Immunogenic composition targeting phosphorylated tau protein

Abstract

The present invention provides an immunogenic composition comprising an antigenic peptide, which is capable of inducing the production of an antibody against a phosphorylated tau protein, and a carrier protein, wherein the antigenic peptide is a human tau protein fragment containing (1) YSpSPGpSPG (SEQ ID NO: 2), (2) AKpSpTPpTAE (SEQ ID NO: 5), (3) AKpSpTPTAE (SEQ ID NO: 31), (4) AKpSTPpTAE (SEQ ID NO: 32), (5) AKSpTPpTAE (SEQ ID NO: 33) or (6) IVpYKpSPV (SEQ ID NO: 24).

Description

Technical Field

[0001] The present invention relates to an immunogenic composition targeting phosphorylated tau protein.

Background Art

[0002] Dementia does not refer to phenomena such as memory decline and mental function decline associated with aging, but rather refers to a pathological state in which intelligence is pathologically reduced due to organic disorders of the brain. Alzheimer's disease is known as a causative disease of dementia. Pathological features of Alzheimer's disease include senile plaques and neurofibrillary tangles (NFT). Senile plaques are formed by the deposition of a peptide called amyloid-β outside nerve cells. NFT is an aggregation and fibrosis of tau protein.

[0003] Tau protein is a protein expressed in nerve cells (neurons) and glial cells in the central and peripheral nervous systems. It was discovered as a type of microtubule-associated protein (MAP) and regulates the polymerization and stabilization of microtubules. In addition to microtubules, it also binds to various other proteins and is involved in various phenomena occurring in the brain nervous system, such as postnatal brain maturation, axonal transport and regulation of its signal transduction, cellular response to heat stress, and adult neurogenesis. Tau protein is abnormally phosphorylated when triggered by amyloid-β, inflammation, etc. As a result, microtubules depolymerize, tau protein detaches from microtubules, becomes oligomers from monomers, further aggregates and fibrillates to form NFT, damages nerve cells, and causes cell death. Furthermore, pathological tau protein is known to be released or secreted extracellularly and taken up by other nerve cells, where it forms new aggregates within the cells. This phenomenon is called "propagation of tau protein". It is known that tau aggregates spread widely as Alzheimer's disease progresses.

[0004] It is known that tau protein has a large number of phosphorylation sites, and it has been reported that they can be classified into phosphorylation sites detected in the brains of Alzheimer's disease patients, phosphorylation sites detected in the brains of both Alzheimer's disease patients and healthy individuals, and phosphorylation sites detected in the brains of healthy individuals (Non-Patent Document 1). So far, antibodies against phosphorylated tau protein have been prepared and therapeutic experiments have been conducted, but no promising antibodies showing high therapeutic effects have been found. However, antibodies have not been examined for all the phosphorylation sites detected in the brains of Alzheimer's disease patients, and it is expected that a vaccine that inhibits the progression of Alzheimer's disease will be developed by finding epitopes with high therapeutic effects.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0006] An object of the present invention is to provide an immunogenic composition that induces an antibody against phosphorylated tau protein. Furthermore, an object of the present invention is to provide an immunogenic composition that induces an antibody against pathological hyperphosphorylated tau protein present in the brain of an Alzheimer's disease patient.

Means for Solving the Problems

[0007] In order to solve the above problems, the present invention includes the following inventions. [1] An immunogenic composition comprising an antigenic peptide that induces antibody production against phosphorylated tau protein and a carrier protein, wherein the antigenic peptide is (1) YSpSPGpSPG (SEQ ID NO: 2), (2) AKpSpTPpTAE (SEQ ID NO: 5), (3) AKpSpTPTAE (SEQ ID NO: 31), (4) AKpSTPpTAE (SEQ ID NO: 32), (5) AKSpTPpTAE (SEQ ID NO: 33) or (6) IVpYKpSPV (SEQ ID NO: 24) A composition that is a fragment of a human tau protein containing [2] The composition according to [1] above, wherein the antigenic peptide contains at least two phosphorylated amino acids. [3] The composition according to [1] or [2] above, wherein the antigenic peptide is 10 amino acids or less. [4] The composition according to [1] above, wherein the antigenic peptide is any one of the phosphorylated peptides (1) to (6). [5] The composition according to any one of [1] to [4] above, wherein the phosphorylated tau protein is a pathologically hyperphosphorylated tau protein. [6] The composition according to any one of [1] to [5] above, wherein the carrier protein is keyhole limpet hemocyanin. [7] The composition according to any one of [1] to [6] above, containing a conjugate of an antigenic peptide and a carrier protein. [8] The composition according to any one of [1] to [7] above, further containing an adjuvant. [9] The composition according to any one of [1] to [8] above, which is for the treatment of tauopathy.

