Tau and amyloid beta peptide immunogenic compositions and related methods

Abstract

The present disclosure relates to improved peptide immunogen constructs that target portions of tau protein for the treatment and / or prevention of tauopathies. The present disclosure also relates to improved peptide immunogen constructs that target portions of amyloid beta protein for the treatment and / or prevention of tauopathies. The present disclosure also relates to compositions and kits containing the peptide immunogen constructs, particularly compositions and kits that include combinations of various peptide immunogen constructs. Methods for making and using the peptide immunogen constructs, as well as antibodies produced by the peptide immunogen constructs, are also provided herein. In some aspects, the present disclosure relates to compositions comprising peptide immunogen constructs based on portions of tau protein and peptide immunogen constructs based on portions of amyloid beta protein, and the use of these peptide immunogen constructs and compositions in therapeutic methods.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 481,744, filed January 26, 2023, and U.S. Patent Application No. 63 / 453,677, filed March 21, 2023, each of which is incorporated by reference herein in its entirety.

[0002] Sequence Listing Reference This application contains a Sequence Listing that has been filed electronically in ST.26(XML) format, entitled "VXH-11025_SL.XML," created on January 16, 2024, and having a size of 216,916 bytes. The Sequence Listing in ST.26(XML) format is a part of the present specification and is incorporated herein by reference in its entirety.

[0003] The present disclosure relates to peptide immunogenic constructs and compositions based on portions of the tau protein and their formulations for the prevention and treatment of tauopathies. The present disclosure also relates to peptide immunogenic constructs and compositions based on portions of the amyloid beta protein and their formulations for the prevention and treatment of tauopathies. The present disclosure further relates to peptide immunogenic constructs based on portions of the tau protein and compositions comprising peptide immunogenic constructs based on portions of the amyloid beta protein, and the use of these compositions in methods of treatment. [Background technology]

[0004] Tau protein is abundant in neurons of the central nervous system and plays a role in stabilizing microtubules. Neurological pathologies and dementia, such as Alzheimer's disease (AD), are associated with defective tau protein that no longer properly stabilizes microtubules. Additional neurodegenerative disorders, called tauopathies, are characterized by abundant fibrillar tau. These diseases include neurofibrillary tangle dementia, chronic traumatic encephalopathy (CTE), argyrophilic grain disease, progressive supranuclear palsy, corticobasal degeneration, glioglobular tauopathy, and Pick's disease.

[0005] Amyloid beta (Aβ) peptides are the major component of amyloid plaques found in the brains of people with Alzheimer's disease, and oligomers of these peptides are toxic to nerve cells.

[0006] There is a need for the development of tau and amyloid beta directed therapies for use in treating patients suffering from diseases such as Alzheimer's disease and tauopathies. Summary of the Invention

[0007] The present disclosure relates to individual peptide immunogenic constructs that target portions of the tau protein for the treatment and / or prevention of tauopathies. The disclosure also relates to individual peptide immunogenic constructs that target portions of the amyloid beta protein for the treatment and / or prevention of tauopathies. The disclosure also relates to compositions containing one, two, or more of the peptide immunogenic constructs, methods of making and using the peptide immunogenic constructs, and antibodies produced by the peptide immunogenic constructs. The disclosure further relates to compositions comprising combinations of one, two, or more tau peptide immunogenic constructs, compositions comprising combinations of one, two, or more tau peptide immunogenic constructs and one or more amyloid beta (Aβ) peptide immunogenic constructs, and methods of using these compositions and combinations in the prevention and treatment of tauopathies, such as Alzheimer's disease and other tauopathies described herein.

[0008] The disclosed peptide immunogen constructs contain about 15 or more amino acids. The tau peptide immunogen construct contains a B-cell epitope derived from a portion of the longest isoform of human tau protein (GenBank: AGF19246.1) having the amino acid sequence of SEQ ID NO: 60 shown in Table 6. The amyloid beta (Aβ) peptide immunogen construct contains an Aβ 1-42 (SEQ ID NO: 68), for example, Aβ 1-14(SEQ ID NO: 69) (see Table 3 and SEQ ID NOs: 70 and 71; see also the Aβ peptides described in WO 2014 / 143087, the contents of which are incorporated herein by reference). In some embodiments, the peptide immunogen constructs and / or B cell epitopes contain chemical modifications and / or unnatural amino acids as described herein. The B cell epitope can be linked, for example, via an optional heterologous spacer, to a heterologous T helper cell (Th) epitope derived from a pathogen protein. The disclosed peptide immunogen constructs stimulate the generation of highly specific antibodies directed against the B cell epitope. The disclosed peptide immunogen constructs can be used as immunotherapy for patients suffering from tauopathies (e.g., Alzheimer's disease; see also other tauopathies listed herein).

[0009] The B cell epitope portion of the tau peptide immunogen construct has the amino acid sequence of full-length tau protein (SEQ ID NO: 60). In some embodiments, the tau peptide immunogen construct has any B cell epitope sequence from tau protein described herein. In some embodiments, the tau peptide immunogen construct has a B cell epitope sequence from unmodified tau protein. In some embodiments, the tau peptide immunogen construct has a B cell epitope sequence from tau protein that has been modified, for example, as described herein (e.g., by substitution or chemical modification of one or more amino acids). Contemplated modifications include, but are not limited to, substitution of one or more amino acids with cysteine ​​to create a disulfide bond, acetylation of one or more amino acids, or N-methylation, as described herein. In some embodiments, the tau B cell epitope has any of the sequences shown in Table 1. In some embodiments, the tau B cell epitope has any of the sequences set forth in Tables 7-10 (particularly, an immunogenic tau B cell epitope provided in any one of Tables 7-10). In some embodiments, the B cell epitope has any one of SEQ ID NOs: 1-8, 17-30, or 63-67 set forth in Table 1, or any one of the sequences set forth in Tables 7-10, or a combination thereof, e.g., a sequence containing SEQ ID NOs: 2 and 7. In some embodiments, the B cell epitope has a sequence containing any one of SEQ ID NOs: 72-76 set forth in Table 1.

[0010] The B cell epitope portion of the Aβ peptide immunogen construct is a full-length Aβ 1-42(SEQ ID NO: 68). In some embodiments, the Aβ peptide immunogen construct has any B cell epitope sequence from the Aβ protein described herein. In some embodiments, the Aβ peptide immunogen construct has a B cell epitope sequence from an unmodified Aβ protein. In some embodiments, the Aβ peptide immunogen construct has a B cell epitope sequence from an Aβ protein that has been modified (e.g., by substitution or chemical modification of one or more amino acids), for example, as described herein. Contemplated modifications include, but are not limited to, substitution of one or more amino acids with cysteine ​​to create a disulfide bond, acetylation of one or more amino acids, or N-methylation, as described herein. In some embodiments, the Aβ B cell epitope has any of the sequences shown in Table 3. In some embodiments, the Aβ B cell epitope has any of the sequences disclosed in WO2014 / 143087 and / or US9,102,752B2, each of which is incorporated by reference in its entirety, particularly for the disclosure of Aβ B cell epitopes (particularly the immunogenic Aβ B cell epitopes provided in WO2014 / 143087 and / or US9,102,752B2). In some embodiments, the B cell epitope is an Aβ B cell epitope. 1-14 (SEQ ID NO: 69) or any other Aβ peptide sequence described in WO2014 / 143087. In some embodiments, the amyloid beta B-cell epitope has SEQ ID NO: 69 or SEQ ID NO: 82.

[0011] The peptide immunogen constructs of the present disclosure may contain a heterologous Th epitope amino acid sequence derived from a pathogenic protein (e.g., any one of ID Nos. 31-59), as shown in Table 5. In certain embodiments, the heterologous Th epitope is derived from a natural pathogen, such as Diphtheria toxin (SEQ ID NO: 35), Plasmodium falciparum (SEQ ID NO: 36), or cholera toxin (SEQ ID NO: 38). In other embodiments, the heterologous Th epitope is an idealized artificial Th epitope derived from measles virus fusion protein (MVF1-5) or hepatitis B surface antigen (HBsAg1-3), either in the form of a single sequence or a combinatorial sequence (e.g., any one of SEQ ID NOs: 32, 39, or 41-45). In some embodiments, the Th epitope sequence is SEQ ID NO: 44. In some embodiments, the Th epitope sequence is SEQ ID NO: 42. In some embodiments, the Th epitope sequence is SEQ ID NO: 45. In some embodiments, the Th epitope sequence is SEQ ID NO: 97. In any one of the peptide immunogen constructs described herein, the provided Th epitope can be substituted for the Th epitope of any of SEQ ID NOs: 42, 44, 45, or 97, e.g., SEQ ID NO: 44.

[0012] In some embodiments, the peptide immunogen construct contains a B-cell epitope derived from tau or Aβ covalently linked to a heterologous T-helper cell (Th) epitope. In some embodiments, the peptide immunogen construct contains a B-cell epitope derived from tau or Aβ linked to a heterologous T-helper cell (Th) epitope via an optional heterologous spacer. In some embodiments, the peptide immunogen construct contains a B-cell epitope derived from tau or Aβ covalently linked directly to a heterologous T-helper cell (Th) epitope.

[0013] In certain embodiments, a tau peptide immunogen construct contains a B cell antigenic site having an amino acid sequence derived from tau (e.g., any one of SEQ ID NOS: 1-8, 17-30, or 63-67, or 202) linked to a heterologous Th epitope derived from a pathogenic protein (e.g., any one of SEQ ID NOS: 31-59 and 97) via an optional heterologous spacer. In some embodiments, a tau peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 2. In some embodiments, a tau peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 7. In some embodiments, the optional heterologous spacer is a molecule or chemical structure capable of linking two amino acids and / or peptides together. In certain embodiments, the spacer is a natural amino acid, a non-natural amino acid, or a combination thereof. In a specific embodiment, a tau peptide immunogen construct has the amino acid sequence of any one of SEQ ID NOS: 9-16 (e.g., SEQ ID NO: 10 or 15) shown in Table 2 (or a construct in which the positions of the tau peptide and Th peptide are reversed).

[0014] In certain embodiments, a tau peptide immunogen construct contains a B cell antigenic site having an amino acid sequence derived from tau (e.g., any one of SEQ ID NOS: 72-76 or 96) linked to a heterologous Th epitope derived from a pathogenic protein (e.g., any one of SEQ ID NOS: 31-59 and 97) via an optional heterologous spacer. In some embodiments, a tau peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 72. In some embodiments, a tau peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 73. In some embodiments, a tau peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 74. In some embodiments, a tau peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 75. In some embodiments, a tau peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 76. In some embodiments, the optional heterologous spacer is a molecule or chemical structure capable of linking two amino acids and / or peptides together. In certain embodiments, the spacer is a natural amino acid, an unnatural amino acid, or a combination thereof. In specific embodiments, the tau peptide immunogen construct has the amino acid sequence of any one of SEQ ID NOS: 77-81 shown in Table 2 (or a construct in which the positions of the tau peptide and Th peptide are reversed).

[0015] In certain embodiments, the Aβ peptide immunogen construct comprises an Aβ peptide linked to a heterologous Th epitope derived from a pathogenic protein (e.g., any one of SEQ ID NOS: 31-59 and 97) via an optional heterologous spacer. 1-42The peptide immunogen construct contains a B cell antigen site having an amino acid sequence derived from SEQ ID NO: 68 (e.g., SEQ ID NO: 68). In some embodiments, the optional heterologous spacer is a molecule or chemical structure capable of linking two amino acids and / or peptides together. In particular embodiments, the spacer is a natural amino acid, an unnatural amino acid, or a combination thereof. In a specific embodiment, the peptide immunogen construct has the amino acid sequence of one of SEQ ID NOs: 70 or 71 shown in Table 4 (or a construct in which the positions of the Aβ peptide and Th peptide are reversed).

[0016] In certain embodiments, the Aβ peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of one of SEQ ID NOs: 68, 69, 82-88 shown in Table 3 linked to a heterologous Th epitope derived from a pathogenic protein (e.g., any one of SEQ ID NOs: 31-59 and 97) via an optional heterologous spacer. In some embodiments, the Aβ peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 69. In some embodiments, the Aβ peptide immunogen construct contains a B cell antigenic site having the amino acid sequence of SEQ ID NO: 82. In some embodiments, the optional heterologous spacer is a molecule or chemical structure capable of linking two amino acids and / or peptides together. In certain embodiments, the spacer is a natural amino acid, an unnatural amino acid, or a combination thereof. In a specific embodiment, the Aβ peptide immunogen construct has the amino acid sequence of SEQ ID NO: 70 or 89 shown in Table 4 (or a construct in which the positions of the Aβ peptide and the Th peptide are reversed).

[0017] The present invention also relates to compositions comprising tau peptide immunogen constructs. In some embodiments, the disclosed compositions contain two or more tau peptide immunogen constructs (e.g., two, three, four, or more tau peptide immunogen constructs) to cover multiple B epitopes from tau. In certain embodiments, the compositions contain a mixture of tau peptide immunogen constructs with two or more heterologous Th epitopes derived from pathogenic proteins (e.g., any combination of two or more of SEQ ID NOS: 9-16, or any combination of two or more of SEQ ID NOS: 9-16 and 77-81) to cover a wide range of patient genetic backgrounds. Compositions containing a mixture of peptide immunogen constructs may result in a higher percentage of response rates upon immunization for the treatment of tauopathies compared to compositions containing only a single Th peptide immunogen construct. In some embodiments, the peptide immunogen constructs targeting tau target different regions of tau (e.g., the N-terminal region of tau for one peptide immunogen construct and the repeat domain for a second peptide immunogen construct, e.g., SEQ ID NOS: 10 and 15, respectively). In some embodiments, the N-terminal region of tau targeted by one or more tau peptide immunogen constructs is within amino acids 1-44 of SEQ ID NO:60. In some embodiments, the N-terminal region of tau targeted by one or more tau peptide immunogen constructs is within amino acids 13-22 of SEQ ID NO:60. In some embodiments, the region targeted by one or more tau peptide immunogen constructs is within amino acids 244-372 of SEQ ID NO:60. In some embodiments, the region targeted by one or more tau peptide immunogen constructs is an amino acid region that is repeated (e.g., repeated 2, 3, or 4 times) within amino acids 244-372 of SEQ ID NO:60. In some embodiments, the region targeted by one or more tau peptide immunogen constructs is within amino acids 250-320 of SEQ ID NO:60. In some embodiments, the region targeted by one or more tau peptide immunogen constructs is an amino acid region that is repeated (e.g., repeated 2, 3, or 4 times) within amino acids 250-320 of SEQ ID NO:60.In some embodiments, the region targeted by one or more tau peptide immunogen constructs is an amino acid region within the 1R, 2R, 3R, and / or 4R repeat region of tau, where the repeat region is known in the art or described herein. In some embodiments, one or more peptide immunogen constructs target a region of tau within amino acids 1-44 or 13-22 of SEQ ID NO:60, and one or more peptide immunogen constructs target a region of tau within amino acids 244-372 or 250-320 of SEQ ID NO:60 (or the amino acid region repeated within amino acids 244-372 or 250-320 of SEQ ID NO:60 as described herein).

[0018] In some embodiments, a composition comprising one or more tau peptide immunogen constructs also comprises one or more Aβ peptide immunogen constructs (e.g., a peptide immunogen construct comprising SEQ ID NO: 70, a peptide immunogen construct comprising SEQ ID NO: 71, or a combination of SEQ ID NOs: 70 and 71). In some embodiments, a composition comprising one or more tau peptide immunogen constructs also comprises one or more Aβ peptide immunogen constructs (e.g., a peptide immunogen construct comprising SEQ ID NO: 70, a peptide immunogen construct comprising SEQ ID NO: 89, or a combination of SEQ ID NOs: 70 and 89).

[0019] The present disclosure also relates to pharmaceutical compositions for the treatment and / or prevention of tauopathies. In some embodiments, the pharmaceutical composition contains one, two, or more of the disclosed peptide immunogen constructs in the form of a stabilized immunostimulatory complex formed through electrostatic association by mixing a CpG oligomer (e.g., CpG1, CpG2, or CpG3) with a composition containing a peptide immunogen complex. Such stabilized immunostimulatory complexes can further enhance the immunogenicity of the peptide immunogen construct. In some embodiments, the pharmaceutical composition contains an adjuvant, such as a mineral salt, such as alum gel (ALHYDROGEL), aluminum phosphate (ADJUPHOS), or a water-in-oil emulsion containing MONTANIDE ISA51 or 720.

[0020] The present invention also relates to antibodies against the disclosed tau peptide immunogen constructs. In particular, the disclosed tau peptide immunogen constructs, when administered to a subject, can stimulate the production of highly specific antibodies that cross-react with tau amino acid sequences (e.g., any one or more of SEQ ID NOS: 1-8, 17-30, 63-67, or 202). The highly specific antibodies produced by the peptide immunogen constructs cross-react with recombinant tau-containing proteins. The disclosed antibodies bind to tau with high specificity, with little or no targeting of heterologous Th epitopes used for immunogenicity enhancement. This contrasts significantly with conventional proteins or other biological carriers used for such peptide antigenic enhancement.

[0021] The present disclosure also relates to antibodies directed against the disclosed amyloid beta peptide immunogen constructs. In particular, the disclosed amyloid beta peptide immunogen constructs, when administered to a subject, can stimulate the production of highly specific antibodies that cross-react with amyloid beta amino acid sequences (e.g., any one or more of SEQ ID NOS: 68, 69, or 83-88). The highly specific antibodies produced by the peptide immunogen constructs cross-react with recombinant amyloid beta-containing proteins. The disclosed antibodies bind to tau with high specificity, with little, if any, directed against heterologous Th epitopes used for immunogenicity enhancement, in stark contrast to conventional proteins or other biological carriers used for such peptide antigenic enhancement.

[0022] The present disclosure also includes methods for treating and / or preventing tauopathies using the disclosed peptide immunogen constructs and / or antibodies against the peptide immunogen constructs. In some embodiments, the methods for treating and / or preventing tauopathies include administering to a subject (e.g., a mammal) a composition containing one, two, or more of the disclosed peptide immunogen constructs. In certain embodiments, the compositions utilized in the methods contain any one or more of the disclosed peptide immunogen constructs in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, via electrostatic association, optionally further supplemented with mineral salts or oils as adjuvants for administration to patients with tauopathies. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogen constructs to a host at risk for or suffering from a tauopathy. In some embodiments, the subject or patient is human.

[0023] In some aspects, the disclosure provides a composition or kit comprising a first tau peptide immunogen construct comprising a first tau B-cell epitope comprising or consisting of about 6 to about 40 amino acid residues from a full-length tau protein of SEQ ID NO: 60; and (i) a second tau peptide immunogenic construct comprising a second tau B-cell epitope comprising, or consisting of, about 6 to about 40 amino acid residues from the full-length tau protein of SEQ ID NO: 60, wherein the second B-cell epitope is not the same as the first B-cell epitope; and / or (ii) A composition or kit is provided that includes an amyloid beta (Aβ) peptide immunogen construct that includes an amyloid beta B-cell epitope that includes or consists of about 6 to about 42 amino acid residues derived from the full-length amyloid beta protein of SEQ ID NO: 68.

