

[0490] In rhesus monkeys, a booster dose of liposomal vaccine induced a shift to a "liposomal-like" epitope profile (ABBA regimen).

[0491] Rhesus monkeys (n=3 males and 3 females per group) were subcutaneously immunized as follows: 1800 μg of tetrapalmitoyl acetate-containing phosphorylated Tau peptide / dose liposome Z vaccine on day 1, 15 μg / dose conjugate X vaccine on days 29 and 85, and again on day 169 with 1800 μg of tetrapalmitoyl acetate-containing phosphorylated Tau peptide / dose liposome Z vaccine (ABBA regimen). As described in Example 6, the epitope recognition profile of the induced antibodies was determined by epitope mapping ELISA one week before the final booster with the liposome vaccine (day 162, ABB regimen) and three weeks after (day 190, ABBA regimen). Figure 2 The study showed that liposome initiation and boosting with a conjugate vaccine induced a combination of N- and C-terminal IgG antibodies (ABB regimen), while a final boost with a liposome vaccine resulted in a shift to a “liposome-like” epitope profile in rhesus monkeys (ABBA regimen). This data demonstrates a high degree of variability in the induction of anti-Tau antibody responses using a heterologous vaccination strategy employing the novel anti-Tau vaccine described herein.

[0492] In summary, the study in rhesus monkeys showed that heterologous vaccination with a sequential immunization schedule using vaccines (liposome Z and conjugate X) not only induced antibody titers against pTau and pathological Tau extracted from the brains of AD patients, but also increased the epitope coverage of antibodies induced in the antigenic sequences used in the two second-generation vaccines.

[0493] Example 5

[0494] Heterologous vaccination with liposome and conjugate vaccines induced ePHF-specific IgG titers in serum and Tau phosphopeptide-specific IgG antibodies in rhesus monkey cerebrospinal fluid (CSF).

[0495] Adult rhesus monkeys (n=3 males and 3 females per group) were immunized subcutaneously or intramuscularly using the following methods: i) 1800 μg of tetrapalmitoyl acetate-containing phosphorylated tau peptide / dose liposome Z vaccine (SEQ ID NO: 2) on days 1, 29, 85, and 169 (AAAA regimen); ii) 1800 μg of tetrapalmitoyl acetate-containing phosphorylated tau peptide / dose liposome Z+ vaccine (SEQ ID NO: 2) on days 1, 29, 85, and 169 (AAAA regimen); iii) 1800 μg of tetrapalmitoyl acetate-containing phosphorylated tau peptide / dose liposome Z vaccine on day 1, 15 μg / dose conjugate X vaccine co-injected with aluminum adjuvant and CpG oligonucleotide CpG2006 on days 29 and 85, and 1800 μg of tetrapalmitoyl acetate-containing SEQ ID NO: 2 on day 169. The following vaccines were administered: iv) a phosphorylated tau peptide / dose liposome Z vaccine of SEQ ID NO: 2 (ABBA regimen); or v) a 1800 μg tetrapalmitoyl acetate SEQ ID NO: 2 phosphorylated tau peptide / dose liposome Z+ vaccine on days 1 and 29, and a 15 μg / dose conjugate X vaccine on days 85 and 169 (AABB regimen); or v) a 15 μg / dose conjugate X vaccine on days 1 and 29, and a 1800 μg tetrapalmitoyl acetate SEQ ID NO: 2 phosphorylated tau peptide / dose liposome Z+ vaccine on days 89 and 169 (BBAA regimen). Blood was drawn on day 190, and serum was separated.

[0496] Using a modified method by Greenberg and Davies (Greenberg and Davies, 1991, Proc Natl Acad Sci USA, 87(15):5827-31), enriched preparations of paired helical filaments (ePHF) were obtained from postmortem brain tissue of histologically confirmed AD subjects via sarcosyl extraction of insoluble tau. Antibody titers specific to ePHF were assessed using the Mesoscale Discovery (MSD) platform and ePHF as the coating antigen. Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% skim milk) and plated in 96-well MSD plates. After two hours of incubation, the samples were removed, and the plates were washed in PBST (PBS, 0.05% Tween 20). Antibodies were detected using SulfoTag-labeled anti-human / monkey IgG antibodies, followed by a fixation step with 1% paraformaldehyde (PFA) and then the addition of Read Buffer T. Plates were analyzed using a Sector Imager (MSD). All samples were run at eight 2-fold serial dilutions, and each plate included both positive and negative control samples. Antibody titer expressed in arbitrary units (AU) per mL was calculated for each monkey. Fold change (for each liposome composition) compared to the geometric mean of titers obtained using AAAA is expressed as the geometric mean for each monkey and for each group.