[10] The composition according to [9] above, wherein the tauopathy is Alzheimer's disease.

[11] A vaccine for the treatment of tauopathy, containing the composition according to any one of [1] to [8] above.

[12] The vaccine according to

[11] above, wherein the tauopathy is Alzheimer's disease. [Advantages of the Invention]

[0008] According to the present invention, an immunogenic composition can be provided that induces antibodies against phosphorylated tau protein present in the brains of Alzheimer's disease patients. [Brief Description of the Drawings]

[0009]

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Mode for Carrying Out the Invention

[0010] The present invention provides an immunogenic composition comprising an antigenic peptide that induces antibody production against phosphorylated tau protein and a carrier protein. The antigenic peptide contained in the immunogenic composition of the present invention may be a fragment of human tau protein containing the following phosphorylated peptides (1) to (6). (1) YSpSPGpSPG (SEQ ID NO: 2) (2) AKpSpTPpTAE (SEQ ID NO: 5) (3) AKpSpTPTAE (SEQ ID NO: 31) (4) AKpSTPpTAE (SEQ ID NO: 32) (5) AKSpTPpTAE (SEQ ID NO: 33) (6) IVpYKpSPV (SEQ ID NO: 24)

[0011] The human tau gene is located on the long arm of chromosome 17 and consists of 16 exons. The human tau protein has six isoforms due to alternative splicing of exon 2, exons 2 and 3, and exon 10. 2N4R (without exon skipping) has 441 amino acids, 1N4R (exon 3 skipping) has 412 amino acids, 0N4R (exons 2 and 3 skipping) has 383 amino acids, 2N3R (exon 10 skipping) has 410 amino acids, 1N3R (exons 3 and 10 skipping) has 381 amino acids, and 0N3R (exons 2, 3, and 10 skipping) has 352 amino acids. The amino acid sequence of human tau protein 2N4R is shown in SEQ ID NO: 34. When exon 3 is skipped, aspartic acid (D) at position 74 to threonine (T) at position 102 in SEQ ID NO: 34 are deleted. When exons 2 and 3 are skipped, glutamic acid (E) at position 45 to threonine (T) at position 102 in SEQ ID NO: 34 are deleted. When exon 10 is skipped, valine (V) at position 275 to serine (S) at position 305 in SEQ ID NO: 34 are deleted.

[0012] (1)'s phosphorylated peptide is a peptide in which serine (S) at position 199 and serine (S) at position 202 are phosphorylated in a peptide consisting of 8 amino acids from positions 197 to 204 of SEQ ID NO: 34. (2)'s phosphorylated peptide is a peptide in which serine (S) at position 68, threonine (T) at position 69, and threonine (T) at position 71 are phosphorylated in a peptide consisting of 8 amino acids from positions 66 to 73 of SEQ ID NO: 34. (3)'s phosphorylated peptide is a peptide in which serine (S) at position 68 and threonine (T) at position 69 are phosphorylated in a peptide consisting of 8 amino acids from positions 66 to 73 of SEQ ID NO: 34. (4)'s phosphorylated peptide is a peptide in which serine (S) at position 68 and threonine (T) at position 71 are phosphorylated in a peptide consisting of 8 amino acids from positions 66 to 73 of SEQ ID NO: 34. (5)'s phosphorylated peptide is a peptide in which threonine (T) at position 69 and threonine (T) at position 71 are phosphorylated in a peptide consisting of 8 amino acids from positions 66 to 73 of SEQ ID NO: 34. (6)'s phosphorylated peptide is a peptide in which tyrosine (Y) at position 394 and serine (S) at position 396 are phosphorylated in a peptide consisting of 7 amino acids from positions 392 to 398 of SEQ ID NO: 34.

[0013] The antigenic peptide contained in the immunogenic composition of the present invention may be a fragment of a human tau protein containing any of the phosphorylated peptides (1) to (6) above. Preferably, it is a fragment of a human tau protein containing any of the phosphorylated peptides (1), (2), and (6), and more preferably, it is a fragment of a human tau protein containing the phosphorylated peptide of (1) or (2). It is preferable that the antigenic peptide contains at least 2 phosphorylated amino acids. The phosphorylated amino acids contained in the antigenic peptide may be 3 or more, 4 or more, or 5 or more.