[0024] In some embodiments of the foregoing or related aspects, a composition or kit disclosed herein comprises a second tau peptide immunogenic construct. In some embodiments of the foregoing or related aspects, a composition or kit disclosed herein comprises an amyloid beta peptide immunogenic construct. In some embodiments of the foregoing or related aspects, a composition or kit disclosed herein comprises a second tau peptide immunogenic construct and an amyloid beta peptide immunogenic construct.

[0025] In some embodiments of the aforementioned or related aspects, the first tau B-cell epitope comprises or consists of about 6-40 amino acid residues from the N-terminal amino acids 1-44 of SEQ ID NO: 60, and the second tau B-cell epitope comprises or consists of about 6-40 amino acid residues from the repeat domain amino acids 244-372 of SEQ ID NO: 60. In some embodiments of the aforementioned or related aspects, the amyloid beta B-cell epitope comprises at least 6 amino acid residues from amino acids 1-16 of SEQ ID NO: 68. In some embodiments of the aforementioned or related aspects, the amyloid beta B-cell epitope comprises at least 8 amino acid residues from amino acids 1-16 of SEQ ID NO: 68.

[0026] In some embodiments of the foregoing or related aspects, the first tau B-cell epitope, the second tau B-cell epitope, and the amyloid beta B-cell epitope are each or independently about 8-40 amino acids in length. In some embodiments of the foregoing or related aspects, the first tau B-cell epitope, the second tau B-cell epitope, and the amyloid beta B-cell epitope are each or independently about 8-15 amino acids in length. In some embodiments of the foregoing or related aspects, the first tau B-cell epitope, the second tau B-cell epitope, and the amyloid beta B-cell epitope are each or independently about 10-14 amino acids in length.

[0027] In some embodiments of the aforementioned or related aspects, the first tau B cell epitope, and, if present, the second tau B cell epitope, are independently selected from SEQ ID NOs: 1-8, 17-30, 63-67, 72-76, 96, and 202. In some embodiments of the aforementioned or related aspects, the first tau B cell epitope comprises or consists of any one of SEQ ID NOs: 1-8, 17-30, 63-67, 72-76, 96, and 202, and the second tau B cell epitope comprises or consists of any one of SEQ ID NOs: 1-8, 17-30, 63-67, 72-76, 96, and 202. In some embodiments, the first tau B cell epitope, and, if present, the second tau B cell epitope, are independently selected from SEQ ID NOs: 2, 4, 7, 72-76, and 202. In some embodiments of the aforementioned or related aspects, the first tau B cell epitope comprises or consists of SEQ ID NO: 2. In some embodiments of the aforementioned or related aspects, the first tau B cell epitope comprises or consists of SEQ ID NO: 7. In some embodiments of the aforementioned or related aspects, the first tau B cell epitope comprises or consists of SEQ ID NO: 202. In some embodiments of the aforementioned or related aspects, the first tau B cell epitope is selected from SEQ ID NO: 1 or SEQ ID NO: 2, and the second tau B cell epitope is selected from SEQ ID NOs: 7 and 72-76. In some embodiments of the aforementioned or related aspects, the first tau B cell epitope comprises or consists of SEQ ID NO: 2, and the second tau B cell epitope comprises or consists of SEQ ID NO: 7. In some embodiments of the aforementioned or related aspects, the amyloid beta B cell epitope is selected from SEQ ID NOs: 69 and 82-88. In some embodiments of the aforementioned or related aspects, the amyloid beta B-cell epitope comprises or consists of SEQ ID NO:69 or SEQ ID NO:82.

[0028] In some embodiments of the foregoing or related aspects, the first tau peptide immunogen construct comprises a first heterologous T helper (Th) epitope, the second tau peptide immunogen construct, if present, comprises a second heterologous Th epitope, and the amyloid beta peptide immunogen construct, if present, comprises a third heterologous Th epitope. In some embodiments, the first heterologous Th epitope, the second heterologous Th epitope, and the third heterologous Th epitope are each independently selected from SEQ ID NOs: 31-59 and 97. In some embodiments, the first heterologous Th epitope, the second heterologous Th epitope, and the third heterologous Th epitope are each independently selected from SEQ ID NOs: 42, 44, 45, and 97. In some embodiments, the first heterologous Th epitope, the second heterologous Th epitope, and the third heterologous Th epitope each have SEQ ID NO: 44.

[0029] In some embodiments of the foregoing or related aspects, the first heterologous Th epitope and the first tau B cell epitope, the second heterologous Th epitope and the second tau B cell epitope, and the third heterologous Th epitope and the amyloid beta B cell epitope are each covalently linked, either directly or via a heterologous spacer. In some embodiments, a composition or kit disclosed herein comprises a heterologous spacer linking the first heterologous Th epitope and the first tau B cell epitope, the second heterologous Th epitope and the second tau B cell epitope, and / or the third heterologous Th epitope and the amyloid beta B cell epitope. In some embodiments, the heterologous spacer comprises one or more amino acids. In some embodiments, one or more amino acids are Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95), or ε-Lys-Lys-Lys-Lys (SEQ ID NO: 62). In some embodiments, one or more amino acids are Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95) or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62).

[0030] In some embodiments of the aforementioned or related aspects, the first tau peptide immunogen construct, and, if present, the second tau peptide immunogen construct, have the formula: (Th) m -(A) n -(Tau fragment)-X or (Tau fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (tau fragment) is a B-cell epitope having about 6 to about 40 amino acid residues (optionally, about 10 to 40 amino acid residues) derived from the full-length tau protein of SEQ ID NO: 60; X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0031] In some embodiments of the aforementioned or related aspects, the amyloid beta peptide immunogen construct has the formula: (Th) m -(A) n -(Aβ fragment)-X or (Aβ fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (Aβ fragment) is a B-cell epitope having about 6 to about 42 amino acid residues (optionally, about 10 to 42 amino acids are present) from the full-length tau protein of SEQ ID NO: 68; X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0032] In some embodiments of the foregoing or related aspects, the first tau peptide immunogen construct comprises a first heterologous Th epitope covalently linked to the carboxyl terminus of the first tau B-cell epitope, the second tau peptide immunogen construct comprises a second heterologous Th epitope covalently linked to the amino terminus of the second tau B-cell epitope, and / or the amyloid beta peptide immunogen construct comprises a third heterologous Th epitope covalently linked to the carboxyl terminus of the amyloid beta B-cell epitope.

[0033] In some embodiments of the foregoing or related aspects, the first and second tau peptide immunogen constructs are selected from SEQ ID NOs:9-16 and 77-81, or are constructs in which the positions of the peptide cellular epitope and Th epitope are reversed. In some embodiments of the foregoing or related aspects, the first tau peptide immunogen construct comprises or consists of the amino acid sequence of SEQ ID NO:10 or SEQ ID NO:15. In some embodiments of the foregoing or related aspects, the first tau peptide immunogen construct comprises or consists of the amino acid sequence of SEQ ID NO:10 and the second tau peptide immunogen construct comprises or consists of the amino acid sequence of SEQ ID NO:15. In some embodiments of the foregoing or related aspects, the first and / or second tau peptide immunogen constructs are selected from SEQ ID NOs:77-81, optionally wherein the first tau peptide immunogen construct has the amino acid sequence of SEQ ID NO:10 and the second tau peptide immunogen construct is selected from SEQ ID NOs:77-81. In some embodiments of the aforementioned or related aspects, the amyloid beta peptide immunogen construct is selected from SEQ ID NOs: 70, 71, and 89, or is a construct in which the positions of the B cell epitope and the Th epitope are reversed. In some embodiments, the amyloid beta peptide immunogen construct comprises or consists of SEQ ID NO: 70. In some embodiments, the amyloid beta peptide immunogen construct comprises or consists of SEQ ID NO: 89.

[0034] In some aspects, the present disclosure provides a tau peptide immunogen construct comprising a B-cell epitope comprising the amino acid sequence of SEQ ID NO:202.

[0035] In some aspects, the disclosure provides a tau peptide immunogenic construct comprising a B cell epitope comprising an amino acid sequence selected from SEQ ID NOs: 72 to 76. In some embodiments of the foregoing or related aspects, the tau peptide immunogenic construct disclosed herein has the formula: (Th) m -(A) n -(B cell epitope)-X or (B cell epitope)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (B cell epitope) comprises, consists essentially of, or consists of an amino acid sequence selected from any one of SEQ ID NOs: 72-76; X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0036] In some embodiments, the tau peptide immunogen constructs disclosed herein comprise, consist essentially of, or consist of the amino acid sequence of any one of SEQ ID NOs: 77-81.

[0037] In some aspects, the disclosure provides an amyloid beta peptide immunogenic construct comprising a B-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO: 82. In some embodiments of the foregoing or related aspects, the amyloid beta peptide immunogenic construct disclosed herein has the formula: (Th) m -(A) n -(B cell epitope)-X or (B cell epitope)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (B-cell epitope) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 82; X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0038] In some embodiments, the amyloid beta peptide immunogen constructs disclosed herein comprise, consist essentially of, or consist of the amino acid sequence of SEQ ID NO:89.

[0039] In some aspects, the present disclosure provides compositions or kits comprising the tau peptide immunogenic constructs disclosed herein.

[0040] In some aspects, the present disclosure provides compositions or kits comprising the amyloid beta peptide immunogen constructs disclosed herein.

[0041] In some embodiments of the foregoing or related aspects, a composition or kit disclosed herein is a single composition comprising a first tau peptide immunogenic construct and at least one (or both) of (i) a second tau peptide immunogenic construct and (ii) an amyloid beta peptide immunogenic construct. In other embodiments, a composition comprising a first tau peptide immunogenic construct may be combined or co-administered with one or more compositions comprising one or more of (i) a second tau peptide immunogenic construct and (ii) an amyloid beta peptide immunogenic construct.

[0042] In some embodiments of the foregoing or related aspects, a composition or kit disclosed herein is a kit comprising a first tau peptide immunogenic construct and at least one of a second tau peptide immunogenic construct and an amyloid beta peptide immunogenic construct. In some embodiments, prior to administration to a subject, the components of the kit are mixed to create a single composition comprising the first tau peptide immunogenic construct and at least one of the second tau peptide immunogenic construct and an amyloid beta peptide immunogenic construct. In some embodiments, the components of the kit are used to create a first composition comprising the first tau peptide immunogenic construct and a second composition comprising at least one of the second tau peptide immunogenic construct and an amyloid beta peptide immunogenic construct.

[0043] In some aspects, the present disclosure provides a pharmaceutical composition comprising a composition disclosed herein and a pharmaceutically acceptable delivery vehicle and / or adjuvant. In some embodiments of the foregoing or related aspects, a first tau peptide immunogen construct, a second tau peptide immunogen construct, and / or an amyloid beta peptide immunogen construct are mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex. In some embodiments, the pharmaceutical composition disclosed herein comprises three or four of the tau peptide immunogen constructs. In some embodiments, the pharmaceutical composition disclosed herein comprises two or more amyloid beta peptide immunogen constructs.

[0044] In some aspects, the present disclosure provides an isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of a tau peptide immunogenic construct or an amyloid beta peptide immunogenic construct, obtained by administering to a mammal a composition(s) disclosed herein, or a peptide immunogenic construct disclosed herein. In some embodiments of the foregoing or related aspects, the isolated antibody or epitope-binding fragment thereof disclosed herein binds to the tau peptide immunogenic construct or the amyloid beta peptide immunogenic construct.

[0045] In some aspects, the present disclosure provides compositions comprising an isolated antibody or epitope-binding fragment thereof disclosed herein.

[0046] In some aspects, the disclosure provides methods of preventing, inhibiting, reducing the severity of, delaying, or treating a tauopathy in a subject, the methods comprising administering to the subject a tau peptide immunogenic construct, amyloid beta peptide immunogenic construct, or composition(s) disclosed herein. In some embodiments of the foregoing or related aspects, the methods comprise administering a composition, wherein the first tau peptide immunogenic construct and at least one of the second tau peptide immunogenic construct and the amyloid beta peptide immunogenic construct are comprised within a single composition.

[0047] In some aspects, the disclosure provides a method of preventing, inhibiting, reducing the severity of, delaying, or treating a tauopathy in a subject, the method comprising administering to the subject a first tau peptide immunogen construct comprising a first tau B-cell epitope comprising or consisting of about 6 to about 40 (e.g., 6-40, 8-40, or 8-15) amino acid residues from a full-length tau protein of SEQ ID NO: 60; (i) a second tau peptide immunogen construct comprising a second tau B-cell epitope comprising or consisting of about 6 to about 40 (e.g., 6 to 40, 8 to 40, or 8 to 15) amino acid residues from the full-length tau protein of SEQ ID NO: 60, wherein the second B-cell epitope is not the same as the first B-cell epitope; and / or (ii) administering an amyloid beta (Aβ) peptide immunogen construct comprising an amyloid beta B-cell epitope comprising or consisting of about 6 to about 42 (e.g., 6 to 40, 8 to 40, or 8 to 15) amino acid residues derived from the full-length amyloid beta protein of SEQ ID NO: 68; The first tau peptide immunogenic construct and the second tau peptide immunogenic construct and / or amyloid beta peptide immunogenic construct are contained within separate compositions that are administered separately. In other embodiments of the method, the first tau peptide immunogenic construct and the second tau peptide immunogenic construct and / or amyloid beta peptide immunogenic construct are contained within a single composition.

[0048] In some embodiments of the aforementioned or related aspects, the tauopathy is chosen from Alzheimer's disease, Lewy body disease, frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia combined, dementia pugilistica, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy. In some embodiments, the tauopathy is Alzheimer's disease. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. [Brief explanation of the drawings]

[0049] [Figure 1] FIG. 1 is a schematic diagram showing a Th peptide carrier bound to a target-specific B-cell epitope (top) and the primary structure of tau with functional domains and targeting peptides annotated (bottom). [Figure 2A] 1 is a dot blot, a set of graphs, and a table showing the results of antibody binding characterization. [Figure 2B] 1 is a dot blot, a set of graphs, and a table showing the results of antibody binding characterization. [Figure 3]1 is a set of graphs showing the results of an aggregation assay using a tau biosensor line. [Figure 4] 1 is a set of graphs showing the results of a tau uptake assay. [Figure 5A] 1 is a graph showing the results of a tau uptake assay. [Figure 5B] 1 is a graph showing the results of a tau uptake assay. [Figure 5C] 1 is a graph showing biolayer interference analysis of IgG from p5555kb, IgG from p5187kb, and mixed IgG from p5555kb and p5187kb. [Figure 5D] Anti-tau IgG isotype EC50 titers are shown. [Figure 6A] 1 shows graphs and photographic images showing the results of vaccination of WT and P301L mice with p5555kb. Arrowheads indicate injections. [Figure 6B] 10 shows graphs and photographic images showing the results of vaccination of WT and P301L mice with p5555kb. [Figure 6C] 10 shows graphs and photographic images showing the results of vaccination of WT and P301L mice with p5555kb. [Figure 7A] Figure 1 shows graphs depicting IgG titers in rats immunized with p5555kb, p5187kb, or VXX-301. VXX-301 refers to a combination vaccine of p5555kb and p5187kb in one formulation. Figure 2 shows IgG titers against monomeric tau after vaccination with p5555kb, p5187kb, or VXX-301. [Figure 7B] Figure 1B is a graph showing IgG titers in rats immunized with p5555kb, p5187kb, or VXX-301. VXX-301 refers to a combination vaccine of p5555kb and p5187kb in one formulation. Figure 1C shows IgG titers against fibrillar tau after vaccination with p5555kb, p5187kb, or VXX-301. [Figure 7C]Figure 1 shows graphs depicting IgG titers in rats immunized with p5555kb, p5187kb, or VXX-301. VXX-301 refers to a combination vaccine of p5555kb and p5187kb in one formulation. Figure 2 shows IgG titer curves over time against monomeric tau after vaccination with p5555kb. [Figure 7D] Figure 1 shows graphs depicting IgG titers in rats immunized with p5555kb, p5187kb, or VXX-301. VXX-301 refers to a combination vaccine of p5555kb and p5187kb in one formulation. Figure 2 shows IgG titer curves over time against monomeric tau after vaccination with p5187kb. [Figure 7E] Figure 1 shows graphs depicting IgG titers in rats immunized with p5555kb, p5187kb, or VXX-301. VXX-301 refers to a combination vaccine of p5555kb and p5187kb in one formulation. Figure 2 shows IgG titer curves over time against monomeric tau after vaccination with VXX-301. [Figure 8] Figure 1 is a graph showing biolayer interference analysis of IgG from p5555kb, p5187kb, or VXX-301. IgG1 and IgG2b were enriched after vaccination with lower titers for IgG2a and IgG2c. [Figure 9] Figure 1 shows the results of Western blot analysis of anti-tau antibodies from rats vaccinated with p5555kb, p5187kb, or VXX-301. Various recombinant preparations of tau were examined for binding, including full-length preparations 2N4R, 2N3R, WT preformed fibrils (PFF), P301S, and P301L K18 (a C-terminal fragment of tau containing the four repeat domains). White arrows indicate that antibodies from p5187kb and VXX-301 were able to bind to all tau preparations. [Figure 10] 1 is a graph showing inhibition of tau aggregation by anti-tau mAb bepranemab or anti-tau antibodies from rats vaccinated with p5555kb, p5187kb, or VXX-301. [Figure 11] 1 is a graph showing the results of a pHrodo-PFF tau uptake assay in b103 cells using anti-tau mAbs semolinemab (Sem), bepranemab (Bep), or anti-tau antibodies from rats vaccinated with p5555kb, p5187kb, or VXX-301. [Figure 12] Figures 12A-12B are graphs showing protection against tau-associated pathology and death, as well as antibody titers, in female P301L mice vaccinated with saline control, p5555kb, p6187kb, or VXX-301. Kaplan-Meier curves for the observed onset of ataxia (Figure 12A) and survival of P301L mice (Figure 12B) are shown. Figures 12C-12D show terminal anti-tau antibody titers against monomeric tau (Figure 12C) or fibrillar tau (Figure 12D). Arrowheads in Figures 12A-12B indicate injections. [Figure 13] 1 is a graph showing the immunogenicity of Aβ (p3102kb) and tau (p5555kb, p5404kb, or p5137kb) combination vaccines against monomeric tau, fibrillar tau, fibrillar P301S tau, or a control B cell epitope peptide not conjugated to a Th peptide (a-peptide). DETAILED DESCRIPTION OF THE INVENTION

[0050] The present disclosure relates to individual peptide immunogenic constructs that target portions of the tau protein for the treatment and / or prevention of Alzheimer's disease and tauopathies. The disclosure also relates to compositions containing the tau peptide immunogenic constructs, methods of making and using the peptide immunogenic constructs, and antibodies produced by the peptide immunogenic constructs. The disclosure further relates to compositions comprising two or more (e.g., two, three, or four) tau peptide immunogenic constructs (e.g., one or more of SEQ ID NOS: 9-16, e.g., 10 and 15, or one or more of SEQ ID NOS: 77-81, in any combination; see also below), and methods of using such compositions or combinations for the prevention and / or treatment of Alzheimer's disease and tauopathies. The present disclosure further provides compositions comprising one or more (e.g., 1, 2, 3, or 4) tau peptide immunogenic constructs, and one or more (e.g., 1, 2, 3, or 4) Aβ peptide immunogenic constructs (e.g., SEQ ID NO: 70, SEQ ID NO: 71, SEQ ID NO: 89, or any combination thereof, such as SEQ ID NO: 70 and SEQ ID NO: 71, or SEQ ID NO: 70 and SEQ ID NO: 89), and methods of using such compositions or combinations for the prevention and / or treatment of Alzheimer's disease and tauopathies. In some embodiments, SEQ ID NO: 10 is combined with SEQ ID NOs: 70 and 71. In some embodiments, SEQ ID NO: 11 is combined with SEQ ID NOs: 70 and 71. In some embodiments, SEQ ID NO: 12 is combined with SEQ ID NOs: 70 and 71. In some embodiments, SEQ ID NO: 13 is combined with SEQ ID NOs: 70 and 71. In some embodiments, SEQ ID NO: 14 is combined with SEQ ID NOs: 70 and 71. In some embodiments, SEQ ID NO: 15 is combined with SEQ ID NOs: 70 and 71. In some embodiments, SEQ ID NO: 16 is combined with SEQ ID NOs: 70 and 71. In some embodiments, an additional different sequence of any one of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, or 16 is added to the above-listed combinations (such as any additional tau peptide immunogen construct sequences described herein). In some embodiments, only SEQ ID NO: 70 or only SEQ ID NO: 71 is included in the above-listed combinations.In some embodiments, SEQ ID NO: 10 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 11 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 12 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 13 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 14 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 15 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 16 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 77 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 78 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 79 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 80 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, SEQ ID NO: 81 is combined with SEQ ID NO: 70 and / or 89. In some embodiments, an additional, different tau peptide immunogen construct sequence is added to any of the combinations listed above (such as any additional tau peptide immunogen construct sequence described herein). In some embodiments, only SEQ ID NO: 70 or only SEQ ID NO: 89 is included in any of the combinations listed above.