[0497] Antibody titers specific to the phosphorylated tau peptide of SEQ ID NO: 2 were evaluated using the MSD platform. Gold small spot streptavidin 96-well plates (MSD) pre-saturated with PBS containing 1% Blocker A were used to coat the N-terminally biotinylated phosphorylated tau peptide of SEQ ID NO: 2. CSF from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% Blocker A) and applied to 96-well MSD plates. After two hours of incubation, the samples were removed, and the plates were washed with PBST (PBS, 0.05% Tween 20). Antibodies were detected using SulfoTag-labeled anti-human / monkey IgG antibodies, followed by readout buffer T. Plates were analyzed using a SectorImager (MSD). All samples were run in eight 2-fold serial dilutions, and each plate included both positive and negative control samples. Antibody titer expressed in AU / mL was calculated for each individual monkey. The fold change compared to the geometric mean of titers obtained using AAAA (for each liposome composition) is expressed as the geometric mean for each monkey and for each group. Samples with high red blood cell counts were removed from the analysis to avoid any bias in antibody titer due to blood contamination.

[0498] like Figure 3 As shown, monkeys given the heterologous regimens ABBA and AABB (where A is a liposomal vaccine with encapsulated T50, TLR4 ligand and lipotropic CpG oligonucleotide (phosphorylated tau peptide with tetrapalmitoyl acetate SEQ ID NO: 2), and B is a conjugate vaccine co-injected with aluminum adjuvant and CpG oligonucleotide (phosphopeptide SEQ ID NO: 2 linked to CRM197)) showed ePHF-specific titers on day 190 (three weeks after the fourth injection), which were similar to or higher than those of monkeys given homologous treatment (AAAA) (fold change ≥1).

[0499] However, all monkeys injected with the heterologous BBAA regimen showed lower ePHF-specific potency (fold change ≤1) than monkeys given homologous treatment (AAAA).

[0500] Figure 6Similar conclusions were reached when IgG antibody responses were monitored in the cerebrospinal fluid of these monkeys on day 183 (two weeks after the fourth injection). Monkeys administered heterologous regimens ABBA and AABB (where A is a liposomal vaccine (containing a phosphorylated tau peptide of SEQ ID NO: 2 with tetrapalmitoyl acetate, an encapsulated T50 (TLR4 ligand), and a lipid-modified CpG oligonucleotide), and B is a conjugate vaccine co-injected with an aluminum adjuvant and a CpG oligonucleotide (a phosphopeptide SEQ ID NO: 2 linked to CRM197)) showed IgG titers specific to the phosphorylated tau peptide of SEQ ID NO: 2, which were generally higher than those of monkeys administered homologous treatment (AAAA) (fold change ≥1). However, all monkeys injected with the heterologous regimen BBAA showed lower IgG titers specific to the phosphorylated tau peptide of SEQ ID NO: 2 (fold change ≤1) than those administered homologous treatment (AAAA).

[0501] Overall, the results of this study indicate that, for both systemic and local (in CSF) antibody responses, the heterologous regimen using a liposomal vaccine as the initiating composition induced higher ptau-specific IgG titers compared to the regimen using a conjugate vaccine as the initiating composition.

[0502] Example 6

[0503] In rhesus monkeys, heterologous vaccination with liposomal vaccines induced higher IgG titers specific to Tau phosphopeptide and ePHF compared to homologous regimens.