[0014] The number of amino acids of the antigenic peptide is not particularly limited, but may be 30 or less, 25 or less, 20 or less, 18 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, or 10 or less. Preferably, it is 10 amino acids or less, and may be 9 amino acids, 8 amino acids, or 7 amino acids.

[0015] The antigenic peptide contained in the immunogenic composition of the present invention is preferably any of the phosphorylated peptides of (1) to (6) above. Among them, it is preferably a phosphorylated peptide of any of (1), (2), and (6), and more preferably a phosphorylated peptide of (1) or (2).

[0016] The phosphorylated tau protein targeted by the immunogenic composition of the present invention is preferably a pathological hyperphosphorylated tau protein present in the brain of Alzheimer's disease patients. The pathological hyperphosphorylated tau protein present in the brain of Alzheimer's disease patients means a hyperphosphorylated human tau protein having so-called seeding activity. The hyperphosphorylated human tau protein having seeding activity is considered to be a hyperphosphorylated human tau protein having the ability to aggregate and spread.

[0017] The carrier protein contained in the immunogenic composition of the present invention is not particularly limited, and can be appropriately selected from known carrier proteins that can be used in vaccines. Examples of known carrier proteins include albumin, ovalbumin, keyhole limpet hemocyanin (KLH), Pseudomonas aeruginosa exotoxin, tetanus toxin, ricin toxin, diphtheria toxin, cholera toxin, heat-labile enterotoxin, epidermal growth factor, fibroblast growth factor, transferrin, platelet-derived growth factor, poly-L-lysine, poly-L-glutamine, mannose-6-phosphate, hepatitis B virus core protein, and the like. Among them, keyhole limpet hemocyanin (KLH) is preferred.

[0018] Alternatively, as the carrier protein, the AJ peptide (ELKLIFLHRLKRLRKRLKRK, SEQ ID NO: 35) discovered by the present inventors may be used. Since the AJ peptide contains a T cell epitope, it is useful in that the target antibody can be induced without using an adjuvant in combination (Tenma A et al., FASEB BioAdvances, 2019; 1: 760-772).

[0019] The antigenic peptide and the carrier protein are preferably linked to form a conjugate. The antigenic peptide and the carrier protein may be directly linked like a fusion protein, or may be linked via a linker (synonymous with a spacer). The linker is not particularly limited as long as it can link the antigenic peptide and the carrier protein. For example, amino carboxylic acids such as β-alanine, γ-aminobutyric acid, ε-aminocaproic acid, 7-aminoheptanoic acid, 12-aminolauric acid, glutamic acid, and p-aminobenzoic acid can be used. Also, L-amino acids present in natural proteins and their D-amino acids can be used. Further, cross-linkers such as EMCS (N-(6-Maleimidocaproyloxy)succinimide), glutaraldehyde, and sulfo GMBS (N-γ-maleimidobutyryl-oxysulfosuccinimide ester) can be used.

[0020] When using the above-mentioned KLH as the carrier protein, when the antigenic peptide does not contain lysine (K), glutaraldehyde can preferably be used. Even when the antigenic peptide contains lysine, when lysine is contained only at the N-terminus or C-terminus of the antigenic peptide, glutaraldehyde can preferably be used. When the antigenic peptide contains lysine at a position other than the N-terminus or C-terminus, it is preferable to introduce cysteine (C) at the N-terminus or C-terminus of the antigenic peptide and / or to link using EMCS. Also, regardless of the sequence of the antigenic peptide, cysteine can be introduced at the C-terminus and linked using sulfo GMBS.

[0021] The linking order in the conjugate of the antigenic peptide and the carrier protein is not particularly limited. The N-terminal side may be the carrier protein and the C-terminal side may be the antigenic peptide, or conversely, the N-terminal side may be the antigenic peptide and the C-terminal side may be the carrier protein. Preferably, the order is such that the N-terminal side is the carrier protein and the C-terminal side is the antigenic peptide.

[0022] It is preferable that the amino acid at the N-terminus of the conjugate of the antigenic peptide and the carrier protein is acetylated. Also, it is preferable that the amino acid at the C-terminus of the conjugate is amidated. More preferably, the amino acid at the N-terminus of the conjugate is acetylated and the amino acid at the C-terminus is amidated.