[0051] The disclosed peptide immunogen constructs contain about 15 or more amino acids, hi some embodiments, the disclosed peptide immunogen constructs comprise no more than about 80, no more than about 75, no more than about 70, no more than about 60, no more than about 50, no more than about 40, or no more than about 35 amino acids.

[0052] In some embodiments, a tau peptide immunogen construct contains a B cell epitope derived from a portion of the longest isoform of human tau protein (GenBank: AGF19246.1) having the amino acid sequence shown in Table 6. For example, the tau B cell epitopes provided in Tables 1 and 7-10 can be used in the peptide immunogen constructs described herein. In some embodiments, an amyloid beta peptide immunogen construct contains a B cell epitope derived from a portion of Aβ (see, e.g., Table 3). For example, the amyloid beta B cell epitopes provided in Table 3, WO 2014 / 143087, or US Pat. No. 9,102,752 B2 (each of which is incorporated herein by reference) can be used in the peptide immunogen constructs described herein. Each B cell epitope can be linked to a heterologous T helper cell (Th) epitope derived from a pathogen protein via an optional heterologous spacer. The disclosed peptide immunogenic constructs stimulate the production of highly specific antibodies directed against tau or Aβ and can be used as immunotherapies for patients suffering from Alzheimer's disease and / or tauopathies.

[0053] The B cell epitope portion of the tau peptide immunogen construct may have the amino acid sequence of a full-length tau protein (Table 6). In some embodiments, the B cell epitope has a sequence containing any one of the target sequences listed in Table 1 (e.g., SEQ ID NOS: 1-8, 202). In some embodiments, the tau B cell epitope has a sequence provided in any one of Tables 7-10. In some embodiments, the tau B cell epitope has any immunogenic sequence (a sequence capable of eliciting anti-tau antibodies) provided in any one of Tables 1 and 7-10. In some embodiments, the tau B-cell epitope has the sequence of p5543a, p5546a, p5547a, p5555a, p5557a, p5558a, p5559a, p5564a, p5565a, p5566a, p5569a, p5695a, p5696, or p5535a, which are SEQ ID NOs: 17-30, respectively, or the sequence of p5556a, p5456a, p4579a, p5541a, or p4062a, which are SEQ ID NOs: 63-67, respectively, also shown in Table 1.

[0054] The B cell epitope portion of the Aβ peptide immunogen construct is 1-42 , e.g., Aβ 1-14(See Table 3, also see additional Aβ peptides in WO2014 / 143087). In some embodiments, the B-cell epitope portion of the Aβ peptide immunogen construct has any one of the amino acid sequences shown in Table 3. In some embodiments, the B-cell epitope portion of the Aβ peptide immunogen construct has any one of the Aβ peptide amino acid sequences in WO2014 / 143087 and / or US9,102,752B2, each of which is incorporated herein by reference in its entirety. The disclosure of amyloid beta B-cell epitopes, and amyloid beta peptide immunogen constructs comprising such B-cell epitopes, in WO2014 / 143087 and / or US9,102,752B2 is specifically incorporated herein by reference. In some embodiments, the Aβ B cell epitope has any Aβ B cell epitope sequence provided in WO2014 / 143087 and / or US9,102,752B2. In some embodiments, the Aβ B cell epitope has any immunogenic Aβ B cell epitope sequence (a sequence capable of eliciting anti-Aβ antibodies) provided in WO2014 / 143087 and / or US9,102,752B2.

[0055] Tau and Aβ peptide immunogen constructs of the present disclosure may contain heterologous Th epitope amino acid sequences derived from pathogenic proteins (e.g., any one of SEQ ID NOS: 31-59 and 97), as shown in Table 5. In certain embodiments, the heterologous Th epitope is derived from a naturally occurring pathogen, such as Diphtheria toxin (SEQ ID NOS: 35), Plasmodium falciparum (SEQ ID NOS: 36), or cholera toxin (SEQ ID NOS: 38). In other embodiments, the heterologous Th epitope is an idealized artificial Th epitope derived from measles virus fusion protein (MVF1-5) or hepatitis B surface antigen (HBsAg1-3), either in the form of a single sequence or a combinatorial sequence (e.g., any one of SEQ ID NOS: 32, 39, or 41-45). In some embodiments, the heterologous Th epitope has an amino acid sequence selected from SEQ ID NOS: 42, 44, 45, and 97. In some embodiments, the heterologous Th epitope has the amino acid sequence of SEQ ID NO:44.

[0056] In some embodiments, the peptide immunogen construct contains a B cell epitope derived from tau or Aβ linked to a heterologous T helper cell (Th) epitope via an optional heterologous spacer. In certain embodiments, the peptide immunogen construct contains a B cell antigen site having an amino acid sequence derived from tau (see Table 1 (e.g., any one of SEQ ID NOS: 1-8) and Tables 7-10 (e.g., any one of SEQ ID NOS: 17-30 or 63-67)) or Aβ (e.g., SEQ ID NOS: 68 or 69) linked to a heterologous Th epitope derived from a pathogenic protein (e.g., any one of SEQ ID NOS: 31-59) via an optional heterologous spacer. In some embodiments, the optional heterologous spacer is a molecule or chemical structure capable of linking two amino acids and / or peptides together. In certain embodiments, the spacer is a naturally occurring amino acid, a non-naturally ... In a specific embodiment, the tau peptide immunogen construct has the amino acid sequence set forth in Table 2 (e.g., the sequence of any one of SEQ ID NOS: 9-16, or a construct in which the positions of the tau peptide and the Th peptide are reversed). In a specific embodiment, the tau peptide immunogen construct has the amino acid sequence of any one of SEQ ID NOS: 77-81, or a construct in which the positions of the tau peptide and the Th peptide are reversed. In a specific embodiment, the Aβ peptide immunogen construct has the amino acid sequence set forth in Table 4 (e.g., the sequence of SEQ ID NOS: 70, 71, or 89, or a construct in which the positions of the tau peptide and the Th peptide are reversed).

[0057] The present disclosure also relates to compositions comprising tau peptide immunogen constructs. In some embodiments, the disclosed compositions contain two or more tau peptide immunogen constructs to cover multiple B epitopes from tau. In certain embodiments, the compositions contain a mixture of tau peptide immunogen constructs containing two or more heterologous Th epitopes from pathogenic proteins to cover a wide range of patient genetic backgrounds. Compositions containing a mixture of peptide immunogen constructs may result in a higher percentage of response rates upon immunization for the treatment of Alzheimer's disease and / or tauopathy compared to compositions containing only a single peptide immunogen construct. The present disclosure also relates to compositions containing, for example, one or more tau peptide immunogen constructs described herein and, for example, one or more Aβ peptide immunogen constructs described herein.

[0058] The present disclosure also relates to pharmaceutical compositions for the treatment and / or prevention of Alzheimer's disease and / or tauopathies. In some embodiments, the pharmaceutical composition contains the disclosed peptide immunogen constructs or combinations thereof (e.g., a mixture of tau peptide immunogen constructs, or a mixture of tau peptide immunogen constructs and Aβ peptide immunogen constructs) in the form of a stabilized immunostimulatory complex formed via electrostatic association by mixing CpG oligomers with a composition containing the peptide immunogen complex. Such stabilized immunostimulatory complexes can further enhance the immunogenicity of the peptide immunogen constructs. In some embodiments, the pharmaceutical composition contains an adjuvant such as a mineral salt, such as alum gel (ALHYDROGEL), aluminum phosphate (ADJUPHOS), or a water-in-oil emulsion containing MONTANIDE ISA51 or 720. When used in combination, the peptide immunogen constructs described herein can be contained in a single composition or in separate compositions.

[0059] The present disclosure also relates to antibodies directed against the disclosed tau peptide immunogen constructs and / or the disclosed amyloid beta peptide immunogen constructs. In particular, the peptide immunogen constructs of the present disclosure, when administered to a subject, can stimulate the production of highly specific antibodies that cross-react with the target amino acid sequence. For example, the highly specific antibodies produced by the peptide immunogen constructs cross-react with recombinant tau-containing proteins. The disclosed antibodies bind to tau with high specificity, with little or no targeting of heterologous Th epitopes used for immunogenicity enhancement. This contrasts significantly with conventional proteins or other biological carriers used for such peptide antigenicity enhancement.

[0060] The present disclosure also includes methods for treating and / or preventing Alzheimer's disease and / or tauopathy using the disclosed peptide immunogen constructs (including, for example, the combinations described above and elsewhere herein) and / or antibodies directed against the peptide immunogen constructs. In some embodiments, the methods for treating and / or preventing Alzheimer's disease and / or tauopathy include administering to a host a composition containing the disclosed peptide immunogen constructs. In certain embodiments, the compositions utilized in the methods contain one or more of the disclosed peptide immunogen constructs in the form of a stable immunostimulatory complex with a negatively charged oligonucleotide, such as a CpG oligomer, via electrostatic association, optionally further supplemented with mineral salts or oils as adjuvants for administration to patients with Alzheimer's disease and / or tauopathy. The disclosed methods also include dosing regimens, dosage forms, and routes for administering the peptide immunogen constructs to a host at risk for or suffering from Alzheimer's disease and / or tauopathy.

[0061] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All references or portions of references cited in this application are expressly incorporated herein by reference in their entirety for all purposes.

[0062] Unless otherwise explained, 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. The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Thus, "comprising A or B" means including A or B, or A and B. Furthermore, it should be understood that all amino acid sizes and all molecular weight or molecular mass values ​​given for polypeptides are approximate and are provided for illustrative purposes. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the disclosed methods, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including explanations of terms, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0063] term As used herein, the term "about" refers to a range of + / - 10% of the stated value. In some embodiments, the range is + / - 5%, + / - 3%, + / - 2%, + / - 1%, + / - 0.5%, or + / - 0.1% of the stated value.

[0064] As used herein, the term "peptide immunogen" or "peptide immunogen construct" refers to a peptide comprising a heterologous T helper cell epitope covalently linked to a peptide containing a B cell epitope. The covalent linkage may be via a covalent peptide bond, a thioester, or a heterologous spacer. In some embodiments, the heterologous spacer is or comprises an amino acid sequence.

[0065] The term "artificial" as used herein in reference to a peptide immunogen or Th epitope refers to an amino acid sequence that does not occur in nature.

[0066] The term "promiscuous" as used herein in reference to Th epitopes refers to Th epitopes that are reactive across species and across individuals of a single species.

[0067] As used herein, the term "preventing" a disease or condition in a subject refers to administering an agent (e.g., a peptide immunogen construct described herein) to a subject before the onset of a disease or condition, if administration of the agent to a statistical sample before the onset of the disease or condition reduces the occurrence of the disease or condition in the treated sample relative to an untreated control sample, or delays the onset of, or reduces the occurrence or severity of, one or more symptoms of the disease or condition relative to an untreated control sample.

[0068] As used herein, the term "treating" a disease or condition in a subject refers to administering an agent (e.g., a peptide immunogen construct described herein) to a subject having or suspected of having a disease or condition (i.e., after onset of the disease or condition) so as to reduce or prevent the worsening of at least one symptom of the disease or condition.

[0069] Peptide immunogen constructs The present disclosure provides peptide immunogen constructs containing a B cell epitope having an amino acid sequence derived from tau or amyloid beta (Aβ) covalently linked, either directly or via an optional heterologous spacer, to a heterologous T helper cell (Th) epitope.

[0070] In some embodiments, the peptide immunogen constructs described herein are modified as described herein, for example, by acetylation of amino acids, N-methylation of amino acids, substitution of one or two amino acids with cysteine ​​for disulfide bond formation, and / or use of N-terminal pyroglutamic acid. Acceptable modifications are any of those described herein (see, e.g., Tables 2 and 4). In some embodiments, modifications contemplated herein protect the peptide immunogen constructs from protease degradation.

[0071] As used herein, the phrase "tau peptide immunogen construct" refers to a peptide containing (a) a B cell epitope having about six or more amino acid residues (e.g., about 6-40, 8-40, 9-30, 10-25, 8-20, 9-18, 8-15, 10-15, 8-14, or 10-14, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) from the full-length sequence of the longest tau isoform (SEQ ID NO: 60), (b) a heterologous Th epitope, and (c) an optional heterologous spacer.

[0072] In certain embodiments, the tau peptide immunogen construct has the formula: (Th) m -(A) n -(Tau fragment)-X or (Tau fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (tau fragment) is a B cell epitope having about 6 to about 40 amino acid residues derived from SEQ ID NO: 60 (e.g., about 6 to 40, 8 to 40, 9 to 30, 10 to 25, 8 to 20, 9 to 18, 8 to 15, 10 to 15, 8 to 14, or 10 to 14, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids), X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0073] In some embodiments, the tau peptide immunogenic construct is selected from any one of SEQ ID NOs: 9-16 and 77-81, or any combination of two, three, or more thereof is used (see Table 2). In some embodiments, the tau peptide immunogenic construct is selected from any one of SEQ ID NOs: 9-16, or any combination of two, three, or more thereof is used (see Table 2). In some embodiments, the tau peptide immunogenic construct is selected from any one of SEQ ID NOs: 77-81, or any combination of two, three, or more thereof is used (see Table 2).

[0074] In some embodiments, two or more (e.g., two, three, four, or more) tau peptide immunogen constructs are used (e.g., in the compositions, kits, and methods described herein) to elicit antibodies that target multiple B epitopes from tau (e.g., two, three, four, or more epitopes each). In some embodiments, the two or more tau peptide immunogen constructs comprise B cell epitopes from different regions of tau (e.g., the N-terminal region of tau for one peptide immunogen construct and the repeat domain for a second peptide immunogen construct, e.g., SEQ ID NOS: 10 and 15, respectively). In some embodiments, the N-terminal region of tau is within amino acids 1-44 (e.g., amino acids 13-22) of SEQ ID NO: 60. In some embodiments, the repeat domain of tau is within amino acids 244-372 of SEQ ID NO: 60. In some embodiments, a tau peptide immunogenic construct comprising a B cell epitope within amino acids 1-44 (e.g., amino acids 13-22) of SEQ ID NO: 60 is used together (e.g., in the same composition, the same kit, or co-administered together) with another tau peptide immunogenic construct comprising a B cell epitope within amino acids 244-372 of SEQ ID NO: 60. In some embodiments, one of the tau peptide immunogenic constructs comprises a B cell epitope from the N-terminal region of tau of SEQ ID NO: 60 (having a sequence within amino acids 1-44, e.g., from amino acids 13-22). In some embodiments, another tau peptide immunogenic construct comprises a B cell epitope from a repeated (e.g., repeated 2, 3, or 4 times) amino acid region within amino acids 244-372 of SEQ ID NO: 60. In some embodiments, the repeated amino acid region is an amino acid region within the 1R, 2R, 3R, and / or 4R repeat region of tau, where the repeat region is known in the art or described herein. In some embodiments, one tau peptide immunogen construct comprises a B cell epitope from the N-terminal region and one tau peptide immunogen construct comprises a B cell epitope from a region other than the N-terminal region (e.g., one or both such constructs elicit antibodies that bind to each of the four, five, or six isoforms of tau provided in Table 6).In some embodiments, one tau peptide immunogen construct comprises a B cell epitope from the repeat domain on tau and one tau peptide immunogen construct comprises a B cell epitope from a region other than the repeat domain of tau (e.g., one or both such constructs elicit antibodies that bind to each of the four, five, or six isoforms of tau provided in Table 6).

[0075] In some embodiments, at least one tau peptide immunogenic construct selected from any one of SEQ ID NOs: 9 and 10 is used together (e.g., in the same composition, kit, or co-administered) with at least one tau peptide immunogenic construct selected from any one of SEQ ID NOs: 15 and 77-81.

[0076] In some embodiments, at least one tau peptide immunogenic construct selected from any one of SEQ ID NOs: 9-16 is used together (e.g., in the same composition, kit, or co-administered) with at least one tau peptide immunogenic construct selected from any one of SEQ ID NOs: 77-81.

[0077] In some embodiments, at least one tau peptide immunogenic construct of SEQ ID NO: 10 is used together (e.g., in the same composition, kit, or co-administered) with at least one tau peptide immunogenic construct selected from any others provided herein.

[0078] In some embodiments, at least one tau peptide immunogenic construct selected from any one of SEQ ID NOs: 15 and 77-81 is used together (e.g., in the same composition, kit, or co-administered) with at least one tau peptide immunogenic construct selected from any others provided herein.

[0079] In some embodiments, at least one tau peptide immunogenic construct of SEQ ID NO: 10 is used together (e.g., in the same composition, kit, or co-administered) with at least one tau peptide immunogenic construct selected from any one of SEQ ID NOs: 15 and 77-81.

[0080] In some embodiments, a composition or kit comprises at least one tau peptide immunogenic construct comprising or consisting of SEQ ID NO: 10, and at least one tau peptide immunogenic construct comprising or consisting of SEQ ID NO: 15 (or a modified version thereof containing modified amino acids or chemical groups). Permissible modifications are any of those described herein (such as in SEQ ID NOs: 77-81). In some embodiments, a composition or kit comprises a tau peptide immunogenic construct of SEQ ID NO: 10, and a tau peptide immunogenic construct of SEQ ID NO: 15. In some embodiments, a composition or kit comprises a tau peptide immunogenic construct of SEQ ID NO: 10, and at least one tau peptide immunogenic construct of any one of SEQ ID NOs: 77-81.