[0504] Adult rhesus monkeys (n=3 males and 3 females per group) were immunized subcutaneously or intramuscularly using the following methods: i) 1800 μg of SEQ ID NO: 2 phosphorylated tau peptide / dose liposome Z vaccine with tetrapalmitoyl acetate on days 1, 29, and 85 (AAA regimen); ii) 1800 μg of SEQ ID NO: 2 phosphorylated tau peptide / dose liposome Z+ vaccine with tetrapalmitoyl acetate (AAA regimen); iii) 1800 μg of SEQ ID NO: 2 phosphorylated tau peptide / dose liposome Z vaccine with tetrapalmitoyl acetate on day 1, and 15 μg / dose conjugate X vaccine on days 29 and 85 (ABB regimen); or iv) 1800 μg of SEQ ID NO: 2 phosphorylated tau peptide / dose liposome Z+ vaccine with tetrapalmitoyl acetate on days 1 and 29, and 15 μg / dose conjugate X vaccine on day 85 (AAB regimen). Blood was drawn on day 106, and serum was separated.

[0505] Phosphorylated tau peptide of SEQ ID NO: 2 was used as the coating antigen to determine the titer of phosphorylated tau peptide-specific IgG antibodies by ELISA. Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% BSA) and applied to 96-well plates pre-coated with the relevant peptide. After two hours of incubation, the samples were removed and the plates were washed in PBST (PBS, 0.05% Tween 20). The antibodies were detected using HRP-conjugated anti-monkey IgG followed by ABTS substrate (Roche). All samples were run in eight 2-fold serial dilutions, and each plate contained both positive and negative control samples. Data are expressed as individual endpoint titers (last serum dilution inducing a positive response) along with the geometric mean for each group. One-way ANOVA and Tukey multiple comparisons were used for statistical analysis.

[0506] The titer of ePHF-specific antibodies was evaluated using the MSD platform. Small dot streptavidin 96-well plates (MSD) pre-saturated with PBS containing 1% BSA were coated with biotinylated anti-tau capture antibody (HT7-Biotin, ThermoScientific) and then incubated with ePHF isolated from the brains of Alzheimer's disease patients. After one hour of incubation, the plates were washed with PBST, and serially diluted serum was added and incubated for two hours. The bound antibodies were detected using SulfoTag-labeled anti-human / monkey IgG antibody, followed by a fixation step with 1% PFA and then readout buffer T. Plates were analyzed using a Sector Imager (MSD). All samples were run at eight 2-fold serial dilutions, and each plate included both positive and negative control samples. Results are co-expressed as AU / mL per monkey individual and geometric mean for each group. The figure represents the ePHF-specific antibody titer at day 106. One-way ANOVA and Tukey multiple comparisons were used for statistical analysis.

[0507] Figure 4A The results showed that in rhesus monkeys, the heterologous regimens AAB and ABB induced statistically significantly higher levels of IgG antibodies specific to phosphorylated tau peptide than the homologous regimen AAA.

[0508] Figure 4B The results showed that the heterologous regimen AAB induced statistically significantly higher levels of ePHF-specific IgG antibodies in rhesus monkeys than the homologous regimen AAA, while ABB showed a trend toward higher levels of ePHF-specific IgG titers than the homologous regimen AAA.

[0509] Overall, the data show that the heterologous immunization regimen using liposomal vaccine (A) as the initiating composition induced higher antibody titers against phosphorylated tau peptide and ePHF of SEQ ID NO: 2 than the homologous regimen AAA.

[0510] Example 7

[0511] In rhesus monkeys, the quality of ePHF-specific IgG antibodies induced by the heterologous protocol was similar to or better than that induced by the homologous protocol.

[0512] Adult rhesus monkeys (n=3 males and 3 females per group) were subcutaneously immunized with: i) 1800 μg of tetrapalmitoyl acetate-containing phosphorylated tau peptide / dose liposome Z vaccine of SEQ ID NO: 2 (AAAA regimen) on days 1, 29, 85, and 169; or ii) 1800 μg of tetrapalmitoyl acetate-containing phosphorylated tau peptide / dose liposome Z vaccine of SEQ ID NO: 2 (ABBA regimen) on day 1, 15 μg / dose conjugate X vaccine on days 29 and 85, and 1800 μg of tetrapalmitoyl acetate-containing phosphorylated tau peptide / dose liposome Z vaccine of SEQ ID NO: 2 (ABBA regimen) on day 169; or intramuscularly immunized with: iii) 1800 μg of tetrapalmitoyl acetate-containing SEQ ID NO: 2 (ABBA regimen) on days 1, 29, 85, and 169. Phosphorylated tau peptide / dose liposome Z+ vaccine (AAAA regimen), or (iv) 1800 μg tetrapalmitoyl acetate SEQ ID NO: 2 phosphorylated tau peptide / dose liposome Z+ vaccine on days 1 and 29, and 15 μg / dose conjugate X vaccine on days 85 and 169 (AABB regimen). Blood was drawn on day 190 and serum was separated.

