Heterologous administration of tau vaccine

Abstract

A method for inducing an immune response against tau protein in a subject suffering from a neurodegenerative disease, disorder, or condition, such as Alzheimer's disease, is described, comprising administering a priming liposomal composition containing a tau peptide, preferably a phosphorylated tau peptide, and a boosting conjugate composition containing a tau peptide, preferably a phosphorylated tau peptide, conjugated to an immunogenic carrier.

Description

[Technical Field]

[0001] The present invention is in the field of medicine, and particularly relates to a method for inducing an immune response against tau protein in a subject suffering from a neurodegenerative disease, disorder, or condition using a priming liposomal composition containing a tau peptide and a boosting conjugate composition comprising a tau peptide conjugated to an immunogenic carrier. [Background technology]

[0002] Alzheimer's disease (AD) is a progressive, debilitating neurodegenerative disorder that affects an estimated 44 million people worldwide (Alzheimers.net). Currently available AD treatments in the clinic aim to slow the progression of clinical symptoms but do not target the pathogenic processes underlying the disease. Unfortunately, these treatments are only minimally effective, and therefore, there is an urgent need to develop and test additional preventive and therapeutic measures.

[0003] The hallmark pathology of Alzheimer's disease is the accumulation of extracellular plaques containing aggregated amyloid beta protein and intracellular "tangles," or aggregates, of hyperphosphorylated tau protein. The molecular events leading to the accumulation of these proteins are poorly characterized. In amyloid, abnormal cleavage of the amyloid precursor protein is hypothesized to result in the accumulation of aggregation-prone fragments containing amino acids 1–42. In tau, dysregulation of kinases, phosphatases, or both is hypothesized to result in the abnormal phosphorylation of tau. After hyperphosphorylation, tau loses its ability to effectively bind and stabilize microtubules and instead accumulates in the cytoplasm of affected neurons. Unbound, hyperphosphorylated tau is thought to form initial oligomers and then higher-order aggregates, the presence of which is presumed to negatively impact the function of the neurons in which it forms, possibly through disruption of normal axonal transport.

[0004] In developed countries, individuals diagnosed with Alzheimer's disease or other dementing tauopathies are commonly treated with cholinesterase inhibitors (e.g., Aricept®) or memantine (e.g., Namenda™). These drugs are reasonably well tolerated but have very modest efficacy. For example, Aricept® delays symptom deterioration by 6 to 12 months in approximately 50% of treated individuals. The remaining treatments are non-pharmacological and focus on enabling patients to manage their daily tasks as their cognitive abilities decline.

[0005] 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., Alzheimers Res Ther. 2014 Aug 1;6(4):44) have demonstrated that active vaccines containing tau peptides can induce anti-tau immune responses in mice or rats, reduce the accumulation of pathological tau aggregates in rodent brains, and slow the rate of cognitive decline in animal models of Alzheimer's disease. Active vaccines against pathological tau protein have been shown to be immunogenic in human patients with Alzheimer's disease (Novak P et al., Lancet Neurology 2017, 16:123-134). WO 2010 / 115843 and WO 2019 / 084118 describe antigenic phosphopeptides that mimic the major pathological phosphoepitopes of the protein tau, as well as related compositions for therapeutic and diagnostic use in the treatment of tauopathies, including Alzheimer's disease. However, currently, there is still no approved effective vaccine on the market to prevent the onset of tau-mediated diseases. There is also no effective drug on the market to interrupt or slow the progression of the disease after it has begun. Therefore, there is an urgent need to identify new preventive measures (e.g., vaccines) that can prevent these diseases. Summary of the Invention [Means for solving the problem]

[0006] Surprisingly, it has been discovered in the present invention that heterologous vaccination with liposomal and conjugate compositions, each of which contains a tau phosphopeptide, increased the epitope coverage of tau phosphopeptide-specific antibodies. In addition, it was found that heterologous vaccination with a priming liposomal composition induced a stronger immune response than that with a priming conjugate composition.

[0007] In one general aspect, the present invention provides a method of inducing an immune response against tau protein, preferably inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF), in a subject in need thereof, preferably a subject suffering from a neurodegenerative disorder, comprising: (i) To the subject, a. a first tau phosphopeptide; b. helper T cell epitopes; c. lipidated CpG oligonucleotides; d. an adjuvant containing a Toll-like receptor 4 ligand; Including, administering a priming composition comprising an immunologically effective amount of liposomes, wherein the liposomes display tau phosphopeptides on their surface, and further comprising a pharmaceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide comprising an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of formula (I):

[0008] [ka] or a structure of formula (II):

[0009] [ka] [In the formula, x is an integer from 0 to 10, preferably from 2 to 6, and most preferably 3; n is an integer of 3 to 15, preferably 3 to 12; Tau peptide represents a second tau phosphopeptide, The carrier represents an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and outer membrane protein mixture (OMP) from N. meningitidis, or derivatives thereof. an immunologically effective amount of a conjugate having administering a first boosting composition further comprising a pharmaceutically acceptable carrier; Including, The present invention relates to a method, wherein the first tau phosphopeptide and the second tau phosphopeptide each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 3 and SEQ ID NO: 5 to SEQ ID NO: 12.

[0010] In certain embodiments, the liposome comprises: a. a first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 39 to SEQ ID NO: 44, preferably a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17; c. A lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, containing one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker. d. Monophosphoryl lipid A (MPLA) and Including, The conjugate comprises a second tau phosphopeptide having an amino acid sequence selected from the group consisting of 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.

[0011] In certain embodiments, the conjugate has the following structure:

[0012] [ka] [In the formula, n is an integer of 3 to 7.] It has.

[0013] According to one embodiment of the present invention, a method for inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof comprises: (i) To the subject, a. a first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 27 to SEQ ID NO: 29 and SEQ ID NO: 31 to SEQ ID NO: 38; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 39 to SEQ ID NO: 44, preferably a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17; c. A lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, containing one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker. d. Monophosphoryl lipid A (MPLA) and administering a priming composition comprising an immunologically effective amount of liposomes, wherein the first tau phosphopeptide is displayed on the surface of the liposomes, and further comprising a pharmaceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide comprising an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure:

[0014] [ka] [In the formula, n is an integer of 3 to 7.] administering a first boosting composition comprising an immunologically effective amount of a conjugate having the formula: Includes.

[0015] According to another embodiment of the present application, a method for inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof comprises: (i) To the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-deacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) at least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering a priming composition comprising an immunologically effective amount of liposomes, wherein the first tau phosphopeptide is displayed on the surface of the liposomes, and further comprising a pharmaceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide comprising an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure:

[0016] [ka] [In the formula, n is an integer of 3 to 7.] administering a first boosting composition comprising an immunologically effective amount of a conjugate having the formula: Includes.

[0017] In certain embodiments, the method further comprises administering the first boosting composition to the subject at least once after the initial administration of the first boosting composition.

[0018] In certain embodiments, the method further comprises administering to the subject a second boosting composition comprising an immunologically effective amount of liposomes and a pharmaceutically acceptable carrier. The second boosting composition can be administered before or after the initial administration of the first boosting composition.

[0019] In certain embodiments, the method further comprises administering a second boosting composition to the subject at least once after the initial administration of the second boosting composition.

[0020] According to one embodiment of the present application, a method for inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof comprises: (i) To the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-deacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) at least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering a priming composition comprising an immunologically effective amount of liposomes, wherein the first tau phosphopeptide is displayed on the surface of the liposomes, and further comprising a pharmaceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide comprising an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure:

[0021] [ka] [In the formula, n is an integer of 3 to 7.] administering a first boosting composition comprising an immunologically effective amount of a conjugate having the formula: (iii) administering to the subject a first boosting composition or a second boosting composition comprising an immunologically effective amount of liposomes and a pharmaceutically acceptable carrier; Includes.

[0022] In certain embodiments, step (ii) is performed before step (iii).

[0023] In other embodiments, step (ii) is performed after step (iii).

[0024] In certain embodiments, the method comprises administering to the subject at least two doses of a first boosting composition to boost the immune response.

[0025] In certain embodiments, the method comprises administering to the subject at least one second boosting composition to boost the immune response.

[0026] In certain embodiments, the first boosting composition is administered about 27-32 days after the priming composition is administered. Preferably, the method further comprises re-administering the first boosting composition about 82-87 days after the priming composition is initially administered, and optionally further comprises administering the second boosting composition about 167-172 days after the priming composition is administered.

[0027] In certain embodiments, the first boosting composition is administered about 82 to 87 days after the priming composition is administered. Preferably, the method further comprises administering the second boosting composition about 27 to 32 days after the priming composition is administered, and optionally further comprises re-administering the first boosting composition about 167 to 172 days after the priming composition is administered.

[0028] In certain embodiments, the immunologically effective amount of liposomes contains about 25 nmole to about 750 nmole of first tau phosphopeptide per dose, e.g., about 29.7 nmole to about 742.5 nmole per dose, preferably about 90 nmole to about 715 nmole per dose, e.g., about 89.1 nmole to about 712.8 nmole, or about 90 nmole to about 535 nmole per dose, e.g., about 89.1 nmole to about 534.6 nmole per dose, or about 90 nmole to about 275 nmole per dose, e.g., about 89.1 nmole to about 267.3 nmole per dose, and the first tau phosphopeptide is displayed on the surface of the liposome. In one embodiment, an effective amount of liposomes comprises the first tau phosphopeptide in an amount of about 265 to about 275 nmoles per dose, e.g., about 265, about 266, about 267, about 268, about 269, about 270, about 271, about 272, about 273, about 274, or about 275 nmoles per dose, or any value therebetween, e.g., about 267.3 nmoles per dose. In another embodiment, an immunologically effective amount of liposomes contains the first tau phosphopeptide in an amount of about 530 to about 540 nmoles per dose, e.g., about 530, about 531, about 532, about 533, about 534, about 535, about 536, about 537, about 538, about 539, or about 540 nmoles per dose, or any value therebetween, e.g., about 534.6 nmoles per dose. In another embodiment, the effective amount of liposomes comprises the first tau phosphopeptide in an amount of about 710 to about 720 nmoles per dose, e.g., about 710, about 711, about 712, about 713, about 714, about 715, about 716, about 717, about 718, about 719, or about 720 nmoles per dose, or any value therebetween, e.g., about 712.8 nmoles per dose.

[0029] In certain embodiments, the first tau phosphopeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 29 and 31 to 38, and preferably consists 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, where the tetrapalmitoylated tau phosphopeptide is presented on the surface of the liposome and is administered in an amount of about 100 μg to about 2500 μg per dose, corresponding to about 29.7 nmoles to about 742.5 nmoles per dose, preferably about 300 μg to about 2400 μg per dose, corresponding to about 89.1 nmoles to about 712.8 nmoles per dose, e.g., about 300 μg, about 900 μg, about 1800 μg, or about 2400 μg per dose, corresponding to about 89.1 nmoles, about 267.3 nmoles, about 534.6 nmoles, or about 712.8 nmoles per dose.

[0030] In certain embodiments, an immunologically effective amount of liposomes comprises helper T cell epitopes in an amount of from about 2 nmole to about 110 nmole per dose, e.g., from about 4.02 nmole to about 100.44 nmole per dose, or from about 4 nmole to about 75 nmole per dose, e.g., from about 4.02 nmole to about 72.32 nmole per dose, or from about 10 nmole to about 105 nmole per dose, e.g., from about 12.06 nmole to about 100.44 nmole per dose, or from about 70 to about 105 nmole per dose, e.g., from about 72.32 nmole to about 100.44 nmole per dose. In certain embodiments, an effective amount of liposomes comprises a helper T cell epitope, preferably a T50 helper T cell epitope consisting of the amino acid sequence of SEQ ID NO:13, in an amount of about 2 nmole to about 110 nmole, for example, about 12.06 nmole to about 100.44 nmole.

[0031] The present invention also relates to vaccine combinations, such as kits, of the liposomes and conjugates of the present invention for use in inducing an immune response against tau protein in a subject suffering from a neurodegenerative disorder, or for use in treating or preventing a neurodegenerative disease or disorder in a subject in need thereof.

[0032] The present invention also relates to the use of a vaccine combination of liposomes and conjugates of the 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 thereof.

[0033] Further aspects, features, and advantages of the present invention will be better understood by reading the following detailed description and claims.

[0034] The foregoing summary, as well as the following detailed description of preferred embodiments of the present application, will be better understood when read in conjunction with the accompanying drawings. It is to be understood, however, that the present application is not limited to the specific embodiments shown in the drawings. [Brief explanation of the drawings]

[0035] [Figures 1A-1C] Figures 1A-1C show the epitope recognition profiles of antibodies induced by allogeneic AA (Figure 1A) or BB (Figure 1B) vaccination, or heterologous AB (Figure 1C) vaccination, as determined by epitope mapping ELISA on short 8-mer overlapping peptides covering the phosphopeptide of sequence number 2 and the peptide of sequence number 4. [Figure 2]Figure 2 shows the epitope recognition profile of antibodies induced after heterologous ABB vaccination (day 162) compared with that induced after heterologous ABBA vaccination (day 190) as determined by epitope mapping ELISA for short 8-mer overlapping peptides covering the phosphopeptide of SEQ ID NO: 2 and the peptide of SEQ ID NO: 4. A: Liposome vaccine containing a TLR4 ligand and lipidated CpG oligonucleotide as adjuvant, 1800 μg / dose of acetate tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 28, corresponding to SEQ ID NO: 28, and an encapsulated T50 helper T cell epitope. B: Conjugate vaccine containing 15 μg / dose of CRM197 conjugated to the phosphorylated tau peptide of SEQ ID NO: 2 via a linker, which was co-injected with alum and CpG oligonucleotide. [Figure 3] FIG. 3 shows IgG titers specific to enriched paired helical fibrils (ePHFs) isolated from postmortem brains of Alzheimer's disease patients at 190 days after heterologous vaccination (ABBA, AABB, or BBAA, where A is a liposomal vaccine comprising a TLR4 ligand and lipidated CpG oligonucleotide as adjuvant, 1800 μg / dose of tetrapalmitoylated acetate phosphorylated tau peptide of SEQ ID NO: 2, and an encapsulated T50 helper T cell epitope; and B is a conjugate vaccine containing 15 μg / dose of CRM197 conjugated to the phosphorylated tau peptide of SEQ ID NO: 2 via a linker, which was co-injected with alum and CpG oligonucleotide), or homologous vaccination (AAAA), with fold change relative to AAAA plotted. [Figure 4A-4B]Figure 4A shows IgG titers specific to the phosphopeptide (T3.5) having the sequence of SEQ ID NO: 2 in rhesus macaques on day 106 after allogeneic (AAA) and heterogeneous (AAB and ABB) immunization regimens, measured by ELISA, with data presented as endpoint titers (EPTs). Figure 4B shows IgG titers specific to ePHF in rhesus macaques on day 106 after allogeneic (AAA) and heterogeneous (AAB and ABB) immunization regimens, presented as arbitrary units (AU) / mL. [Figure 5] Figure 5 shows the quality of ePHF-specific IgG antibodies induced by the heterologous regimens ABBA (Figure 5A) and AABB (Figure 5B) compared with those induced by the allogeneic AAAA regimen, as measured by binding to ePHFs under limited coating conditions. Expressed in AU / mL, ePHF-specific IgG titers measured by MSD for limited coating of ePHFs are plotted. [Figure 6] Figure 6 shows IgG titers specific for an N-terminally biotinylated phosphopeptide having the sequence of SEQ ID NO: 2 in the cerebrospinal fluid (CSF) of rhesus macaques 183 days after the first immunization following heterologous vaccination (ABBA, AABB, or BBAA, where A is a liposomal vaccine comprising a TLR4 ligand and lipidated CpG oligonucleotide as adjuvant, 1800 μg / dose of acetate tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2, and an encapsulated T50 helper T cell epitope; and B is a conjugate vaccine containing 15 μg / dose of CRM197 conjugated to the phosphorylated tau peptide of SEQ ID NO: 2 via a linker and co-injected with alum and CpG oligonucleotide), with the fold change relative to the homologous vaccination AAAA plotted. [Figure 7]Figure 7 shows the ratio of IgG titers specific to the phosphopeptide having the sequence of SEQ ID NO: 2 (T3.5) to the IgG titers specific to the T50 peptide in rhesus macaques 190 days after the first immunization following allogeneic (AAAA) and heterogeneous (AABB and ABBA) immunizations, as measured by ELISA. Statistical analysis was performed using the Mann-Whitney test. (**p<0.001). DETAILED DESCRIPTION OF THE INVENTION

[0036] Various publications, articles, and patents are cited or described throughout the Background and Specification, and each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles, and the like which has been included herein is for the purpose of providing a context for the present invention. Such discussion is not an admission that any of these items, or the whole thereof, form part of the prior art with respect to any invention(s) disclosed or claimed.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Unless otherwise defined, certain terms used herein have the meanings set forth herein.

[0038] It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "th" include plural referents unless the context clearly dictates otherwise.

[0039] Unless otherwise stated, any numerical values, such as concentrations or concentration ranges, set forth herein are understood to be modified in all instances by the term "about." Thus, numerical values ​​typically 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). As used herein, the use of numerical ranges expressly includes all possible subranges, all individual numerical values ​​within such ranges (including integers within such ranges), and fractions of values, unless the context clearly dictates otherwise.