[0023] The immunogenic composition of the present invention may further contain one or more adjuvants. The adjuvant can be appropriately selected from known adjuvants and used. Specifically, for example, aluminum adjuvants (such as aluminum salts like aluminum hydroxide, aluminum phosphate, aluminum sulfate, or combinations thereof), Freund's adjuvant (complete or incomplete), TLR ligands (such as CpG, Poly(I:C), Pam3CSK4, etc.), BAY, DC-chol, pcpp, monophosphoryl lipid A, QS-21, cholera toxin, formylmethionyl peptide, and the like can be mentioned. Preferably, it is an aluminum adjuvant, a TLR ligand, or a combination thereof. When the immunogenic composition of the present invention contains an adjuvant, the blending amount of the adjuvant is not particularly limited and can be appropriately selected according to the type of adjuvant and the like.

[0024] By administering the immunogenic composition of the present invention to a human, antibodies against pathogenic hyperphosphorylated tau protein are produced, so that tauopathy can be treated. Therefore, the immunogenic composition of the present invention can be suitably used as a vaccine for treating tauopathy. In addition, the treatment of tauopathy includes delaying or inhibiting the progression of tauopathy. Therefore, the vaccine for treating tauopathy containing the immunogenic composition of the present invention may be referred to as a vaccine for delaying the progression of tauopathy or a vaccine for inhibiting the progression of tauopathy.

[0025] Tauopathy is a general term for diseases in which it is considered that the tau protein undergoes excessive phosphorylation to become insoluble and abnormally accumulates in cells. Examples of tauopathy include Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, Down syndrome, FTDP-17 (frontotemporal dementia with parkinsonism linked to chromosome 17), argyrophilic grain dementia, neurofibrillary change-predominant dementia, subacute sclerosing panencephalitis, and the like. The immunogenic composition of the present invention is preferably used as a vaccine for treating Alzheimer's disease.

[0026] The immunogenic composition of the present invention can be administered by oral administration or parenteral administration. Examples of parenteral administration include intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, intranasal administration, transdermal administration, transmucosal administration, sublingual administration, inhalation administration, and the like. Preferably, it is parenteral administration, and more preferably, it is intradermal administration, subcutaneous administration or intramuscular administration. In addition, means of parenteral administration include microneedle injection, needle-free injection, stamp, and the like.

[0027] The immunogenic composition of the present invention can be formulated by appropriately blending an antigenic peptide that induces antibody production against phosphorylated tau protein, a carrier protein, a pharmaceutically acceptable carrier, and further additives. Specifically, it can be made into oral administration preparations such as tablets, coated tablets, pills, powders, granules, capsules, solutions, suspensions, emulsions, etc.; parenteral administration preparations such as injections, infusions, suppositories, ointments, patches, etc. The blending ratio of the carrier or additive can be appropriately set based on the range commonly adopted in the pharmaceutical field. The carriers or additives that can be blended are not particularly limited. For example, various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases; various additives such as excipients, binders, pH adjusters, disintegrants, absorption promoters, lubricants, colorants, flavoring agents, fragrances, etc.

[0028] Examples of additives used in solid preparations for oral administration include excipients such as lactose, mannitol, glucose, microcrystalline cellulose, corn starch, etc.; binders such as hydroxypropyl cellulose, polyvinylpyrrolidone, magnesium aluminometasilicate, etc.; dispersants such as corn starch, etc.; disintegrants such as calcium carboxymethyl cellulose, etc.; lubricants such as magnesium stearate, etc.; solubilizing agents such as glutamic acid, aspartic acid, etc.; stabilizers; water-soluble polymers such as celluloses such as hydroxypropyl cellulose, hydroxypropylmethyl cellulose, methyl cellulose, etc., synthetic polymers such as polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, etc.; sweeteners such as sucrose, powdered sugar, sucrose, fructose, glucose, lactose, reduced maltose syrup, powdered reduced maltose syrup, grape fructose liquid sugar, fructose glucose liquid sugar, honey, sorbitol, maltitol, mannitol, xylitol, erythritol, aspartame, saccharin, sodium saccharin, etc., coating agents such as sucrose, gelatin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose phthalate, etc.

[0029] Liquid preparations for oral administration are formulated by dissolving, suspending, or emulsifying them in commonly used diluents. Examples of diluents include purified water, ethanol, mixtures thereof, and the like. Furthermore, this liquid preparation may contain wetting agents, suspending agents, emulsifying agents, sweeteners, flavoring agents, fragrances, preservatives, buffers, and the like.