[0513] The quality of antibodies specific to enriched paired helical fibers (ePHF) was assessed using the MSD platform for binding to low ePHF coating concentrations (also referred to herein as under limited coating conditions). Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% skim milk) and plated in 96-well MSD plates. After two hours of incubation, the samples were removed and the plates were washed in PBST (PBS, 0.05% Tween 20). Antibodies were detected using SulfoTag-labeled anti-human / monkey IgG antibodies, followed by a fixation step with 1% PFA, and then readout buffer T. Plates were analyzed using a Sector Imager (MSD). All samples were run in eight 2-fold serial dilutions, and each plate included both positive and negative control samples. Antibody titers expressed in AU / mL were calculated for each monkey individual.

[0514] Phosphorylated tau peptide of SEQ ID NO: 2 was used as the coating antigen to determine the titer of phosphorylated tau peptide-specific IgG antibodies by ELISA. Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% BSA) and plated in 96-well plates pre-coated with the relevant peptide. After two hours of incubation, the samples were removed and the plates were washed in PBST (PBS, 0.05% Tween 20). The antibodies were detected using HRP-conjugated anti-monkey IgG followed by ABTS substrate (Roche). All samples were run in eight 2-fold serial dilutions, and each plate included both positive and negative control samples. Data are co-expressed as individual endpoint titers (last serum dilution inducing a positive response) along with the geometric mean for each group.

[0515] Using the T50 peptide as the coating antigen, the titer of universal T-cell epitope (T50)-specific IgG antibodies was determined by ELISA. Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% skim milk) and applied to 96-well plates pre-coated with the relevant peptide. After two hours of incubation, the samples were removed, and the plates were washed in PBST (PBS, 0.05% Tween 20). The antibodies were detected using HRP-conjugated anti-monkey IgG followed by ABTS substrate (Roche). All samples were run in eight 2-fold serial dilutions, and each plate included both positive and negative control samples. Data are co-expressed as individual endpoint titers (the final serum dilution inducing a positive response) along with the geometric mean for each group.

[0516] Geometric mean values ​​for each monkey and group represent the fold change between phosphorylated tau peptide-specific IgG antibody titers and universal T-cell epitope (T50)-specific IgG antibody titers. The Mann-Whitney test was used for statistical analysis.

[0517] Figure 5A and 5B The ePHF-specific IgG titer, as shown under limited coating conditions, reflects an antibody with high binding capacity. Figure 5A The results showed that the ABBA regimen induced a slightly higher antibody titer than the AAAA regimen, while the AABB regimen induced a similar IgG titer. Furthermore, as... Figure 3 As shown in the data, compared to the homologous regimen (AAAA regimen) using liposome vaccines alone, the heterologous regimens ABBA and AABB induced similar or higher overall ePHF-specific antibody titers (fold change ≥1). Overall, these data indicate that the quality of ePHF-specific antibodies induced by heterologous vaccination in rhesus monkeys is similar to or better than that induced by the homologous AAAA vaccination regimen.

[0518] While not wishing to be bound by theory, it is believed that the heterologous protocols according to this application diversify CD4 T cell responses. Due to limited space within the germinal center, if immunization is maintained with the same CD4 helper epitope / protein (e.g., tetanus), tetanus-specific B and T cells will displace tau-specific B cells from the germinal center. On the other hand, if CD4 stimulation is alternating, tau-specific B cells (which are also aided by tetanus or diphtheria-specific T cells) will have better access to them. In this invention, it was observed that when using the AAAA protocol, the antibody titer against the T50 helper peptide was significantly increased. When using the heterologous protocols of A and B, the antibody titer against the T50 helper peptide was much lower. Figure 7 The results showed that the ratio of IgG antibody titers against the phosphorylated tau peptide of SEQ ID NO: 2 to those against the T50 peptide was higher in the heterologous regimens (ABBA and AABB) compared to their respective homologous regimens (AAAA). This reflects the diversity of CD4 T cell responses and also confirms that the T50-specific B cell response does not completely dominate the anti-pTau response.