[0040] Unless otherwise specified, the term "at least" preceding a series of elements is understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0041] As used herein, the terms "comprise," "include," "includes," "including," "including," "having," "having," "containing," or "containing," or any other variation thereof, shall be understood to imply the inclusion of a stated integer or group of integers, but not the exclusion of any other integer or group of integers, and shall be intended to be non-exclusive or open-ended. For example, a composition, mixture, process, method, article, or apparatus that includes a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent in such composition, mixture, process, method, article, or apparatus. Furthermore, unless expressly stated to the contrary, "or" refers to an inclusive or, not an exclusive or. For example, condition 1 or 2 can be satisfied by any one of the following: 1 is true (or present) and 2 is false (or absent), 1 is false (or absent) and 2 is true (or present), and both 1 and 2 are true (or present).

[0042] Those skilled in the art will understand that the terms "about," "approximately," "generally," "substantially," and similar terms used herein when referring to dimensions or characteristics of components of the preferred invention indicate that the described dimensions / characteristics are not precise boundaries or parameters, but rather functionally the same or similar, and do not exclude minor variations therefrom. At a minimum, such references involving numerical parameters will include variations that do not alter the least significant digit using mathematical and industrial principles recognized in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.).

[0043] According to an embodiment of the present application, an effective amount of liposomes contains a tau phosphopeptide in an amount of about 25 nmoles to about 750 nmoles per dose, e.g., about 29.7 nmoles to about 742.5 nmoles per dose, preferably about 90 nmoles to about 715 nmoles per dose, e.g., about 89.1 nmoles to about 712.8 nmoles per dose, or about 90 nmoles to about 535 nmoles per dose, e.g., about 89.1 nmoles to about 534.6 nmoles per dose, or about 90 nmoles to about 275 nmoles per dose, e.g., about 89.1 nmoles to about 267.3 nmoles per dose, comprising a tau phosphopeptide comprising the amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12. Preferably, the tau phosphopeptide consists of the amino acid sequence of one of SEQ ID NOs: 27-38. More preferably, the tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 28. In one embodiment, an effective amount of liposomes comprises a toll-like receptor 4 agonist and a tetrapalmitoylated tau phosphopeptide consisting of the amino acid sequence of 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, corresponding to about 29.7 nmole to about 742.5 nmole, preferably about 89.1 nmole to about 712.8 nmole.

[0044] According to an embodiment of the present application, an effective amount of liposomes is 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, 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 1600 μg, about 1700 μg, about 1800 μg, or about 1900 μg per dose. The composition contains 500 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 of tau phosphopeptide, or any value therebetween, for example, a tau phosphopeptide comprising the amino acid sequence of one of SEQ ID NOs: 1 to 3 or 5 to 12, preferably a tau phosphopeptide consisting of the amino acid sequence of one of SEQ ID NOs: 27 to 38, more preferably SEQ ID NO: 28.

[0045] In certain embodiments, an effective amount of liposomes comprises about 30 μg to about 900 μg, preferably about 100 μg to about 585 μg, of a Toll-like receptor 4 agonist per dose. In certain embodiments, an effective amount of liposomes comprises 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 of a monophosphoryl hexa-acyl lipid A, 3-deacylated Toll-like receptor agonist per dose.

[0046] In certain embodiments, an effective amount of liposomes comprises lipidated CpG oligonucleotides in an amount of about 50 μg to about 1250 μg per dose, preferably about 150 μg to about 800 μg per dose. For example, an effective amount of liposomes comprises lipidated 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 a specific embodiment, an 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, preferably about 150 μg to about 800 μg per dose.

[0047] According to an embodiment of the present application, tau phosphopeptides are displayed on the surface of liposomes.

[0048] As used herein, the immunologically effective amount of conjugate is defined by the amount of immunogenic carrier conjugated with the second tau phosphopeptide in the conjugate.For example, 15 μg of conjugate refers to a conjugate composition that contains 15 μg of immunogenic carrier conjugated with a tau phosphopeptide.One or more tau phosphopeptides can be conjugated with one immunogenic carrier.

[0049] According to embodiments of the present application, the effective amount of the conjugate can be determined using methods known in the art in light of the present disclosure, such as clinical experience.

[0050] The tau phosphopeptide comprises one of the amino acid sequences of SEQ ID NOs: 1 to 3 or 5 to 12. Preferably, the tau phosphopeptide present on the liposome consists of one of the amino acid sequences of SEQ ID NOs: 27 to 29 and 31 to 38. More preferably, the tau phosphopeptide present on the liposome consists of the amino acid sequence of SEQ ID NO: 28. Preferably, the tau phosphopeptide present in the conjugate consists of one of the amino acid sequences of SEQ ID NOs: 1 to 3 or 5 to 12, preferably SEQ ID NO: 2.

[0051] As used herein, the term "tau" or "tau protein," also known as microtubule-associated protein tau, MAPT, neurofibrillary tangle protein, paired helical filament-tau, PHF-tau, MAPTL, and MTBT1, refers to a protein abundant in the central and peripheral nervous system, with multiple isoforms. In the human central nervous system (CNS), six major tau isoforms exist, ranging in size from 352 to 441 amino acids in length, due to alternative splicing (Hanger et al., Trends Mol Med. 15:112-9, 2009). Examples of tau include, but are not limited to, the longest tau isoform (4R2N) of 441 amino acids, with four repeats and two insertions, and the shortest (fetal) isoform (3R0N) of 352 amino acids, with three repeats and no insertions. Examples of tau also include the "big tau" isoform expressed in peripheral nerves, which contains 300 additional residues (exon 4a). Friedhoff et al., Biochimica et Biophysica Acta 1502 (2000) 122-132. Examples of tau include human big tau, a 758 amino acid protein encoded by a 6762 nucleotide long mRNA transcript (NM_016835.4), or its isoforms. The amino acid sequence of an exemplary human big tau is represented in GenBank accession number NP_058519.3. As used herein, the term "tau" includes tau homologs from non-human species, such as cynomolgous monkeys (Macaca Fascicularis) or chimpanzees (Pan troglodytes). As used herein, the term "tau" includes proteins containing mutations of full-length wild-type tau, such as point mutations, fragments, insertions, deletions, and splice variants. The term "tau" also encompasses post-translational modifications of the tau amino acid sequence. Post-translational modifications include, but are not limited to, phosphorylation.

[0052] As used herein, the term "phosphorylated tau" refers to tau protein or a fragment or peptide thereof that contains at least one phosphorylated residue.

[0053] As used herein, the term "enriched paired helical filaments" or "ePHFs" refers to preparations in which tau protein is enriched in paired helical filaments. PHFs are a prominent component of Alzheimer's disease neurofibrillary tangles.

[0054] As used herein, the term "peptide" or "polypeptide" refers to a polymer composed of amino acid residues linked by peptide bonds, related naturally occurring structural variants, and synthetic, non-naturally occurring analogs thereof. The term refers to peptides of any size, structure, or function. Typically, peptides are at least three amino acids in length. Peptides can be naturally occurring, recombinant, or synthetic, or any combination thereof. Synthetic peptides can be synthesized, for example, using an automated polypeptide synthesizer. Examples of tau peptides include any peptide of tau protein that is about 5 to about 30 amino acids in length, preferably about 10 to about 25 amino acids in length, and more preferably about 16 to about 21 amino acids in length. In this disclosure, peptides are listed from N to C-terminus using standard three-letter or one-letter amino acid abbreviations, with phosphate residues indicated by "p." Examples of tau peptides useful in the present invention include, but are not limited to, tau peptides comprising the amino acid sequence of any of SEQ ID NOs: 1-12, or tau peptides having an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of any of SEQ ID NOs: 1-12.

[0055] As used herein, the term "phosphopeptide" or "phosphoepitope" refers to a peptide phosphorylated at one or more amino acid residues. Examples of tau phosphopeptides include any tau peptide containing one or more phosphorylated amino acid residues. Examples of tau phosphopeptides useful in the present invention include, but are not limited to, tau phosphopeptides comprising the amino acid sequence of any of SEQ ID NOS: 1-3 or 5-12, or tau phosphopeptides having an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of any of SEQ ID NOS: 1-3 or 5-12.

[0056] The tau peptides of the present invention can be synthesized by solid-phase peptide synthesis or recombinant expression systems. Automated peptide synthesizers are commercially available from a number of suppliers, including Applied Biosystems (Foster City, CA). Recombinant expression systems can include bacteria such as E. coli, yeast, insect cells, or mammalian cells. Procedures for recombinant expression are described by Sambrook et al., Molecular Cloning: A Laboratory Manual (CSHP Press, NY 2d ed., 1989).

[0057] Tau is a human "self" protein. This means that, in principle, all lymphocytes bearing tau-specific receptors should be lost during development (central tolerance) or rendered refractory by peripheral tolerance mechanisms. This problem has proven to be a significant obstacle in the development of vaccines against self or "altered self" proteins (e.g., tumor antigens).

[0058] The production of high-quality antibodies against antigens (self or infectious) depends not only on antibody-producing B lymphocytes, but also on CD4 + It also requires the action of T "helper" lymphocytes. CD4 + T cells provide crucial survival and maturation signals to B lymphocytes, and CD4+ T cell-deficient animals are severely immunosuppressed. + T cells are susceptible to tolerance mechanisms, and a further obstacle to generating a strong anti-self (e.g., anti-tau) antibody response is the tau-reactive CD4 + It is also likely that T cells are rare or absent in the human / animal repertoire.

[0059] Without wishing to be bound by theory and in any way limiting the scope of the invention, it is believed that this problem is circumvented by the vaccine compositions of the present invention.

[0060] In one embodiment, liposomes containing tau peptides are produced that also contain T cell epitopes capable of binding to most or all of the HLA DR (human leukocyte antigen-antigen D related) molecules. The T cell epitopes are then bound to CD4 + T cells can be activated and provide essential maturation and survival signals to tau-specific B cells. In another embodiment, conjugates of tau peptides with carrier proteins are generated, which generate strong helper T cell responses. This embodiment uses "untethered recognition," in which carrier-specific T cells provide survival and maturation signals to autoreactive B cells. Thus, tau-specific B cells receive critical signals to initiate affinity maturation, immunoglobulin class switching, and establish long-term memory pools. Tau liposomes and tau conjugates can be used to generate high-quality antibodies against tau antigens in allogeneic or heterogeneous immunization schemes, using either liposomes or conjugates in the prime and / or boost phases.

[0061] Liposomes Liposomes are used in the present application in the priming composition and, optionally, in the boosting composition. Liposomes useful in the methods of the present invention include: a tau peptide, preferably a tau phosphopeptide; Helper T cell epitopes and wherein the tau peptide is displayed on the surface of the liposome.

[0062] Liposomes according to embodiments of the present invention are also referred to herein as "improved liposomes," "improved liposomal vaccines," or "liposomal vaccines according to embodiments of the present invention," or "tau liposomes," or "optimized liposomal vaccines," or "second generation liposomes."

[0063] As used herein, the term "liposome" generally refers to a lipid vesicle made from materials with a high lipid content, such as phospholipids and cholesterol. The lipids in these vesicles are generally organized in the form of lipid bilayers. The lipid bilayers are generally interspersed between multiple onion-like layers of lipid bilayers to form multilamellar lipid vesicles (MLVs), or encapsulate a volume contained within an amorphous central cavity. Lipid vesicles with an amorphous central cavity are unilamellar lipid vesicles, i.e., have a single peripheral bilayer surrounding the cavity. Large unilamellar vesicles (LUVs) generally have diameters of 100 nm to several micrometers, e.g., 100-200 nm or more, while small unilamellar lipid vesicles (SUVs) generally have diameters of less than 100 nm, e.g., 20-100 nm, typically 15-30 nm.

[0064] According to certain embodiments, the liposomes comprise one or more tau peptides. According to certain embodiments, the tau peptides in the liposomes can be the same or different.

[0065] Any suitable tau peptide known to one of skill in the art can be used in the present invention in light of the present disclosure. According to certain embodiments, one or more of the tau peptides comprises the amino acid sequence of one of SEQ ID NOs: 1-12. In other embodiments, one or more of the tau peptides comprises an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of one of SEQ ID NOs: 1-12, wherein none of the amino acid residues are phosphorylated, or one or more amino acid residues are phosphorylated.

[0066] According to certain embodiments, one or more of the tau peptides are tau phosphopeptides. According to certain embodiments, the one or more tau phosphopeptides comprise the amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, or an amino acid sequence at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, wherein one or more of the indicated amino acid residues are phosphorylated. Preferably, the tau phosphopeptide comprises the amino acid sequence of one of SEQ ID NOs: 1-3. The tau peptide may be amidated at the C-terminus.

[0067] According to an embodiment of the present application, tau peptides are presented on the surface of liposomes. Tau peptides, preferably tau phosphopeptides, can be presented on the surface of liposomes using methods known in the art in light of the present disclosure. 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 certain embodiments, one or more tau peptides, including phosphopeptides, further comprise one or more modifications, such as palmitoylation or dodecyl modification, that enable the tau peptides to be presented on the surface of liposomes. To facilitate the modification, additional amino acid residues, such as Lys, Cys, or, in some cases, Ser or Thr, can be added to the tau peptide. It has been reported that the position of the lipid anchor induces various conformations of the peptide sequence (Hickman et al., J. Biol. Chem. vol. 286, NO. 16, pp. 13966-13976, April 22, 2011). Without wishing to be bound by theory, it is believed that adding hydrophobic moieties to both termini can increase the pathological beta-sheet conformation of tau peptides. Thus, one or more tau peptides further comprise hydrophobic moieties at both termini. The modified tau peptide may be C-terminally amidated. Preferably, the tau peptide displayed on the surface of the liposome consists of one of the amino acid sequences set forth in SEQ ID NOs: 27 to 38. More preferably, the peptide is a tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 29 or SEQ ID NOs: 31 to 38.

[0068] As used herein, the term "helper T cell epitope" refers to a polypeptide that contains an epitope capable of recognition by a helper T cell. 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 toxin B, toxoid, diphtheria toxoid, measles virus F protein, Chlamydia trachomatis major outer membrane protein, Plasmodium falciparum circumsporozite T, P. falciparum CS antigen, Schistosoma mansoni triosephosphate isomerase, Bordetella pertussis, Clostridium tetani, Pertusaria trachythallina, Escherichia coli, coli TraT, and influenza virus hemagglutinin (HA).

[0069] Any suitable helper T cell epitope known to those of skill in the art can be used in the present invention in light of the present disclosure. According to certain embodiments, the helper T cell epitope comprises at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 23 to 26. Preferably, the helper T cell epitope comprises two or more of the amino acid sequences of SEQ ID NOs: 23 to 26 fused together via a linker, such as a peptide linker comprising one or more amino acids, e.g., Val (V), Ala (A), Arg (R), Gly (G), Ser (S), or Lys (K). The length of the linker can vary, preferably from 1 to 5 amino acids. Preferably, the helper T cell epitope comprises three or more of the amino acid sequences of SEQ ID NOs: 23 to 26 fused together via one or more linkers selected from the group consisting of VVR, GS, RR, and RK. The helper T cell epitope may be amidated at its C-terminus.

[0070] According to an embodiment of the present application, helper T cell epitopes can be incorporated onto the liposome surface, for example, by tethering them via a covalently attached hydrophobic moiety, where the hydrophobic moiety is an alkyl group, a fatty acid, a triglyceride, a diglyceride, a steroid, a sphingolipid, a glycolipid, or a phospholipid, particularly an alkyl group or a fatty acid, particularly one having a carbon backbone 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, and particularly at least 16 carbon atoms. In one embodiment of the present invention, the hydrophobic moiety is palmitic acid. Alternatively, the helper T cell epitope can be encapsulated in the liposome. According to a specific embodiment, the helper T cell epitope is encapsulated in the liposome.

[0071] The helper T cell epitope can be modified at its desired location in the liposome using methods known in the art in light of this disclosure. According to certain embodiments, the helper T cell epitope useful in the present invention comprises the amino acid sequence of one of SEQ ID NOs: 39 to 44. Preferably, the helper T cell epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17.

[0072] According to certain embodiments, the liposome comprises tau peptide and helper T cell epitope in a weight ratio of 1:1, 2:1, 3:1, 4:1, 5:1, or 6:1.

[0073] In one embodiment, the liposome further comprises at least one adjuvant comprising a Toll-like receptor ligand. Thus, in another general aspect, the present invention provides a liposome comprising: a tau peptide, preferably a tau phosphopeptide; a helper T cell epitope, i. Toll-like receptor 9 ligand, and Toll-like receptor 4 ligand with at least one of The present invention relates to a liposome comprising:

[0074] As used herein, the term "Toll-like receptor" or "TLR" refers to a class of pattern recognition receptor (PRR) proteins that play a major role in the innate immune response. TLRs recognize pathogen-associated molecular patterns (PAMPs) from microbial pathogens, such as bacteria, fungi, parasites, and viruses, which can be distinguished from host molecules. TLRs are transmembrane proteins that typically 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, namely, TLR1-TLR10, and at least 12 murine TLR family members, namely, TLR1-TLR9 and TLR11-TLR13, which differ in the types of antigens they recognize. For example, TLR4 recognizes lipopolysaccharide (LPS), a component present in many Gram-negative bacteria, as well as viral proteins, polysaccharides, and endogenous proteins such as low-density lipoproteins, beta-defensins, and heat shock proteins. TLR9 is a nucleotide-sensing TLR activated by unmethylated cytosine-phosphate-guanine (CpG) single- or double-stranded dinucleotides, which are abundant in prokaryotic genomes but rare in vertebrate genomes. TLR activation leads to a series of signaling events that result in the production of type I interferons (IFNs), inflammatory cytokines, and chemokines, and the induction of immune responses. Ultimately, this inflammation also activates the adaptive immune system, resulting in the subsequent elimination of invading pathogens and infected cells.