[0030] Examples of additives used in injections for parenteral administration include isotonic agents such as sodium chloride, potassium chloride, glycerin, mannitol, sorbitol, boric acid, borax, glucose, propylene glycol, etc.; buffers such as phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, epsilon-aminocaproic acid buffer, etc.; preservatives such as methyl paraben, ethyl paraben, propyl paraben, butyl paraben, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, borax, etc.; thickening agents such as hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, polyethylene glycol, etc.; stabilizers such as sodium bisulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, dibutylhydroxytoluene, etc.; pH adjusters such as hydrochloric acid, sodium hydroxide, phosphoric acid, acetic acid, etc. Injections may also be further formulated with appropriate solubilizing aids such as alcohols like ethanol; polyalcohols such as propylene glycol, polyethylene glycol, etc.; nonionic surfactants such as polysorbate 80, polyoxyethylene hydrogenated castor oil 50, lysophosphatidylcholine, pluronic polyol, etc. Liquid preparations such as injections can also be stored by removing moisture through freeze preservation or lyophilization, etc. Lyophilized preparations are reconstituted with distilled water for injection or the like before use and then used.

[0031] The immunogenic composition of the present invention can be administered to any animal (human, non-human) having an immune system. For example, mammals such as humans, monkeys, cows, horses, pigs, sheep, goats, dogs, cats, guinea pigs, rats, mice, etc.; birds such as chickens, ducks, geese, etc. The immunogenic composition of the present invention is preferably intended for human children and adults.

[0032] The number of administrations and the administration intervals of the immunogenic composition of the present invention are not particularly limited. For example, a single administration may be used, or multiple administrations may be performed at intervals of about 2 days to about 8 weeks. The dosage of the immunogenic composition varies depending on the administration subject, administration method, etc., but the single dosage is preferably about 0.01 μg to about 10 mg as the amount of antigenic peptide, more preferably about 0.1 μg to about 1 mg, and even more preferably about 1 μg to about 0.1 mg.

[0033] The present invention includes the following inventions. A method for treating tauopathy, comprising administering the immunogenic composition of the present invention to an animal. A method for treating Alzheimer's disease, comprising administering the immunogenic composition of the present invention to an animal. The immunogenic composition of the present invention for use in the treatment of tauopathy. The immunogenic composition of the present invention for use in the treatment of Alzheimer's disease. Use of the immunogenic composition of the present invention for the manufacture of a vaccine or medicament for treating tauopathy. Use of the immunogenic composition of the present invention for the manufacture of a vaccine or medicament for treating Alzheimer's disease.

Examples

[0034] Hereinafter, the present invention will be described in detail by way of examples, but the present invention is not limited thereto.

[0035] 〔Example 1: Screening of phosphorylated tau epitopes〕 (1) Primary screening Thirty phosphorylated tau epitopes (YAV-1 to YAV-30, SEQ ID NOs: 1 to 30) consisting of 5 to 9 amino acids and containing at least 1 phosphorylated amino acid were subjected to primary screening. The numbers indicating the positions of the phosphorylated amino acids correspond to the amino acid positions in the amino acid sequence (SEQ ID NO: 34) of the 2N4R isoform of human tau protein. A conjugate peptide (see Table 1, hereinafter referred to as "AJ conjugate") in which each phosphorylated tau epitope and the AJ peptide (SEQ ID NO: 35) were linked via ε-aminocaproic acid (ε-Acp) was prepared and administered intradermally to BALB / c mice (male, 7 weeks old) (500 μg / 100 μL / mouse, N = 4). A second administration was performed 2 weeks later, blood was collected 5 weeks later, serum was separated, and used for measurement of antibody titer.

[0036]

Table 1

[0037] ELISA was used to measure the antibody titer. A 96-well plate was coated with 10 μg / ml of each AJ conjugate and incubated overnight at 4°C. After blocking with PBS containing 5% skim milk for 2 hours, diluted serum was added and incubated overnight at 4°C. The plate was washed with PBS containing 0.05% tween20, HRP-labeled anti-mouse IgG antibody was added and incubated for 3 hours. TMB (3,3’,5,5’-tetramethylbenzine) was added to develop color of HRP, and sulfuric acid was added after 30 minutes to stop the reaction. The absorbance at a wavelength of 450 nm was measured using a microplate reader (product name iMark, Bio-Rad).

[0038] The results are shown in Figure 1. The antibody titer was shown as the half maximum value. The half maximum value is the dilution ratio of the serum showing the absorbance that is half of the maximum absorbance in the measuring instrument, and was calculated by creating a sigmoid curve of the serum dilution ratio and absorbance. From among the AJ conjugates subjected to the primary screening, 11 phosphorylated tau epitopes (YAV-2, 5, 6, 9, 12, 15, 16, 17, 18, 24, and 26) showing a certain degree of increase in antibody titer were selected.