[0081] In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:9. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:10. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:11. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:12. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:13. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:14. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:15. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:16. In some embodiments, the tau peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO:77. In some embodiments, the tau peptide immunogenic construct comprises, consists essentially of, or consists of SEQ ID NO: 78. In some embodiments, the tau peptide immunogenic construct comprises, consists essentially of, or consists of SEQ ID NO: 79. In some embodiments, the tau peptide immunogenic construct comprises, consists essentially of, or consists of SEQ ID NO: 80. In some embodiments, the tau peptide immunogenic construct comprises, consists essentially of, or consists of SEQ ID NO: 81.

[0082] As used herein, the phrase "amyloid beta or Aβ peptide immunogen construct" refers to a peptide containing (a) a B cell epitope having about six or more amino acid residues (e.g., about 6-40, 8-40, 9-30, 10-25, 8-20, 9-18, 8-15, 10-15, 8-14, or 10-14, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) from the full-length sequence of amyloid beta protein of SEQ ID NO: 68, (b) a heterologous Th epitope, and (c) an optional heterologous spacer.

[0083] In certain embodiments, the Aβ peptide immunogen construct has the formula: (Th) m -(A) n -(Aβ fragment)-X or (Aβ fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (Aβ fragment) is a B cell epitope having about 6 to about 40 amino acid residues derived from SEQ ID NO: 68 (e.g., about 6 to 40, 8 to 40, 9 to 30, 10 to 25, 8 to 20, 9 to 18, 8 to 15, 10 to 15, 8 to 14, or 10 to 14, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids), X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0084] In some embodiments, the Aβ peptide immunogen construct is selected from SEQ ID NO: 70 and SEQ ID NO: 71, or a combination thereof is used (see Table 4). In some embodiments, the Aβ peptide immunogen construct is selected from SEQ ID NO: 70, SEQ ID NO: 71, and SEQ ID NO: 89, or any combination of two or three thereof is used (see Table 4).

[0085] See also WO2014 / 143087 for additional Aβ peptides and Aβ peptide immunogenic constructs that can be used in combination with the tau peptide immunogenic constructs described herein. Peptide immunogenic constructs falling within the formulas disclosed in WO2014 / 143087 can also be used and are included in the present disclosure. In some embodiments, the Aβ peptide immunogenic construct can be represented by any of the formulas for Aβ peptide immunogenic constructs presented in WO2014 / 143087 and / or US Pat. No. 9,102,752 B2, the disclosures of which are specifically incorporated herein by reference with respect to such formulas.

[0086] In some embodiments, the Aβ peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO: 70 or a modified version thereof, including modified amino acids or chemical groups. Permissible modifications are any of those described herein (such as in SEQ ID NO: 89). In some embodiments, the Aβ peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO: 70. In some embodiments, the Aβ peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO: 71. In some embodiments, the Aβ peptide immunogen construct comprises, consists essentially of, or consists of SEQ ID NO: 89.

[0087] In some embodiments, the C-terminus of the peptide immunogen constructs described herein is amidated.

[0088] In some embodiments, a combination of one or more (e.g., one, two, or three) tau peptide immunogen constructs and one or more (e.g., one, two, or three) amyloid beta peptide immunogen constructs are used in the compositions, kits, and methods described herein. In some embodiments, a combination of one tau peptide immunogen construct and one amyloid beta peptide immunogen construct is used in the compositions, kits, and methods described herein. In some embodiments, a composition or kit comprises at least one tau peptide immunogen construct, e.g., comprising or consisting of SEQ ID NO: 10 and / or comprising or consisting of SEQ ID NO: 15 (or a modified version thereof containing modified amino acids or chemical groups, e.g., any of SEQ ID NOs: 77-81), and at least one amyloid beta peptide immunogen construct having an amino acid sequence, e.g., comprising or consisting of SEQ ID NO: 70 (or a modified version thereof containing modified amino acids or chemical groups, e.g., SEQ ID NO: 89). Acceptable modifications are any of those described herein (such as in SEQ ID NOs: 77-81 and 89). In some embodiments, the composition or kit comprises at least one tau peptide immunogenic construct of SEQ ID NO: 10 and / or SEQ ID NO: 15 and at least one amyloid beta peptide immunogenic construct of SEQ ID NO: 70.

[0089] In any of the constructs and combinations described herein, the heterologous spacer and heterologous Th epitope can be replaced by another heterologous spacer and / or heterologous Th epitope described herein. For example, but not limited to, the heterologous spacer can be any of SEQ ID NOs: 62 and 95. Similarly, for example, but not limited to, the heterologous Th epitope can be any of SEQ ID NOs: 42, 44, 45, and 97.

[0090] The various components of the disclosed peptide immunogen constructs are described below.

[0091] a. Tau and Aβ fragments In some embodiments, the disclosed peptide immunogenic constructs contain a B cell epitope of about 6 or more amino acids. In some embodiments, the disclosed peptide immunogenic constructs contain a B cell epitope of about 8 or more amino acids. In some embodiments, the tau or amyloid beta B cell epitopes provided herein are about 6-40 amino acids. In some embodiments, the tau or amyloid beta B-cell epitopes provided herein are about 8-40, 9-30, 10-25, 8-20, 9-18, 8-15, 10-15, 8-14, or 10-14 amino acids in length, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or any number of amino acids therebetween.

[0092] In some embodiments, the B-cell epitopes described herein are modified as described herein, e.g., by acetylation of amino acids, N-methylation of amino acids, substitution of one or two amino acids with cysteine ​​for disulfide bond formation, and / or use of N-terminal pyroglutamic acid. Acceptable modifications are any of those described herein (see, e.g., Tables 1 and 3).

[0093] The tau peptide immunogen constructs contain a B cell epitope derived from a portion of the longest isoform of human tau protein (GenBank: AGF19246.1), having the amino acid sequence of SEQ ID NO: 60 shown in Table 6. The B cell epitope can be linked to a heterologous T helper cell (Th) epitope derived from a pathogen protein via an optional heterologous spacer. The disclosed peptide immunogen constructs stimulate the production of highly specific antibodies against tau. The disclosed peptide immunogen constructs can be used as immunotherapy for patients suffering from Alzheimer's disease and / or tauopathies.

[0094] The B-cell epitope portion of the tau peptide immunogen construct has the amino acid sequence of the full-length tau protein (SEQ ID NO: 60).

[0095] In some embodiments, the tau B-cell epitope has a sequence set forth in Table 1 (any one of SEQ ID NOs: 1-8) or Tables 7-10 (e.g., any one of SEQ ID NOs: 17-30 or 63-67).

[0096] In some embodiments, the tau peptide (or tau B-cell epitope) of the tau peptide immunogen construct comprises or consists of a peptide from any one of Tables 1 and 7-10.

[0097] In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 1. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 2. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 3. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 4. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 5. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 6. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 7. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 8. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 202. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 17. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 18. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 19. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 20. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 21. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 22. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 23. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 24.In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 25. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 26. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 27. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 28. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 29. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 30. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 63. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 64. In some embodiments, the tau B-cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 65. In some embodiments, the tau B-cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 66. In some embodiments, the tau B-cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 67.

[0098] In some embodiments, the tau peptide (tau B-cell epitope) of the tau peptide immunogenic construct comprises or consists of a peptide of any one of Tables 1 and 7-10, wherein the peptide comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 60 on the N-terminus of the peptide of Tables 1 or 7-10. In some embodiments, the tau peptide (tau B-cell epitope) of the tau peptide immunogenic construct comprises or consists of a peptide of any one of Tables 1 and 7-10, wherein the peptide comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 60 on the C-terminus of the peptide of Tables 1 or 7-10. In some embodiments, the tau peptide (tau B-cell epitope) of the tau peptide immunogen construct comprises or consists of a peptide of any one of Tables 1 and 7-10, wherein the peptide comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 60 on the N-terminus and / or C-terminus of the peptide of Tables 1 or 7-10. The additional amino acids in the latter peptides should be understood to be amino acids of the tau protein of SEQ ID NO: 60 that are contiguous with the peptide sequence of Tables 1 and 7-10.

[0099] In some embodiments, the tau B cell epitope has the sequence of any one of SEQ ID NOs: 72-76. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 72. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 73. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 74. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 75. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 76. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 77. In some embodiments, the tau B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 96.

[0100] In some embodiments, the tau B-cell epitope of the tau peptide immunogenic construct comprises or consists of the peptide of any one of SEQ ID NOs: 72-76 or 96, and the B-cell epitope comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 60 on the N-terminus of the peptide. In some embodiments, the tau B-cell epitope of the tau peptide immunogenic construct comprises or consists of the peptide of any one of SEQ ID NOs: 72-76 or 96, and the B-cell epitope comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 60 on the C-terminus of the peptide. In some embodiments, the tau B-cell epitope of the tau peptide immunogenic construct comprises or consists of the peptide of any one of SEQ ID NOs: 72-76 or 96, and the B-cell epitope comprises 1, 2, 3, 4, or 5 additional amino acids of the tau protein of SEQ ID NO: 60 on the N-terminus and / or C-terminus of the peptide. The additional amino acids should be understood to be amino acids of the tau protein of SEQ ID NO: 60 that are contiguous with the peptide sequence of any one of SEQ ID NOs: 72-76 and 96, respectively.

[0101] In some embodiments, the tau peptide (B cell epitope) of the tau peptide immunogen construct comprises or consists of about 8 to about 40 amino acid residues (e.g., about 8 to 40, 9 to 30, 10 to 25, 8 to 20, 9 to 18, or 10 to 15, or about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) from SEQ ID NO: 60. In some embodiments, the tau peptide of the peptide immunogen construct is any one of Tables 1 and 7-10, e.g., any one of SEQ ID NOs: 1-8, 17-30, or 63-67, optionally with one or more of the additional amino acids described above. In some embodiments, the tau peptide (B-cell epitope) of the tau peptide immunogen construct is any one of SEQ ID NOs: 72-76, optionally with one or more of the additional amino acids described above.

[0102] The B-cell epitope portion of the amyloid beta peptide immunogen construct has an amino acid sequence derived from the full-length amyloid beta protein (SEQ ID NO: 68).

[0103] In some embodiments, the amyloid beta peptide (or amyloid beta B-cell epitope) of the amyloid beta peptide immunogen construct comprises or consists of any one of the peptides in Table 3.

[0104] In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 69. In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 82. In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 83. In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 84. In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 85. In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 86. In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 87. In some embodiments, the amyloid beta B cell epitope comprises, consists essentially of, or consists of SEQ ID NO: 88. In some embodiments, the amyloid beta B-cell epitope comprises, consists essentially of, or consists of any of the sequences of amyloid beta peptides or B-cell epitopes described in WO2014 / 143087 or US Pat. No. 9,102,752 B2.

[0105] In some embodiments, the B cell epitope of the amyloid beta peptide immunogen construct comprises or consists of a peptide of any one of SEQ ID NOs: 69 and 82-88, wherein the B cell epitope comprises 1, 2, 3, 4, or 5 additional amino acids of the amyloid beta protein of SEQ ID NO: 68 on the N-terminus of the peptide. In some embodiments, the B cell epitope of the amyloid beta peptide immunogen construct comprises or consists of a peptide of any one of SEQ ID NOs: 69 and 82-88, wherein the B cell epitope comprises 1, 2, 3, 4, or 5 additional amino acids of the amyloid beta protein of SEQ ID NO: 68 on the C-terminus of the peptide. In some embodiments, the B cell epitope of the amyloid beta peptide immunogen construct comprises or consists of the peptide of any one of SEQ ID NOs: 69 and 82-88, and the B cell epitope comprises 1, 2, 3, 4, or 5 additional amino acids of the amyloid beta protein of SEQ ID NO: 68 on the N-terminus and / or C-terminus of the peptide, where the additional amino acids should be understood to be amino acids of the amyloid protein of SEQ ID NO: 68 that are contiguous with the peptide sequence of any one of SEQ ID NOs: 69 and 82-88, respectively.

[0106] As noted above, in some embodiments, the Aβ peptide immunogen construct is selected from SEQ ID NO: 70 and SEQ ID NO: 71, or a combination thereof is used (see Table 4). See also WO2014 / 143087 for additional Aβ peptides and Aβ peptide immunogenic constructs that can be used in combination with the tau peptide immunogen constructs described herein. Peptide immunogen constructs falling within the formula of WO2014 / 143087 can also be used and are included in the present disclosure.

[0107] In some embodiments, the C-terminus of the peptide immunogenic constructs described herein is amidated.

[0108] b. Heterologous T helper cell epitopes (Th epitopes) The present disclosure provides peptide immunogen constructs containing a B cell epitope derived from tau or Aβ covalently linked, either directly or via an optional heterologous spacer, to a heterologous T helper cell (Th) epitope.

[0109] Heterologous Th epitopes in peptide immunogen constructs enhance the immunogenicity of tau or Aβ fragments, which facilitates the production of specific, high-titer antibodies directed against optimized target B cell epitopes (e.g., tau fragments) through rational design.

[0110] The term "heterologous" as used herein refers to an amino acid sequence derived from a portion of or a non-homologous amino acid sequence of the wild-type sequence of tau or Aβ. Thus, in the case of tau, a heterologous Th epitope is a Th epitope derived from an amino acid sequence not naturally found in tau (i.e., the Th epitope is not autologous to tau). Because a Th epitope is heterologous to tau, when a heterologous Th epitope is covalently attached to a tau fragment, the native amino acid sequence of tau is not extended either N- or C-terminally. Similarly, in the case of Aβ, a heterologous Th epitope is a Th epitope derived from an amino acid sequence not naturally found in Aβ (i.e., the Th epitope is not autologous to Aβ). Because a Th epitope is heterologous to Aβ, when a heterologous Th epitope is covalently attached to an Aβ fragment, the native amino acid sequence of Aβ is not extended either N- or C-terminally.

[0111] The heterologous Th epitope of the present disclosure can be any Th epitope that does not have an amino acid sequence naturally found in tau (or Aβ). The Th epitope can have an amino acid sequence derived from any species (e.g., human, pig, cow, dog, rat, mouse, guinea pig, etc.). The Th epitope can also have promiscuous binding motifs for MHC class II molecules of multiple species. In certain embodiments, the Th epitope contains multiple promiscuous MHC class II binding motifs, allowing for maximal activation of helper T cells, which lead to the initiation and regulation of the immune response. The Th epitope is preferably immunosilent by itself, i.e., few, if any, antibodies generated by the peptide immunogen construct are directed against the Th epitope, allowing for a highly focused immune response directed against the targeted B-cell epitope of the tau or Aβ fragment.

[0112] Epitopes of the present disclosure include, but are not limited to, amino acid sequences derived from foreign pathogens exemplified in Table 5 (e.g., any one of SEQ ID NOS: 31-59 or 97). Furthermore, Th epitopes include idealized artificial Th epitopes and combinatorial idealized artificial Th epitopes (e.g., any one of SEQ ID NOS: 32 and 39-45). Heterologous Th epitope peptides presented as combinatorial sequences (e.g., any one of SEQ ID NOS: 40-43) contain a mixture of amino acid residues represented at specific positions within the peptide framework based on the variable residues of homologs to that particular peptide. A collection of combinatorial peptides can be synthesized in a single process by adding a mixture of designated protected amino acids in place of one specific amino acid to a designated position during the synthesis process. Such a combinatorial collection of heterologous Th epitope peptides can cover a wide range of Th epitopes for animals with diverse genetic backgrounds. Representative combinatorial sequences of heterologous Th epitope peptides include SEQ ID NOs: 40 to 43 shown in Table 5. The epitope peptides of the present invention provide broad reactivity and immunogenicity to animals and patients from genetically diverse populations.

[0113] In some embodiments, in Th epitopes shown in Table 5 that include an N-terminal lysine (such as an N-terminal "KKK" amino acid sequence), the N-terminal lysine may be removed for use in the peptide immunogen constructs described herein (e.g., for use in peptide immunogen constructs that include a spacer comprising one or more lysines). In some embodiments, in Th epitopes shown in Table 5 that include an N-terminal "KKK" amino acid sequence, the "KKK" amino acid sequence may be removed for use in the peptide immunogen constructs described herein (e.g., for use in peptide immunogen constructs that include a spacer comprising a "KKK" amino acid sequence). See, for example, the peptide immunogen construct of SEQ ID NO: 71, where the sequence of the UBI Th®2 Th epitope in the peptide immunogen construct lacks three N-terminal lysines.

[0114] In some embodiments, the heterologous Th epitope comprises or has the amino acid sequence of SEQ ID NO: 44. In some embodiments, the heterologous Th epitope comprises or has the amino acid sequence of SEQ ID NO: 42. In some embodiments, the heterologous Th epitope comprises or has the amino acid sequence of SEQ ID NO: 45. In some embodiments, the heterologous Th epitope comprises or has the amino acid sequence of SEQ ID NO: 97.

[0115] Peptide immunogen constructs containing Th epitopes are or can be produced in tandem with B cell epitope-containing fragments in a single solid-phase peptide synthesis. Th epitopes also include immunological analogs of Th epitopes. Immunological Th analogs include immunopotentiating analogs, cross-reactive analogs, and segments of any one of these Th epitopes sufficient to enhance or stimulate an immune response to the B cell epitope-containing fragment.

[0116] Functional immunological analogs of Th epitope peptides are also useful and are included as part of the present invention. Functional immunological Th analogs can include conservative substitutions, additions, deletions, and insertions of one to about five amino acid residues in the Th epitope that do not substantially modify the Th stimulatory function of the Th epitope. Conservative substitutions, additions, and insertions can be achieved using natural or unnatural amino acids. Table 5 identifies additional variations of functional analogs to Th epitope peptides. In particular, SEQ ID NOS: 32 and 39 of MvF1 and MvF2 Th are functional analogs of SEQ ID NOS: 42 and 44 of MvF4 and MvF5 in that they differ in the amino acid frame by the deletion (SEQ ID NOS: 32 and 39) or inclusion (SEQ ID NOS: 42 and 44) ​​of two amino acids at the N-terminus and C-terminus, respectively. The differences between these two sets of similar sequences will not affect the function of the Th epitopes contained within these sequences. Thus, functional immunological Th analogs include versions of Th epitopes from measles virus fusion protein MvF1-4Th (any one of SEQ ID NOS: 32, 39, 40, 42, and 44) ​​and from hepatitis surface protein HBsAg1-3Th (any one of SEQ ID NOS: 41, 43, and 45). In some embodiments, the sequence of SEQ ID NO: 44 is used. In some embodiments, the sequence of SEQ ID NO: 42 is used.

[0117] The Th epitope in the peptide immunogen construct can be covalently linked to either the N-terminus or C-terminus of the B cell epitope-containing peptide. In some embodiments, the Th epitope is covalently linked to the N-terminus of the B cell epitope-containing peptide. In other embodiments, the Th epitope is covalently linked to the C-terminus of the B cell epitope-containing peptide. In certain embodiments, two or more Th epitopes are covalently linked to the B cell epitope-containing fragment. When two or more Th epitopes are linked to the B cell epitope-containing fragment, each Th epitope can have the same or different amino acid sequence. In addition, when two or more Th epitopes are linked to the B cell epitope-containing fragment, the Th epitopes can be arranged in any order. For example, the Th epitopes can be contiguously linked to the N-terminus of the B cell epitope-containing fragment, or contiguously linked to the C-terminus of the B cell epitope-containing fragment, or the Th epitopes can be covalently linked to the N-terminus of the B cell epitope-containing fragment while separate Th epitopes are covalently linked to the C-terminus of the B cell epitope-containing fragment. There are no limitations on the arrangement of the Th epitopes with respect to the B cell epitope-containing fragment. For the immunogenic constructs shown in Tables 2 and 4, it should be understood that the present disclosure includes corresponding immunogenic constructs in which the positions of the B cell epitope-containing peptides and Th epitopes are reversed from the orientation set forth in Tables 2 and 4. It should also be understood that in some embodiments, alternative Th epitopes can be used for the immunogenic constructs shown in Tables 2 and 4 (i.e., the provided Th epitopes can be replaced by alternatives described herein or known in the art).