[0075] 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 certain embodiments, the Toll-like receptor ligand is a Toll-like receptor agonist.

[0076] 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, an agonist can induce, stimulate, increase, activate, promote, enhance, or upregulate the activity of the receptor. Such activity is referred to as "agonism." For example, a TLR4 or TLR9 agonist can activate or increase cell signaling through the bound 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 receptors. Agonists can mimic the activity of natural receptor ligands. Agonists can be homologous to these natural receptor ligands in terms of sequence, conformation, charge, or other characteristics so that they can be recognized by the receptor. This recognition can result in physiological and / or biochemical changes within the cell such that the cell responds to the presence of the agonist in the same manner as if the natural receptor ligand were present. According to certain embodiments, the Toll-like receptor agonist is at least one of a Toll-like receptor 4 agonist and a Toll-like receptor 9 agonist.

[0077] As used herein, the term "Toll-like receptor 4 agonist" refers to any compound that acts as an agonist of TLR4. Any suitable Toll-like receptor 4 agonist known to one of skill in the art can be used in the present invention in light of the present disclosure. Examples of Toll-like receptor 4 ligands useful in the present invention include TLR4 agonists, including, but not limited to, monophosphoryl lipid A (MPLA). As used herein, the term "monophosphoryl lipid A" or "MPLA" refers to a modified form of lipid A, the biologically active portion of Gram-negative bacterial lipopolysaccharide (LPS) endotoxin. MPLA is less toxic than LPS while retaining 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'-monophosphoryl lipid A, monophosphoryl hexa-acyl lipid A, 3-deacylated, monophosphoryl 3-deacylated lipid A, and structurally related variants thereof. MPLA useful in the present 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 certain embodiments, the Toll-like receptor 4 agonist is MPLA.

[0078] As used herein, the term "Toll-like receptor 9 agonist" refers to any compound that acts as an agonist of TLR9. Any suitable Toll-like receptor 9 agonist known to those skilled in the art can be used in the present invention in light of this disclosure. Examples of Toll-like receptor 9 ligands useful in the present invention include TLR9 agonists, including, but not limited to, CpG oligonucleotides.

[0079] As used herein, the term "CpG oligonucleotide," "CpG oligodeoxynucleotide," or "CpG ODN" refers to an oligonucleotide containing at least one CpG motif. As used herein, "oligonucleotide," "oligodeoxynucleotide," or "ODN" refers to a polynucleotide formed from a plurality of linked nucleotide units. Such oligonucleotides can be obtained from existing nucleic acid sources or can be produced by synthetic methods. As used herein, the term "CpG motif" refers to a nucleotide sequence containing unmethylated cytosine-phosphate-guanine (CpG) dinucleotides (i.e., cytosine (C) followed by guanine (G)) linked by a phosphate bond or a phosphodiester backbone or other internucleotide bond.

[0080] According to certain embodiments, the CpG oligonucleotide is lipidated, ie, conjugated (covalently linked) to a lipid moiety.

[0081] As used herein, the term "lipid moiety" refers to a moiety containing a lipophilic structure. When attached to highly hydrophilic molecules such as nucleic acids, lipid moieties such as alkyl groups, fatty acids, triglycerides, diglycerides, steroids, sphingolipids, glycolipids, or phospholipids, particularly sterols such as cholesterol or fatty acids, can substantially enhance plasma protein binding and consequently the circulatory half-life of hydrophilic molecules. Furthermore, binding to specific plasma proteins such as lipoproteins has been shown to increase uptake into specific tissues expressing the corresponding lipoprotein receptors (e.g., LDL receptor, HDL receptor, or scavenger receptor SR-B1). In particular, lipid moieties conjugated with phosphopeptides and / or CpG oligonucleotides allow the peptides and / or oligonucleotides to be tethered to the membrane of liposomes via the hydrophobic moiety.

[0082] According to certain embodiments, in view of the present disclosure, the CpG oligonucleotide can include any suitable internucleotide linkage.

[0083] As used herein, the term "internucleotide bond" refers to a chemical bond that connects two nucleotides through their sugars, consisting of a phosphorus atom and a charged or neutral group between adjacent nucleosides. Examples of internucleotide bonds include phosphodiester (po), phosphorothioate (ps), phosphorodithioate (ps2), methylphosphonate (mp), and methylphosphorothioate (rp). Phosphorothioate, phosphorodithioate, methylphosphonate, and methylphosphorothioate are stabilized internucleotide bonds, while phosphodiester is a naturally occurring internucleotide bond. Oligonucleotide phosphorothioates are typically synthesized as a random racemic mixture of Rp and Sp phosphorothioate linkages.

[0084] Any suitable CpG oligonucleotide known to those skilled in the art can be used in the present invention in light of the present disclosure. Examples of such CpG oligonucleotides include, but are not limited to, CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336.

[0085] CpG oligonucleotides can be lipidated using methods known in the art in light of the present disclosure. In some embodiments, the 3' end of the CpG oligonucleotide is covalently linked to a cholesterol molecule via a phosphate bond, optionally via a PEG linker. Other lipophilic moieties can also be covalently linked to the 3' end of the CpG oligonucleotide. For example, the CpG oligonucleotide can be covalently linked, optionally via a PEG linker, to a lipid anchor of the same length as the phospholipid from the liposome, i.e., one palmitic acid chain (using Pal-OH or similar, activated for coupling) or two palmitic acids (e.g., 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) or similar, activated for coupling). See, for example, the related disclosure in U.S. Pat. No. 7,741,297, the contents of which are incorporated herein by reference. The length of the PEG can vary, for example, from 1 to 5 PEG units.

[0086] Other linkers can also be used to covalently attach CpG oligonucleotides to lipophilic moieties (such as cholesterol molecules), examples of which include, but are not limited to, alkyl spacers having 3 to 12 carbons. A short linker compatible with oligonucleotide chemistry, such as an aminodiol, is required. In some embodiments, no linker is used for covalent attachment. See, for example, Ries et al., "Convenient synthesis 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.

[0087] According to certain embodiments, lipidated CpG oligonucleotides useful in the present invention comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the nucleotide sequence comprises one or more phosphorothioate internucleotide linkages and 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 the cholesterol molecule. Preferably, the linker comprises polyethylene glycol (PEG).

[0088] According to certain embodiments, the liposomes comprise: Tau phosphopeptide, a helper T cell epitope, a lipidated CpG oligonucleotide; Toll-like receptor 4 ligand and wherein the tau phosphopeptide is displayed on the surface of the liposome and the helper T cell epitope is encapsulated within the liposome.

[0089] According to certain embodiments, the liposomes comprise: a tau peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 29 or SEQ ID NOs: 31 to 38; a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 44, preferably a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17; a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, containing one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker; Monophosphoryl lipid A (MPLA) and Includes.

[0090] According to certain embodiments, the liposome further comprises one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol.

[0091] According to certain embodiments, the liposome further comprises a buffer. Any suitable buffer known to those skilled in the art can be used in the present invention in light of this disclosure. In one embodiment, the liposome comprises phosphate buffered saline. According to certain embodiments, the buffer comprises histidine and sucrose.

[0092] According to certain embodiments, the liposome comprises DMPC, DMPG, cholesterol, tau phosphopeptide, and helper T cell epitope in a molar ratio of 9:1:7:0.07:0.04.

[0093] Liposomes of the present invention can be made using methods known in the art in light of this disclosure.

[0094] An exemplary liposome of the present application comprises a tau tetrapalmitoylated phosphopeptide (pTau peptide T3, SEQ ID NO: 28) displayed on the surface of the liposome via two palmitic acids at each end of the tau peptide, a TLR-9 ligand incorporated within the liposome membrane via covalently attached cholesterol, including lipidated CpG (adjuvant CpG7909-Chol), a TLR-4 ligand incorporated within the membrane (adjuvant 3D-(6-acyl)PHAD®), and an encapsulated helper T cell epitope (PAN-DR binder T50).

[0095] Conjugates The conjugates are used in the boosting compositions of the present application. The conjugates useful in the methods of the present invention comprise a tau peptide, preferably a tau phosphopeptide, conjugated thereto with an immunogenic carrier.

[0096] According to a particular embodiment, the conjugate has the following structure:

[0097] [ka] or a structure of formula (II):

[0098] [ka] [In the formula, x is an integer from 0 to 10, n is an integer of 2 to 15, preferably 3 to 11; Carrier refers to an immunogenic carrier, Tau peptide refers to tau phosphopeptide.] It has.

[0099] According to certain embodiments, x is an integer from 1 to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, or from 2 to 3. According to certain embodiments, x is 3.

[0100] According to certain embodiments, n is 2-15, 3-11, 3-9, 3-8, or 3-7.

[0101] According to certain embodiments, the conjugate comprises one or more tau peptides. According to certain embodiments, the tau peptides of the conjugate may be the same or different.

[0102] According to certain embodiments, any suitable tau peptide may be used in the present invention in light of the present disclosure. According to certain embodiments, one or more of the tau peptides comprises the amino acid sequence of one of SEQ ID NOs: 1-12, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of one of SEQ ID NOs: 1-12, wherein none, one, or more of the amino acid residues are phosphorylated.

[0103] According to certain embodiments, one or more of the tau peptides is a tau phosphopeptide. According to certain embodiments, the one or more tau phosphopeptides comprise the amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, or an amino acid sequence that is at least 75%, 80%, 85%, 90%, or 95% identical to the amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, wherein one or more of the indicated amino acid residues are phosphorylated.

[0104] According to certain embodiments, the tau phosphopeptide consists of the amino acid sequence of one of SEQ ID NOs: 1-3.

[0105] As used herein, the term "immunogenic carrier" refers to an immunogenic substance that can be coupled to a tau peptide. An immunogenic moiety coupled to a tau peptide can induce an immune response and elicit the production of antibodies capable of specifically binding to the tau peptide. An immunogenic moiety is an operable moiety, including proteins, polypeptides, glycoproteins, complex polysaccharides, particles, nucleic acids, polynucleotides, etc., that is recognized as foreign and thus elicits an immune response from the host. Any suitable immunogenic carrier known to those skilled in the art can be used in the present invention in light of the present disclosure. According to certain embodiments, the immunogenic carrier is keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197 (a non-toxic form of diphtheria toxin), outer membrane protein mixture (OMP) from Neisseria meningitidis, or a derivative thereof. According to certain embodiments, the immunogenic carrier is KLH or CRM197.

[0106] According to certain embodiments, the tau peptide is conjugated to the carrier via a linker. As used herein, the term "linker" refers to a chemical moiety that connects the immunogenic carrier to the tau peptide. Any suitable linker known to those skilled in the art can be used in the present invention in light of the present disclosure. The linker can be, for example, a single covalent bond, a substituted or unsubstituted alkyl, a substituted or unsubstituted heteroalkyl moiety, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, a cleavable linker such as a disulfide bond or a protease cleavage site, an amino acid, or a combination thereof. Exemplary linkers can include one or more of polyethylene glycol (PEG), succinimidyl 3-(bromoacetamido)propionate (SBAP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), one or more amino acids such as Cys, Lys, or, in some cases, Ser or Thr, or a combination thereof.

[0107] According to certain embodiments, the linker comprises (C2H4O)x-cysteine-acetamidopropionamide or m-maleimidobenzoyl-N-hydroxysuccinimide ester-cysteine-(C2H4O)x, where x is an integer between 0 and 10, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.

[0108] According to certain embodiments, the carrier is covalently attached to the N-terminus of the tau peptide via a linker.

[0109] According to other particular embodiments, the carrier is covalently attached to the C-terminus of the tau peptide via a linker.

[0110] According to certain embodiments, the conjugate has the following structure:

[0111] [ka] [In the formula, n is an integer of 2 to 15, preferably 3 to 11, and more preferably 3 to 7.] It has.

[0112] The conjugates of the present invention can be made by methods known in the art in light of this disclosure. For example, the conjugates can be made by reacting succinimidyl-3-(bromoacetamido)propionate (SBAP):

[0113] [ka] with the amino group of CRM197 to form an amide bond. This CRM197 precursor can then be reacted with a tau peptide (e.g., a phosphorylated tau peptide of SEQ ID NO: 2) conjugated to a PEG-cysteine ​​linker bearing a free nucleophilic thiol group at its N-terminus or its C-terminus to form a tau phosphopeptide conjugate.

[0114] An exemplary conjugate according to one embodiment of the present application comprises multiple tau phosphopeptides (pTau peptide T3.76) covalently linked to the carrier protein CRM197.

[0115] Pharmaceutical Composition The liposomes and conjugates are administered to a subject in a pharmaceutical composition. A pharmaceutical composition is a composition containing a therapeutically effective amount of liposomes or a therapeutically effective amount of the conjugate of the present invention, respectively, together with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include excipients and / or carriers known in the art (see Remington's Pharmaceutical Science (15th ed.), Mack Publishing Company, Easton, Pa., 1980). The preferred formulation of a pharmaceutical composition depends on the intended mode of administration and therapeutic application. The composition may contain a pharmaceutically acceptable, non-toxic carrier or diluent, defined as a vehicle commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solution, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, etc. It will be understood that the characteristics of the carrier, excipient, or diluent will depend on the route of administration for a particular application, for example, intramuscular, subcutaneous, oral, intradermal, cutaneous, intramucosal (e.g., intestinal), intranasal, or intraperitoneal routes. Preferably, the pharmaceutically acceptable carrier included in the pharmaceutical composition is suitable for intramuscular administration.

[0116] Pharmaceutical compositions can be formulated as vaccines (also called "immunogenic compositions"), such as priming or boosting compositions, by methods well known in the art.

[0117] A pharmaceutical composition can contain a mixture of the same immunogenic tau peptide, or alternatively, a pharmaceutical composition can contain a mixture of different immunogenic tau peptides of the invention.

[0118] Another problem associated with vaccines against neurological diseases is the potentially necessary need 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 the passage of substances from the circulation into the brain, thereby preventing toxins, microorganisms, and other substances from entering the central nervous system. The BBB also has the potentially more undesirable effect of preventing the efficient entry of immune mediators (such as antibodies) into the interstitial and cerebrospinal fluids that surround the brain.

[0119] Approximately 0.1% of antibodies present in the systemic circulation cross the BBB and enter the brain, meaning that the systemic titers induced by vaccines targeting CNS antigens must be at least 1000-fold higher than the minimal effective titer to be effective in the brain.

[0120] According to certain embodiments, the pharmaceutical compositions of the present invention further comprise one or more suitable adjuvants. Therefore, the tau peptides of the present invention present in liposomes or conjugates can be administered in combination with a suitable adjuvant to achieve a desired immune response in a subject. A suitable adjuvant can be administered before, after, or simultaneously with the administration of the liposomes or conjugates of the present invention. A preferred adjuvant enhances the response to the intrinsic immunogen without causing conformational changes in the immunogen that affect the qualitative form of the response. Examples of adjuvants include aluminum salts (alum), 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. Such adjuvants can be used with or without other specific immunostimulants, such as the MPLA class (3 De-O-acylated monophosphoryl lipid A (MPL™), monophosphoryl hexa-acyl lipid A, 3-deacylated synthetic (3D-(6-acyl) PHAD™), PHAD™, PHAD™-504, 3D-PHAD™ lipid A, polymeric or monomeric amino acids such as polyglutamic acid or polylysine, etc. Such adjuvants can be used with or without other specific immunostimulants, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr)), or other adjuvants 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-isoglutaminyl-L-alanine-2-(1'-2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucosaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™), or other bacterial cell wall components.Oil-in-water emulsions include MF59 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) (see WO 90 / 14837), SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic block polymer L121, and thr-MDP (either microfluidized into a submicron emulsion or vortexed to produce a larger particle size emulsion), Ribi™ Adjuvant System (RAS) (Ribi ImmunoChem, Hamilton, Montana) 0.2% Tween 80 and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL™), trehalose dimycolate (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).

[0121] As used herein, the term "therapeutically effective amount" refers to that amount of an active ingredient or component that elicits a desired biological or pharmaceutical response in a subject. A therapeutically effective amount can be determined empirically and routinely in connection with the stated purpose. For example, in vitro assays can optionally be used to help identify optimal dosage ranges. Selection of a specific effective dose can be determined (e.g., through clinical trials) by one of ordinary skill in the art based on consideration of several factors, 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 those of skill in the art. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the disease, and should be determined according to the judgment of the practitioner and each patient's circumstances. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model test systems.