[0519] It should be understood that the embodiments and implementations described herein are for illustrative purposes only, and changes may be made to the above-described embodiments without departing from their broad inventive concept. Therefore, it should be understood that this invention is not limited to the specific embodiments disclosed, but rather is contemplated to be covered within the spirit and scope of the invention as defined in the appended claims.

[0520] sequence list

[0521] SEQ ID NO: 1 - Phosphorylated tau peptide (7.1)

[0522] GDRSGYS[pS]PG[pS]PG[pT]PGSRSRT

[0523] SEQ ID NO: 2 - Phosphorylated tau peptide (T3.5)

[0524] VYK[pS]PVVSGDT[pS]PRHL

[0525] SEQ ID NO: 3 - Phosphorylated tau peptide (22.1)

[0526] SSTGSIDMVD[pS]PQLA[pT]LA

[0527] SEQ ID NO: 4 - tau peptide

[0528] VYKSPVVSGDTSPRHL

[0529] SEQ ID NO: 5 - Phosphorylated tau peptide

[0530] RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHL

[0531] SEQ ID NO: 6 - Phosphorylated tau peptide

[0532] RQEFEVMEDHAGT[pY]GL

[0533] SEQ ID NO: 7 - Phosphorylated tau peptide

[0534] PGSRSR[pT]P[pS]LPTPPTR

[0535] SEQ ID NO: 8 - Phosphorylated tau peptide

[0536] GYSSPG[pS]PG[pT]PGSRSR

[0537] SEQ ID NO: 9 - Phosphorylated tau peptide

[0538] GDT[pS]PRHL[pS]NVSSTGSID

[0539] SEQ ID NO: 10 - Phosphorylated tau peptide

[0540] PG[pS]PG[pT]PGSRSR[pT]P[pS]LP

[0541] SEQ ID NO: 11 - Phosphorylated tau peptide

[0542] HL[pS]NVSSTGSID

[0543] SEQ ID NO: 12 - Phosphorylated tau peptide

[0544] VSGDT[pS]PRHL

[0545] SEQ ID NO: 13 - T50 T cell epitope

[0546] AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE-NH2

[0547] SEQ ID NO: 14 - T46 T cell epitope

[0548] AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEK(Pal)K(Pal)-NH2

[0549] SEQ ID NO: 15 - T48 helper T cell epitope

[0550] AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ-NH2

[0551] SEQ ID NO: 16 - T51 helper T cell epitope

[0552] AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLE-NH2

[0553] SEQ ID NO: 17 - T52 helper T cell epitope

[0554] AKFVAAWTLKAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLE-NH2

[0555] SEQ ID NO: 18 - CpG2006 (also known as CpG7909)

[0556] 5'-tcgtcgttttgtcgttttgtcgtt-3'

[0557] The lowercase letters represent the internucleotide bonds of phosphate thioesters (ps).

[0558] SEQ ID NO: 19 - CpG1018

[0559] 5'-tgactgtgaacgttcgagatga-3'

[0560] The lowercase letters represent the internucleotide bonds of thiophosphate esters.

[0561] SEQ ID NO: 20 – CpG2395

[0562] 5'-tcgtcgttttcggcgcgcgcgccg-3'

[0563] The lowercase letters represent the internucleotide bonds of thiophosphate esters.

[0564] SEQ ID NO: 21 – CpG2216

[0565] 5'-ggGGGACGATCGTCgggggg-3'

[0566] The lowercase letters represent the internucleotide bonds of thiophosphate esters, and the uppercase letters represent the phosphodiester bonds (po).

[0567] SEQ ID NO:22 – CpG2336

[0568] 5'-ggggGACGACGTCGTGgggggg-3',

[0569] Lowercase letters represent thiophosphate nucleotide bonds, and uppercase letters represent phosphodiester bonds.