[0118] In some embodiments, the Th epitope is covalently linked directly to the B cell epitope-containing fragment, while in other embodiments, the Th epitope is covalently linked to the B cell epitope-containing fragment via a heterologous spacer, as described in more detail below.

[0119] c. heterologous spacer The disclosed B-cell epitope-containing peptide immunogen constructs optionally contain a heterologous spacer that covalently links the B-cell epitope from the B-cell epitope-containing peptide to a heterologous T-helper cell (Th) epitope.

[0120] As discussed above, the term "heterologous" refers to an amino acid sequence that is derived from an amino acid sequence that is not a part of or homologous to the wild-type sequence of tau or Aβ. Thus, the native amino acid sequence of tau or Aβ is not extended in either the N- or C-terminal direction when a heterologous spacer is covalently attached to a B-cell epitope derived from tau or Aβ, since the spacer is heterologous to the tau or Aβ sequence.

[0121] A spacer is any molecule or chemical structure that can link two amino acids and / or peptides together. Spacers can vary in length or polarity depending on the application. Spacer attachment can be via an amide bond or a carboxyl bond, although other functional groups are possible as well. Spacers can include chemical compounds, natural amino acids, or unnatural amino acids.

[0122] Spacers can provide structural features to tau or beta amyloid peptide immunogen constructs. Structurally, spacers provide physical separation of Th epitopes from B cell epitopes of tau or beta amyloid fragments. This physical separation can disrupt any artificial secondary structure created by the binding of Th epitopes to B cell epitopes. Furthermore, physical separation of epitopes by spacers can eliminate interference between Th cell responses and / or B cell responses. Furthermore, spacers can be designed to create or modify secondary structure in peptide immunogen constructs. For example, spacers can be designed to act as flexible hinges to enhance the separation of Th and B cell epitopes. Flexible hinge spacers can also enable more efficient interaction between the presented peptide immunogen and appropriate Th and B cells, enhancing immune responses to Th and B cell epitopes. Examples of sequences encoding flexible hinges are found in immunoglobulin heavy chain hinge regions, which are often proline-rich. One particularly useful flexible hinge that can be used as a spacer is provided by the sequence Pro-Pro-Xaa-Pro-Xaa-Pro (SEQ ID NO: 61), where Xaa is any amino acid, preferably aspartic acid.

[0123] Spacers can also provide functional characteristics to peptide immunogen constructs. For example, spacers can be designed to alter the overall charge of the peptide immunogen construct, which can affect the solubility of the peptide immunogen construct. Furthermore, altering the overall charge of the peptide immunogen construct can affect the ability of the peptide immunogen construct to associate with other compounds and reagents. As discussed in more detail below, peptide immunogen constructs can form stable immunostimulatory complexes with highly charged oligonucleotides, such as CpG oligomers, through electrostatic association. The overall charge of the peptide immunogen construct is important for the formation of these stable immunostimulatory complexes.

[0124] In some embodiments, the heterologous spacer comprises or consists of one of the following amino acids: Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, Lys-Lys-Lys-εLys, ε-N-Lys-Lys-Lys-Lys, and Pro-Pro-Xaa-Pro-Xaa-Pro, where Xaa is any amino acid or aspartic acid. In some embodiments, the spacer comprises or consists of the amino acid sequence Lys-Lys-Lys, ε-Lys-Lys-Lys-Lys, or Lys-Lys-Lys-ε-Lys. In some embodiments, the sequence of the spacer comprises or consists of ε-Lys-Lys-Lys-Lys or Lys-Lys-Lys-ε-Lys. In some embodiments, the heterologous spacer is Lys-Lys-Lys-εLys. In some embodiments, the heterologous spacer is εLys-Lys-Lys-Lys.

[0125] As is understood in the art, for constructs including ε-Lys as or within the spacer, the C-terminus of the first peptide sequence is conjugated to the epsilon-amino group of the lysine to form a first amide bond, and the N-terminus of the second peptide sequence is conjugated to the carboxyl group of the lysine to form a second amide bond.

[0126] The structure of the Lys-ε-Lys (K-εK) bond is shown below: [ka]

[0127] Compounds that can be used as spacers include, but are not limited to, (2-aminoethoxy)acetic acid (AEA), 5-aminovaleric acid (AVA), 6-aminocaproic acid (Ahx), 8-amino-3,6-dioxaoctanoic acid (AEEA, mini-PEG1), 12-amino-4,7,10-trioxadodecanoic acid (mini-PEG2), 15-amino-4,7,10,13-tetraoxapentadecanoic acid (mini-PEG3), trioxatridecane-succinic acid (Ttds), 12-amino-dodecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), and the like.

[0128] Naturally occurring amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine.

[0129] Unnatural amino acids include, but are not limited to, ε-N-lysine, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-aminobutyric acid, homoserine, citrulline, aminobenzoic acid, 6-aminocaproic acid (Aca, 6-aminohexanoic acid), hydroxyproline, mercaptopropionic acid (MPA), 3-nitro-tyrosine, pyroglutamic acid, and the like.

[0130] The spacer in the peptide immunogen construct can be covalently attached to either the N- or C-terminus of the Th epitope- and B cell epitope-containing peptide. In some embodiments, the spacer is covalently attached to the C-terminus of the Th epitope and the N-terminus of the B cell epitope-containing peptide. In other embodiments, the spacer is covalently attached to the C-terminus of the B cell epitope-containing peptide and the N-terminus of the Th epitope. In certain embodiments, for example, when two or more Th epitopes are present in the peptide immunogen construct, two or more spacers can be used. When two or more spacers are used, each spacer can be the same as or different from the other spacers. Furthermore, when two or more Th epitopes are present in the peptide immunogen construct, the Th epitopes can be separated from the B cell epitope by a spacer that can be the same as or different from the spacer used to separate the Th epitope from the B cell epitope. There are no limitations on the placement of the spacer with respect to the Th epitope or B cell epitope-containing fragment.

[0131] In certain embodiments, the heterologous spacer is a natural amino acid or an unnatural amino acid. In other embodiments, the spacer contains two or more natural or unnatural amino acids. In some embodiments, the spacer includes at least three or at least four amino acids (e.g., lysine). In some embodiments, the spacer includes fewer than seven, six, or five amino acids. In specific embodiments, the spacer is Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62).

[0132] d. Modified B-cell epitope and peptide immunogen constructs In some embodiments, modified tau and Aβ B-cell epitopes are provided herein. Specific modified B-cell epitopes provided herein are shown in Tables 1 and 3.

[0133] In some embodiments, modified tau and Aβ peptide immunogen constructs are provided herein. Specific modified peptide immunogen constructs provided herein are shown in Tables 2 and 4.

[0134] In some embodiments, the tau B-cell epitope comprises N-methylated amino acid(s). The N-methylated amino acid is monomethylated and is attached to the following amino acid by an amide bond between the N-methylated amine of the amino acid and the carboxylic acid of the following amino acid. One, two, or more amino acids can be N-methylated. In some embodiments, the N-methylated amino acid is isoleucine and / or lysine. In some embodiments, one isoleucine in the B-cell epitope is N-methylated. In some embodiments, one lysine in the B-cell epitope is N-methylated. In some embodiments, one isoleucine and one lysine in the B-cell epitope are N-methylated. In some embodiments, the N-methylated amino acid appears at the N-terminus of the B-cell epitope (such as a tau B-cell epitope). N-methylated amino acids are present in the tau B-cell epitopes of SEQ ID NOs: 73-75. Examples of N-methylated amino acid analogs are shown below: [ka]

[0135] In some embodiments, the tau B cell epitope comprises acetylated amino acid(s). In some embodiments, an isoleucine (e.g., one isoleucine of the B cell epitope) is acetylated. In some embodiments, an acetylated amino acid is at the N-terminus of the B cell epitope (e.g., a tau B cell epitope). An acetylated amino acid is present in the tau B cell epitope of SEQ ID NO: 72. In some embodiments, the Th epitope comprises acetylated amino acid(s). In some embodiments, an acetylated amino acid is at the N-terminus of the Th epitope. In some embodiments, an isoleucine (e.g., one isoleucine of the Th epitope) is acetylated. Examples of acetylated amino acids contemplated herein are shown below (particularly N-acetylated isoleucine): [ka]

[0136] In some embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct comprises, instead of an acetylated amino acid, another modification (as an alternative to acetylation) that protects the N-terminus from protease degradation. In some embodiments, such another modification is any modification known in the art (that can protect the N-terminus from protease degradation). In some embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct comprises one of the following modifications at the N-terminus: one or more additional glycines (e.g., three additional glycines or GGG), propionylation, a benzyloxycarbonyl group, a short PEG, or a D-amino acid. In some embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct comprises three glycines (that are not part of the B cell epitope) at the N-terminus. In some embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct is propionated at the N-terminus. In some embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct comprises a benzyloxycarbonyl at the N-terminus. In some embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct comprises a PEG (e.g., a short PEG) at the N-terminus. In some embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct comprises a D-amino acid at the N-terminus. In other embodiments, the tau B cell epitope, Th epitope, or peptide immunogen construct is acetylated at the N-terminus.

[0137] In some embodiments, the tau B-cell epitope comprises one or two amino acid substitutions, where a non-cysteine ​​amino acid is replaced with a cysteine ​​such that the resulting B-cell epitope contains two cysteines that can form disulfide bonds (resulting in cyclization via cysteine ​​disulfide bonds). Such substituted amino acids are present in the tau B-cell epitope of SEQ ID NO: 76.

[0138] In some embodiments, the amyloid beta B cell epitope comprises pyroglutamic acid. In some embodiments, the amyloid beta B cell epitope comprises pyroglutamic acid at the N-terminus of the B cell epitope. Such a modification is present in the amyloid beta B cell epitope of SEQ ID NO: 82. Pyroglutamic acid is a derivative of glutamic acid in which the side chain is dehydrated, resulting in an amide bond between the side chain acid group and the alpha amino group, as shown below. [ka]

[0139] The specific B cell epitope modifications described herein (such as in SEQ ID NOs: 72-76 and 82) are based on the predicted structures and / or empirically determined tryptic digestion products of tau and amyloid beta peptide sequences.

[0140] In some embodiments, the modifications described herein improve or confer protease stability to the peptide.

[0141] e. Specific Embodiments of Tau and Aβ Peptide Immunogen Constructs and Combinations The tau peptide immunogen construct has the formula: (Th) m -(A) n -(Tau fragment)-X or (Tau fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (tau fragment) is a B-cell epitope having 6 to about 40, or about 8 to about 40 amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or any length range between these values) derived from the full-length tau protein of SEQ ID NO: 60, X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0142] In certain embodiments, the heterologous Th epitope in the tau peptide immunogen construct has an amino acid sequence selected from any one of those set forth in Table 5. In certain embodiments, the tau peptide immunogen construct comprises two or more Th epitopes. In certain embodiments, the tau peptide immunogen construct comprises two or more Th epitopes, for example, of SEQ ID NO: 44.

[0143] In certain embodiments, the optional heterologous spacer is selected from any one of Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62), or Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95), and combinations thereof. In a specific embodiment, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62). In a specific embodiment, the heterologous spacer is Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95).

[0144] In certain embodiments, the tau fragment has about 8 to about 40 (e.g., 10 to 30, 10 to 25, 12 to 20, or 12 to 15) amino acid residues from the full-length tau protein of SEQ ID NO: 60. In some embodiments, the tau fragment has about 8 to 20, 8 to 15, 10 to 15, or 10 to 14 amino acid residues from the full-length tau protein of SEQ ID NO: 60. In specific embodiments, the tau fragment has an amino acid sequence set forth in Table 1 (e.g., one of SEQ ID NOs: 1-8) or one of Tables 7-10 (e.g., one of SEQ ID NOs: 17-30 or 63-67). In other specific embodiments, the tau fragment has the amino acid sequence of any one of SEQ ID NOs: 72-76 set forth in Table 1.

[0145] In some embodiments, the tau peptide immunogen construct is selected from the following: [Table 12]

[0146] In some embodiments, the T cell epitope of any one of SEQ ID NOs: 9-16 is replaced with the T cell epitope of any one of SEQ ID NOs: 31-59. In some embodiments, the locations of the B cell epitope and T cell epitope of any one of SEQ ID NOs: 9-16 are reversed within the molecule (i.e., if the B cell epitope is at the N-terminus of the original molecule, it is positioned at the C-terminus of the molecule in the reversed molecule, etc.).

[0147] In some embodiments, the tau peptide immunogen construct is selected from any one of SEQ ID NOs: 77-81. In some embodiments, the T cell epitope in any one of SEQ ID NOs: 77-81 is replaced with the T cell epitope in any one of SEQ ID NOs: 31-59. In some embodiments, the locations of the B cell epitope and T cell epitope in any one of SEQ ID NOs: 77-81 are reversed within the molecule (i.e., if the B cell epitope is at the N-terminus of the original molecule, it is positioned at the C-terminus of the molecule in the reversed molecule, etc.).

[0148] The Aβ peptide immunogen construct has the formula: (Th) m -(A) n -(Aβ fragment)-X or (Aβ fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (Aβ fragment) is a B-cell epitope having 6 to about 40, or about 8 to about 40, amino acid residues (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids, or any length range between these values) derived from the full-length Aβ protein of SEQ ID NO: 68, X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; n is 0 to about 10.

[0149] In certain embodiments, the heterologous Th epitope in the Aβ peptide immunogen construct has an amino acid sequence selected from any one of those listed in Table 5. In certain embodiments, the Aβ peptide immunogen construct comprises two or more Th epitopes, e.g., two or more Th epitopes of SEQ ID NO: 44.

[0150] In certain embodiments, the optional heterologous spacer is selected from any one of Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62), or Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95), and combinations thereof. In a specific embodiment, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62). In a specific embodiment, the heterologous spacer is Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95).

[0151] In certain embodiments, the Aβ fragment has about 8 to about 40 (e.g., 10 to 30, 10 to 25, 12 to 20, or 12 to 15) amino acid residues from the full-length tau protein of SEQ ID NO: 60. In some embodiments, the Aβ fragment has about 8 to 20, 8 to 15, 10 to 15, or 10 to 14 amino acid residues from the full-length Aβ protein of SEQ ID NO: 68. In specific embodiments, the Aβ fragment has an amino acid sequence set forth in Table 3 (e.g., one of SEQ ID NOs: 68 or 69). In other specific embodiments, the Aβ fragment has the amino acid sequence of any one of SEQ ID NOs: 83 to 88 set forth in Table 3.

[0152] In some embodiments, the Aβ peptide immunogen construct is selected from SEQ ID NOs: 70, 71, and 89.

[0153] In some embodiments, the T cell epitope of any one of SEQ ID NOs: 70, 71, and 89 is replaced with the T cell epitope of any one of SEQ ID NOs: 31-59. In some embodiments, the locations of the B cell epitope and T cell epitope of any one of SEQ ID NOs: 70, 71, and 89 are reversed within the molecule (i.e., if the B cell epitope is at the N-terminus of the original molecule, it is located at the C-terminus of the molecule in the reversed molecule, etc.).

[0154] In some embodiments, provided herein are combinations of any two, three, or more tau peptide immunogen constructs described herein.

[0155] In some embodiments, provided herein is a combination of any one, two, or more tau peptide immunogen constructs described herein with any one or more Aβ peptide immunogen constructs described herein. In some embodiments, the combination is formulated into a single composition as described herein. In some embodiments, such two or more peptide immunogen constructs are present in the same kit as described herein.

[0156] f. Variants, homologs, and functional analogs Variants and analogs of the above immunogenic peptides that induce and / or cross-react with preferred epitopes of tau or Aβ proteins can also be used. Analogs, including allelic, species, and induced variants, typically differ from the native peptide at one, two, or several positions, often by conservative substitutions. Analogs typically exhibit at least 80 or 90% sequence identity with the native peptide. Some analogs also include unnatural amino acids or N- or C-terminal amino acid modifications at one, two, or several positions.

[0157] Variants that are functional analogs can have conservative substitutions of amino acid positions, changes in overall charge, covalent linkage to another moiety, or amino acid additions, insertions, or deletions, and / or any combination thereof.

[0158] Conservative substitutions are when one amino acid residue is replaced with another amino acid residue having similar chemical properties. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

[0159] In certain embodiments, a functional analog has at least 50% identity to the original amino acid sequence. In other embodiments, a functional analog has at least 80% identity to the original amino acid sequence. In yet other embodiments, a functional analog has at least 85% identity to the original amino acid sequence. In yet other embodiments, a functional analog has at least 90% identity to the original amino acid sequence.

[0160] Variants also include mutations in phosphorylated residues. For example, variants may include different residues in the peptide that are phosphorylated. Variant immunogenic tau or Aβ peptides may also include pseudophosphorylated peptides. Pseudophosphorylated peptides are generated by replacing one or more of the phosphorylated serine, threonine, and tyrosine residues of tau or Aβ peptides with acidic amino acid residues such as glutamic acid and aspartic acid.

[0161] Tau isoforms targeted by tau peptide immunogen constructs and combinations Tau is expressed in six isoforms, reflecting alternative splicing of the same gene. These isoforms include splice variants at the N-terminus (0N, 1N, and 2N forms) and within the repeat domain (3R and 4R forms), resulting in the combinations 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R (see, e.g., Wu et al., 2022, Frontiers in Aging Science 14:Article 945875, doi:10.3389 / fnagi.2022.945875, which is incorporated herein by reference in its entirety, particularly for its disclosure of tau splice variants). Because specific tau isoforms are differentially enriched in various tauoplasias, important considerations must be taken into account when targeting tau. Ideally, an effective tau-targeting therapy would be effective across all tau isoforms. Although 4R tau-targeting therapies may be effective in AD and other tauopathies associated with 4R enrichment, therapies targeting 4R rather than 3R isoforms of tau are unlikely to be effective in treating Pick's disease, where 3R tau is present.

