Immunogenic composition that targets phosphorylated tau protein

Abstract

To provide an immunogenic composition that induces an antibody to phosphorylated tau protein.SOLUTION: An immunogenic composition contains an antigenic peptide that induces antibody production to phosphorylated tau protein and carrier protein, where the antigenic peptide is a fragment of human tau protein including (1) YSpSPGpSPG (sequence number 2), (2) AKpSpTPpTAE (sequence number 5), (3) AKpSpTPTAE (sequence number 31), (4) AKpSTPpTAE (sequence number 32), (5) AKSpTPpTAE (sequence number 33) or (6) IVpYKpSPV (sequence number 24).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to immunogenic compositions that target phosphorylated tau protein. [Background technology]

[0002] Dementia is not a phenomenon of memory or mental function decline that accompanies aging, but a pathological state of decreased intelligence due to organic brain damage. Alzheimer's disease is known to be a cause of dementia. Pathological characteristics of Alzheimer's disease include senile plaques and neurofibrillary tangles (NFTs). Senile plaques are formed when a peptide called amyloid beta is deposited outside of nerve cells. NFTs are aggregated fibrils 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. It also binds to various proteins other than microtubules and is involved in various phenomena occurring in the brain and nervous system, such as postnatal brain maturation, regulation of axonal transport and its signal transduction, cellular response to heat stress, and adult neurogenesis. Tau protein is abnormally phosphorylated, triggered by amyloid beta and inflammation. As a result, microtubules are depolymerized, tau protein is detached from microtubules and becomes oligomers from monomers, and further aggregates and fibrils to become NFTs, damaging nerve cells and causing cell death. In addition, it is known that pathological tau protein is released or secreted outside the cell and taken up by other nerve cells, forming new aggregates within those cells. This phenomenon is called "tau protein propagation." It is known that tau aggregates also spread widely as Alzheimer's disease becomes more severe.

[0004] It is known that tau protein has many phosphorylation sites, and it has been reported that they can be divided 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). Up to now, antibodies against phosphorylated tau protein have been produced and therapeutic experiments have been conducted, but promising antibodies that show high therapeutic effects have not been found. However, antibodies have not been examined for all of 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 literature]

[0005] [Non-Patent Document 1] Simic G et al, Biomolecules. 2016 Jan 6;6(1):6. doi: 10.3390 / biom6010006. Summary of the Invention [Problem to be solved by the invention]

[0006] An objective of the present invention is to provide an immunogenic composition that induces antibodies against phosphorylated tau protein. Another objective of the present invention is to provide an immunogenic composition that induces antibodies against pathological hyperphosphorylated tau protein present in the brains of Alzheimer's disease patients. [Means for solving the problem]

[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) 23. A composition comprising a fragment of human tau protein comprising: [2] The composition described in [1] above, wherein the antigenic peptide contains at least two phosphorylated amino acids. [3] The composition described in [1] or [2] above, wherein the antigenic peptide has 10 amino acids or less. [4] The composition described in [1] above, wherein the antigenic peptide is a phosphorylated peptide of any one of (1) to (6). [5] The composition according to any one of [1] to [4] above, wherein the phosphorylated tau protein is a pathological 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, which comprises a conjugate of an antigenic peptide and a carrier protein. [8] The composition according to any one of [1] to [7] above, further comprising an adjuvant. [9] The composition according to any one of [1] to [8] above, which is for treating tauopathy.