[0039] (2) Secondary screening A conjugate peptide (hereinafter referred to as "KLH conjugate") in which 11 phosphorylated tau epitopes selected in the primary screening were linked to KLH (Keyhole limpet hemocyanin) was prepared, and administered twice to BALB / c mice in the same manner as in the primary screening. Five weeks after the first administration, blood was collected to separate serum, which was then subjected to measurement of antibody titer and Western blotting. The phosphorylated tau epitope and KLH were linked via glutaraldehyde or EMCS according to the sequence of the phosphorylated tau epitope.

[0040] ELISA was used for the measurement of antibody titer. The antibody titer was measured in the same manner as in the primary screening, except that the 96-well plate was coated with the KLH conjugate. The results are shown in Figure 2. The antibody titer was shown as the half maximum value. Eight of the 11 phosphorylated tau epitopes (YAV-2, 5, 6, 9, 15, 16, 24, and 26) showed high antibody titers.

[0041] (3) Tertiary screening Brains of 4-month-old PS19 Tg mice (Jackson Laboratory) or wild-type mice (WT littermates of PS19 Tg mice with a genetic background of C57 / B6xC3H) were harvested and homogenized in ice-cold PBS containing protease inhibitor (Roche). The homogenate was sonicated and centrifuged at 15,000×g for 5 minutes. The supernatant was collected and stored at -80°C. PS19 Tg mice are transgenic mice in which a gene encoding the P301S-1N4R isoform of human tau protein is integrated downstream of the prion promoter, and they are tauopathy model mice in which brain atrophy and the formation of intracellular tau aggregates have been confirmed at 6-8 months of age (see https: / / www.alzforum.org / research-models / tau-p301s-line-ps19).

[0042] Brain extracts (2 μg each) from wild-type mice, PS19 Tg mice, and recombinant tau protein (100 ng) were boiled at 95°C for 5 minutes and separated by SDS-PAGE. The separated proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour. As primary antibodies, sera collected from mice immunized with KLH conjugates of YAV-2, 5, 6, 9, 15, 16, 24, and 26, anti-tau antibody (Tau-5), and anti-phosphorylated tau antibody (p396) were used. The membrane was incubated with the sera or antibodies at 4°C overnight. After washing the membrane with PBS containing 0.05% tween 20, the membrane was incubated with an HRP-labeled anti-rabbit IgG antibody or an HRP-labeled anti-mouse IgG antibody at room temperature for 1 hour, and the binding of the antibody was evaluated by chemiluminescence (Chemi-Lumi One L, Nacalai Tesque).

[0043] The results are shown in Figure 3. Sera from mice immunized with YAV-2, YAV-5, and YAV-24 recognized only hyperphosphorylated tau from the brain extracts of PS19 Tg mice and did not recognize tau in the brain extracts of wild-type mice.

[0044] [Example 2: Evaluation of the Binding between an Antibody Induced by a Phosphorylated Tau Epitope and a Hyperphosphorylated Tau Protein with Seeding Activity] Rabbits were immunized with KLH conjugates of YAV-2, YAV-5, and YAV-24, and antibodies specific for phosphorylated Tau epitopes were purified. The KLH conjugate of each epitope and Freund's adjuvant were administered to rabbits four times at two-week intervals. After confirming the antibody titer, blood was collected, serum was separated, and the antibody was purified using an epitope-specific affinity column.

[0045] Three types of rabbit-purified antibodies prepared, control IgG as a negative control, anti-Tau antibody (Tau-5), and anti-phosphorylated Tau antibody (p396) as a positive control were used. The antibody was bound to dynabeads protein G (Thermo Fisher) and incubated with brain extracts of PS19 Tg mice at room temperature for 10 minutes. Tau that binds to the antibody was immunoprecipitated and removed, and the supernatant was collected as a sample. HEK293 cells expressing CFP- / YFP-TauRD (P301S) (Proc Natl Acad Sci USA. 2014 Oct 14;111(41):E4376-4385. doi: 10.1073 / pnas.1411649111., Nat Commun. 2015 Oct 13;6:8490. doi: 10.1038 / ncomms9490.) were seeded in 384-well plates, and the next day, the sample was added to the cells together with Lipofectamine 2000 (Life Technologies). The cells were fixed with 4% paraformaldehyde (PFA) and stained with Hoechst for nuclei. Confocal microscope images were acquired through the FRET channel (excited with a 458 nm laser and fluorescence captured with a 500-550 nm filter). Tau aggregation was calculated from the fluorescence intensity and normalized by the number of cells.