[0162] In some embodiments, the tau peptide immunogenic constructs provided herein elicit antibodies capable of binding to at least two, three, four, five, or each of tau isoforms selected from 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R. The amino acid sequences of such tau isoforms are set forth in Table 6 (particularly, the 2N4R isoform has SEQ ID NO: 60, the 0N3R isoform has SEQ ID NO: 90, the 1N3R isoform has SEQ ID NO: 91, the 2N3R isoform has SEQ ID NO: 92, the 0N4R isoform has SEQ ID NO: 93, and the 1N4R isoform has SEQ ID NO: 94). In some embodiments, the tau peptide immunogenic constructs provided herein elicit antibodies capable of binding to each of the tau isoforms of SEQ ID NOs: 60 and 90-94. In some embodiments, tau peptide immunogenic constructs provided herein that include a B-cell epitope derived from the N-terminal region of tau (within amino acids 1-44) elicit antibodies that bind to at least two, three, four, five, or each isoform of tau selected from ON3R, 1N3R, 2N3R, ON4R, 1N4R, and 2N4R. In some embodiments, tau peptide immunogenic constructs provided herein that include a B-cell epitope derived from the N-terminal region of tau (within amino acids 1-44) elicit antibodies that bind to at least two, three, four, five, or each of SEQ ID NOs: 60 and 90-94. In some embodiments, tau peptide immunogenic constructs provided herein that include a B-cell epitope derived from the N-terminal region of tau (within amino acids 1-44) elicit antibodies that bind to each of SEQ ID NOs: 60 and 90-94. In some embodiments, the tau peptide immunogen constructs provided herein that include a B cell epitope from the repeat domain of tau (within amino acids 244-372, or within amino acids 250-320) elicit antibodies that bind to at least two, three, four, five, or each isoform of tau selected from 0N3R, 1N3R, 2N3R, 0N4R, 1N4R, and 2N4R.In some embodiments, tau peptide immunogenic constructs provided herein that include B cell epitopes from the repeat domain of tau (within amino acids 244-372) elicit antibodies that bind to at least two, three, four, five, or each of SEQ ID NOs: 60 and 90-94. In some embodiments, tau peptide immunogenic constructs provided herein that include B cell epitopes from the repeat domain of tau (within amino acids 244-372) elicit antibodies that bind to each of SEQ ID NOs: 60 and 90-94. In some embodiments, tau peptide immunogenic constructs provided herein that include B cell epitopes from the repeat domain of tau (within amino acids 244-372) elicit antibodies that can bind to two epitopes in 4R-containing tau isoforms and one epitope in 3R-containing tau isoforms.

[0163] In some embodiments, a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 10 elicits antibodies that bind to each of SEQ ID NOs: 60 and 90-94. In some embodiments, a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 elicits antibodies that bind to each of SEQ ID NOs: 60 and 90-94. In some embodiments, a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 elicits antibodies that can bind to at least one epitope in a 4R-containing tau isoform. In some embodiments, a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 elicits antibodies that can bind to two epitopes in a 4R-containing tau isoform. In some embodiments, a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 elicits antibodies that can bind to one epitope in a 3R-containing tau isoform. In some embodiments, a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 elicits antibodies capable of binding to the P301S mutant of tau. In some embodiments, a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 elicits antibodies capable of binding to the P301L mutant of tau. In some embodiments, a composition or combination comprising a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 10 elicits antibodies capable of binding to 4R-containing tau isoforms. In some embodiments, a composition or combination comprising a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 10 peptide elicits antibodies capable of binding to 3R-containing tau isoforms.In some embodiments, a composition or combination comprising a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 10 elicits antibodies capable of binding to the P301S mutant of tau. In some embodiments, a composition or combination comprising a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 15 and a tau peptide immunogenic construct comprising, consisting essentially of, or consisting of SEQ ID NO: 10 elicits antibodies capable of binding to the P301L mutant of tau.

[0164] composition The present disclosure also provides compositions comprising one, two, or more of the disclosed tau peptide immunogenic constructs, and / or one or more of the disclosed tau peptide immunogenic constructs in combination with one or more Aβ peptide immunogenic constructs.

[0165] a. Peptide composition Compositions containing one, two, or more of the disclosed tau peptide immunogen constructs, optionally in combination with one or more Aβ peptide immunogen constructs, can be in liquid or solid form. Liquid compositions can contain water, buffers, solvents, salts, and / or any other acceptable reagents that do not alter the structural or functional properties of the peptide immunogen constructs. The peptide compositions can include one or more of the disclosed peptide immunogen constructs.

[0166] b. Pharmaceutical Compositions The present invention also relates to pharmaceutical compositions containing the disclosed peptide immunogenic constructs.

[0167] Pharmaceutical compositions may contain carriers and / or other additives in a pharmaceutically acceptable delivery system. Thus, pharmaceutical compositions may contain a pharmaceutically effective amount of one or more peptide immunogen constructs together with pharmaceutically acceptable carriers, adjuvants, and / or other excipients, such as diluents, additives, stabilizers, preservatives, solubilizers, buffers, etc.

[0168] The pharmaceutical composition may contain one or more adjuvants that act to accelerate, prolong, or enhance the immune response to the peptide immunogen construct without having any specific antigenic effect itself. Adjuvants used in the pharmaceutical composition may include oils, aluminum salts, virosomes, aluminum phosphate (e.g., ADJU-PHOS®), aluminum hydroxide (e.g., ALHYDROGEL®), liposin, saponin, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA35, ISA206, ISA50V, ISA51, ISA720, and other adjuvants and emulsifiers.

[0169] In some embodiments, the pharmaceutical compositions described herein include an adjuvant that is an aluminum adjuvant. In some embodiments, the adjuvant is an aluminum salt. In some embodiments, the aluminum salt is aluminum phosphate (ADJU-PHOS®) or AlPO4. In some embodiments, the aluminum salt is aluminum hydroxide (ALHYDROGEL®) or Al(OH)3. In some embodiments, the peptide immunogen is adsorbed onto aluminum gel. In some embodiments, the peptide immunogen is precipitated with an aluminum adjuvant.

[0170] In some embodiments, the pharmaceutical composition is a mineral salt suspension. In some embodiments, the pharmaceutical composition is a gel. In some embodiments, the pharmaceutical composition is a polymeric gel. In some embodiments, the pharmaceutical composition is a wet gel suspension.

[0171] In some embodiments, the pharmaceutical composition contains MONTANIDE™ ISA51 (an oil adjuvant composition composed of vegetable oil and mannide oleate to produce a water-in-oil emulsion), Tween® 80 (also known as polysorbate 80 or polyoxyethylene (20) sorbitan monooleate), CpG oligonucleotides, and / or any combination thereof. In other embodiments, the pharmaceutical composition is a water-in-oil-in-water (i.e., w / o / w) emulsion that includes Emulsigen or Emulsigen D as an adjuvant.

[0172] In some embodiments, the pharmaceutical compositions described herein comprise a peptide immunogen and a CpG oligonucleotide. In some embodiments, the pharmaceutical composition comprises CpG1, CpG2, and / or CpG3. In some embodiments, the pharmaceutical composition comprises a CpG1 oligonucleotide. In some embodiments, the pharmaceutical composition comprises a CpG2 oligonucleotide. In some embodiments, the pharmaceutical composition comprises a CpG3 oligonucleotide. In some embodiments, the CpG oligonucleotide acts as a peptide immunogen stabilizer. In some embodiments, the peptide immunogen and CpG oligonucleotide are in the form of particles. In some embodiments, the peptide immunogen and CpG oligonucleotide are formulated as a suspension. In some embodiments, the CpG oligonucleotide is as described in WO 03 / 068169, which is incorporated herein by reference in its entirety, particularly as it relates to CpG oligonucleotides. In some embodiments, the CpG comprises or consists of the sequence of CpG1 (tcgtcgtttt gtcgttttgt cgttttgtcg tt), CpG2 (tcgtcgtttt gtcgttttgt cgtt), or CpG3 (tcgtcgtttt gtcgttttgt cgtt). In some embodiments, the CpG oligonucleotide (e.g., CpG1, CpG2, or CpG3) is a phosphorothioate oligonucleotide. In some embodiments, CpG1 comprises the fully phosphorothioated sequence 5'TCgTCgTTTTgTCgTTTTgTCgTTTTgTCgTT3'. Non-limiting examples of CpG oligonucleotides are provided in Table 11.

[0173] The pharmaceutical compositions can be formulated as immediate-release or sustained-release formulations. Furthermore, the pharmaceutical compositions can be formulated to induce systemic or local mucosal immunity through entrapment of immunogens and co-administration with microparticles. Such delivery systems are readily determined by those skilled in the art.

[0174] Pharmaceutical compositions can be prepared as injections, either as liquid solutions or suspensions. A liquid vehicle containing the peptide immunogen construct can also be prepared prior to injection. Pharmaceutical compositions can be administered by any suitable mode of administration, such as intradermal (ID), intravenous (IV), intraperitoneal (IP), intramuscular (IM), intranasal, oral, subcutaneous, etc., and with any suitable delivery device. In certain embodiments, pharmaceutical compositions are formulated for intravenous, subcutaneous, intradermal, or intramuscular administration. Pharmaceutical compositions suitable for other administration methods, including oral and intranasal administration, can also be prepared.

[0175] Pharmaceutical compositions can also be formulated into suitable dosage units. In some embodiments, the pharmaceutical composition contains about 0.5 μg to about 1 mg of peptide immunogen construct(s) per kg body weight. The effective dose of the pharmaceutical composition will vary depending on many factors, including the means of administration, the target site, the physiological condition of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is human, although non-human mammals, including transgenic mammals, can also be treated. When delivered in multiple doses, the pharmaceutical composition can be conveniently divided into appropriate amounts per dosage unit. The dosage will vary depending on the age, weight, and general health of the subject, as is well known in the therapeutic field.

[0176] In some embodiments, the pharmaceutical composition contains two or more peptide immunogen constructs. The pharmaceutical composition may contain a mixture of two or more peptide immunogen constructs to synergistically enhance the immunological efficacy of the constructs. The pharmaceutical composition containing two or more peptide immunogen constructs may be more effective in a larger genetic population due to broader MHC class II coverage, thus providing an improved immune response to the peptide immunogen constructs.

[0177] In some embodiments, the pharmaceutical composition contains a tau peptide immunogen construct shown in Table 2, or a homolog, analog, and / or combination thereof. In some embodiments, the pharmaceutical composition comprises a tau peptide immunogen construct comprising a tau peptide from one of Tables 1 and 7-10. In some embodiments, the pharmaceutical composition comprises at least two (e.g., two or three) tau peptide immunogen constructs having a B-cell epitope shown in Table 1. In some embodiments, the pharmaceutical composition comprises at least two (e.g., two or three) tau peptide immunogen constructs shown in Table 2. In some embodiments, the peptide immunogen constructs targeting tau target different regions of tau (e.g., the N-terminal region of tau for one peptide immunogen construct and the repeat domain for a second peptide immunogen construct, e.g., SEQ ID NOs: 10 and 15, respectively).

[0178] In some embodiments, the pharmaceutical composition contains an amyloid beta peptide immunogen construct as set forth in Table 4, as well as homologs, analogs, and / or combinations thereof. In some embodiments, the pharmaceutical composition contains an amyloid beta peptide immunogen construct comprising an amyloid beta peptide as set forth in Table 3.

[0179] In some embodiments, the pharmaceutical composition contains a tau peptide immunogen construct (or a homolog, analog, and / or combination thereof) and an amyloid beta peptide immunogen construct (or a homolog, analog, and / or combination thereof). In some embodiments, the pharmaceutical composition contains at least one (or, e.g., two or three) tau peptide immunogen construct having a B-cell epitope shown in Table 1 and at least one (or, e.g., two or three) amyloid beta peptide immunogen construct having a B-cell epitope shown in Table 3. In some embodiments, the pharmaceutical composition contains at least one (or, e.g., two or three) tau peptide immunogen construct shown in Table 2 and at least one (or, e.g., two or three) amyloid beta peptide immunogen construct shown in Table 4. Pharmaceutical compositions containing the peptide immunogen constructs can be used to elicit an immune response and produce antibodies in a host upon administration.

[0180] c. Immune stimulating complex The present disclosure also relates to pharmaceutical compositions containing one or more peptide immunogen constructs in the form of an immune stimulating complex with a CpG oligonucleotide. Such immune stimulating complexes are specifically adapted to act as adjuvants and stabilizers for peptide immunogens. The immune stimulating complexes are in the form of particles, which can efficiently present the peptide immunogen to cells of the immune system to generate an immune response. The immune stimulating complexes may also be formulated as suspensions for parenteral administration. The immune stimulating complexes may also be formulated in the form of w / o emulsions, as suspensions combined with inorganic salts or in situ gelling polymers, to efficiently deliver the peptide immunogen to cells of the host's immune system after parenteral administration.

[0181] The stabilized immunostimulatory complex can be formed by complexing a peptide immunogen construct with an anionic molecule, an oligonucleotide, a polynucleotide, or a combination thereof via electrostatic association. The stabilized immunostimulatory complex can be incorporated into a pharmaceutical composition as an immunogen delivery system.

[0182] In certain embodiments, peptide immunogen constructs are designed to contain cationic moieties that are positively charged at a pH ranging from 5.0 to 8.0. The net charge of the cationic moieties of a peptide immunogen construct or mixture of constructs is calculated by assigning a +1 charge to each lysine (K), arginine (R), or histidine (H) in the sequence, a -1 charge to each aspartic acid (D) or glutamic acid (E), and a 0 charge to other amino acids. The charges are summed within the cationic moieties of the peptide immunogen construct and expressed as a net average charge. Suitable peptide immunogens have cationic moieties with a net average positive charge of +1. Preferably, peptide immunogens have a net positive charge in the range of greater than +2. In some embodiments, the cationic moieties of the peptide immunogen construct are heterologous spacers. In certain embodiments, the cationic portion of the peptide immunogen construct has a charge of +4 when the spacer sequence is (α,ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62).

[0183] As used herein, the term "anionic molecule" refers to any molecule that is negatively charged at a pH in the range of 5.0 to 8.0. In certain embodiments, the anionic molecule is an oligomer or polymer. The net negative charge on the oligomer or polymer is calculated by assigning a charge of -1 to each phosphodiester or phosphorothioate group in the oligomer. Suitable anionic oligonucleotides are single-stranded DNA molecules having 8 to 64 nucleotide bases, with the number of repeating CpG motifs ranging from 1 to 10. Preferably, the CpG immunostimulatory single-stranded DNA molecule contains 18 to 48 nucleotide bases, with the number of repeating CpG motifs ranging from 3 to 8.

[0184] More preferably, the anionic oligonucleotide has the formula: 5′X 1 CGX 2 3', where C and G are unmethylated and X 1 is selected from A (adenine), G (guanine), and T (thymine); X 2is C (cytosine) or T (thymine). Alternatively, the anionic oligonucleotide may have the formula: 5'(X 3 )2CG(X 4 ) 23′, wherein C and G are not methylated, and X 3 is selected from A, T, or G, and X 4 is C or T.

[0185] The resulting immunostimulating complexes are typically in the form of particles with sizes ranging from 1 to 50 microns, a function of many factors, including the relative charge stoichiometry and molecular weight of the interacting species. Micronized immunostimulating complexes have the advantage of providing in vivo adjuvantage and upregulation of specific immune responses. Furthermore, stabilized immunostimulating complexes are suitable for preparing pharmaceutical compositions by a variety of processes, including water-in-oil emulsions, mineral salt suspensions, and polymer gels.

[0186] antibody Also provided herein is an isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of the disclosed peptide immunogen. In some embodiments, such an antibody is an IgG antibody. In some embodiments, such an antibody is an IgG1 antibody.

[0187] Antibodies for use in therapy can be produced using standard methods in the art and include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and trispecific antibodies), and antibody fragments (as long as the desired antigen-binding activity and specificity are maintained). Antibody fragments include, for example, Fv, single-chain Fv (scFv), Fab, Fab', dis-scFv, sdAb (single-domain antibody), and (Fab')2 (including chemically conjugated F(ab')2). Antibodies also include, for example, chimeric antibodies, humanized antibodies, and antibodies from various species such as mouse, human, and cynomolgus monkey.

[0188] In certain embodiments, provided herein are methods for producing antibodies, or epitope-binding fragments thereof, that specifically bind to a B-cell epitope of a disclosed peptide immunogen. In some embodiments, such methods comprise administering (e.g., parenterally, e.g., intramuscularly) the peptide immunogen to a subject (e.g., a mammal such as a mouse, rat, non-human primate, or human). In some embodiments, such methods further comprise recovering and isolating the antibodies produced after a period sufficient to generate antibodies (e.g., about 1 week to about 16 weeks after administration, or about 6, 9, and / or 12 weeks after administration).

[0189] In certain embodiments, the present disclosure provides antibodies elicited by any of the tau peptide immunogen constructs.

[0190] The disclosed tau peptide immunogen constructs, comprising a tau fragment, a heterologous Th epitope, and an optional heterologous spacer, can elicit an immune response and the production of anti-tau antibodies when administered to a subject. The design of the tau peptide immunogen constructs can break tolerance to self-tau and elicit the production of site-specific antibodies that recognize conformational, rather than linear, epitopes.

[0191] Antibodies generated by tau peptide immunogen constructs recognize and bind to tau in its monomeric, dimeric, trimeric, and oligomeric forms.

[0192] Antibodies elicited by tau peptide immunogen constructs are surprisingly able to prevent tau aggregation (anti-aggregation activity) and also dissociate already formed tau aggregates (disaggregation activity).

[0193] The immune responses obtained from animals immunized with the tau peptide immunogen constructs of the present invention demonstrated the ability of the constructs to generate potent site-specific antibodies reactive with monomeric, dimeric, trimeric, and oligomeric forms of tau.

[0194] In certain embodiments, the present disclosure provides antibodies induced by any of the amyloid beta peptide immunogen constructs. The disclosed amyloid beta peptide immunogen constructs, comprising a tau fragment, a heterologous Th epitope, and an optional heterologous spacer, can induce an immune response and the production of anti-amyloid beta antibodies when administered to a subject.

[0195] kit Also provided herein, in certain embodiments, are kits comprising, consisting essentially of, or consisting of one, two, three, or more peptide immunogen constructs described herein (e.g., comprising two or more tau peptide immunogen constructs, or a tau peptide immunogen construct and an amyloid beta immunogen construct), optionally one or more adjuvants described herein (e.g., one or more aluminum salts described herein), optionally a CpG oligonucleotide described herein, and optionally one or more excipients. In some embodiments, such kits comprise a single container containing two, three, or more peptide immunogen constructs described herein (e.g., comprising two or more tau peptide immunogen constructs, or a tau peptide immunogen construct and an amyloid beta immunogen construct) in a single formulation. In some embodiments, such kits comprise these components in separate containers. In some embodiments, the kits further comprise instructions for use, such as directions for making a peptide immunogen composition using the provided components. In some embodiments, the kit further comprises a delivery vehicle (eg, a syringe) for administering the peptide immunogenic composition to a subject.

[0196] method The present invention also relates to methods for making and using the peptide immunogen constructs, compositions, and pharmaceutical compositions.

[0197] a. Methods for producing peptide immunogen constructs The peptide immunogen constructs of the present disclosure can be produced by chemical synthesis methods well known to those skilled in the art (see, for example, Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, W.H. Freeman & Co., New York, NY, 1992, p. 77). Peptide immunogen constructs can be synthesized, for example, using automated Merrifield techniques of solid-phase synthesis on an Applied Biosystems Peptide Synthesizer Model 430A or 431, using α-NH2 protected by either t-Boc or F-moc chemistry using side-chain protected amino acids. Preparation of peptide immunogen constructs, including combinatorial library peptides against Th epitopes, can be achieved by providing a mixture of alternative amino acids for coupling at a given variable position.

[0198] After the desired peptide immunogen construct is fully assembled, the resin can be treated according to standard procedures to cleave the peptide from the resin and deblock the functional groups on the amino acid side chains. The free peptide can be purified by HPLC and characterized biochemically, for example, by amino acid analysis or sequencing. Methods for purifying and characterizing peptides are well known to those skilled in the art. The quality of the peptides produced by this chemical process can be controlled and defined, thereby ensuring the reproducibility, immunogenicity, and yield of the peptide immunogen construct.