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

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

[12] The vaccine described in

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

[0008] INDUSTRIAL APPLICABILITY The present invention can provide an immunogenic composition that induces antibodies against phosphorylated tau protein present in the brains of Alzheimer's disease patients. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the results of primary screening of 30 types of phosphorylated tau epitope candidates, in which conjugates of each phosphorylated tau epitope candidate and AJ peptide were administered to mice and antibody titers against the epitope candidates were measured. [Diagram 2] This figure shows the results of secondary screening of 11 types of phosphorylated tau epitope candidates selected in the primary screening, and shows the results of administering conjugates of each phosphorylated tau epitope candidate with keyhole limpet hemocyanin (KLH) to mice and measuring the antibody titers against the epitope candidates. [Diagram 3] FIG. 1 shows the results of tertiary screening of eight phosphorylated tau epitope candidates selected in the secondary screening. Conjugates of each phosphorylated tau epitope candidate and KLH were administered to mice, and Western blotting was used to confirm whether the induced antibodies do not recognize tau protein in brain extracts of wild-type mice, but recognize hyperphosphorylated tau protein in brain extracts of PS19 Tg mice, a tauopathy model mouse. [Figure 4] FIG. 13 shows the results of administering conjugates of three types of phosphorylated tau epitope candidates selected in the tertiary screening with KLH to rabbits, and examining whether the induced antibodies bind to hyperphosphorylated tau protein with agglutinating activity in brain extracts from PS19 Tg mice. [Diagram 5] FIG. 1 shows the experimental protocol of Example 3. [Figure 6] FIG. 13 shows the time course of serum neutralizing antibody titers in PS19 Tg mice and wild-type mice administered a KLH conjugate of phosphorylated tau epitope YAV-5. [Figure 7] FIG. 13 shows the relationship between serum neutralizing antibody titers and cerebrospinal fluid neutralizing antibody titers in PS19 Tg mice administered a KLH conjugate of phosphorylated tau epitope YAV-5. [Figure 8]FIG. 1 shows the results of measuring the seeding activity of PBS-soluble brain extracts from PS19 Tg mice administered a KLH conjugate of phosphorylated tau epitope YAV-5. (A) is a representative image of intracellular tau aggregates in tau biosensor cells, and (B) is a graph showing the number of intracellular tau aggregates normalized by cell number (number of DAPI-positive cells). [Figure 9] FIG. 13 shows the results of measuring tau aggregates contained in the insoluble fraction of the brain of PS19 Tg mice administered a KLH conjugate of phosphorylated tau epitope YAV-5. [Figure 10] FIG. 1 shows the results of observing NFT-like lesions in the brain of PS19 Tg mice administered a KLH conjugate of phosphorylated tau epitope YAV-5. (A) is a representative image of brain tissue sections immunostained with anti-AT8 antibody, and (B) is the result of counting the number of AT8-positive cells in the hippocampus region. [Figure 11] FIG. 13 shows the results of measuring tau concentrations in the cerebrospinal fluid of PS19 Tg mice administered a KLH conjugate of phosphorylated tau epitope YAV-5. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[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 comprising any of 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. Human tau protein has six isoforms due to alternative splicing of exon 2, exons 2 and 3, and exon 10. 2N4R (no exon skip) has 441 amino acids, 1N4R (exon 3 skip) has 412 amino acids, 0N4R (exon 2 and 3 skip) has 383 amino acids, 2N3R (exon 10 skip) has 410 amino acids, 1N3R (exon 3 and 10 skip) has 381 amino acids, and 0N3R (exon 2, 3, and 10 skip) 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 of SEQ ID NO: 34 are deleted. Skipping of exons 2 and 3 results in a deletion of glutamic acid (E) at position 45 to threonine (T) at position 102 of SEQ ID NO: 34. Skipping of exon 10 results in a deletion of valine (V) at position 275 to serine (S) at position 305 of SEQ ID NO: 34.

[0012] The phosphopeptide (1) is a peptide consisting of 8 amino acids from positions 197 to 204 of SEQ ID NO: 34, in which serine (S) at position 199 and serine (S) at position 202 are phosphorylated. The phosphopeptide (2) is a peptide consisting of 8 amino acids from positions 66 to 73 of SEQ ID NO: 34, in which serine (S) at position 68, threonine (T) at position 69, and threonine (T) at position 71 are phosphorylated. The phosphopeptide (3) is a peptide consisting of 8 amino acids from positions 66 to 73 of SEQ ID NO: 34, in which serine (S) at position 68 and threonine (T) at position 69 are phosphorylated. The phosphopeptide (4) is a peptide consisting of 8 amino acids from positions 66 to 73 of SEQ ID NO: 34, in which serine (S) at position 68 and threonine (T) at position 71 are phosphorylated. The phosphorylated peptide (5) is a peptide consisting of eight amino acids from positions 66 to 73 of SEQ ID NO: 34, in which threonine (T) at position 69 and threonine (T) at position 71 are phosphorylated. The phosphorylated peptide (6) is a peptide consisting of seven amino acids from positions 392 to 398 of SEQ ID NO: 34, in which tyrosine (Y) at position 394 and serine (S) at position 396 are phosphorylated.

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

[0014] The number of amino acids in the antigenic peptide is not particularly limited, and 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. It is preferably 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 one of the phosphorylated peptides (1) to (6) above. Of these, any one of the phosphorylated peptides (1), (2), and (6) is preferred, and the phosphorylated peptide (1) or (2) is more preferred.