[0122] In certain embodiments, a "therapeutically effective amount" is an "immunologically effective amount," which refers to an amount of a composition sufficient to induce a desired immune effect or immune response in a subject in need thereof. In one embodiment, an immunogenically effective amount refers to an amount sufficient to induce an immune response in a subject in need thereof. In another embodiment, an immunogenically effective amount refers to an amount sufficient to induce immunity in a subject in need thereof, e.g., to provide a therapeutic effect against a neurodegenerative disease, disorder, or condition. An immunogenically effective amount can vary depending on various factors, such as the physical condition, age, weight, health status, etc. of the subject. An immunogenically effective amount can be readily determined by one of skill in the art in light of the present disclosure.

[0123] In one embodiment, the immunogenic composition is a priming composition used to prime an immune response, administered before administration of a boosting composition. According to an embodiment of the present invention, the priming composition comprises an immunologically effective amount of liposomes described herein. According to an embodiment of the present invention, the immunogenic composition is a boosting composition used to boost an immune response, administered after administration of a priming composition. According to an embodiment of the present invention, the boosting composition comprises an immunologically effective amount of a conjugate described herein. According to an embodiment of the present application, an immunogenic composition comprising an immunologically effective amount of liposomes described herein can be used as both a priming composition for priming an immune response and a boosting composition for boosting an immune response. A priming composition can be used in combination with one or more boosting compositions. A boosting composition can be administered two or more times.

[0124] As used herein, the term "in combination" in the context of administering two or more therapies to a subject refers to the use of more than one therapeutic agent. The use of the term "in combination" does not restrict the order in which the therapeutic agents are administered to a subject. For example, a first therapeutic agent (e.g., a liposome composition described herein) can be administered prior to (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, 12 weeks, or more before), simultaneously with, or subsequent to (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, 12 weeks, or more after) administration of a second therapeutic agent (e.g., a conjugate composition described herein) to a subject.

[0125] The pharmaceutical compositions of the present invention can be formulated by methods well known in the art. The optimal ratio of each component in the composition can be determined by techniques well known to those skilled in the art in light of the present disclosure.

[0126] In certain embodiments, the pharmaceutical composition comprises a liposome described herein and a buffer comprising one or more amino acids, such as histidine or glycine, and / or one or more carbohydrates, such as glucose or sucrose.

[0127] In other embodiments, the pharmaceutical composition comprises a conjugate described herein and a buffer comprising one or more amino acids, such as histidine or glycine, one or more carbohydrates, such as glucose or sucrose, and / or a surfactant, such as polysorbate 80, polysorbate 20.

[0128] How to use The present invention provides methods for priming and boosting immune responses against tau protein in subjects suffering from neurodegenerative disorders using liposomal vaccines containing tau peptides and conjugate vaccines comprising tau peptides conjugated to immunogenic carriers.

[0129] In one general aspect, a method of inducing an immune response against tau protein in a subject suffering from a neurodegenerative disorder comprises: Target, a first tau phosphopeptide; and a helper T cell epitope, a lipidated CpG oligonucleotide; Adjuvants containing Toll-like receptor 4 ligands administering a priming composition comprising an immunologically effective amount of liposomes, wherein the tau phosphopeptide is displayed on the surface of the liposomes, and further comprising a pharmaceutically acceptable carrier; The subject is administered a second tau phosphopeptide comprising an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of formula (I):

[0130] [ka] or a structure of formula (II):

[0131] [ka] [In the formula, x is an integer from 0 to 10, preferably from 2 to 6, and most preferably 3; n is an integer of 3 to 15, preferably 3 to 12; the carrier represents an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and outer membrane protein mixture (OMP) from N. meningitidis, or a derivative thereof; Tau peptide represents the second tau phosphopeptide.] an immunologically effective amount of a conjugate having administering a first boosting composition further comprising a pharmaceutically acceptable carrier; Including, The first tau phosphopeptide and the second tau phosphopeptide each independently have an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3 and SEQ ID NO:5 to SEQ ID NO:12.

[0132] In certain embodiments, an immunologically effective amount of the conjugate is administered with one or more adjuvants, such as those described herein. In one embodiment, an immunologically effective amount of the conjugate is administered with one or more aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate, and / or one or more CpGs, such as CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336.

[0133] In certain 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 they share at least one common epitope.

[0134] In certain embodiments, the method further comprises administering to the subject a second boosting composition comprising an immunogenically effective amount of liposomes.

[0135] According to a particular embodiment, the immune response is induced by antibodies against phosphorylated tau protein, preferably ePHF.

[0136] In certain embodiments, the first boosting composition is administered about 27-32 days after the priming composition, e.g., about 27, 28, 29, 30, 31, or 32 days after administration. In certain embodiments, the first boosting composition is re-administered about 82-87 days after the priming composition, e.g., about 82, 83, 84, 85, 86, or 87 days after administration. In certain embodiments, the second boosting composition comprising an immunogenically effective amount of liposomes is administered about 167-172 days after the priming composition, e.g., about 167, 168, 169, 170, 171, or 172 days after administration.

[0137] In certain embodiments, the first boosting composition is administered about 82-87 days after the priming composition, e.g., about 82, 83, 84, 85, 86, or 87 days after administration. In certain embodiments, the second boosting composition is administered about 27-32 days after the priming composition, e.g., about 27, 28, 29, 30, 31, or 32 days after administration. In certain embodiments, the first boosting composition is re-administered about 167-172 days after the priming composition, e.g., about 167, 168, 169, 170, 171, or 172 days after administration.

[0138] Those skilled in the art will be able to vary the exact timing of the priming and boosting compositions, their frequency of administration, their dosages, etc. based on the teachings herein and clinical experience.

[0139] Any of the priming and boosting compositions described herein can be used in methods of inducing an immune response against tau protein in a subject suffering from a neurodegenerative disorder. Embodiments of priming compositions, boosting compositions, liposomes, and / or conjugates, etc. that can be used in the methods of the invention are discussed in detail above and in the illustrative examples below.

[0140] As used herein, the terms "induce" and "stimulate" and variations thereof refer to any amount of measurable increase in cellular activity. Induction of an immune response can include, for example, activation, proliferation, or maturation of immune cell populations, increased cytokine production, and / or other indicators of increased immune function. In certain embodiments, induction of an immune response can include increased proliferation of B cells, production of antigen-specific antibodies, increased proliferation of antigen-specific T cells, improved dendritic cell antigen presentation, and / or increased expression of certain cytokines, chemokines, and costimulatory markers.

[0141] The ability to induce or stimulate an anti-tau immune response upon administration to an animal or human organism can be assessed either in vitro or in vivo using a variety of assays that are standard in the art. For a general description of techniques available for assessing the initiation and activation of immune responses, see, for example, Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & Sons Inc, National Institutes of Health). Measurement of cellular immunity can be performed by methods readily known in the art, for example, by measuring CD4 + and CD8 + This can be done by measuring the cytokine profile secreted by activated effector cells, including those derived from T cells (e.g., quantification of IL-4 or IFN-gamma producing cells by ELISPOT), by determining the activation state of immune effector cells (e.g., classical [H]thymidine incorporation T cell proliferation assays), or by assaying antigen-specific T lymphocytes in sensitized subjects (e.g., peptide-specific lysis in cytotoxicity assays).

[0142] The ability to stimulate a cellular and / or humoral response can be determined by testing a biological sample (e.g., blood, plasma, serum, PBMCs, urine, saliva, feces, CSF, or lymph) from the subject for the presence of antibodies directed against the immunogenic tau peptide administered in the pharmaceutical composition (see, e.g., Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, the titer of antibodies produced in response to administration of a composition providing an immunogen can be measured by enzyme-linked immunosorbent assay (ELISA), Mesoscale Discovery (MSD), dot blot, SDS-PAGE gel, ELISPOT, or antibody-dependent cellular phagocytosis (ADCP) assay.

[0143] The present invention provides methods of treating or preventing neurodegenerative diseases or disorders in a subject in need thereof using liposomal vaccines containing tau peptides and conjugate vaccines comprising tau peptides conjugated to an immunogenic carrier.

[0144] Those skilled in the art will be able to vary the exact timing of the boosting composition, prime and boosting compositions, their frequency of administration, their doses, etc. based on the teachings herein and clinical experience.

[0145] Any of the priming and boosting compositions described herein can be used in methods of treating or preventing a neurodegenerative disease or disorder in a subject in need thereof. Embodiments of priming compositions, boosting compositions, liposomes, and / or conjugates, etc. that can be used in the methods of the invention are discussed in detail above and in the illustrative examples below.

[0146] As used herein, the term "subject" refers to an animal. According to certain embodiments, the subject is a mammal, including a non-primate (e.g., a camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, or mouse) or a primate (e.g., a monkey, chimpanzee, or human). According to certain embodiments, the subject is a human.

[0147] As used herein, the terms "treat," "treating," and "treatment" are all intended to refer to the amelioration or reversal of at least one measurable physical parameter associated with a neurodegenerative disease, disorder, or condition, which need not necessarily be discernible in the subject, but which can be discernible in the subject. The terms "treat," "treating," and "treatment" can also refer to causing regression, preventing progression, or at least slowing progression of the disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the alleviation, prevention of the onset or development, or shortening of the duration of one or more symptoms associated with a neurodegenerative disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the prevention of recurrence of the disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to increasing the survival of a subject having a disease, disorder, or condition. In certain embodiments, "treat," "treating," and "treatment" refer to the elimination of the disease, disorder, or condition in a subject.

[0148] According to certain embodiments, a therapeutically effective amount refers to an amount of a therapeutic agent sufficient to achieve one, two, three, four, or more of the following effects: (i) reducing the severity or ameliorating the disease, disorder, or condition being treated, or the symptoms associated therewith; (ii) shortening the duration of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iii) preventing the progression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (iv) causing the regression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (v) preventing the onset or progression of the disease, disorder, or condition being treated, or the symptoms associated therewith; (vi) preventing the recurrence of the disease, disorder, or condition being treated, or symptoms associated therewith; (vii) reducing the hospitalization of a subject having the disease, disorder, or condition being treated, or symptoms associated therewith; (viii) reducing the length of hospitalization of a subject having the disease, disorder, or condition being treated, or symptoms associated therewith; (ix) increasing the survival of a subject having the disease, disorder, or condition being treated, or symptoms associated therewith; (xi) inhibiting or alleviating the disease, disorder, or condition being treated, or symptoms associated therewith, in a subject; and / or (xii) enhancing or improving the prophylactic or therapeutic efficacy of another therapeutic agent.

[0149] As used herein, "neurodegenerative disease, disorder, or condition" includes any neurodegenerative disease, disorder, or condition known to one of skill in the art in light of the present disclosure. Examples of neurodegenerative diseases, disorders, or conditions include neurodegenerative diseases or disorders caused by or associated with the formation of neurofibrillary lesions, such as the tau-related diseases, disorders, or conditions referred to as tauopathies. According to certain embodiments, neurodegenerative diseases, disorders, or conditions include, but are not limited to, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia pugilistica, Down's syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Parkinsonism-dementia complex of Guam, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcifications, and the like. Included are any of the diseases or disorders exhibiting coexistence of tau and amyloid pathology, including tangles, frontotemporal dementia, preferably frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobe dementia, Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), cerebrovascular disease, or Lewy body dementia (LBD). According to certain embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease or another tauopathy.

[0150] The clinical course of Alzheimer's disease can be divided into multiple stages based on the progression pattern of cognitive and functional impairment.Stages can be defined using assessment scales known in the art, including, for example, the NIA-AA diagnostic criteria.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, the entire contents of each of which are incorporated herein by reference.

[0151] According to a preferred embodiment, the neurodegenerative disease, disorder, or condition is early Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.

[0152] In some embodiments, the subject in need of treatment is amyloid-positive in the brain, but has not yet shown significant cognitive impairment.Amyloid deposits in the brain can be detected using methods known in the art, such as PET scan, immunoprecipitation mass spectrometry, or other methods.

[0153] The present invention also provides a method for promoting clearance of tau aggregates from the brain of a subject, comprising administering to the subject a pharmaceutical composition according to an embodiment of the present invention under conditions effective to promote clearance of tau aggregates from the brain of the subject. According to certain embodiments, the tau aggregates are neurofibrillary tangles or pathological tau precursors thereof.

[0154] The present invention also provides a method of delaying the progression of a behavioral phenotype associated with tau pathology in a subject, comprising administering to the subject a pharmaceutical composition according to an embodiment of the present invention under conditions effective to delay the progression of the behavioral phenotype associated with tau pathology in the subject.

[0155] In a preferred embodiment of the invention, administration of tau peptides via administration of a pharmaceutical composition according to one embodiment of the invention induces an active immune response in a subject against tau peptides and pathological forms of tau, thus facilitating clearance of associated tau aggregates, slowing the progression of behaviors associated with tau pathology, and / or treating the underlying tauopathy. According to this aspect of the invention, the immune response comprises the beneficial generation of a humoral (antibody-mediated) response directed against the tau peptide and a cellular (mediated by antigen-specific T cells or their secreted products) response directed against the T cell epitope or immunogenic carrier.

[0156] As used herein, behavioral phenotypes associated with tau pathology include, but are not limited to, cognitive impairment, early personality changes and disinhibition, apathy, apraxia, mutism, apraxia, perseveration, stereotyped movements / behaviors, hyper-mouthiness, confusion, inability to plan or structure sequential tasks, egocentric / numbness, antisocial traits, lack of empathy, stammering, agrammatic speech with frequent erroneous errors but relatively preserved comprehension, impaired comprehension and lexical finding deficits, slowly progressive gait unsteadiness, retropulsion, freezing, frequent falls, levodopa-refractory axial rigidity, supranuclear discontinuation palsy, square wave eye movements, slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory loss, and tremor.

[0157] In carrying out the method of the present invention, it is preferable to select subjects who are suffering from or at risk of suffering from Alzheimer's disease or other tauopathies, who have tau aggregates in their brains, or who exhibit tangle-related behavioral phenotypes before administering the immunogenic peptide or antibody of the present invention. Subjects amenable to treatment include individuals who are at risk of the disease but do not show symptoms, as well as patients who currently show symptoms. In the case of Alzheimer's disease, virtually everyone is at risk of developing Alzheimer's disease. Therefore, the method can be administered prophylactically to the general population without any risk assessment of the subject patient. The method is particularly useful for individuals with a known genetic risk of Alzheimer's disease. Such individuals include those who have relatives who have experienced the disease and those whose risk has been determined by analysis of genetic or biochemical markers.

[0158] In asymptomatic patients, treatment can begin at any age (e.g., 10, 20, or 30 years old). However, it is usually not necessary to begin treatment until the patient reaches 40, 50, 60, or 70 years of age. Treatment typically involves multiple doses over a period of time. Treatment can be monitored by assaying the response of activated T cells or B cells to the antibody or therapeutic agent over time. If the response decreases, a booster dose is indicated.

[0159] In prophylactic applications, pharmaceutical compositions containing tau peptides are administered to patients susceptible to or otherwise at risk of Alzheimer's disease or other tauopathies in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the onset of the disease, including biochemical, histological, and / or behavioral symptoms of the disease, its complications, and intermediate pathological phenotypes that present during the development of the disease. In therapeutic applications, pharmaceutical compositions containing tau peptides are administered to patients suspected of or already suffering from such diseases in an amount sufficient to cure or at least partially arrest the symptoms of the disease (biochemical, histological, and / or behavioral), including its complications and intermediate pathological phenotypes in the development of the disease.

[0160] The effective dose of the pharmaceutical composition of the present invention for preventing and / or treating a neurodegenerative disease, disorder, or condition will vary depending on many different factors, including the mode of administration, the target site, the patient's physiological condition, other administered pharmaceuticals, and whether the treatment is prophylactic or therapeutic. The amount of the pharmaceutical composition will depend on whether an adjuvant is also administered, with higher doses being required in the absence of an adjuvant. In the methods of the present invention, a subject is administered at least one priming composition and at least one boosting composition. The antigens in each priming and boosting composition need not be identical, regardless of how many boosting compositions are used, but should share antigenic determinants or be substantially similar to each other.

[0161] Those skilled in the art will readily appreciate that the regimen for priming and boosting can be adjusted based on the measured immune response after administration. For example, the boosting composition is generally administered several weeks or months after administration of the priming composition, e.g., 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 1-2 years after administration of the priming composition.

[0162] The pharmaceutical composition can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intracranially, intraperitoneally, intradermally, intranasally, or intramuscularly for prophylactic and / or therapeutic treatment. The most typical route of administration of the immunogenic agent is subcutaneous or intramuscular injection. This latter type of injection is most typically performed intramuscularly in the arm or leg.

[0163] The composition can be presented in a kit, pack, or dispenser, which can contain one or more unit dosage forms containing the active ingredient, if desired. The kit can, for example, comprise a metal or plastic foil such as a blister pack. The kit, pack, or dispenser can be accompanied by instructions for administration.

[0164] According to a particular embodiment, the kit comprises a pharmaceutical composition comprising a liposome according to an embodiment of the invention and a pharmaceutical composition comprising a conjugate according to an embodiment of the invention.

[0165] Embodiment The present invention also provides the following non-limiting embodiments.