[0570] SEQ ID NO:23 - PanDR epitope (PADRE) peptide

[0571] AKFVAAWTLKAAA

[0572] SEQ ID NO:24 – P2

[0573] QYIKANSKFIGITEL

[0574] SEQ ID NO:25 – P30

[0575] FNNFTVSFWLRVPKVSASHLE

[0576] SEQ ID NO: 26 - TT586–605

[0577] LINSTKIYSYFPSVISKVNQ

[0578] SEQ ID NO: 27 – Palmitoylated phosphorylated tau peptide (palmitoylated 7.1)

[0579] K(pal)K(pal)GDRSGYS[pS]PG[pS]PG[pT]PGSRSRTK(pal)K(pal)

[0580] SEQ ID NO: 28 - Palmitoylated phosphorylated tau peptide (T3, palmitoylated T3.5, palmitoylated SEQ ID NO: 2)

[0581] K(pal)K(pal)VYK[pS]PVVSGDT[pS]PRHLK(pal)K(pal)

[0582] SEQ ID NO: 29 - Palmitoylated phosphorylated tau peptide (palmitoylated 22.1)

[0583] K(pal)K(pal)SSTGSIDMVD[pS]PQLA[pT]LAK(pal)K(pal)

[0584] SEQ ID NO: 30 - Palmitoylated tau peptide

[0585] K(pal)K(pal)VYKSPVVSGDTSPRHLK(pal)K(pal)

[0586] SEQ ID NO: 31 - Palmitoylated phosphorylated tau peptide

[0587] K(pal)K(pal)RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHLK(pal)K(pal)

[0588] SEQ ID NO: 32 - Palmitoylated phosphorylated tau peptide

[0589] K(pal)K(pal)RQEFEVMEDHAGT[pY]GLK(pal)K(pal)

[0590] SEQ ID NO: 33 - Palmitoylated phosphorylated tau peptide

[0591] K(pal)K(pal)PGSRSR[pT]P[pS]LPTPPTRK(pal)K(pal)

[0592] SEQ ID NO: 34 - Palmitoylated phosphorylated tau peptide

[0593] K(pal)K(pal)GYSSPG[pS]PG[pT]PGSRSRK(pal)K(pal)

[0594] SEQ ID NO: 35 - Palmitoylated phosphorylated tau peptide

[0595] K(pal)K(pal)GDT[pS]PRHL[pS]NVSSTGSIDK(pal)K(pal)

[0596] SEQ ID NO: 36 - Palmitoylated phosphorylated tau peptide

[0597] K(pal)K(pal)PG[pS]PG[pT]PGSRSR[pT]P[pS]LPK(pal)K(pal)

[0598] SEQ ID NO: 37 - Palmitoylated phosphorylated tau peptide

[0599] K(pal)K(pal)HL[pS]NVSSTGSIDK(pal)K(pal)

[0600] SEQ ID NO: 38 - Palmitoylated phosphorylated tau peptide

[0601] K(pal)K(pal)VSGDT[pS]PRHLK(pal)K(pal)

[0602] SEQ ID NO:39 - T50 without C-terminal amide

[0603] AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE

[0604] SEQ ID NO: 40 - T46 without -Lys(Pal)-Lys(Pal)-NH2 at the C-terminus

[0605] AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLE

[0606] SEQ ID NO: 41 - T48 without C-terminal amide

[0607] AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ

[0608] SEQ ID NO: 42 - T51 without C-terminal amide

[0609] AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLE

[0610] SEQ ID NO: 43 - T52 without C-terminal amide

[0611] AKFVAAWTLKAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLE

[0612] SEQ ID NO: 44 - T57 (56, 57)

[0613] AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE-K(Pal)K(Pal)-NH2

[0614] References

[0615] Asuni AA et al., J Neurosci. 2007 Aug 22; 27(34):9115-29

[0616] Bentebibel et al., 2013, Sci Transl Med., 5(176):176ra32

[0617] Crotty, 2011, Annual Reviews of Immunology. Vol 29:p621-663

[0618] Friedhoff et al., Biochimica et Biophysica Acta 1502 (2000) 122-132

[0619] Greenberg and Davies, 1991, Proc Natl Acad Sci USA, 87(15):5827-31

[0620] Hanger et al., Trends Mol Med. 15:112-9, 2009

[0621] Hickman et al., J. Biol. Chem. vol. 286, NO. 16, pp. 13966–13976, April 22, 2011