[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 an Alzheimer's disease patient. The pathological hyperphosphorylated tau protein present in the brain of an Alzheimer's disease patient 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 propagate.

[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, and hepatitis B virus core protein. Among these, keyhole limpet hemocyanin (KLH) is preferred.

[0018] In addition, the AJ peptide (ELKLIFLHRLKRLRKRLKRK, SEQ ID NO: 35) discovered by the present inventors may be used as a carrier protein. The AJ peptide contains a T cell epitope, so it is useful in that it can induce the desired antibody without the use of an adjuvant (Tenma A et al., FASEB BioAdvances, 2019; 1: 760-772).

[0019] It is preferable that the antigenic peptide and the carrier protein are 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 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-aminoalanine, γ-aminobutyric acid, ε-aminocaproic acid, 7-aminoheptanoic acid, 12-aminolauric acid, glutamic acid, and p-aminobenzoic acid can be used. L-amino acids present in natural proteins and their D-amino acids can also be used. Crosslinkers such as EMCS (N-(6-Maleimidocaproyloxy)succinimide), glutaraldehyde, and sulfo-GMBS (N-γ-maleimidobutyryl-oxysulfosuccinimide ester) can also be used.

[0020] When the above-mentioned KLH is used as the carrier protein, glutaraldehyde can be preferably used if the antigenic peptide does not contain lysine (K). Even if the antigenic peptide contains lysine, glutaraldehyde can be preferably used if the antigenic peptide contains lysine only at the N-terminus or C-terminus. 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 the peptide using EMCS. Moreover, regardless of the sequence of the antigenic peptide, cysteine ​​can be introduced at the C-terminus and linked using sulfo-GMBS.

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

[0022] The conjugate of an antigenic peptide and a carrier protein is preferably one in which the N-terminal amino acid is acetylated. Also, the conjugate is preferably one in which the C-terminal amino acid is amidated. More preferably, the conjugate is one in which the N-terminal amino acid is acetylated and the C-terminal amino acid 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. Specific examples include aluminum adjuvants (e.g., aluminum salts such as aluminum hydroxide, aluminum phosphate, and aluminum sulfate, or combinations thereof), Freund's adjuvant (complete or incomplete), TLR ligands (e.g., CpG, Poly(I:C), Pam3CSK4, etc.), BAY, DC-chol, pcpp, monophosphoryl lipid A, QS-21, cholera toxin, and formylmethionyl peptide. Aluminum adjuvants, TLR ligands, or combinations thereof are preferred. When the immunogenic composition of the present invention contains an adjuvant, the amount of the adjuvant is not particularly limited and may be appropriately selected depending on the type of adjuvant, etc.

[0024] By administering the immunogenic composition of the present invention to a human, antibodies against pathological hyperphosphorylated tau protein are produced, and thus tauopathy can be treated. Therefore, the immunogenic composition of the present invention can be suitably used as a vaccine for treating tauopathy. Note that the treatment of tauopathy includes delaying or inhibiting the progression of tauopathy. Therefore, a vaccine for treating tauopathy comprising the immunogenic composition of the present invention may be called 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 the important pathogenesis is thought to be the excessive phosphorylation of tau protein, which makes it insoluble and abnormally accumulates in cells. Examples of tauopathy include Alzheimer's disease, Pick's disease, progressive supranuclear palsy, cerebral cortical degeneration, Down's syndrome, FTDP-17 (frontotemporal dementia with parkinsonism associated with chromosome 17), argyrophilic grain dementia, neurofibrillary tangle-predominant dementia, and subacute sclerosing panencephalitis. 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 orally or parenterally. Examples of parenteral administration include intraperitoneal administration, subcutaneous administration, intradermal administration, intramuscular administration, intravenous administration, intranasal administration, transdermal administration, transmucosal administration, sublingual administration, and inhalation administration. Parenteral administration is preferred, and intradermal, subcutaneous, or intramuscular administration is more preferred. In addition, parenteral administration means include microneedle injection, needleless injection, stamps, and the like.