[0166] Embodiment 1 is a method of inducing an immune response against tau protein, preferably inducing antibodies against tau phosphopeptides and / or ePHF, in a subject suffering from a neurodegenerative disorder, comprising: Target, a. a first tau peptide; b. Helper T cell epitopes and administering a priming composition comprising an immunologically effective amount of liposomes, wherein the tau peptide is displayed on the surface of the liposomes, and a pharmaceutically acceptable carrier; The subject is administered a second tau phosphopeptide comprising an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of formula (I):

[0167] [ka] or a structure of formula (II):

[0168] [ka] [In the formula, x is an integer from 0 to 10, preferably from 2 to 6, and most preferably 3; n is an integer of 3 to 15, preferably 3 to 12; the carrier represents an immunogenic carrier, preferably the immunogenic carrier is selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and outer membrane protein mixture (OMP) from N. meningitidis, or a derivative thereof; Tau peptide represents the second tau phosphopeptide.] an immunologically effective amount of a conjugate having administering a first boosting composition further comprising a pharmaceutically acceptable carrier; The method includes:

[0169] Embodiment 2 is the method of embodiment 1, wherein the tau peptide of the liposome is a tau phosphopeptide.

[0170] Embodiment 3 is the method of embodiment 1 or 2, wherein the liposome further comprises a toll-like receptor ligand.

[0171] Embodiment 4 is the method of embodiment 3, wherein the toll-like receptor ligand comprises at least one of a toll-like receptor 4 ligand and a toll-like receptor 9 ligand.

[0172] Embodiment 5 is the method of embodiment 3 or 4, wherein the toll-like receptor ligand is a toll-like receptor 4 ligand.

[0173] Embodiment 6 is the method of embodiment 5, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA).

[0174] Embodiment 7 is the method of embodiment 3 or 4, wherein the toll-like receptor ligand is a toll-like receptor 9 ligand.

[0175] Embodiment 8 is the method of embodiment 7, wherein the toll-like receptor 9 ligand comprises a lipidated CpG oligonucleotide.

[0176] Embodiment 9 is a method for preparing a priming composition comprising: a. tau peptide; b. helper T cell epitopes; ci Toll-like receptor 9 ligand, and ii. Toll-like receptor 4 ligand with at least one of 2. The method of embodiment 1, comprising an immunologically effective amount of liposomes comprising:

[0177] Embodiment 10 is the method of embodiment 9, wherein the tau peptide is a tau phosphopeptide.

[0178] Embodiment 11 is the method of embodiment 9 or 10, wherein the toll-like receptor 9 ligand is a lipidated CpG oligonucleotide.

[0179] Embodiment 12 is the method of any of embodiments 9 to 11, comprising a Toll-like receptor 4 ligand and a Toll-like receptor 9 ligand.

[0180] Embodiment 13 is the method of embodiment 12, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA).

[0181] Embodiment 14 is a method for preparing a priming composition comprising: Tau phosphopeptide, a helper T cell epitope, a lipidated CpG oligonucleotide; Adjuvants containing Toll-like receptor 4 ligands an immunologically effective amount of liposomes comprising 2. The method of embodiment 1, wherein the tau phosphopeptide is displayed on the surface of a liposome.

[0182] Embodiment 15 is the method of embodiment 14, wherein the Toll-like receptor 4 ligand comprises monophosphoryl lipid A (MPLA).

[0183] Embodiment 16 is the method of any one of embodiments 1 to 15, wherein the helper T cell epitope is encapsulated in a liposome.

[0184] Embodiment 16a is the method of any one of embodiments 1 to 15, wherein the helper T cell epitope is incorporated into the membrane of the liposome.

[0185] Embodiment 16b is the method of any one of embodiments 1 to 15, wherein the helper T cell epitope is displayed on the surface of the liposome.

[0186] Embodiment 17 is a method for preparing a priming composition comprising: Tau phosphopeptide, a helper T cell epitope, a lipidated CpG oligonucleotide; Monophosphoryl lipid A (MPLA) and an immunologically effective amount of liposomes comprising Tau phosphopeptides are displayed on the surface of the liposomes; T cell epitopes are encapsulated within liposomes. The method according to any one of embodiments 1 to 16b.

[0187] Embodiment 17a is the method of embodiment 17, wherein the MPLA is 3-O-deacylated-4'-monophosphoryl lipid A, preferably MPL™.

[0188] Embodiment 17b is the method of embodiment 17, wherein the MPLA is monophosphoryl hexa-acyl lipid A, 3-desacylated, preferably 3D-(6-acyl) PHAD®.

[0189] Embodiment 17c is the method of embodiment 17, wherein the MPLA is monophosphoryl 3-desacylated lipid A, preferably 3D-PHAD®.

[0190] Embodiment 18 is the method of any of embodiments 1 to 17c, wherein the liposome composition of the priming composition further comprises one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol.

[0191] Embodiment 19 is the method of any one of embodiments 1 to 18, wherein the tau peptide of the liposome has an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:12, or an amino acid sequence that is at least 85%, 90%, or 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:12.

[0192] Embodiment 19-1 is the method of embodiment 19, wherein the tau peptide of the liposome is a phosphopeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-12.

[0193] Embodiment 19-2 is the method of embodiment 19-1, wherein the tau phosphopeptide of the liposome comprises the amino acid sequence of SEQ ID NO:1.

[0194] Embodiment 19-3 is the method of embodiment 19-1, wherein the tau phosphopeptide of the liposome comprises the amino acid sequence of SEQ ID NO:2.

[0195] Embodiment 19-4 is the method of embodiment 19-1, wherein the tau phosphopeptide of the liposome comprises the amino acid sequence of SEQ ID NO:3.

[0196] Embodiment 19a is the method of any one of embodiments 19, 19-1, 19-2, 19-3, and 19-4, wherein the amino acid sequence further comprises one or more modifications that enable the tau peptide to be displayed on the surface of the liposome.

[0197] Embodiment 19b is the method of embodiment 19a, wherein the one or more modifications include at least one of palmitoylation and dodecyl modification.

[0198] Embodiment 19c is the method of embodiment 19a or 19b, wherein the tau peptide of the liposome is modified at its N-terminus with one or more modifications.

[0199] Embodiment 19d is the method of any of embodiments 19a to 19c, wherein the tau peptide of the liposome is modified at its C-terminus with one or more modifications.

[0200] Embodiment 19e is the method of embodiment 19d, wherein the tau peptide of the liposome is palmitoylated at both its N-terminus and C-terminus.

[0201] Embodiment 19f is the method of any of embodiments 19a to 19e, wherein the tau peptide of the liposome further comprises one or more additional amino acids to facilitate one or more modifications.

[0202] Embodiment 19g is the method of embodiment 19f, wherein the one or more additional amino acids are selected from the group consisting of Lys, Cys, Ser, and Thr.

[0203] Embodiment 19h is the method of any of embodiments 19 to 19g, wherein the liposomal tau peptide is amidated at its C-terminus.

[0204] Embodiment 19i is the method of any of embodiments 19 to 19h, wherein the tau peptide of the liposome consists of an amino acid sequence selected from the group consisting of SEQ ID NO:27 to SEQ ID NO:38.

[0205] Embodiment 19j is the method of any of embodiments 19 to 19i, wherein the tau peptide of the liposome consists of the amino acid sequence of SEQ ID NO:27.

[0206] Embodiment 19k is the method of any of embodiments 19 to 19i, wherein the tau peptide of the liposome consists of the amino acid sequence of SEQ ID NO:28.

[0207] Embodiment 191 is the method of any of embodiments 19 to 19i, wherein the tau peptide of the liposome consists of the amino acid sequence of SEQ ID NO:29.

[0208] Embodiment 20 is the method of any one of embodiments 1 to 191, wherein the helper T cell epitope of the liposome comprises at least one amino acid sequence selected from the group consisting of SEQ ID NO:23 to SEQ ID NO:26.

[0209] Embodiment 20a is the method of embodiment 20, wherein the helper T cell epitope comprises at least two amino acid sequences selected from the group consisting of SEQ ID NO:23 to SEQ ID NO:26.

[0210] Embodiment 20b is the method of embodiment 20, wherein the helper T cell epitope comprises at least three amino acid sequences selected from the group consisting of SEQ ID NO:23 to SEQ ID NO:26.

[0211] Embodiment 20c is the method of embodiment 20, wherein the helper T cell epitope comprises the four amino acid sequences of SEQ ID NO:23 to SEQ ID NO:26.

[0212] Embodiment 20d is the method of any of embodiments 20a to 20c, wherein two or more amino acid sequences selected from the group consisting of SEQ ID NO:23 to SEQ ID NO:26 are covalently linked by a linker.

[0213] Embodiment 20e is the method of embodiment 20d, wherein the linker comprises one or more amino acids selected from the group consisting of Val (V), Ala (A), Arg (R), Gly (G), Ser (S), and Lys (K).

[0214] Embodiment 20f is the method of embodiment 20e, wherein the linker comprises an amino acid sequence selected from the group consisting of VVR, GS, RR, and RK.

[0215] Embodiment 20g is the method of any of embodiments 20 to 20f, wherein the helper T cell epitope is amidated at its C-terminus.

[0216] Embodiment 20h is the method of any of embodiments 20 to 20g, wherein the helper T cell epitope is modified for insertion into the membrane of the liposome, presentation on the surface of the liposome, or encapsulation within the liposome, depending on the intended location of the helper T cell epitope.

[0217] Embodiment 20i is the method of any of embodiments 20 to 20h, wherein the helper T cell epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NO:13 to SEQ ID NO:17.

[0218] Embodiment 20j is the method of any of embodiments 1 to 20i, comprising the tau peptide and the helper T cell epitope in a weight ratio of 6:1.

[0219] Embodiment 20k is the method of any of embodiments 1 to 20i, comprising the tau peptide and the helper T cell epitope in a weight ratio of 5:1.

[0220] Embodiment 201 is the method of any of embodiments 1 to 20i, comprising the tau peptide and the helper T cell epitope in a weight ratio of 4:1.

[0221] Embodiment 20m is the method of any of embodiments 1 to 20i, comprising the tau peptide and the helper T cell epitope in a weight ratio of 3:1.

[0222] Embodiment 20n is the method of any one of embodiments 1 to 20i, comprising the tau peptide and the helper T cell epitope in a weight ratio of 2:1.

[0223] Embodiment 20o is the method of any of embodiments 1 to 20i, comprising the tau peptide and the helper T cell epitope in a 1:1 ratio by weight.

[0224] Embodiment 21 is the method of any one of embodiments 1 to 20o, wherein the lipidated CpG oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22.

[0225] Embodiment 21a is the method of embodiment 21, wherein the CpG oligonucleotide has one or more phosphorothioate internucleotide linkages.

[0226] Embodiment 21b is the method of embodiment 21a, wherein all of the CpG oligonucleotides have phosphorothioate internucleotide linkages.

[0227] Embodiment 21c is the method of any of embodiments 21 to 21b, wherein the lipidated CpG oligonucleotide comprises a CpG oligonucleotide covalently linked to at least one lipophilic group via a linker.

[0228] Embodiment 21d is the method of embodiment 21c, wherein the linker comprises (C2H4O)n, where n is an integer from 0 to 10.

[0229] Embodiment 21e is the method of embodiment 21c, wherein the linker comprises an alkyl spacer having 3 to 12 carbons.

[0230] Embodiment 21f is the method of any of embodiments 21 to 21e, wherein at least one lipophilic group is cholesterol.

[0231] Embodiment 21g is the method of any of embodiments 21 to 21f, wherein the lipidated CpG oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19 covalently attached to a cholesterol molecule via a linker comprising (CHO), where n is an integer from 3 to 5.

[0232] Embodiment 22 is a method of inducing an immune response against tau protein in a subject suffering from a neurodegenerative disorder, comprising: Target, a first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 29 and SEQ ID NOs: 31 to 38; a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 44, preferably a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17; a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, containing one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker; Monophosphoryl lipid A (MPLA) and an immunologically effective amount of liposomes comprising administering a priming composition comprising a pharmaceutically acceptable carrier; The subject is administered a second tau phosphopeptide comprising an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of formula (I):

[0233] [ka] or a structure of formula (II):

[0234] [ka] [In the formula, x is an integer from 0 to 10, preferably from 2 to 6, and most preferably 3; n is an integer of 3 to 15, preferably 3 to 12; Carrier refers to an immunogenic carrier, Tau peptide represents the second tau phosphopeptide.] an immunologically effective amount of a conjugate having administering a first boosting composition comprising a pharmaceutically acceptable carrier; The method includes:

[0235] Embodiment 22a is a method for preparing liposomes comprising: a. a first tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29; b. a helper T cell epitope consisting of the amino acid sequence of SEQ ID NO: 13; c. A lipidated CpG oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19, which is covalently bound to cholesterol via a linker comprising (C2H4O)n, where n is an integer of 3 to 7; d. Monophosphoryl lipid A (MPLA) and 23. The method of embodiment 22, comprising:

[0236] Embodiment 22b is the method of embodiment 22 or 22a, wherein the MPLA is 3-O-deacylated-4'-monophosphoryl lipid A, preferably MPL™.

[0237] Embodiment 22c is the method of embodiment 22 or 22a, wherein the MPLA is monophosphoryl hexa-acyl lipid A, 3-desacylated, preferably 3D-(6-acyl) PHAD®.

[0238] Embodiment 22d is the method of embodiment 22 or 22a, wherein the MPLA is preferably 3D-PHAD®.

[0239] Embodiment 23 is the method of any of embodiments 22 to 22d, wherein the helper T cell epitope is encapsulated in a liposome.

[0240] Embodiment 24 is the method of any one of embodiments 1 to 23, wherein x is an integer from 2 to 6.

[0241] Embodiment 25 is the method of any one of embodiments 1 to 24, wherein x is 3.

[0242] Embodiment 26 is a conjugate according to any one of embodiments 1 to 25, wherein n is 3 to 7.

[0243] Embodiment 27 is the method of any one of embodiments 1 to 26, wherein the carrier is an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and outer membrane protein mixture (OMP) from N. meningitidis, or a derivative thereof.

[0244] Embodiment 28 is the method of any one of embodiments 1 to 27, wherein the second tau phosphopeptide of the conjugate consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:3 and SEQ ID NO:5 to SEQ ID NO:12.

[0245] Embodiment 28a is the method of embodiment 28, wherein the first tau phosphopeptide of the liposome and the second tau phosphopeptide of the conjugate are the same.

[0246] Embodiment 28b is the method of embodiment 28, wherein the first tau phosphopeptide of the liposome and the second tau phosphopeptide of the conjugate are different.

[0247] Embodiment 28c is the method of embodiment 28b, wherein the first tau phosphopeptide of the liposome and the second tau phosphopeptide of the conjugate share at least one common epitope.

[0248] Embodiment 29 is the method of any one of embodiments 28 to 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.

[0249] Embodiment 30 is the method of any one of embodiments 1 to 29, wherein the carrier is CRM197.

[0250] Embodiment 31 is a method for producing a conjugate having the following structure:

[0251] [ka] [In the formula, n is 3 to 7.] 31. The method according to any one of embodiments 1 to 30, comprising:

[0252] Embodiment 32 is directed to a compound having the following structure:

[0253] [ka] [In the formula, the tau peptide consists of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3; x is an integer from 0 to 10, n is an integer from 2 to 15, Tau peptide refers to tau phosphopeptide, KLH stands for keyhole limpet hemocyanin. 30. The conjugate of any one of embodiments 1 to 29, having the formula:

[0254] Embodiment 33 is the method of any of embodiments 1 to 32, further comprising administering to the subject a second boosting composition comprising an immunogenically effective amount of liposomes.

[0255] Embodiment 33a is the method of embodiment 33, wherein the priming composition, the first boosting composition, and / or the second boosting composition further comprises an adjuvant.

[0256] Embodiment 33b is the method of embodiment 33a, wherein the adjuvant comprises at least one of a TLR-4 ligand and a TLR-9 ligand.

[0257] Embodiment 34 is the method of any of embodiments 1 to 33b, wherein the first boosting composition is administered about 27 to 32 days, preferably about 29 days, after the first administration of the priming composition.

[0258] Embodiment 35 is the method of embodiment 34, further comprising re-administering the first boosting composition about 82 to 87 days, preferably about 85 days, after the priming composition is first administered.

[0259] Embodiment 36 is the method of embodiment 35, further comprising administering a second boosting composition about 167 to 172 days, preferably about 169 days, after the first administration of the priming composition.

[0260] Embodiment 37 is the method of any of embodiments 1 to 33b, wherein the first boosting composition is administered about 82 to 87 days, preferably about 85 days, after the first administration of the priming composition.

[0261] Embodiment 38 is the method of embodiment 37, further comprising administering a second boosting composition about 27 to 32 days, preferably about 29 days, after the first administration of the priming composition.

[0262] Embodiment 39 is the method of embodiment 35, further comprising re-administering the first boosting composition about 167 to 172 days, preferably about 169 days, after the priming composition is first administered.

[0263] Embodiment 40 is a method of treating or preventing a neurodegenerative disease or disorder in a subject in need thereof, comprising: Target, a first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 27 to 29 and SEQ ID NOs: 31 to 38; a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 44, preferably a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 17; a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, containing one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker; Monophosphoryl lipid A (MPLA) and administering a priming composition comprising an immunologically effective amount of liposomes, wherein the tau phosphopeptide is displayed on the surface of the liposomes, and a pharmaceutically acceptable carrier; The subject includes a tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure:

[0264] [ka] [In the formula, n is an integer of 3 to 7.] an immunologically effective amount of a conjugate having administering a first boosting composition comprising a pharmaceutically acceptable carrier; The method includes:

[0265] Embodiment 41 is the method of embodiment 40, wherein the first boosting composition is administered about 27 to 32 days, preferably about 29 days, after the first administration of the priming composition.