[0046] The results are shown in Figure 4. The seeding activity indicates the Tau aggregation rate of each antibody when the Tau aggregation rate of control IgG is set to 100%. The antibodies induced by YAV-2 and YAV-5 showed significantly lower aggregation rates, indicating that they can remove hyperphosphorylated Tau proteins with seeding activity.

[0047] [Example 3: Evaluation of the Vaccine Effect of Phosphorylated Tau Epitope] The KLH conjugate of YAV-5 was administered intradermally to 9-week-old PS19 Tg mice (Jackson Laboratory) or wild-type mice (WT littermates of PS19 Tg mice (genetic background C57 / B6xC3H)). 200 μg per mouse was administered in two divided sites (100 μg / 50 μL / site). Control IgG was administered as a control. The second administration was performed at 11 weeks of age, and the third administration was performed at 15 weeks of age. Blood was collected at 9 weeks, 13 weeks, 15 weeks, 19 weeks, 23 weeks, 27 weeks, and 31 weeks of age and used for quantification of antibody titers. The mice were euthanized at 31 weeks of age, and the brain and cerebrospinal fluid were collected and subjected to biochemical analysis and histological analysis. The experimental protocol is shown in Figure 5.

[0048] (1) Serum neutralizing antibody titer against YAV-5 The serum neutralizing antibody titer against YAV-5 was measured by ELISA (see Example 1). The transition of the neutralizing antibody titer from the first administration (9 weeks of age) to 31 weeks of age is shown in Figure 6. The antibody titer is shown as the half maximum value. Strong induction of neutralizing antibodies against YAV-5 was observed in both PS19 Tg mice and wild-type mice by administration of the KLH conjugate of YAV-5. After the third administration at 15 weeks of age, the antibody titer in PS19 Tg mice decreased slightly, but very high antibody titers were maintained until 31 weeks of age.

[0049] (2) Relationship between serum neutralizing antibody titer and cerebrospinal fluid (CSF) neutralizing antibody titer The antibody titer of CSF was measured using the same ELISA method as the measurement of serum neutralizing antibody titer. The correlation between serum neutralizing antibody titer and CSF neutralizing antibody titer is shown in Fig. 7. In Fig. 7, the serum neutralizing antibody titer is represented by the numerical value of AUC (Area under the curve) calculated based on the antibody titers measured at each time point of 9, 13, 15, 19, 23, and 31 weeks of age, and the CSF neutralizing antibody titer is represented by the measured value (OD value) in ELISA at 31 weeks of age. The two showed a statistically significant positive correlation (R2 = 0.90, p < 0.05, n = 8, Pearson), suggesting that the neutralizing antibody against the p-tau antibody induced peripherally enters the central nervous system in a dose-dependent manner.

[0050] (3) Effects on tau species involved in tau propagation The present inventors have reported that high molecular weight phosphorylated tau involved in tau propagation is detected in the PBS-soluble fraction of brain extracts (Nature communications 6:8490, The American journal of pathology 187(6):1399-1412, PloS one 12(5):e0177914, Nature medicine 26(8):1256-1263). Therefore, PBS-soluble brain extracts were prepared from brains collected at 31 weeks of age, and the seeding activity of tau was evaluated using tau biosensor cells (see Takeda et al., Nature communications 6:8490). Tau biosensor cells are HEK293 cells stably expressing CFP- / YFP-TauRD(P301S) (Holmes, B. B. et al., Proc. Natl Acad. Sci. USA 111, E4376?E4385 (2014).), and were purchased from ATCC. Tau biosensor cells were seeded in a 96-well PDL-coated plate (3×10 4(Cells / well), and cultured overnight. PBS-soluble brain extract was added to Opti-MEM (Life technologies) containing Lipofectamine 2000 (Life technologies), added to the medium of tau biosensor cells, and cultured for 48 hours. Cells were fixed with 4% paraformaldehyde (PFA), and the proportion of tau aggregate-positive cells was counted using flow cytometry (FRET channel). Welch's t-test was used for statistical analysis.

[0051] The results are shown in Figure 8. (A) is a representative image of intracellular tau aggregates in tau biosensor cells, and the white arrow indicates intracellular tau aggregates. (B) is a graph showing the number of intracellular tau aggregates normalized by the number of cells (DAPI-positive cell number). Administration of the KLH conjugate of YAV-5 was shown to reduce the seeding activity of tau in the brains of PS19 Tg mice by approximately 60% (* p<0.05). This result was considered that the antibody against YAV-5 decreased the amount and activity of tau species involved in propagation.