[0199] The range of structural variability that allows for the retention of intended immunological activity has been found to be much more flexible than the range of structural variability that allows for the retention of specific drug activity by small molecule drugs or the desired activity and undesired toxicity observed in large molecule drugs co-produced with biologically derived drugs. Thus, intentionally designed peptide analogs, or those inevitably produced by errors in the synthesis process as a mixture of deletion sequence by-products with similar chromatographic and immunological properties to the intended peptide, are often as effective as purified preparations of the desired peptide. Mixtures of designed and unintended analogs are effective if discriminatory QC procedures are developed to monitor both the manufacturing process and the product evaluation process to ensure the reproducibility and efficacy of final products using these peptides.

[0200] Peptide immunogen constructs can also be produced using recombinant DNA technology, including nucleic acid molecules, vectors, and / or host cells. Thus, nucleic acid molecules encoding peptide immunogen constructs and their immunologically functional analogs are also included in this disclosure as part of the present invention. Similarly, vectors, such as expression vectors, containing nucleic acid molecules, and host cells harboring the vectors are also included in this disclosure as part of the present invention.

[0201] Various exemplary embodiments also encompass methods for producing peptide immunogen constructs and immunologically functional analogs thereof. For example, the methods may include incubating host cells containing an expression vector containing a nucleic acid molecule encoding the peptide immunogen construct and / or its immunologically functional analog under conditions such that the peptide and / or analog is expressed. Longer synthetic peptide immunogens can be synthesized by well-known recombinant DNA techniques. Such techniques are provided in well-known standard manuals with detailed protocols. To construct a gene encoding a peptide of the invention, the amino acid sequence is reverse-translated to obtain a nucleic acid sequence encoding the amino acid sequence, preferably with optimal codons for the organism in which the gene will be expressed. A synthetic gene is then typically created by synthesizing oligonucleotides encoding the peptide and any necessary regulatory elements. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions appropriate for the selected expression system and host. The peptide is purified and characterized by standard methods.

[0202] b. Methods of producing immunostimulating complexes Various exemplary embodiments also include a method for producing an immune stimulating complex (ISC) comprising a peptide immunogen construct and a CpG oligodeoxynucleotide (ODN) molecule. The stabilized immune stimulating complex (ISC) is derived from the cationic portion of the peptide immunogen construct and a polyanionic CpG ODN molecule. The self-assembly system is driven by electrostatic neutralization of charges. The degree of association is determined by the stoichiometry of the molar charge ratio of the cationic portion of the peptide immunogen construct to the anionic oligomer. The non-covalent electrostatic association of the tau peptide immunogen construct and the CpG ODN is a completely reproducible process. The peptide / CpG ODN immune stimulating complex aggregates facilitate presentation to "professional" antigen-presenting cells (APCs) of the immune system, thus further enhancing the immunogenicity of the complex. These complexes are easily characterized for quality control during manufacturing. The peptide / CpG ISC is well tolerated in vivo. A particle system containing CpG ODN and B cell epitope-containing fragments derived from the immunogen construct was designed to take advantage of the generalized B cell mitogenicity associated with the use of CpG ODN and further promote a balanced Th-1 / Th-2 type response.

[0203] The CpG ODN in the disclosed pharmaceutical composition binds 100% to the immunogen in a process mediated by electrostatic neutralization of opposite charges, resulting in the formation of micron-sized particles. This microparticulate form allows the CpG dose to be significantly reduced compared to the conventional use of CpG adjuvants, reducing the possibility of harmful innate immune responses and promoting alternative immunogen processing pathways involving antigen-presenting cells (APCs). Thus, such formulations are conceptually novel and offer potential advantages by promoting the stimulation of immune responses through alternative mechanisms.

[0204] c. Methods for producing pharmaceutical compositions Various exemplary embodiments also encompass pharmaceutical compositions containing the peptide immunogen constructs described herein. In certain embodiments, pharmaceutical compositions employ water-in-oil emulsions and mineral salt suspensions.

[0205] Since pharmaceutical compositions are used by many people and the prevention of tau or amyloid aggregation is also part of the purpose of administration, safety becomes another important factor to consider. Although water-in-oil emulsions have been used in many formulations in humans in clinical trials, alum remains the main adjuvant used in formulations for safety reasons. Therefore, alum or its inorganic salt, aluminum phosphate (ADJUPHOS), is frequently used as an adjuvant in preparations for clinical use.

[0206] Other adjuvants and immunostimulants that may be used include polymeric or monomeric amino acids such as 3 de-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polyglutamic acid or polylysine. Such adjuvants can be used with or without other specific immunostimulants, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-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. The oil-in-water emulsions included MF59 (Van Nest et al.) 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. al., WO 90 / 14837, which is incorporated herein by reference in its entirety); SAF containing 10% squalene, 0.4% Tween® 80, 5% Pluronic®-block polymer L121, and thr-MDP, microfluidized into a submicron emulsion or vortexed to produce a larger particle size emulsion; and Ribi™ Adjuvant System (RAS) (Ribi ImmunoChem, Hamilton, Mont.), containing 2% squalene, 0.2% Tween® 80, and one or more bacterial cell wall components selected from monophosphorylated lipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL+CWS (Detox™).Other adjuvants include complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IFA), and cytokines such as interleukins (IL-1, IL-2, and IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF).

[0207] The choice of adjuvant depends on the stability of the immunogenic preparation containing the adjuvant, the route of administration, the administration schedule, and the effectiveness of the adjuvant for the species to be vaccinated. For humans, a pharmaceutically acceptable adjuvant is an adjuvant that has been approved or can be approved by appropriate regulatory agencies for administration to humans. For example, alum, MPL, or incomplete Freund's adjuvant (Chang et al., Advanced Drug Delivery Reviews 32:173-186 (1998), which is incorporated herein by reference in its entirety), alone or optionally in combination, are suitable for administration to humans.

[0208] The composition may contain a pharmaceutically acceptable non-toxic carrier or diluent, defined as a vehicle commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents include 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.

[0209] Pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acid, polyglycolic acid and copolymers (e.g., latex-functionalized Sepharose, agarose, cellulose, etc.), polymeric amino acids, amino acid copolymers, and lipid aggregates (e.g., oil droplets or liposomes). Additionally, these carriers can function as immunostimulating agents (adjuvants).

[0210] The pharmaceutical compositions of the present invention may further comprise a suitable delivery vehicle, including, but not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and cochleates.

[0211] d. Methods of Using the Pharmaceutical Composition The present disclosure also includes methods of using pharmaceutical compositions containing tau peptide immunogenic constructs, and methods of using pharmaceutical compositions containing one or more tau peptide immunogenic constructs and / or one or more Aβ peptide immunogenic constructs.

[0212] In some embodiments, the methods employ one or more compositions comprising one or more tau peptide immunogenic constructs and one or more separate compositions comprising one or more Aβ peptide immunogenic constructs, hi some embodiments, the one or more tau peptide immunogenic constructs and the one or more Aβ peptide immunogenic constructs are in a single composition.

[0213] In some embodiments, the methods use one, two, or more compositions comprising two or more tau peptide immunogenic constructs. In some embodiments, the two or more tau peptide immunogenic constructs are in separate compositions. In some embodiments, the two or more tau peptide immunogenic constructs are in a single composition.

[0214] In certain embodiments, pharmaceutical compositions containing peptide immunogen constructs may be used for: (a) inhibiting tau aggregation in the host; (b) induce disaggregation of already formed tau aggregates in the host; (c) reducing neurodegeneration caused by exogenous tau aggregates in the host; (d) reducing neurodegeneration in tau-overexpressing cells; (e) reducing tau concentrations in the host's serum; (f) reducing the level of oligomeric tau in the host brain; (g) reduction of neuropathology and restoration of motor activity in the host; (h) reducing the formation of amyloid plaques;

[0215] In some embodiments, administration of a tau peptide immunogen construct or any combination composition described herein to a subject (e.g., a mammal) reduces, inhibits, or prevents tau aggregation (e.g., pathological aggregation) in the brain of the subject.

[0216] In some embodiments, administration of a tau peptide immunogen construct or any combination composition described herein to a subject (e.g., a mammal) reduces, inhibits, or prevents the formation of neurofibrillary tangles in the brain of the subject.

[0217] In some embodiments, administration of a tau peptide immunogen construct or any combination composition described herein to a subject (e.g., a mammal) reduces the level of oligomeric tau in the brain of the subject.

[0218] In some embodiments, administration of a tau peptide immunogenic construct or any combination composition described herein to a subject (e.g., a mammal) reduces the level of tau fibrils in the subject's brain. In some embodiments, the tau fibrils comprise a p301S mutation. In some embodiments, the tau fibrils comprise a p301L mutation. In some embodiments, administration of a tau peptide immunogenic construct or any combination composition described herein to a subject (e.g., a mammal) reduces the level of tau carrying the p301S or p301L mutation (e.g., fibrillar tau) in the subject's brain.

[0219] In some embodiments, administration of a tau peptide immunogen construct or any combination composition described herein to a subject (e.g., a mammal) reduces monomeric tau levels in the serum or brain of the subject.

[0220] In some embodiments, administration of an amyloid beta peptide immunogen construct or any combination composition described herein to a subject (e.g., a mammal) reduces, inhibits, or prevents the accumulation of amyloid aggregates comprising amyloid beta peptide in the brain of the subject.

[0221] In some embodiments, administration of an amyloid beta peptide immunogen construct described herein to a subject (e.g., a mammal) reduces, inhibits, or prevents the formation of amyloid plaques in the brain of the subject.

[0222] In some embodiments, administration of an amyloid beta peptide immunogen construct or any combination composition described herein to a subject (e.g., a mammal) reduces the level of oligomeric amyloid beta in the brain of the subject.

[0223] In some embodiments, administration of an amyloid beta peptide immunogen construct or any combination composition described herein to a subject (e.g., a mammal) reduces the level of amyloid beta fibrils in the brain of the subject.

[0224] The method comprises administering to a subject in need thereof a pharmaceutical composition comprising a pharmacologically effective amount of one or more peptide immunogen constructs.

[0225] In some embodiments, the methods described herein are for the treatment and / or prevention of a tauopathy.

[0226] As used herein, "tauopathy" includes any neurodegenerative disease that involves pathological aggregation of the microtubule protein tau in the brain. Thus, in addition to both familial and sporadic Alzheimer's disease, other tauopathies that may be treated using the methods of the present invention include, but are not limited to, frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia combined, dementia pugilistica, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy. The methods can also be used to prevent and treat Lewy body diseases.

[0227] In some embodiments, the methods described herein are for the treatment and / or prevention of Alzheimer's dementia.

[0228] In some embodiments, the methods described herein are for the treatment and / or prevention of Alzheimer's disease.

[0229] In some embodiments, the methods described herein are for inhibiting the formation of or reducing the levels of misfolded or pathological tau, e.g., before the clinical onset of Alzheimer's disease.

[0230] In some embodiments, the methods described herein are for inhibiting the formation of or reducing the level of amyloid plaques, for example, prior to the clinical onset of Alzheimer's disease.

[0231] Another aspect of the present disclosure relates to methods for promoting clearance of tau and / or Aβ aggregates from the brain of a subject, comprising administering to the subject any one or more immunogenic tau or Aβ peptide immunogen constructs described herein (see, e.g., Tables 2 or 4), or one or more antibodies that recognize an epitope of one of such peptides, under conditions effective to promote clearance of tau or Aβ aggregates from the brain of the subject. In some embodiments, these methods comprise, for example, administration of one or more tau peptide immunogen constructs and one or more Aβ peptide immunogen constructs described herein.

[0232] Clearance of tau aggregates includes clearance of neurofibrillary tangles and / or pathological tau precursors to neurofibrillary tangles. Neurofibrillary tangles are often associated with neurodegenerative diseases, including, for example, sporadic and familial Alzheimer's disease, amyotrophic lateral sclerosis, argyrophilic grain dementia, dementia pugilistica, chronic traumatic encephalopathy, diffuse neurofibrillary tangles with calcifications, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, hereditary frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, Pick's disease, prion protein cerebral amyloid angiopathy, sporadic corticobasal degeneration, progressive supranuclear palsy, subacute sclerosing panencephalitis, myotonic dystrophy, motor neuron disease with neurofibrillary tangles, neurofibrillary tangle dementia, and progressive subcortical gliosis.

[0233] Another aspect of the present disclosure relates to a method of slowing the progression of a tau or Aβ pathology-associated behavioral phenotype in a subject, the method comprising administering to the subject any one or more immunogenic tau or Aβ peptide immunogenic constructs described herein (see, e.g., Tables 2 or 4), or one or more antibodies that recognize an immunogenic epitope of one of such peptides, under conditions effective to slow the tau or Aβ pathology-associated behavioral phenotype in the subject. The method can also include, for example, administering one or more tau peptide immunogenic constructs and one or more Aβ peptide immunogenic constructs described herein.

[0234] As used herein, tau pathology or Aβ pathology associated behavioral phenotypes include, but are not limited to, cognitive impairment, early personality changes and incontrol, emotional numbness, apraxia, mutism, apraxia, perseveration, stereotyped movements / behaviors, lip-syncope, confusion, inability to plan or organize sequential tasks, selfishness / surliness, antisocial traits, lack of empathy, stuttering, agrammatic speech with frequent errors but relatively preserved comprehension, decreased comprehension and word-finding disorders, slowly progressive gait unsteadiness, retropulsion, freezing, frequent falls, non-levodopa-responsive axial rigidity, supranuclear gaze palsy, square wave seizures, slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory loss, and tremor.

[0235] According to the methods of the present disclosure, in one embodiment, an immunogenic tau peptide, a combination of immunogenic tau peptides, or a combination of one or more tau peptide immunogenic constructs and one or more Aβ immunogenic constructs is administered to a subject in need thereof. In some embodiments, the subject (e.g., a human) has (e.g., has been diagnosed with) a disease, condition, or pathology described herein or treated according to the methods provided herein. In some embodiments, the subject (e.g., a human) is at risk for a disease, condition, or pathology described herein.

[0236] Suitable immunogenic peptide fragments of proteins include one or more antigenic epitopes that mimic the pathological form of the corresponding protein. Exemplary immunogenic tau epitopes are phosphorylated at one or more amino acids that are phosphorylated in pathological forms of tau but not in normal or non-pathological forms of tau.

[0237] In some embodiments, administration of the immunogenic tau or Aβ peptide induces an active immune response in the subject against the immunogenic tau or Aβ peptide and pathological forms of tau or Aβ, thereby promoting clearance of associated tau or Aβ aggregates, slowing the progression of tau or Aβ pathology-related behaviors, and treating the underlying disease. According to this aspect of the invention, the immune response involves the generation of beneficial humoral (antibody-mediated) and / or cellular (mediated by antigen-specific T cells or their secretory products) responses directed against the immunogenic tau or Aβ peptide.

[0238] The presence of a humoral immune response can be determined and monitored by testing a biological sample (e.g., blood, plasma, serum, urine, saliva, feces, CSF, or lymph) from a subject for the presence of antibodies directed against immunogenic tau or Aβ peptides. Methods for detecting antibodies in biological samples are well known in the art, such as ELISA, dot blot, SDS-PAGE gel, or ELISPOT. The presence of a cell-mediated immune response can be determined by proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocyte) assays, which are readily known in the art.

[0239] In some embodiments of any of the methods described herein, the administration is parenteral. In some embodiments, the administration is intravenous (IV), subcutaneous (SC), intramuscular (IM), intradermal (ID), or intraperitoneal (IP). In some embodiments, the administration is intramuscular. In other embodiments, the administration is intranasal.

[0240] Isolated immunogenic tau peptides of the invention that may be used in the tau peptide immunogen constructs in any of the methods described herein include any one of the amino acid sequences in Tables 1 and 7-10 below.

[0241] Isolated immunogenic Aβ peptides that may be used in the amyloid beta peptide immunogen constructs in any of the methods described herein include any one of the amino acid sequences in Table 3, as well as any of those described in WO2014 / 143087 and / or US9,102,752B2 (including peptides within that formula).