[0266] Embodiment 42 is the method of embodiment 41, further comprising re-administering the first boosting composition about 82 to 87 days, preferably about 85 days, after the priming composition is first administered.

[0267] Embodiment 43 is the method of embodiment 42, further comprising administering a second boosting composition comprising an immunogenically effective amount of liposomes about 167 to 172 days, preferably about 169 days, after the first administration of the priming composition.

[0268] Embodiment 44 is the method of embodiment 40, wherein the first boosting composition is administered about 82 to 87 days, preferably about 85 days, after the first administration of the priming composition.

[0269] Embodiment 45 is the method of embodiment 44, further comprising administering a second boosting composition comprising an immunogenically effective amount of liposomes about 27 to 32 days, preferably about 29 days, after the first administration of the priming composition.

[0270] Embodiment 46 is the method of embodiment 45, further comprising re-administering the first boosting composition about 167 to 172 days, preferably about 169 days, after the priming composition is first administered.

[0271] Embodiment 46a is the method of any one of embodiments 1 to 46, wherein an immunologically effective amount of the conjugate is administered with one or more adjuvants.

[0272] Embodiment 46b is the method of embodiment 46a, wherein the adjuvant comprises an aluminum salt, such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate.

[0273] Embodiment 46c is the method of embodiment 46a or 46b, wherein the adjuvant comprises a CpG such as CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336.

[0274] Embodiment 46d is a method according to embodiment 46c, in which an immunologically effective amount of the conjugate is administered together with an aluminum salt, such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate, and a CpG, such as CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, CpG1826, or CpG2336.

[0275] Embodiment 46e is the method of embodiment 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum phosphate and CpG1018.

[0276] Embodiment 46f is the method of embodiment 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum hydroxide and CpG7909.

[0277] Embodiment 46g is the method of embodiment 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum sulfate and CpG2395.

[0278] Embodiment 46h is the method of embodiment 46d, wherein an immunologically effective amount of the conjugate is administered together with aluminum hydroxide and CpG1826.

[0279] Embodiment 47 is the method of any of embodiments 1 to 46h, wherein the neurodegenerative disease or disorder is caused by or associated with the formation of neurofibrillary lesions.

[0280] Embodiment 48 is a method for treating a neurodegenerative disease or disorder, comprising administering to a subject therapies including but not limited to Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia pugilistica, Down's syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Parkinsonism-Dementia Complex of Guam, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia, or the like. 48. The method of any of embodiments 1 to 47, wherein the dementia is preferably frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobe dementia, Hallevorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), cerebrovascular disease, or Lewy body dementia (LBD).

[0281] Embodiment 49 is the method of any of embodiments 1 to 48, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down's syndrome, progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), Pick's disease, and PART (primary age-related tauopathy), corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, myotonic dystrophy (dysphasy), chronic traumatic encephalopathy (CTE), cerebrovascular disease, or dementia with Lewy bodies (LBD).

[0282] Embodiment 50 is the method of any of embodiments 1 to 49, wherein the neurodegenerative disease or disorder is Alzheimer's disease, progressive supranuclear palsy (PSP), frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), or Pick's disease, and PART (primary age-related tauopathy).

[0283] Embodiment 51 is the method of any of embodiments 1 to 50, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down's syndrome, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, myotonic dystrophy (dysphasy), chronic traumatic encephalopathy (CTE), cerebrovascular disease, or Lewy body dementia (LBD).

[0284] Embodiment 51a is the method of any of embodiments 1 to 51, wherein the neurodegenerative disease or disorder is early Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease, mild Alzheimer's disease, or mild to moderate Alzheimer's disease.

[0285] Embodiment 52 is a combination, such as a kit, comprising a priming composition used in any of embodiments 1 to 51a and a first boosting composition.

[0286] Embodiment 53 is an immunologically effective amount of liposomes of about 25 nmole to about 750 nmole per dose, preferably about 90 nmole to about 715 nmole per dose or about 90 nmole to about 535 nmole per dose, such as about 25 nmole, about 30 nmole, about 35 nmole, about 40 nmole, about 45 nmole, about 50 nmole, about 55 nmole, about 60 nmole, about 65 nmole, about 70 nmole, about 75 nmole, about 80 nmole, about 85 nmole, about 90 nmole, about 95 nmole, about 100 nmole, about 125 nmole, about 150 nmole, about 175 nmole, about 200 nmole per dose. The method of any one of embodiments 1 to 51a, or the combination of embodiment 52, wherein the first tau phosphopeptide is about 225 nmole, about 250 nmole, about 275 nmole, about 300 nmole, about 325 nmole, about 350 nmole, about 375 nmole, about 400 nmole, about 425 nmole, about 450 nmole, about 475 nmole, about 500 nmole, about 525 nmole, about 550 nmole, about 575 nmole, about 600 nmole, about 625 nmole, about 650 nmole, about 675 nmole, about 700 nmole, about 725 nmole, about 750 nmole, or any value therebetween.

[0287] Embodiment 53a is the method of any one of embodiments 1 to 51a, or the combination of embodiment 52, wherein the immunologically effective amount of liposomes contains from about 25 nmole to about 750 nmole per dose, for example, from about 29.7 nmole to about 742.5 nmole per dose, preferably from about 90 nmole to about 715 nmole per dose, for example, from about 89.1 nmole to about 712.8 nmole per dose, or from about 90 nmole to about 535 nmole per dose, for example, from about 89.1 nmole to about 534.6 nmole per dose, or from about 90 nmole to about 268 nmole per dose, for example, from about 89.1 nmole to about 267.3 nmole per dose, of the first tau phosphopeptide.

[0288] In embodiment 53b, the immunologically effective amount of liposomes is about 100 μg to about 2500 μg, preferably about 300 μg to about 2400 μg per dose, 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, or the like. 53. The method or combination of embodiment 53 or 53a, wherein the first tau phosphopeptide is 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, about 2400 μg, about 2500 μg, or any value therebetween.

[0289] Embodiment 53c is a method or combination according to any one of embodiments 53 to 53b, wherein the first tau phosphopeptide consists of the amino acid sequence of one of SEQ ID NO:27 to SEQ ID NO:38.

[0290] Embodiment 53d is a method or combination according to embodiment 53c, wherein the first tau phosphopeptide consists of the amino acid sequence of SEQ ID NO:28.

[0291] Embodiment 54 is a method or combination according to any one of embodiments 53 to 53d, wherein the second tau phosphopeptide consists of the amino acid sequence of SEQ ID NOs: 1-3 or 5-12.

[0292] Embodiment 54a is the method or combination of embodiment 54, wherein the second tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 1, 2, or 3. [Example]

[0293] [Example 1] Preparation of liposomal vaccines Preparation of control liposomal vaccine (ethanol injection technique) A control liposomal vaccine was produced by ethanol (EtOH) injection followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, The Netherlands), and MPLA (Avanti Polar Lipids, Alabama, USA) were solubilized in a 20:1 (V / V) mixture of EtOH and tert-butanol (t-BuOH) at a molar ratio of 9:1:7:0.05 at 60°C. The lipid / ethanol solution was diluted with phosphate buffered saline (PBS), pH 7.4, at 60°C to maintain a 10% EtOH concentration, resulting in the formation of multilamellar liposomal vesicles (MLVs). The MLVs were then subjected to five consecutive extrusions through three polycarbonate filters (Whatman) with 0.08 μm pore size in series using an EmulsiFlex-C5 (Avestin, Canada). The resulting liposomes were diluted with PBS, pH 7.4, and heated to 60°C to obtain the liposome solution before adding tau peptide.

[0294] The acetate tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2 (Bachem AG, Switzerland), hereafter referred to as the active pharmaceutical ingredient (API), corresponding to SEQ ID NO: 28, was dissolved in PBS, pH 11.4, containing 2.0% octyl β-D-glucopyranoside (Sigma-Aldrich, USA) at a concentration of 1 mg / mL. The peptide solution was injected into the liposome solution at 60°C and then stirred for 30 minutes at 60°C. Concentration was performed by ultrafiltration to the target final volume, and buffer exchange was performed 10 times with PBS, pH 7.4 during diafiltration. The resulting liposomes, with the API displayed on their surface, were then sterile filtered through two 0.2 μm polycarbonate syringe filters in series, and the final product was stored at 5°C.

[0295] Liposomes Z and Z + Vaccine preparation Liposome Z with a final API concentration of 1200 μg / ml and a final T50 concentration of 1200 μg / ml + The vaccine was produced by the ethanol injection technique followed by extrusion, and 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 the lipid thin layer technique followed by homogenization and extrusion.

[0296] Preparation of liposomal Z vaccine by lipid thin layer technique. The liposomal Z vaccine was produced by a lipid thin layer technique, followed by homogenization and extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, The Netherlands), and monophosphoryl hexa-acyl lipid A3-deacylated synthetic (3D-(6-acyl)PHAD®) (Avanti Polar Lipids, Alabama, USA) were solubilized in EtOH at 60°C in a molar ratio of 9:1:7:0.05. The ethanol was evaporated in a rotavapor under vacuum to obtain a lipid thin layer.

[0297] The lipid film was rehydrated with PBS, pH 7.4, 5% DMSO (all Sigma-Aldrich) containing 0.15 mg / mL T50 peptide (Peptides & Elephants, Germany). The sample was gently agitated for 15 min and then vigorously vortexed to dissolve the lipid thin layer. The resulting multilamellar vesicles were subjected to 10 freeze-thaw cycles (liquid N2 and a water bath at 37°C), homogenized, and then sequentially extruded through a polycarbonate membrane with a 0.08 μm pore size (Whatman, UK). Both the homogenization and extrusion steps were performed in an EmulsiFlex-C5 (Avestin, Canada). The extruded liposomes with encapsulated T50 peptide were concentrated by ultrafiltration and buffer exchanged into PBS, pH 7.4, by diafiltration. The resulting liposomes were diluted with PBS, pH 7.4, and heated to 60°C to obtain a liposome solution before the addition of tau peptide and adjuvant.

[0298] CpG2006-cholesterol (CpG2006-Chol) (Microsynth, Switzerland) is a DNA oligonucleotide with all internucleotide bonds as thiophosphates, and the 5' end is modified with a cholesterol molecule via a phosphate bond via a PEG spacer. CpG2006-cholesterol (CpG2006-Chol) (Microsynth, Switzerland) was dissolved in PBS, pH 7.4, at 1 mg / mL and injected into the liposome solution. After 15 minutes of incubation, the API was inserted.

[0299] The API (Bachem AG, Switzerland) was dissolved in PBS, pH 11.4, containing 2% octyl β-D-glucopyranoside (Sigma-Aldrich, USA) at a concentration of 1 mg / mL. The peptide solution was injected into the liposome solution at 60°C and then stirred for 30 minutes at 60°C. Concentration was performed by ultrafiltration to obtain the target values ​​(400 μg / mL API and 100 μg / mL T50 for Liposome Z), and buffer exchange was performed 10 times with PBS, pH 7.4 during diafiltration. The resulting liposomes, with the API displayed on the surface of the liposomes, were then sterile filtered through a 0.2 μm polycarbonate syringe filter, and the final product was stored at 5°C.

[0300] Liposome Z by ethanol injection + Vaccine preparation The liposome Z+ vaccine was produced using an ethanol injection-based process. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, The Netherlands), and 3D-(6-acyl)PHAD® (Avanti Polar Lipids, Alabama, USA) were solubilized in EtOH at 60°C in a molar ratio of approximately 9:1:7:0.04. T50 peptide (Bachem AG, Switzerland) was dissolved in 10 mM His / 270 mM sucrose (pH 5.8-6.0). The lipid ethanol solution was then injected into the solution containing the T50 peptide and gently stirred for 15 minutes to generate multilamellar vesicles (MLVs). The MLVs were then homogenized (liposome Z). + six times), followed by continuous extrusion (Liposome Z) through a polycarbonate membrane (Whatman, UK) with a pore size of 0.08 μm. + Liposome Z was subjected to the same procedure (5 times in the case of the 5th generation). +In this study, both the homogenization and extrusion steps were performed in an EmulsiFlex-C5 (Avestin, Canada). The extruded liposomes were concentrated by ultrafiltration and the buffer was exchanged into 20 mM His / 145 mM NaCl, pH 7.4 by diafiltration. The resulting liposomes with encapsulated T50 peptide were diluted with 20 mM His / 145 mM NaCl, pH 7.4 and heated to 60°C to obtain the liposome solution before addition of API and adjuvants.

[0301] CpG2006-Chol (Liposome Z + Microsynth (Switzerland) was dissolved at 1 mg / mL in 20 mM His / 145 mM NaCl, pH 7.4, and injected into the liposome solution, followed by a 15-minute incubation before the API was inserted.

[0302] The API (Bachem AG, Switzerland) was dissolved in carbonate buffer, pH 10.2, together with 1% octyl β-D-glucopyranoside (Sigma-Aldrich, USA) at a concentration of 1 mg / mL, and the peptide solution was incubated at 60 °C for 1 h to form liposome Z. + The peptide solution was mixed with Liposome Z at 60°C using T-Line Mixing. + The solution was mixed and then stirred for 30 minutes at 60°C. Concentration was performed by ultrafiltration to the target value (liposome Z). + The resulting concentration of API was 1200 μg / ml and T50 was 1200 μg / ml. Buffer exchange was performed 10 times with 10 mM His / 270 mM sucrose, pH 6.5 during diafiltration. The resulting Z was then used to display the API on the surface of the liposomes. + The liposomes were sterile filtered through a 0.2 μm polycarbonate syringe / capsule filter and the final product was stored at 5°C.

[0303] [Example 2] Preparation of conjugate vaccines Peptides and adjuvants The phosphorylated tau peptide (SEQ ID NO: 2) used in this study was synthetically produced (Pepscan, The Netherlands), with phospho residues added during synthesis. A conjugate comprising a phosphorylated tau peptide having the amino acid sequence of SEQ ID NO: 2 covalently attached to a CRM carrier via a linker is referred to herein as conjugate X.

[0304] The vaccine peptide was conjugated to the carrier protein CRM197 via a polyethylene glycol (PEG)-cysteine-acetamidopropionamide linker. A phosphorylated tau peptide with the amino acid sequence of SEQ ID NO:2 was synthetically produced (Polypeptide Laboratories SAS), and a phospho residue and PEG3 spacer were added during synthesis. Conjugate X was prepared by conjugating the carrier protein CRM197 to the N-terminal cysteine ​​of the peptide via a succinimidyl 3-(bromoacetamido)propionate (SBAP) linker. SBAP was ligated to the primary amine (-NH2) of the CRM197 protein via NHS ester reaction chemistry. Excess SBAP linker was removed using ultrafiltration and diafiltration (UF / DF). The CRM197-SBAP intermediate was conjugated to the phosphorylated tau peptide, and after the reaction was complete, the conjugation reaction was stopped by adding an excess amount of L-cystine to quench the reaction. The crude CRM197-peptide conjugate product was purified using a Capto Q ImpRes (GE Healthcare) chromatography column and eluted using an isocratic salt method. The purified CRM197-peptide product was then formulated in a buffer containing Tris and sucrose, such as 20 mM Tris and 250 mM sucrose, pH 8.1, using UF / DF. A CRM197-tau peptide drug substance (DS) stock solution was generated by adding a PS80 stock buffer, such as 10% polysorbate 80 (PS80) stock buffer, to reach a final PS80 concentration of 0.01%. The solution was mixed thoroughly before filtration. Prior to injection, the stock solution was diluted with PBS and CpG / alum to a first CRM197-tau peptide concentration of, for example, 0.8 mg / mL, and then further diluted with PBS and CpG / alum to a final CRM197-tau peptide concentration of 30 μg / mL for injection. For Examples 3-7, CRM197-tau peptide stock solutions were kept at a concentration of 3.1 mg / mL in 10 mM PBS (pH 7.3) and further diluted in PBS to reach the desired working concentration.CpG oligonucleotides, alum, and PBS were then added to reach a final concentration of 30 ug / mL based on CRM197-tau peptide, and the final formulation was mixed thoroughly before injection.

[0305] One concern about targeting CNS antigens with active vaccines is that non-specific or off-target inflammation may cause undesired neuropathological changes.To investigate this, whole brains from mice immunized with the conjugate composition were collected and stained to visualize perivascular or other cellular infiltrates.None of the immunized animals showed any signs of neuroinflammation, cellular infiltration, or other undesired neuropathological changes (data not shown).This suggests that vaccine-induced antibodies and the natural immune response to vaccination did not cause neuropathological changes in mice.

[0306] The following examples demonstrate different aspects of the immune response induced by different vaccine regimens.

[0307] [Example 3] Heterologous vaccination increases epitope coverage of tau phosphopeptide-specific antibodies in rhesus macaques All animal experiments were approved and conducted in accordance with local regulations regarding animal experimentation. Rhesus macaques (Macaca mulatta) were obtained from Kunming Biomed International Ltd, China, Yunnan Yinmore Bio-Tech Co. LTD, China, and Yunnan Laboratory Primates Inc., China. Animals were 2–5 years old and weighed a minimum of 2.5 kg at the start of immunization. Detailed clinical examinations were performed before the start of treatment and weekly thereafter. Additionally, macaques were observed twice daily, and clinical signs were recorded.