[0027] The immunogenic composition of the present invention can be formulated by appropriately mixing an antigenic peptide that induces antibody production against phosphorylated tau protein, a carrier protein, a pharma- ceutically acceptable carrier, and further additives. Specifically, it can be oral preparations such as tablets, coated tablets, pills, powders, granules, capsules, liquids, suspensions, and emulsions; or parenteral preparations such as injections, infusions, suppositories, ointments, and patches. The mixing ratio of the carrier or additive may be appropriately set based on the range normally adopted in the pharmaceutical field. The carrier or additive that can be mixed is not particularly limited, and examples thereof include various carriers such as water, physiological saline, other aqueous solvents, aqueous or oily bases, and various additives such as excipients, binders, pH adjusters, disintegrants, absorption enhancers, lubricants, colorants, flavorings, and flavors.

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

[0029] Liquid preparations for oral administration are prepared by dissolving, suspending or emulsifying in a commonly used diluent. Examples of diluents include purified water, ethanol, and mixtures thereof. Furthermore, the liquid preparation may contain a wetting agent, a suspending agent, an emulsifying agent, a sweetener, a flavoring agent, an aromatic agent, a preservative, a buffer, 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, and propylene glycol; buffers such as phosphate buffer, acetate buffer, borate buffer, carbonate buffer, citrate buffer, Tris buffer, glutamate buffer, and epsilon aminocaproic acid buffer; preservatives such as methyl parahydroxybenzoate, ethyl parahydroxybenzoate, propyl parahydroxybenzoate, butyl parahydroxybenzoate, chlorobutanol, benzyl alcohol, benzalkonium chloride, sodium dehydroacetate, sodium edetate, boric acid, and borax; thickeners such as hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinyl alcohol, and polyethylene glycol; stabilizers such as sodium hydrogen sulfite, sodium thiosulfate, sodium edetate, sodium citrate, ascorbic acid, and dibutylhydroxytoluene; and pH adjusters such as hydrochloric acid, sodium hydroxide, phosphoric acid, and acetic acid. In addition, the injection may further contain a suitable solubilizing agent, for example, alcohol such as ethanol; polyalcohol such as propylene glycol and polyethylene glycol; nonionic surfactants such as polysorbate 80, polyoxyethylene hydrogenated castor oil 50, lysolecithin, and pluronic polyol. Liquid preparations such as injections can be stored by freezing or freeze-drying to remove water. Freeze-dried preparations are used by adding distilled water for injection to redissolve them when needed.

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

[0032] The number of administrations and the administration interval of the immunogenic composition of the present invention are not particularly limited. For example, it may be administered once, or may be administered multiple times at intervals of about 2 days to about 8 weeks. The dose of the immunogenic composition varies depending on the subject of administration, the administration method, etc., but the dose is preferably about 0.01 μg to about 10 mg, more preferably about 0.1 μg to about 1 mg, and even more preferably about 1 μg to about 0.1 mg, in terms of the amount of antigenic peptide per administration.

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

[0034] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these.

[0035] Example 1: Screening for phosphorylated tau epitopes (1) Primary screening Thirty types of phosphorylated tau epitopes (YAV-1 to YAV-30, SEQ ID NO: 1 to 30) each containing at least one phosphorylated amino acid and consisting of 5 to 9 amino acids 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 of the 2N4R isoform of human tau protein (SEQ ID NO: 34). Conjugate peptides (see Table 1, hereinafter referred to as "AJ conjugates") were prepared by linking each phosphorylated tau epitope and AJ peptide (SEQ ID NO: 35) via ε-aminocaproic acid (ε-Acp), and were administered intradermally to BALB / c mice (male, 7 weeks old) (500 μg / 100 μL / mouse, N=4). A second administration was performed two weeks later, and blood was collected five weeks later, serum was separated, and antibody titers were measured.

[0036] [Table 1]

[0037] ELISA was used to measure antibody titers. 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% Tween 20, and HRP-labeled anti-mouse IgG antibody was added and incubated for 3 hours. TMB (3,3',5,5'-tetramethylbenzine) was added to develop 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. Antibody titers are shown as half-maximum values. The half-maximum value is the serum dilution factor that shows half the maximum absorbance value in the measuring device, and was calculated by creating a sigmoid curve of serum dilution factor and absorbance. From the AJ conjugates subjected to the primary screening, 11 types of phosphorylated tau epitopes (YAV-2, 5, 6, 9, 12, 15, 16, 17, 18, 24, and 26) that showed a certain degree of increase in antibody titers were selected.