[0052] (4) Effect on tau aggregates contained in the insoluble fraction of the brain Mouse brains were homogenized with 5 volumes of PBS (containing protease inhibitor), and after centrifugation (10,000 g, 4 °C, 15 minutes), the supernatant (PBS-soluble fraction) was removed, and the pellet was incubated with 1% sarcosyl at 37 °C for 30 minutes and sonicated for 20 seconds. After centrifugation at 100,000 g, 4 °C for 30 minutes, the supernatant was removed, 100 μl of 8M Urea was added to the remaining pellet for solubilization, and the supernatant after centrifugation at 100,000 g, 4 °C for 30 minutes was used for measurement as the brain insoluble fraction. The amount of phosphorylated tau (T181) in the sample was measured using an ELISA kit (Thermo, KHO0631). Tau concentration was corrected by the total protein concentration in the sample. Mann-Whitney's U test was used for statistical analysis. The results are shown in Figure 9. Administration of the KLH conjugate of YAV-5 was shown to reduce insoluble phosphorylated tau in the brains of PS19 Tg mice by more than approximately 60% (* p<0.05).

[0053] (5) Effect on NFT-like lesions in the brain According to the method described by Takeda et al. (Nature communications 6:8490), brain tissue sections were prepared and immunostained with anti-AT8 antibody (an antibody that recognizes tau phosphorylated at both serine 202 and threonine 205). Also, the number of AT8-positive cells in the hippocampal region was counted. Student's t-test was used for statistical analysis. The results are shown in Figure 10. (A) is a representative image immunostained with anti-AT8 antibody. The upper row shows the whole brain, the middle row shows the olfactory cortex, and the lower row is an enlarged view within the dotted frame in the middle row. (B) is the result of counting the number of AT8-positive cells in the hippocampal region. DG represents the dentate gyrus, and Hip. represents the whole hippocampus (the total of CA1, CA3, and DG). Administration of the KLH conjugate of YAV-5 significantly reduced AT8-positive NFT-like lesions throughout the brain, including the olfactory cortex, in PS19 Tg mice. Also, administration of the KLH conjugate of YAV-5 significantly decreased the number of AT8-positive cells in the dentate gyrus and the whole hippocampus of PS19 Tg mice (* p<0.05, ** p<0.01).

[0054] (6) Effect on tau concentration in cerebrospinal fluid (CSF) The tau concentration in CSF was compared using a highly sensitive ELISA for total human tau. 5 μl of mouse cerebrospinal fluid collected from the cisterna magna was used and diluted 10-fold with the buffer attached to the ELISA kit for measurement. The ELISA used the Tau (Total) Human ELISA Kit (KHB0041) from Thermo. Student's t-test was used for statistical analysis. The results are shown in Figure 11. Administration of the KLH conjugate of YAV-5 was shown to reduce the tau concentration in the CSF of PS19 Tg mice by approximately 60% (* p<0.05). This result suggests that the tau concentration in CSF can be used as a biomarker to track the vaccine effect on tau lesions in the brain.

[0055] Note that the present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope shown in the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments are also included in the technical scope of the present invention. In addition, all of the academic and patent documents described in this specification are incorporated herein by reference.

Claims

**Claim 1** An immunogenic composition comprising an antigenic peptide that induces antibody production against phosphorylated tau protein and a carrier protein, wherein the antigenic peptide is a fragment of human tau protein that is AKpSpTpPpTAE (SEQ ID NO: 5). **Claim 2** The composition according to claim 1, wherein the phosphorylated tau protein is a pathologically hyperphosphorylated tau protein. **Claim 3** The composition according to claim 1 or 2, wherein the carrier protein is keyhole limpet hemocyanin. **Claim 4** The composition according to any one of claims 1 to 3, comprising a conjugate of the antigenic peptide and the carrier protein. **Claim 5** The composition according to any one of claims 1 to 4, further comprising an adjuvant. **Claim 6** The composition according to any one of claims 1 to 5, which is for the treatment of tauopathy. **Claim 7** The composition according to claim 6, wherein the tauopathy is Alzheimer's disease. **Claim 8** A vaccine for the treatment of tauopathy, comprising the composition according to any one of claims 1 to 5. **Claim 9** The vaccine according to claim 8, wherein the tauopathy is Alzheimer's disease.

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