[0308] Liposomal vaccines, such as Liposome Z or Liposome Z+ (both containing the 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 throughout this application as "A," while conjugate vaccines, such as Conjugate X (the phosphorylated tau peptide of SEQ ID NO: 2 linked to CRM197), are referred to throughout this application as "B."

[0309] Groups of rhesus macaques (n=3 males and 3 females per group) were immunized subcutaneously on days 1 and 29 with i) 1800ug of tetrapalmitoylated phosphorylated tau acetate peptide of SEQ ID NO: 2 / dose of liposomal Z vaccine (regimen AA), ii) 15ug / dose of conjugate X vaccine (regimen BB), or iii) 1800ug of tetrapalmitoylated phosphorylated tau acetate peptide of SEQ ID NO: 2 / dose of liposomal Z vaccine on day 1 followed by 15ug / dose of conjugate X vaccine on day 29 (regimen AB). The epitope recognition profile of the antibodies was determined 3 hours after the second immunization (day 50) by epitope mapping ELISA using a library of N-terminally biotinylated 8-mer peptides shifted by one amino acid and covering the entire sequence of the phosphotau peptide of SEQ ID NO: 2, as well as the sequence of SEQ ID NO: 4 (VYKSPVVSGDTSPRHL, the non-phosphorylated peptide of SEQ ID NO: 2).

[0310] Figures 1A-1C show that, although the liposomal and conjugate vaccines contain the same phosphorylated tau peptide of SEQ ID NO: 2, monkeys immunized with the liposomal vaccine (liposome Z) produced IgG antibodies that mostly bound to the N-terminal portion of the peptide (AA regimen, Figure 1A), whereas monkeys immunized with the conjugate vaccine (conjugate X) produced IgG antibodies that mostly bound to the C-terminal portion of the peptide (BB regimen, Figure 1B). The heterologous regimen induced antibodies that bound to both the N- and C-terminal portions of the peptide (AB regimen, Figure 1C), thus increasing the epitope coverage of the induced antibodies. Furthermore, the IgG antibodies induced in monkeys immunized with the liposomal vaccine (liposome Z) did not bind to the non-phosphorylated library, whereas the IgG antibodies induced in monkeys immunized with the conjugate vaccine (conjugate X) also recognized the non-phosphorylated octamer peptide. IgG antibodies induced in monkeys immunized with the heterologous regimen (AB) showed binding to the intermediate, non-phosphorylated library.

[0311] [Example 4] Boosting with liposomal vaccine induced a shift to a "liposome-like" epitope profile in rhesus macaques (ABBA regimen) Groups of rhesus macaques (n=3 males and 3 females / group) were immunized subcutaneously with 1800 μg of liposomal Z vaccine at 1 dose of tetrapalmitoylated phosphorylated tau acetate peptide of SEQ ID NO: 2 on day 1, 15 μg of conjugate X vaccine on days 29 and 85, and again with 1800 μg of liposomal Z vaccine at 1 dose of tetrapalmitoylated phosphorylated tau acetate peptide of SEQ ID NO: 2 on day 169 (regimen ABBA). The epitope recognition profile of the induced antibodies was determined by epitope mapping ELISA one week before (day 162, ABB regimen) and three weeks after (day 190, ABBA regimen) the final boost with liposomal vaccine, as described in Example 3. Figure 2 shows that priming with liposomes and boosting with the conjugate vaccine induced a combination of N-terminal and C-terminal IgG antibodies (ABB regimen), while a final boost with the liposome vaccine resulted in a shift to a "liposome-like" epitope profile in rhesus macaques (ABBA regimen). The data demonstrate high flexibility in inducing anti-tau antibody responses using the heterologous vaccination strategy of the novel anti-tau vaccine described herein.

[0312] In summary, studies in rhesus macaques showed that heterologous vaccination with a sequential immunization schedule using the vaccines, namely liposome Z and conjugate X, not only induced antibody titers against p-tau and pathological tau extracted from the brains of AD patients, but also increased the epitope coverage of the induced antibodies within the antigenic sequences used in both second-generation vaccines.

[0313] [Example 5] Heterologous vaccination with liposome and conjugate vaccines induced ePHF-specific IgG titers in serum and tau phosphopeptide-specific IgG antibodies in cerebrospinal fluid (CSF) of rhesus macaques Adult rhesus macaques (n=3 males and 3 females per group) received i) 1800ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine (regimen AAAA) on days 1, 29, 85, and 169; ii) 1800ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine (regimen AAAA) on days 1, 29, 85, and 169; iii) 1800ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine on day 1 and 15ug / dose of conjugate X vaccine co-injected with alum and the CpG oligonucleotide CpG2006 on days 29 and 85. iv) liposomal Z vaccine at 1800 μg of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose on day 169 (regimen ABBA), iv) liposomal Z vaccine at 1800 μg of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose on days 1 and 29 and 15 μg / dose of conjugate X vaccine on days 85 and 169 (regimen AABB), or v) conjugate X vaccine at 15 μg / dose on days 1 and 29 and 1800 μg of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose on days 85 and 169 (regimen BBAA). Bleeding was performed on day 190 and serum was isolated.

[0314] Preparations of enriched paired helical fibrils (ePHFs) were obtained from postmortem brain tissue of histologically confirmed AD subjects by sarkosyl extraction of insoluble tau using a modified method by Greenberg and Davies (Greenberg and Davies, 1991, Proc Natl Acad Sci USA, 87(15):5827-31). Antibody titers specific to ePHFs 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% nonfat milk) and applied to a 96-well MSD plate. After a 2-hour incubation, samples were removed and the plate was washed with PBST (PBS, 0.05% Tween 20). Antibodies were detected using a sulfo-tagged anti-human / monkey IgG antibody, followed by a fixation step in 1% paraformaldehyde (PFA) followed by the addition of Read Buffer T. Plates were analyzed using a Sector Imager (MSD). All samples were run in eight 2-fold serial dilutions, and positive and negative control samples were included on each plate. Antibody titers, expressed in arbitrary units (AU) / mL, were calculated for each individual monkey. The fold change relative to the geometric mean of titers obtained with AAAA for each liposomal composition is presented for each monkey and geometric mean per group.

[0315] Antibody titers specific for the phosphorylated tau peptide of SEQ ID NO:2 were assessed using the MSD platform. Gold small-spot streptavidin 96-well plates (MSD), previously saturated with 1% Blocker A in PBS, were coated with the phosphorylated tau peptide of SEQ ID NO:2, biotinylated at the N-terminus of the sequence. CSF from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% Blocker A) and applied to the 96-well MSD plate. After a 2-hour incubation, samples were removed, and the plate was washed with PBST (PBS, 0.05% Tween 20). Antibodies were detected using a sulfo-tagged anti-human / monkey IgG antibody, followed by the addition of Read Buffer T. Plates were analyzed using a Sector Imager (MSD). All samples were run in eight two-fold serial dilutions, and positive and negative control samples were included on each plate. Antibody titers, expressed in AU / mL, were calculated for each individual monkey. The fold change relative to the geometric mean of titers obtained with AAAA for each respective liposomal composition is expressed for each monkey and geometric mean per group. Samples with high red blood cell counts were excluded from the analysis to avoid any bias in antibody titers due to blood contamination.

[0316] As presented in Figure 3, monkeys receiving the heterologous regimens ABBA and AABB (A is a liposomal vaccine (having tetrapalmitoylated phosphotau peptide of SEQ ID NO: 2) with encapsulated T50, a TLR4 ligand, and lipidated CpG oligonucleotide; B is a conjugate vaccine (phosphopeptide of SEQ ID NO: 2 linked to CRM197) co-injected with alum and CpG oligonucleotide) showed ePHF-specific titers at day 190 (3 weeks after the fourth injection) that were comparable to or higher (fold change ≥ 1) than monkeys receiving the allogeneic treatment (AAAA).

[0317] However, all monkeys injected with the heterologous regimen BBAA showed lower ePHF-specific titers (fold change ≤ 1) than monkeys receiving the homologous treatment (AAAA).

[0318] Figure 6 shows that similar conclusions were reached when monitoring IgG antibody responses in the cerebrospinal fluid of these monkeys on day 183 (two weeks after the fourth injection). Monkeys administered heterologous regimens ABBA and AABB (A: liposomal vaccine containing tetrapalmitoylated phosphorylated tau peptide of SEQ ID NO: 2, encapsulated T50 (TLR4 ligand), and lipidated CpG oligonucleotide; B: conjugate vaccine (phosphopeptide of SEQ ID NO: 2 linked to CRM197) co-injected with alum and CpG oligonucleotide) showed generally higher IgG titers specific for the phosphorylated tau peptide of SEQ ID NO: 2 (fold change ≥ 1) than monkeys administered the allogeneic treatment (AAAA). However, all monkeys administered heterologous regimen BBAA showed lower IgG titers specific for the phosphorylated tau peptide of SEQ ID NO: 2 (fold change ≤ 1) than monkeys administered the allogeneic treatment (AAAA).

[0319] Overall, the results of this study indicate that, in terms of systemic and local (in CSF) antibody responses, heterologous regimens using liposomal vaccines as priming compositions induced higher p-tau-specific IgG titers compared to regimens using conjugate vaccines as priming compositions.

[0320] [Example 6] Heterologous vaccination with a primer liposomal vaccine induced higher tau phosphopeptide- and ePHF-specific IgG titers than homologous regimens in rhesus macaques Adult rhesus macaques (n=3 males and 3 females per group) were immunized subcutaneously or intramuscularly with i) 1800ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine on days 1, 29, and 85 (regimen AAA); ii) 1800ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine (regimen AAA); iii) 1800ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine on day 1 and 15ug / dose of conjugate X vaccine on days 29 and 85 (regimen ABB); or iv) 1800ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine on days 1 and 29 and 15ug / dose of conjugate X vaccine on day 85 (regimen AAB). Bleeding was performed on day 106 and serum was isolated.

[0321] Phosphotau peptide-specific IgG antibody titers were determined by ELISA using the phosphorylated tau peptide of SEQ ID NO: 2 as the coating antigen. Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% BSA) and applied to 96-well plates previously coated with the relevant peptide. After 2 hours of incubation, samples were removed and plates were washed with PBST (PBS, 0.05% Tween 20). Antibodies were detected using anti-monkey IgG conjugated with HRP followed by ABTS substrate (Roche). All samples were run in eight two-fold serial dilutions, with positive and negative control samples included on each plate. Data were expressed as individual endpoint titers (the last serum dilution eliciting a positive response) along with the geometric mean per group. One-way ANOVA with Tukey's multiple comparisons was used for statistical analysis.

[0322] The titer of ePHF-specific antibodies was assessed using the MSD platform. Gold small-spot streptavidin 96-well plates (MSD), pre-saturated with 1% BSA in PBS, were coated with a biotin-labeled anti-tau capture antibody (HT7-biotin, ThermoScientific) and then incubated with ePHFs isolated from the brains of Alzheimer's disease patients. After 1 hour of incubation, the plates were washed with PBST, and serial dilutions of serum were added and incubated for 2 hours. Bound antibodies were detected using a sulfo-tagged anti-human / monkey IgG antibody, followed by a fixation step in 1% PFA followed by the addition of Read Buffer T. The plates were analyzed using a Sector Imager (MSD). All samples were run at eight 2-fold serial dilutions, and positive and negative control samples were included on each plate. Results are expressed in AU / mL for each individual monkey, along with the geometric mean per group. The ePHF-specific antibody titer at day 106 is shown. One-way ANOVA test with Tukey's multiple comparisons was used for statistical analysis.

[0323] FIG. 4A shows that heterologous regimens AAB and ABB induced statistically significantly higher levels of IgG antibodies specific for phosphotau peptides than did allogeneic regimen AAA in rhesus monkeys.

[0324] Figure 4B shows that heterologous regimen AAB induced statistically significantly higher levels of ePHF-specific IgG antibodies in rhesus macaques than allogeneic regimen AAA, while ABB showed a trend toward higher levels of ePHF-specific IgG titers in rhesus macaques than allogeneic regimen AAA.

[0325] Overall, the data show that the heterologous immunization regimen using liposomal vaccine (A) as the priming composition induced higher titers of antibodies against the phosphorylated tau peptide of SEQ ID NO: 2 and ePHF than the allogeneic regimen AAA.

[0326] [Example 7] The quality of ePHF-specific IgG antibodies induced by a xenogeneic regimen is comparable to or better than that induced by an allogeneic regimen in rhesus macaques Adult rhesus macaques (n=3 males and 3 females per group) were administered i) 1800 ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine on days 1, 29, 85, and 169 (regimen AAAA); or ii) 1800 ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine on day 1, 15 ug / dose of conjugate X vaccine on days 29 and 85, and ...5 ug of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine on day 1. Mice were immunized subcutaneously with 15 μg / dose of liposomal Z vaccine (regimen ABBA), or iii) with 1800 μg of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine (regimen AAAA) on days 1, 29, 85, and 169, or iv) with 1800 μg of tetrapalmitoylated phosphorylated tau peptide acetate of SEQ ID NO: 2 / dose of liposomal Z vaccine (regimen AABB) on days 1 and 29 and 15 μg / dose of conjugate X vaccine (regimen AABB) on days 85 and 169. Bleeding was performed on day 190 and serum was isolated.

[0327] The quality of antibodies specific for enriched helical fibrils (ePHFs) was evaluated using the MSD platform for binding to ePHFs at low coating concentrations (also referred to herein as limited coating conditions). Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% nonfat milk) and applied to a 96-well MSD plate. After a 2-hour incubation, samples were removed and the plate was washed with PBST (PBS, 0.05% Tween 20). Antibodies were detected using a sulfo-tagged anti-human / monkey IgG antibody, followed by a fixation step in 1% PFA followed by the addition of Read Buffer T. Plates were analyzed using a Sector Imager (MSD). All samples were run in eight two-fold serial dilutions, and positive and negative control samples were included on each plate. Antibody titers, expressed in AU / mL, were calculated for each individual monkey.

[0328] Phosphotau peptide-specific IgG antibody titers were determined by ELISA using the phosphorylated tau peptide of SEQ ID NO: 2 as the coating antigen. Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% BSA) and applied to 96-well plates previously coated with the relevant peptide. After a 2-hour incubation, samples were removed and plates were washed with PBST (PBS, 0.05% Tween 20). Antibodies were detected using anti-monkey IgG conjugated with HRP followed by ABTS substrate (Roche). All samples were run in eight 2-fold serial dilutions, with positive and negative control samples included on each plate. Data were expressed as individual endpoint titers (the last serum dilution inducing a positive response) along with the geometric mean per group.

[0329] Pan-T cell epitope (T50)-specific IgG antibody titers were determined by ELISA using the T50 peptide as the coating antigen. Serum from immunized monkeys was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% nonfat milk) and applied to 96-well plates previously coated with the relevant peptide. After a 2-hour incubation, samples were removed and plates were washed with PBST (PBS, 0.05% Tween 20). Antibodies were detected using anti-monkey IgG conjugated to HRP followed by ABTS substrate (Roche). All samples were run in eight 2-fold serial dilutions, with positive and negative control samples included on each plate. Data were expressed as individual endpoint titers (the last serum dilution eliciting a positive response) along with the geometric mean per group.

[0330] The fold change between phosphotau peptide-specific and pan-T cell epitope (T50)-specific IgG antibody titers is expressed for each monkey and geometric mean per group. The Mann-Whitney test was used for statistical analysis.

[0331] Figures 5A and 5B show ePHF-specific IgG titers under limited coating conditions, reflecting antibodies with high binding capacity. Figure 5A shows that the ABBA regimen induced slightly higher antibody titers than the AAAA regimen, while AABB induced comparable IgG titers. Furthermore, as shown in Figure 3, the heterologous regimens ABBA and AABB induced overall comparable or higher ePHF-specific antibody titers (fold change ≥ 1) compared with the allogeneic regimen using liposomal vaccine alone (AAAA regimen). Overall, the data suggest that the quality of ePHF-specific antibodies induced by heterologous vaccination is comparable to or better than that induced by the allogeneic AAAA vaccination regimen in rhesus macaques.

[0332] Without wishing to be bound by theory, the heterologous regimen according to the present application is believed to diversify CD4 T cell responses. Due to the limited space in germinal centers, if immunization is continued using the same CD4 helper epitope / protein (e.g., tetanus), tetanus-specific B and T cells push tau-specific B cells out of the germinal center. On the other hand, if two types of CD4 stimulation are alternated, tau-specific B cells (which also receive help from tetanus- or diphtheria-specific T cells) can better utilize them. In the present invention, we observed that the AAAA regimen significantly increased the antibody titer against the T50 helper peptide. When heterologous regimens A and B were used, the antibody titer against the T50 helper peptide was much lower. Figure 7 shows that the ratio between the IgG antibody titer directed against the phosphorylated tau peptide of SEQ ID NO: 2 and the IgG antibody titer directed against the T50 peptide was higher in the heterologous regimens (ABBA and AABB) compared to the respective allologous regimens (AAAA). This reflects the diversity of CD4 T cell responses and also demonstrates that T50-specific B cell responses do not completely dominate the anti-p-tau response.