[0039] (2) Secondary screening Conjugate peptides (hereafter referred to as "KLH conjugates") were prepared by linking 11 types of phosphorylated tau epitopes selected in the primary screening with KLH (Keyhole limpet hemocyanin), and were administered twice to BALB / c mice in the same manner as in the primary screening. Blood was collected 5 weeks after the first administration, and serum was separated and subjected to measurement of antibody titers and Western blotting. Phosphorylated tau epitopes and KLH were linked via glutaraldehyde or EMCS depending on the sequence of the phosphorylated tau epitope.

[0040] ELISA was used to measure antibody titers. Except for coating the 96-well plate with KLH conjugate, antibody titers were measured in the same manner as in the primary screening. The results are shown in Figure 2. Antibody titers are shown as half maximum values. 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 from 4-month-old PS19 Tg mice (Jackson Laboratory) or wild-type mice (WT littermates of PS19 Tg mice (genetic background C57 / B6xC3H)) were collected and homogenized in chilled PBS containing protease inhibitors (Roche). The homogenates were sonicated and centrifuged at 15,000 × g for 5 min. The supernatants were collected and stored at -80°C. PS19 Tg mice are TG mice that incorporate a gene encoding the P301S-1N4R isoform of human tau protein downstream of the prion promoter. They are a tauopathy model mouse that has been confirmed to show brain atrophy and formation of intraneuronal tau aggregates at 6 to 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 and PS19 Tg mice and recombinant tau protein (100 ng) were boiled at 95°C for 5 min and subjected to SDS-PAGE for separation. The separated proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 h. The primary antibodies used were serum 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). The membrane was incubated with serum or antibody overnight at 4°C. After washing the membrane with PBS containing 0.05% Tween 20, the membrane was incubated with HRP-labeled anti-rabbit IgG antibody or HRP-labeled anti-mouse IgG antibody at room temperature for 1 h, and antibody binding 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 in brain extracts from PS19 Tg mice, but did not recognize tau in brain extracts from wild-type mice.

[0044] Example 2: Evaluation of binding between antibodies induced by phosphorylated tau epitopes and hyperphosphorylated tau proteins with seeding activity Rabbits were immunized with KLH conjugates of YAV-2, YAV-5, and YAV-24, and phosphorylated tau epitope-specific antibodies were purified. The KLH conjugates of each epitope and Freund's adjuvant were administered to the rabbits four times every other week. After confirming the antibody titer, blood was collected and the serum was separated, and the antibodies were purified using epitope-specific affinity columns.

[0045] The three rabbit purified antibodies we prepared, control IgG and anti-tau antibody (Tau-5) as negative controls, and anti-phosphorylated tau antibody (p396) as a positive control were used. The antibodies were bound to dynabeads Protein G (Thermo Fisher) and incubated with brain extracts from PS19 Tg mice at room temperature for 10 minutes. Tau bound to the antibodies was removed by immunoprecipitation, and the supernatant was collected and used 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, samples were added to the cells with Lipofectamine 2000 (Life Technologies). Cells were fixed with 4% paraformaldehyde (PFA) and nuclear stained with Hoechst. Confocal microscope images were acquired via the FRET channel (excitation with a 458 nm laser, fluorescence capture with a 500-550 nm filter). Tau aggregation was calculated based on fluorescence intensity and normalized by cell number.

[0046] The results are shown in Figure 4. The seeding activity was shown as the tau aggregation rate of each antibody, with the tau aggregation rate of control IgG taken as 100%. The aggregation rate of antibodies induced by YAV-2 and YAV-5 was significantly lower, indicating that hyperphosphorylated tau protein with seeding activity can be removed.

[0047] Example 3: Evaluation of vaccine effect of phosphorylated tau epitopes PS19 Tg mice (Jackson Laboratory) or wild-type mice (WT littermates of PS19 Tg mice (genetic background C57 / B6xC3H)) at 9 weeks of age were intradermally administered with the KLH conjugate of YAV-5. 200 μg per mouse was administered in two separate 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, 13, 15, 19, 23, 27 and 31 weeks of age and subjected to quantification of antibody titers. At 31 weeks of age, the mice were euthanized, and brain and cerebrospinal fluid were collected and subjected to biochemical and histological analysis. The experimental protocol is shown in Figure 5.

[0048] (1) Serum neutralizing antibody titer against YAV-5 Serum neutralizing antibody titers against YAV-5 were measured by ELISA (see Example 1). The transition of neutralizing antibody titers from the first administration (9 weeks of age) to 31 weeks of age is shown in FIG. 6. Antibody titers are shown as half maximum values. Administration of the KLH conjugate of YAV-5 strongly induced neutralizing antibodies against YAV-5 in both PS19 Tg mice and wild-type mice. After the third administration at 15 weeks of age, the antibody titers in PS19 Tg mice slightly decreased, but remained very high until 31 weeks of age.