[0622] Kontsekova E et al., Alzheimers Res Ther. 2014 Aug 1;6(4):44

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Claims

1. Use of the initiating composition and the first enhancing composition in the preparation of a medicament for the treatment of neurodegenerative diseases or disorders by inducing antibodies against at least one of phosphorylated Tau and enriched paired helical filaments in subjects in need of the medicament, said use comprising: (i) Administering the initiating composition to a subject comprising an immunologically effective amount of liposomes, the liposomes comprising: a. First tau phosphopeptide; b. Helper T cell epitopes, the amino acid sequences of which are shown in any one of SEQ ID NO: 39 to SEQ ID NO: 44; c. Lipidified CpG oligonucleotides having nucleotide sequences as shown in any one of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; and d. Monophospholipid A; The amino acid sequence of the first tau phosphopeptide is shown in any one of SEQ ID NO: 2, 5, 9, and 12, the first tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further comprises a pharmaceutically acceptable carrier; and (ii) Administering to the subject the first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having a structure of formula (I): Or it may consist of the structure of formula (II): in x is an integer from 0 to 10; n is an integer from 3 to 15; Tau peptide represents the second tau phosphopeptide; and The vector represents an immunogenic vector selected from keyhole hemocyanin and CRM197; and The first enhancement composition further comprises a pharmaceutically acceptable carrier; The amino acid sequence of the second tau phosphopeptide is shown in any one of SEQ ID NO: 2, 5, 9 and 12.

2. The use according to claim 1, wherein x is an integer from 2 to 6, and n is an integer from 3 to 12.

3. The use according to claim 2, wherein x is 3.

4. The use according to claim 1, wherein: The liposomes comprise: a. The first tau phosphopeptide, the amino acid sequence of which is shown in any one of SEQ ID NO: 28, 31, 35 and 38; b. Helper T cell epitopes, the amino acid sequences of which are shown in any one of SEQ ID NO: 13 to SEQ ID NO: 17; c. Lipidified CpG oligonucleotides having nucleotide sequences as shown in any one of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; and d. Monophospholipid A; and The conjugate comprises a second tau phosphopeptide conjugated to CRM197 via a linker, the amino acid sequence of which is shown in any one of SEQ ID NO: 2, 5, 9 and 12.

5. The use according to claim 1, wherein the conjugate has the following structure: ， Where n is an integer from 3 to 7.

6. The use according to claim 1, further comprising administering the first strengthening composition to the subject at least once after the initial administration of the first strengthening composition.

7. The use according to claim 1, further comprising administering to a subject a second enhancement composition comprising an immunologically effective amount of the liposome and a pharmaceutically acceptable carrier.

8. The use according to claim 7, further comprising administering the second strengthening composition to the subject at least once after the initial administration of the second strengthening composition.

9. Use of the initiating composition and the first enhancing composition in the preparation of a medicament for treating neurodegenerative diseases or disorders by inducing antibodies against at least one of phosphorylated Tau and enriched paired helical filaments in subjects in need of the medicament, said use comprising: (i) Administering the initiating composition to a subject comprising an immunologically effective amount of liposomes, the liposomes comprising: a. The first tau phosphopeptide, the amino acid sequence of which is shown in any one of SEQ ID NO: 28, 31, 35 and 38; b. Helper T cell epitopes, the amino acid sequences of which are shown in any one of SEQ ID NO: 39 to SEQ ID NO: 44; c. Lipidified CpG oligonucleotides having nucleotide sequences as shown in any one of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphate thioester nucleotide internucleotide bonds, and the CpG oligonucleotide is covalently linked to at least one cholesterol via a linker; and d. Monophospholipid A; The first tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further comprises a pharmaceutically acceptable carrier; and (ii) Administering to the subject the first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the following structure: ， Where n is an integer from 3 to 7, and the first enhancing composition further comprises a pharmaceutically acceptable carrier.

10. The use according to claim 9, wherein the amino acid sequence of the helper T cell epitope is shown in any one of SEQ ID NO: 13 to SEQ ID NO:

17.