[0333] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that variations of the above-described embodiments may be made without departing from the broad inventive concept thereof. It is understood, therefore, that the invention is not limited to the particular embodiments disclosed, but rather that it is intended to cover modifications within the spirit and scope of the invention as defined by the appended claims.

[0334] [Sequence table] SEQ ID NO:1 - Phosphotau peptide (7.1) GDRSGYS[pS]PG[pS]PG[pT]PGSRSRT SEQ ID NO:2 - Phosphotau peptide (T3.5) VYK[pS]PVVSGDT[pS]PRHL SEQ ID NO:3 - Phosphotau peptide (22.1) SSTGSIDMVD[pS]PQLA[pT]LA SEQ ID NO: 4 - Tau peptide VYKSPVVSGDTSPRHL SEQ ID NO:5 - Phosphotau peptide RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHL SEQ ID NO:6 - Phosphotau peptide RQEFEVMEDHAGT[pY]GL SEQ ID NO:7 - Phosphotau peptide PGSRSR[pT]P[pS]LPTPPTR SEQ ID NO:8 - Phosphotau peptide GYSSPG[pS]PG[pT]PGSRSR SEQ ID NO:9 - Phosphotau peptide GDT[pS]PRHL[pS]NVSSTGSID SEQ ID NO: 10 - Phosphotau peptide PG[pS]PG[pT]PGSRSR[pT]P[pS]LP SEQ ID NO:11 - Phosphotau peptide HL[pS]NVSSTGSID SEQ ID NO: 12 - Phosphotau peptide VSGDT[pS]PRHL SEQ ID NO: 13-T50 T cell epitope AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE-NH2 SEQ ID NO: 14-T46 T cell epitope AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEK(Pal)K(Pal)-NH2 SEQ ID NO: 15 - T48 helper T cell epitope AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ-NH2 SEQ ID NO: 16 - T51 helper T cell epitope AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLE-NH2 SEQ ID NO: 17 - T52 helper T cell epitope AKFVAAWTLKAAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLE-NH2 SEQ ID NO: 18 - CpG2006 (also known as CpG7909) 5'-tcgtcgttttgtcgttttgtcgtt-3' where lowercase letters indicate phosphorothioate (ps) internucleotide linkages. SEQ ID NO: 19-CpG1018 5'-tgactgtgaacgttcgagatga-3' where lowercase letters indicate phosphorothioate internucleotide linkages SEQ ID NO: 20-CpG2395 5'-tcgtcgttttcggcgcgcgccg-3' where lowercase letters indicate phosphorothioate internucleotide linkages SEQ ID NO: 21-CpG2216 5'-ggGGGACGATCGTCgggggg-3' where lowercase letters indicate phosphorothioate internucleotide linkages and uppercase letters indicate phosphodiester (po) linkages SEQ ID NO: 22-CpG2336 5'-gggGACGACGTCGTGgggggg -3', where lowercase letters indicate phosphorothioate internucleotide linkages and uppercase letters indicate phosphodiester linkages SEQ ID NO: 23 - Pan DR epitope (PADRE) peptide AKFVAAWTLKAAA SEQ ID NO:24-P2 QYIKANSKFIGITEL SEQ ID NO:25-P30 FNNFTVSFWLRVPKVSASHLE SEQ ID NO: 26-TT586-605 LINSTKIYSYFPSVISKVNQ SEQ ID NO:27 - Palmitoylated phosphotau peptide (palmitoylated 7.1) K(pal)K(pal)GDRSGYS[pS]PG[pS]PG[pT]PGSRSRTK(pal)K(pal) SEQ ID NO:28 - Palmitoylated phosphotau peptide (T3, palmitoylated T3.5, palmitoylated SEQ ID NO:2) K(pal)K(pal)VYK[pS]PVVSGDT[pS]PRHLK(pal)K(pal) SEQ ID NO: 29 - Palmitoylated phosphotau peptide (palmitoylated 22.1) K(pal)K(pal)SSTGSIDMVD[pS]PQLA[pT]LAK(pal)K(pal) SEQ ID NO:30 - Palmitoylated tau peptide K(pal)K(pal)VYKSPVVSGDTSPRHLK(pal)K(pal) SEQ ID NO:31 - Palmitoylated phosphotau peptide K(pal)K(pal)RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHLK(pal)K(pal) SEQ ID NO:32 - Palmitoylated phosphotau peptide K(pal)K(pal)RQEFEVMEDHAGT[pY]GLK(pal)K(pal) SEQ ID NO: 33 - Palmitoylated phosphotau peptide K(pal)K(pal)PGSRSR[pT]P[pS]LPTPPTRK(pal)K(pal) SEQ ID NO: 34 - Palmitoylated phosphotau peptide K(pal)K(pal)GYSSPG[pS]PG[pT]PGSRSRK(pal)K(pal) SEQ ID NO: 35 - Palmitoylated phosphotau peptide K(pal)K(pal)GDT[pS]PRHL[pS]NVSSTGSIDK(pal)K(pal) SEQ ID NO: 36 - Palmitoylated phosphotau peptide K(pal)K(pal)PG[pS]PG[pT]PGSRSR[pT]P[pS]LPK(pal)K(pal) SEQ ID NO: 37 - Palmitoylated phosphotau peptide K(pal)K(pal)HL[pS]NVSSTGSIDK(pal)K(pal) SEQ ID NO: 38 - Palmitoylated phosphotau peptide K(pal)K(pal)VSGDT[pS]PRHLK(pal)K(pal) SEQ ID NO:39 - T50 without C-terminal amide AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE SEQ ID NO: 40 - T46 without -Lys(Pal)-Lys(Pal)-NH2 at the C-terminus AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLE SEQ ID NO: 41 - T48 without C-terminal amide AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEGSLINSTKIYSYFPSVISKVNQ SEQ ID NO: 42 - T51 without C-terminal amide AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLE SEQ ID NO: 43 - T52 without C-terminal amide AKFVAAWTLKAAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLE SEQ ID NO: 44-T57(56, 57) AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE-K(Pal)K(Pal)-NH2

[0335] References [Table 1] The inventions described in the original claims of this application are listed below. [Invention 1] 1. A method for inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, comprising: (i) to the subject, a. a first tau phosphopeptide; b. helper T cell epitopes; c. lipidated CpG oligonucleotides; d. an adjuvant containing a Toll-like receptor 4 ligand; administering a priming composition comprising an immunologically effective amount of liposomes, wherein the tau phosphopeptide is displayed on the surface of the liposomes, and further comprising a pharmaceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of Formula (I):

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Claims

1. 1. A priming composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, a. a first tau phosphopeptide; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO:39 to SEQ ID NO:44; c. a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:18 to SEQ ID NO:22; d. An adjuvant containing a Toll-like receptor 4 ligand; administering the priming composition comprising an immunologically effective amount of the liposomes, wherein the tau phosphopeptide is presented on the surface of the liposomes, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of formula (I): 【Chemistry 1A】 Or a structure of formula (II): 【Chemistry 2A】 [In the formula, x is an integer from 0 to 10; n is an integer from 3 to 15, tau peptide represents said second tau phosphopeptide; The carrier represents an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and outer membrane protein mixture (OMP) from Neisseria meningitidis, or a derivative thereof. and an immunologically effective amount of a conjugate having the formula: administering a first boosting composition further comprising a pharma- ceutically acceptable carrier; Including, The priming composition, wherein the first tau phosphopeptide and the second tau phosphopeptide each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9 and 12.

2. 1. A first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, a. a first tau phosphopeptide; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO:39 to SEQ ID NO:44; c. a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:18 to SEQ ID NO:22; d. An adjuvant containing a Toll-like receptor 4 ligand; administering a priming composition comprising an immunologically effective amount of said liposomes, wherein said tau phosphopeptide is presented on the surface of said liposomes, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of formula (I): 【Chemistry 1B】 Or a structure of formula (II): 【Chemistry 2B】 [In the formula, x is an integer from 0 to 10; n is an integer from 3 to 15, tau peptide represents said second tau phosphopeptide; The carrier represents an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and outer membrane protein mixture (OMP) from Neisseria meningitidis, or a derivative thereof. and an immunologically effective amount of a conjugate having the formula: administering the first boosting composition, further comprising a pharma- ceutically acceptable carrier; The first boosting composition, wherein the first tau phosphopeptide and the second tau phosphopeptide each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9 and 12.

3. 1. A combination composition of a priming composition and a first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, a. a first tau phosphopeptide; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO:39 to SEQ ID NO:44; c. a lipidated CpG oligonucleotide selected from the group consisting of SEQ ID NO:18 to SEQ ID NO:22; d. An adjuvant containing a Toll-like receptor 4 ligand; administering the priming composition comprising an immunologically effective amount of the liposomes, wherein the tau phosphopeptide is presented on the surface of the liposomes, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the structure of formula (I): 【Chemistry 1C】 Or a structure of formula (II): 【Chemistry 2C】 [In the formula, x is an integer from 0 to 10; n is an integer from 3 to 15, tau peptide represents said second tau phosphopeptide; The carrier represents an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and outer membrane protein mixture (OMP) from Neisseria meningitidis, or a derivative thereof. and an immunologically effective amount of a conjugate having the formula: administering the first boosting composition, further comprising a pharma- ceutically acceptable carrier; A combination composition of the priming composition and the first boosting composition, wherein the first tau phosphopeptide and the second tau phosphopeptide each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9 and 12.

4. The priming composition, first boost composition, or combination composition of any one of claims 1 to 3, wherein x is an integer from 2 to 6 and n is an integer from 3 to 12.

5. The priming composition, first boost composition, or combination composition of any one of claims 1 to 3, wherein x is 3.

6. The liposome is a. the first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 31, 35, and 38; b. the helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 13 to SEQ ID NO: 17; c. a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:18-SEQ ID NO:22, comprising one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker; d. Monophosphoryl lipid A (MPLA) and Including, the conjugate comprises the second tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9 and 12, conjugated to CRM197 via the linker; A priming composition, a first boosting composition, or a combination composition according to any one of claims 1 to 5.

7. The conjugate has the following structure: 【Chemistry 3】 [In the formula, n is an integer from 3 to 7.] 7. The priming composition, first boosting composition, or combination composition of claim 6, having the following structure:

8. 1. A priming composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, a. a first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 31, 35, and 38; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO:39 to SEQ ID NO:44; c. a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:18-SEQ ID NO:22, comprising one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker; d. Monophosphoryl lipid A (MPLA) and administering the priming composition comprising an immunologically effective amount of the liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 4A】 [In the formula, n is an integer from 3 to 7.] administering a first boosting composition comprising an immunologically effective amount of a conjugate having the formula: The priming composition comprising:

9. 1. A first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, a. a first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 31, 35, and 38; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO:39 to SEQ ID NO:44; c. a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:18-SEQ ID NO:22, comprising one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker; d. Monophosphoryl lipid A (MPLA) and administering a priming composition comprising an immunologically effective amount of a liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 4B】 [In the formula, n is an integer from 3 to 7.] administering said first boosting composition comprising an immunologically effective amount of a conjugate having The first boosting composition comprising:

10. 1. A combination composition of a priming composition and a first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, a. a first tau phosphopeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 31, 35, and 38; b. a helper T cell epitope having an amino acid sequence selected from the group consisting of SEQ ID NO:39 to SEQ ID NO:44; c. a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO:18-SEQ ID NO:22, comprising one or more phosphorothioate internucleotide linkages, and covalently linked to at least one cholesterol via a linker; d. Monophosphoryl lipid A (MPLA) and administering the priming composition comprising an immunologically effective amount of the liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 4C】 [In the formula, n is an integer from 3 to 7.] administering said first boosting composition comprising an immunologically effective amount of a conjugate having A combination composition of the priming composition and the first boosting composition comprising:

11. A priming composition, first boosting composition, or combination composition described in any one of claims 8 to 10, wherein the helper T cell epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NO:13 to SEQ ID NO:

17.

12. 1. A priming composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; and (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-desacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) At least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering the priming composition comprising an immunologically effective amount of the liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 5A】 [In the formula, n is an integer from 3 to 7.] administering a first boosting composition comprising an immunologically effective amount of a conjugate having the formula: The priming composition comprising:

13. 1. A first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; and (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-desacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) At least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering a priming composition comprising an immunologically effective amount of a liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 5B】 [In the formula, n is an integer from 3 to 7.] administering said first boosting composition comprising an immunologically effective amount of a conjugate having The first boosting composition comprising:

14. 1. A combination composition of a priming composition and a first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; and (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-desacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) At least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering the priming composition comprising an immunologically effective amount of the liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 5C】 [In the formula, n is an integer from 3 to 7.] administering said first boosting composition comprising an immunologically effective amount of a conjugate having A combination composition of the priming composition and the first boosting composition comprising:

15. The method further comprising: (a) administering the first boosting composition to the subject at least once after the initial administration of the first boosting composition; and / or (b) administering to the subject a second boosting composition comprising an immunologically effective amount of the liposomes and a pharma- ceutically acceptable carrier. Further comprising: The method comprises: further comprising administering to the subject a second boosting composition comprising an immunologically effective amount of the liposomes and a pharma- ceutically acceptable carrier. A priming composition, a first boosting composition, or a combination composition according to any one of claims 1 to 3, 8 to 10, and 12 to 14.

16. 1. A priming composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; and (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-desacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) At least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering the priming composition comprising an immunologically effective amount of the liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 6A】 [In the formula, n is an integer from 3 to 7.] administering a first boosting composition comprising an immunologically effective amount of a conjugate having the formula: (iii) administering to the subject the first boosting composition or a second boosting composition comprising an immunologically effective amount of the liposomes and a pharma- ceutically acceptable carrier; The priming composition comprising:

17. 1. A first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; and (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-desacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) At least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering a priming composition comprising an immunologically effective amount of a liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 6B】 [In the formula, n is an integer from 3 to 7.] administering said first boosting composition comprising an immunologically effective amount of a conjugate having the formula: (iii) administering to the subject the first boost composition or a second boost composition comprising an immunologically effective amount of the liposomes and a pharma- ceutically acceptable carrier.

18. 1. A combination composition of a priming composition and a first boosting composition for use in a method of inducing antibodies against at least one of phosphorylated tau and condensed paired helical filaments (ePHF) in a subject in need thereof, the method comprising: (i) administering to the subject, (1) a first tau phosphopeptide having the amino acid sequence of SEQ ID NO: 28; and (2) a toll-like receptor 4 agonist comprising monophosphoryl hexa-acyl lipid A, 3-desacylated; (3) a helper T cell epitope comprising the amino acid sequence of SEQ ID NO: 39; and (4) a lipidated CpG oligonucleotide comprising the nucleotide sequence of SEQ ID NO: 18; and (5) At least one lipid selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl-3'-rac-glycerol (DMPG), and cholesterol; administering the priming composition comprising an immunologically effective amount of the liposome, wherein the first tau phosphopeptide is presented on the surface of the liposome, and further comprising a pharma- ceutically acceptable carrier; (ii) administering to the subject a second tau phosphopeptide and an immunogenic carrier conjugated thereto via a linker, the immunogenic carrier having the following structure: 【Chemistry 6C】 [In the formula, n is an integer from 3 to 7.] administering said first boosting composition comprising an immunologically effective amount of a conjugate having the formula: (iii) administering to the subject the first boost composition, or a second boost composition comprising an immunologically effective amount of the liposomes and a pharma- ceutically acceptable carrier.

19. (a)(ii) is performed before (iii); or (b)(ii) is carried out after (iii); A priming composition, a first boosting composition, or a combination composition according to any one of claims 16 to 18.

20. (a) administering the first boosting composition to the subject at least once after the initial administration of the first boosting composition; and / or (b) administering the second boosting composition to the subject at least once.

19. The priming composition, first boosting composition, or combination composition of any one of claims 16 to 18, further comprising:

21. the subject is in need of treatment for a neurodegenerative disease or disorder caused by or associated with the formation of neurofibrillary lesions; The neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, dementia pugilistica, Down's syndrome, Gerstmann-Straussler-Scheinker disease, inclusion body myositis, prion protein cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, Parkinson-Dementia Complex of Guam, non-Guam motor neuron disease with neurofibrillary tangles, argyrophilic grain dementia, corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcifications, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, tangle-only dementia, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), cerebrovascular disease, or Lewy body dementia (LBD). A priming composition, a first boosting composition, or a combination composition according to any one of claims 1 to 3, 8 to 10, 12 to 14, and 16 to 18.

22. 22. The priming composition, first boosting composition, or combination composition of claim 21, wherein the subject is in need of treatment for Alzheimer's disease, early Alzheimer's disease, mild cognitive impairment (MCI) due to Alzheimer's disease, or mild to moderate Alzheimer's disease.

23. (a) an immunologically effective amount of the liposome comprises the first tau phosphopeptide in an amount of about 25 nmoles to about 750 nmoles per dose, about 90 nmoles to about 715 nmoles per dose, or about 90 nmoles to about 535 nmoles per dose; and / or (b) an immunologically effective amount of the liposomes is from about 100 μg to about 2500 μg, from about 300 μg to about 2400 μg, 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 per dose; 1000 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, about 2400 μg, about 2500 μg, or any value therebetween of said first tau phosphopeptide. A priming composition, a first boosting composition, or a combination composition according to any one of claims 1 to 3, 8 to 10, 12 to 14, and 16 to 18.

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