[0049] (2) Relationship between serum neutralizing antibody titers and cerebrospinal fluid (CSF) neutralizing antibody titers The antibody titer in the CSF was measured using the same ELISA method as that used to measure the serum neutralizing antibody titer. The correlation between serum neutralizing antibody titer and CSF neutralizing antibody titer is shown in Figure 7. In Figure 7, the serum neutralizing antibody titer is shown as the AUC (area under the curve) value calculated based on the antibody titer measured at each time point of 9, 13, 15, 19, 23 and 31 weeks of age, and the CSF neutralizing antibody titer is shown as the measured value (OD value) by 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 p-tau antibody induced in the periphery 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, which is 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 tau seeding activity was evaluated using tau biosensor cells (Takeda et al., Nature communications 6:8490). The tau biosensor cells were HEK293 cells stably expressing CFP- / YFP-TauRD(P301S) (Holmes, BB et al., Proc. Natl Acad. Sci. USA 111, E4376-E4385 (2014).) and purchased from ATCC. The tau biosensor cells were seeded (3 × 10 4The cells were cultured overnight (cells / well). PBS-soluble brain extract was added to Opti-MEM (Life Technologies) containing Lipofectamine 2000 (Life Technologies), and the mixture was added to the medium of the tau biosensor cells and cultured for 48 hours. The cells were fixed with 4% paraformaldehyde (PFA), and the percentage 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 the intracellular tau aggregates. (B) is a graph showing the number of intracellular tau aggregates normalized by cell number (number of DAPI-positive cells). It was shown that administration of the KLH conjugate of YAV-5 reduced the seeding activity of tau in the brain of PS19 Tg mice by approximately 60% (* p < 0.05). This result suggests that antibodies against YAV-5 reduced 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 brain was homogenized in 5 volumes of PBS (containing protease inhibitors) and centrifuged (10,000g, 4℃, 15 min). After removing the supernatant (PBS-soluble fraction), the pellet was incubated with 1% Sarkosyl at 37℃ for 30 min and sonicated for 20 s. After centrifugation at 100,000g, 4℃ for 30 min, the supernatant was removed and the remaining pellet was solubilized by adding 100μl of 8M urea. After centrifugation at 100,000g, 4℃ for 30 min, the supernatant was used as the brain insoluble fraction for measurement. The amount of phosphorylated tau (T181) in the samples was measured using an ELISA kit (Thermo, KHO0631). The tau concentration was corrected by the total protein concentration in the samples. The Mann-Whitney U test was used for statistical analysis. The results are shown in Figure 9. Administration of the KLH conjugate of YAV-5 reduced insoluble phosphorylated tau in the brains of PS19 Tg mice by more than 60% (*p<0.05).

[0053] (5) Effect on NFT-like lesions in the brain Brain tissue sections were prepared and immunostained with anti-AT8 antibody (antibody that recognizes tau phosphorylated at both serine 202 and threonine 205) according to the method described by Takeda et al. (Nature communications 6:8490). The number of AT8-positive cells in the hippocampus was also 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 is the whole brain, the middle row is the entorhinal cortex, and the lower row is an enlarged view of the dotted frame in the middle row. (B) is the result of counting the number of AT8-positive cells in the hippocampus, where DG represents the dentate gyrus and Hip. represents the whole hippocampus (the sum of CA1, CA3, and DG). Administration of the KLH conjugate of YAV-5 significantly reduced AT8-positive NFT-like lesions in the entire brain, including the entorhinal cortex, of PS19 Tg mice. Furthermore, administration of the KLH conjugate of YAV-5 significantly reduced the number of AT8-positive cells in the dentate gyrus and the entire 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 human total tau. Five microliters of mouse cerebrospinal fluid collected from Cisterna magna was used, diluted 10-fold with the buffer provided with the ELISA kit. The ELISA was performed using Thermo's Tau (Total) Human ELISA Kit (KHB0041). Student's t-test was used for statistical analysis. The results are shown in Figure 11. It was shown that administration of the KLH conjugate of YAV-5 reduced 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 pathology in the brain.

[0055] The present invention is not limited to the above-mentioned embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in the different embodiments. In addition, all academic documents 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 (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) 23. A composition comprising a fragment of human tau protein comprising:

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