11. Use of the initiating composition and the first enhancing composition in the preparation of a medicament for the treatment of neurodegenerative diseases or disorders by inducing antibodies against at least one of phosphorylated Tau and enriched paired helical filaments in subjects in need of the medicament, said use comprising: (i) Administering the initiating composition to a subject comprising an immunologically effective amount of liposomes, the liposomes comprising: (1) The first tau phosphopeptide, whose amino acid sequence is shown in SEQ ID NO:28; (2) Toll-like receptor 4 agonist, which is composed of monophosphoryl hexa-acyl lipid A, 3-deacylated; (3) Helper T cell epitopes, the amino acid sequence of which is shown in SEQ ID NO: 39; (4) Lipid-modified CpG oligonucleotides, the nucleotide sequences of which are shown in SEQ ID NO:18; and (5) At least one lipid selected from 1,2-dimyristic-sn-glycerol-3-phosphocholine, 1,2-dimyristic-sn-glycerol-3-phospho-3'-rac-glycerol, and cholesterol. The first tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further comprises a pharmaceutically acceptable carrier; and (ii) Administering to the subject the first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the following structure: ， Where n is an integer from 3 to 7, and the first enhancing composition further comprises a pharmaceutically acceptable carrier.

12. Use of the initiating composition and the first enhancing composition in the preparation of a medicament for the treatment of neurodegenerative diseases or disorders by inducing antibodies against at least one of phosphorylated Tau and enriched paired helical filaments in subjects in need thereof, said use comprising: (i) Administering the initiating composition to a subject comprising an immunologically effective amount of liposomes, the liposomes comprising: (1) The first tau phosphopeptide, whose amino acid sequence is shown in SEQ ID NO:28; (2) Toll-like receptor 4 agonist, which is composed of monophosphoryl hexa-acyl lipid A, 3-deacylated; (3) Helper T cell epitopes, the amino acid sequence of which is shown in SEQ ID NO: 39; (4) Lipidified CpG oligonucleotides, the nucleotide sequence of which is shown in SEQ ID NO:18; (5) At least one lipid selected from 1,2-dimyristic-sn-glycerol-3-phosphocholine, 1,2-dimyristic-sn-glycerol-3-phospho-3'-rac-glycerol, and cholesterol. The first tau phosphopeptide is present on the surface of the liposomes, and the initiating composition further comprises a pharmaceutically acceptable carrier; and (ii) Administering to the subject the first enhancement composition comprising an immunologically effective amount of the conjugate, said conjugate comprising a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, said conjugate having the following structure: ， Where n is an integer from 3 to 7, and the first enhancing composition further comprises a pharmaceutically acceptable carrier; and (iii) Administer to the subject a first enhancement composition or a second enhancement composition comprising an immunologically effective amount of the liposome and a pharmaceutically acceptable carrier.

13. The use according to claim 12, wherein (ii) is performed before (iii).

14. The use according to claim 12, wherein (ii) is performed after (iii).

15. The use according to claim 12, further comprising administering the first strengthening composition to the subject at least once after the initial administration of the first strengthening composition.

16. The use according to claim 12, comprising administering the second enhancing composition to the subject at least once.

17. The use according to any one of claims 1-16, wherein the neurodegenerative disease or disorder is caused by the formation of neurofibrillary damage.

18. The use according to claim 17, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, boxer's dementia, Down syndrome, Gerstmann's disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Guam Parkinson's-dementia syndrome, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic granular dementia, corticobasal degeneration, Lewy body dementia. Amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcification, frontotemporal dementia associated with chromosome 17 and Parkinson's syndrome (FTDP-17), Hastings-Schwarzman disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangles-only dementia, post-encephalitis Parkinson's syndrome, myotonic dystrophy, chronic traumatic encephalopathy, cerebrovascular disease, or Lewy body dementia.

19. The use according to claim 18, wherein the subject needs to be treated for Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment caused by Alzheimer's disease, or mild to moderate Alzheimer's disease.

20. The use according to any one of claims 1-16, wherein the immunologically effective amount of liposomes comprises a first tau phosphopeptide in an amount of 25 nanomoles to 750 nanomoles per dose.

21. The use according to any one of claims 1-16, wherein the immunologically effective amount of liposomes comprises a first tau phosphopeptide in an amount of 100 µg to 2500 µg per dose.

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