[0242] Specific Numbered Embodiment 1 Specific embodiments of the present disclosure are described in the following numbered paragraphs. 1. A tau peptide immunogen construct comprising the formula: (Th) m -(A) n -(Tau fragment)-X or (Tau fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (tau fragment) is a B cell epitope having about 10 to about 40 amino acid residues derived from the full-length tau protein of SEQ ID NO: 60, X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; The tau peptide immunogen construct, wherein n is 0 to about 10. 2. The tau peptide immunogen construct of paragraph 1, wherein the tau fragment is selected from SEQ ID NOs: 1-8, 17-30, and 63-67. 3. The tau peptide immunogenic construct of any one of paragraphs 1 or 2, wherein the Th epitope is SEQ ID NO: 31-59. 4. The tau peptide immunogen construct described in paragraph 1, wherein the peptide immunogen construct is selected from SEQ ID NOs: 9 to 16, or is a construct in which the positions of the tau peptide and the Th peptide are reversed. 5. A tau peptide immunogen construct, comprising: a B cell epitope comprising about 8 to about 40 amino acid residues (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) derived from the full-length tau protein of SEQ ID NO: 60; A T helper epitope comprising an amino acid sequence selected from SEQ ID NOs: 31 to 59; and an optional heterologous spacer selected from the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62); The tau peptide immunogen construct, wherein the B cell epitope is covalently linked to the T helper epitope directly or via the optional heterologous spacer. 6. The tau peptide immunogenic construct of paragraph 5, wherein the B-cell epitope is selected from SEQ ID NOs: 1-8, 17-30, and 63-67. 7. The tau peptide immunogenic construct of paragraph 5 or 6, wherein the T helper epitope is selected from SEQ ID NOs: 31-59. 8. The tau peptide immunogen construct of any one of paragraphs 5 to 7, wherein the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (sequence number 62). 9. The tau peptide immunogenic construct of any one of paragraphs 5 to 8, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope. 10. The tau peptide immunogen construct of any one of paragraphs 5 to 9, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope via the optional heterologous spacer. 11. A composition comprising a tau peptide immunogenic construct according to any one of paragraphs 1 to 10. 12. a. The tau peptide immunogenic construct of any one of paragraphs 1 to 10; and b. A pharmaceutical composition comprising a pharmaceutically acceptable delivery vehicle and / or adjuvant. 13. a. the tau peptide immunogen construct is selected from SEQ ID NOs: 9 to 16, or a tau peptide immunogen construct in which the positions of the B cell epitope and the T cell epitope are reversed compared to one of SEQ ID NOs: 9 to 16; b. The pharmaceutical composition of paragraph 12, wherein the tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immune stimulatory complex. 14. The composition according to any one of paragraphs 11 to 13, wherein the composition or pharmaceutical composition comprises two or more of the tau peptide immunogenic constructs. 15. The composition of any one of paragraphs 11 to 14, wherein the composition or pharmaceutical composition comprises two, three, or four of the tau peptide immunogenic constructs. 16. The composition of any one of paragraphs 11 to 15, wherein the composition or pharmaceutical composition comprises one or more of SEQ ID NOs: 9 to 16, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 10 and SEQ ID NO: 15, or any combination thereof, or wherein the composition or pharmaceutical composition comprises a first peptide immunogenic construct that targets a first region of tau (e.g., the N-terminal region) and a second peptide immunogenic construct that targets a second region of tau (e.g., a repeat sequence), optionally wherein these peptide immunogenic constructs comprise SEQ ID NOs: 2 and / or 10, and 7 and / or 15, respectively. 17. An isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of the tau peptide immunogenic construct of any one of paragraphs 1 to 10. 18. The isolated antibody or epitope-binding fragment thereof of paragraph 17 bound to said tau peptide immunogenic construct. 19. A composition comprising the isolated antibody or epitope-binding fragment thereof of paragraph 17 or 18. 20. Selected Tau Peptide Immunogen Constructs: [Table 13] 21. A composition comprising the tau peptide immunogenic construct of paragraph 20. twenty two. a. The tau peptide immunogen construct of paragraph 20; and b. A pharmaceutical composition comprising a pharmaceutically acceptable delivery vehicle and / or adjuvant. 23. The pharmaceutical composition of paragraph 22, wherein the tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immune stimulatory complex. 24. The composition of any one of paragraphs 21 to 23, wherein the composition or pharmaceutical composition comprises two or more of the tau peptide immunogenic constructs. 25. The composition of any one of paragraphs 21 to 24, wherein the composition or pharmaceutical composition comprises two, three, or four of the tau peptide immunogenic constructs. 26. The composition according to any of the preceding paragraphs, wherein the composition or pharmaceutical composition further comprises one or more amyloid-beta (Aβ) peptide immunogen constructs. 27. The composition of paragraph 26, wherein the composition comprises an Aβ peptide immunogenic construct of SEQ ID NO: 70, an Aβ peptide immunogenic construct of SEQ ID NO: 71, or an Aβ peptide immunogenic construct of SEQ ID NO: 70 and an Aβ peptide immunogenic construct of SEQ ID NO: 71. 28. The composition comprises any one or more of SEQ ID NOs: 9 to 16, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 10 and SEQ ID NO: 15, or any combination thereof, for example, a combination of SEQ ID NO: 10 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 11 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 12 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 13 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 14 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 15 with SEQ ID NOs: 70 and 71, or a combination of SEQ ID NO: 16 with SEQ ID NOs: 70 and 71. or a combination thereof, or any of the above-listed combinations comprising SEQ ID NO:70 but not SEQ ID NO:71, or SEQ ID NO:71 but not SEQ ID NO:70; or the composition or pharmaceutical composition comprises a first peptide immunogenic construct that targets a first region of tau (e.g., the N-terminal region) and a second peptide immunogenic construct that targets a second region of tau (e.g., a repeat sequence), optionally wherein these peptide immunogenic constructs comprise SEQ ID NOs: 2 and / or 10, and 7 and / or 15, respectively. 29. The composition of any one of paragraphs 26 to 28, wherein the composition comprises an Aβ peptide immunogenic construct of WO2014 / 143087. 30. A method for preventing, inhibiting, reducing the severity of, delaying, or treating a tauopathy in a subject, said method comprising administering to said subject a tau peptide immunogen construct or a composition of any one of the preceding paragraphs. 31. The method of paragraph 30, wherein the tauopathy is selected from Alzheimer's disease, Lewy body disease, frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia combined, dementia pugilistica, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy. 32. The method of paragraph 30 or 31, wherein the tauopathy is Alzheimer's disease. 33. The method of any one of paragraphs 30 to 32, wherein the method comprises administering one or more tau peptide immunogenic constructs, optionally in combination with one or more amyloid beta peptide immunogenic constructs. 34. The method of paragraph 33, wherein the one or more tau peptide immunogenic constructs and / or the one or more amyloid beta peptide immunogenic constructs are comprised within a single composition. 35. The method of paragraph 33, wherein the one or more tau peptide immunogenic constructs and / or the one or more amyloid beta peptide immunogenic constructs are comprised in separate compositions that are administered separately. 36. The method of any one of paragraphs 30 to 35, comprising administration of one or more of the peptide immunogen constructs, compositions, or combinations thereof described in any of the preceding paragraphs.

[0243] Specific Numbered Embodiment 2 Additional specific embodiments of the present disclosure are described in the following numbered paragraphs. 1. A tau peptide immunogen construct comprising the formula: (Th) m -(A) n -(Tau fragment)-X or (Tau fragment)-(A) n -(Th) m -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer, (tau fragment) is a B cell epitope having about 10 to about 40 amino acid residues derived from the full-length tau protein of SEQ ID NO: 60, X is an amino acid α-COOH or α-CONH2, m is from 1 to about 4; The tau peptide immunogen construct, wherein n is 0 to about 10. 2. The tau peptide immunogen construct of paragraph 1, wherein the tau fragment is selected from SEQ ID NOs: 1-8, 17-30, and 63-67. 3. The tau peptide immunogenic construct of any one of paragraphs 1 or 2, wherein the Th epitope is selected from SEQ ID NOs: 31-59. 4. The tau peptide immunogen construct described in paragraph 1, wherein the peptide immunogen construct is selected from SEQ ID NOs: 9 to 16, or is a construct in which the positions of the tau peptide and the Th peptide are reversed. 5. A tau peptide immunogen construct, comprising: a B cell epitope comprising about 8 to about 40 amino acid residues (e.g., about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 amino acids) derived from the full-length tau protein of SEQ ID NO: 60; A T helper epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 31 to 59; and an optional heterologous spacer selected from the amino acids Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62); The tau peptide immunogen construct, wherein the B cell epitope is covalently linked to the T helper epitope directly or via the optional heterologous spacer. 6. The tau peptide immunogenic construct of paragraph 5, wherein the B-cell epitope is selected from SEQ ID NOs: 1-8, 17-30, and 63-67. 7. The tau peptide immunogenic construct of paragraph 5 or 6, wherein the T helper epitope is selected from SEQ ID NOs: 31-59. 8. The tau peptide immunogen construct of any one of paragraphs 5 to 7, wherein the optional heterologous spacer is (α,ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (sequence number 62). 9. The tau peptide immunogenic construct of any one of paragraphs 5 to 8, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope. 10. The tau peptide immunogen construct of any one of paragraphs 5 to 9, wherein the T helper epitope is covalently linked to the amino terminus of the B cell epitope via the optional heterologous spacer. 11. A composition comprising a tau peptide immunogenic construct according to any one of paragraphs 1 to 10. 12. a. The tau peptide immunogenic construct of any one of paragraphs 1 to 10; and b. A pharmaceutical composition comprising a pharmaceutically acceptable delivery vehicle and / or adjuvant. 13. a. the tau peptide immunogen construct is selected from SEQ ID NOs: 9 to 16, or a tau peptide immunogen construct in which the positions of the B cell epitope and the T cell epitope are reversed compared to one of SEQ ID NOs: 9 to 16; b. The pharmaceutical composition of paragraph 12, wherein the tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immune stimulatory complex. 14. The composition according to any one of paragraphs 11 to 13, wherein the composition or pharmaceutical composition comprises two or more of the tau peptide immunogenic constructs. 15. The composition of any one of paragraphs 11 to 14, wherein the composition or pharmaceutical composition comprises two, three, or four of the tau peptide immunogenic constructs. 16. The composition of any one of claims 11 to 15, wherein the composition or pharmaceutical composition comprises one or more of SEQ ID NOs: 9 to 16, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 10 and SEQ ID NO: 15, or any combination thereof, or wherein the composition or pharmaceutical composition comprises a first peptide immunogenic construct that targets a first region of tau (e.g. the N-terminal region) and a second peptide immunogenic construct that targets a second region of tau (e.g. the repeat sequence), optionally wherein these peptide immunogenic constructs comprise SEQ ID NOs: 2 and / or 10, and 7 and / or 15, respectively. 17. An isolated antibody or epitope-binding fragment thereof that specifically binds to a B-cell epitope of the tau peptide immunogenic construct of any one of paragraphs 1 to 10. 18. The isolated antibody or epitope-binding fragment thereof of paragraph 17 bound to said tau peptide immunogenic construct. 19. A composition comprising the isolated antibody or epitope-binding fragment thereof of paragraph 17 or 18. 20. A tau peptide immunogen construct selected from: [Table 14] 21. A composition comprising the tau peptide immunogenic construct of paragraph 20. twenty two. a. The tau peptide immunogen construct of paragraph 20; and b. A pharmaceutical composition comprising a pharmaceutically acceptable delivery vehicle and / or adjuvant. 23. The pharmaceutical composition of paragraph 22, wherein the tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immune stimulatory complex. 24. The composition of any one of paragraphs 21 to 23, wherein the composition or pharmaceutical composition comprises two or more of the tau peptide immunogenic constructs. 25. The composition of any one of paragraphs 21 to 24, wherein the composition or pharmaceutical composition comprises two, three, or four of the tau peptide immunogenic constructs. 26. The composition according to any of the preceding paragraphs, wherein the composition or pharmaceutical composition further comprises one or more amyloid-beta (Aβ) peptide immunogen constructs. 27. The composition of paragraph 26, wherein the composition comprises an Aβ peptide immunogenic construct of SEQ ID NO: 70, an Aβ peptide immunogenic construct of SEQ ID NO: 71, or an Aβ peptide immunogenic construct of SEQ ID NO: 70 and an Aβ peptide immunogenic construct of SEQ ID NO: 71. 28. The composition comprises any one or more of SEQ ID NOs: 9 to 16, SEQ ID NO: 10, SEQ ID NO: 15, SEQ ID NO: 10 and SEQ ID NO: 15, or any combination thereof, for example, a combination of SEQ ID NO: 10 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 11 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 12 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 13 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 14 with SEQ ID NOs: 70 and 71, a combination of SEQ ID NO: 15 with SEQ ID NOs: 70 and 71, or a combination of SEQ ID NO: 16 with SEQ ID NOs: 70 and 71. or a combination thereof, or any of the above-listed combinations comprising SEQ ID NO:70 but not SEQ ID NO:71, or SEQ ID NO:71 but not SEQ ID NO:70; or the composition or pharmaceutical composition comprises a first peptide immunogenic construct that targets a first region of tau (e.g., the N-terminal region) and a second peptide immunogenic construct that targets a second region of tau (e.g., a repeat sequence), optionally wherein these peptide immunogenic constructs comprise SEQ ID NOs: 2 and / or 10, and 7 and / or 15, respectively. 29. The composition of any one of paragraphs 26 to 28, wherein the composition comprises an Aβ peptide immunogenic construct of WO2014 / 143087. 30. A method for preventing, inhibiting, reducing the severity of, delaying, or treating a tauopathy in a subject, said method comprising administering to said subject a tau peptide immunogenic construct or a composition of any one of the preceding claims. 31. The method of paragraph 30, wherein the tauopathy is selected from Alzheimer's disease, Lewy body disease, frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia combined, dementia pugilistica, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy. 32. The method of paragraph 30 or 31, wherein the tauopathy is Alzheimer's disease. 33. The method of any one of paragraphs 30 to 32, wherein the method comprises administering one or more tau peptide immunogenic constructs, optionally in combination with one or more amyloid beta peptide immunogenic constructs. 34. The method of paragraph 33, wherein the one or more tau peptide immunogenic constructs and / or the one or more amyloid beta peptide immunogenic constructs are comprised within a single composition. 35. The method of paragraph 33, wherein the one or more tau peptide immunogenic constructs and / or the one or more amyloid beta peptide immunogenic constructs are comprised in separate compositions that are administered separately. 36. A method according to any one of paragraphs 30 to 35, comprising administration of one or more of the peptide immunogenic constructs, compositions or combinations thereof according to any of the preceding claims. [Example]

[0244] Example 1 Accumulation of misfolded tau in the brain correlates with clinical decline in Alzheimer's disease (AD), but manifests decades earlier than cognitive symptoms (Congdon and Sigurdsson, Nat. Rev. Neurol, 2018). Targeting pathological tau before the clinical onset of AD may help prevent disease and / or progression. Vaccine-based immunotherapy for the prevention and treatment of Alzheimer's disease is described herein. The results of preclinical characterization of the tau vaccine are presented in Table A below. Peptides listed in the figure can be cross-referenced based on the letter codes in column 1 of Table 2. [Table A]

[0245] The vaccine platform is shown in Figure 1 and includes a B cell epitope, a linker, and a Th peptide carrier. B cells recognize the B cell epitope and generate antibodies against the peptide, which mimics the biological target (e.g., tau). The linker chemically attaches the B cell epitope to the Th peptide, thereby optimizing presentation of the B cell epitope to the immune system. The Th peptide carrier activates T helper cells, alerting B cells to initiate antibody production against the B cell epitope while avoiding inflammation and off-target activity. The primary structure of tau, with functional domains and targeting peptides annotated, is shown at the bottom of Figure 1. The structures of the peptide immunogen constructs shown in this figure are also shown (and referenced by letter code) in Table 2.

[0246] The peptide immunogen constructs tested herein were formulated in Adju-Phos CpG1 at 100 μg / mL + 300 μg / 0.25 mL of peptide. Guinea pigs received five intramuscular injections at 3-week intervals, with a terminal bleed at week 15. Antibodies to T-helper peptides, adjuvant components, and tau were quantified by ELISA at serial dilutions. Antibody binding was further characterized by Western blot, dot blot, and biolayer interferometry (BLI) against recombinant and brain-derived tau preparations. In vitro functional assessment was performed by tau FRET aggregation and pHrodo uptake assays in HEK293 and B103 cells.

[0247] The results of antibody binding characterization are shown in Figures 2A and 2B. The binding ability of vaccine-induced antibodies to three forms of recombinant tau (monomer, oligomer, and PFF) and sarkosyl extract (Br) from postmortem brain tissue was characterized via dot blot analysis (Figure 2A). Diverse binding profiles were observed for various forms of tau. BLI results showed that the antibodies bound to tau forms and functioned with Kds in the nM range, and that the three lead-derived antibodies had slower off-rates than bepraneumab (Figure 2B). Representative binding curves for Bep and lead A-derived antibodies to PFF are presented.

[0248] Results of aggregation assays using the tau biosensor line are shown in Figures 3A and 3B. The tau biosensor line (Frost et al., J. Biol. Chem., 2009;284(19):12845-12852) was used to evaluate functional inhibition of tau aggregation by vaccine-induced antibodies compared to the anti-tau mAbs semolinemab and bepranemab. Assay conditions included the addition of lipofectamine (Figure 3A) and no lipofectamine (Figure 3B). Only the lipofectamine-free condition generated Abs that inhibited aggregation.

[0249] The results of the tau uptake assay are shown in Figures 4A and 4B. B103 cells were exposed to pHrodo-labeled tau preparations for 6 hours. Upon uptake, pHrodo-tau emitted fluorescence, which could be quantified using an IncuCyte live-cell imager. Leads A, B, C, and F induced antibodies that inhibited monomeric tau uptake in a dose-responsive manner (Figure 4A). All lead-derived antibodies resulted in a dose-dependent reduction in PFF uptake (Figure 4B). The potencies of the lead-derived antibodies varied; however, all demonstrated stronger inhibition of PFF than monomeric tau uptake.

[0250] Antibodies induced by the tested tau peptide immunogen constructs exhibited diverse binding profiles to various forms of tau. No significant immunogenicity was observed for Th1 peptides or CpG1. Functional assays showed that such antibodies prevented tau aggregation (as indicated by inhibition of tau uptake).

[0251] Example 2 Combinations of anti-tau IgG antibodies generated using p5555kb and p5187kb were tested in activity and binding assays. Figure 5A shows the results of a tau uptake assay in B103 cells treated with pHrodo-PFF tau and anti-tau antibodies from rats vaccinated with p5555kb, p5187kb, or both p5555kb and p5187kb. Antibodies generated by the indicated vaccines in Figures 5A, 5B, and 5C, p5555kb + p5187kb, were mixed together in the assay. The data show that tau uptake is reduced when anti-tau IgG antibodies from rats vaccinated with p5555kb and p5187kb IgG are used. Figure 5B is another presentation of the data from Figure 5A, showing the percent inhibition of tau uptake.

[0252] To investigate whether antibodies derived from p5555kb and p5187kb can bind the same target together, we used biolayer interferometry (BLI). Briefly, BLI allows for quantification of binding by measuring the interference of light reflected from a biosensor coated with the target protein or peptide. As binding occurs, interference increases. If two antibodies can bind together, interference is expected to increase after the introduction of a different antibody following binding of the antibody to the same target. If the antibodies cannot bind together to the same target, interference will not change with the second antibody after binding of the first antibody. Figure 5C shows the results of biolayer interferometry analysis of IgG derived from p5555kb and p5187kb. The data show that IgG derived from p5555kb continued to bind to PFF tau following tau binding by IgG derived from p5187kb. Figure 5D shows the anti-tau IgG isotypes generated by administration of p5555kb and p5187kB, demonstrating enrichment for IgG1 and IgG2b isotypes. These results support the feasibility of a combination vaccine against tau.

[0253] Vaccination studies were conducted in WT and P301L (mutant tau) mice. Figure 6A shows that P301L mice vaccinated with p5555kb generated robust titers against monomeric and PFF tau. Figure 6B shows that vaccination with p5555kb reduced tau accumulation in P301L mice. Figure 6C shows that lysates from the brains of P301L mice vaccinated with p5555kb showed evidence of reduced induction of aggregation in the tau biosensor line. These results indicate that both WT and P301L mice responded positively to vaccination with p5555kb.

[0254] Example 3 A combination tau vaccine, designated VXX-301, was designed by combining the p5555kb and p5187kb tau peptide immunogen constructs into a single formulation. References to VXX-301 in the figures refer to antibodies derived from VXX-301-vaccinated animals. Figures 7A-7B show that rats vaccinated with p5555kb, p5187kb, or the combination vaccine VXX-301 generated robust antibody titers against monomeric or fibrillar PFF tau. For vaccination, 0.3 mg / ml of peptide immunogen was used per 0.25 mL dose, and 0.1 mg of CpG was administered intramuscularly. Figures 7C-7E show anti-monomeric tau IgG titer curves over time after vaccination of rats with p5555kb (Grp1, Figure 7C), p5187kb (Grp2, Figure 7D), and VXX-301 (Grp3, Figure 7E). Figure 8 shows the IgG isotypes of anti-tau antibodies after vaccination with p5555kb, p5187kb, or VXX-301, showing that IgG1 and IgG2b were enriched after vaccination (and lower titers of IgG2a and IgG2c).

[0255] Western blot analysis of anti-tau antibodies from rats vaccinated with p5555kb, p5187kb, or VXX-301 was performed to assess binding to different recombinant preparations of tau. Recombinant preparations of tau investigated for binding included 2N4R full-length tau, 2N3R, WT preformed fibrils (PFF), and P301S mutant tau. The P301L K18 preparation is a C-terminal fragment of tau containing four repeat domains. Figure 9 shows that antibodies derived from p5555kb can bind to tau preparations 2N4R, 2N3R, WT preformed fibrils (PFF), and P301S (but not K18), while antibodies derived from p5187kb and VXX-301 can bind to all forms of tau, including the P301L K18 preparation (see white arrows in Figure 9).

[0256] Anti-tau antibodies from vaccinated rats inhibited tau aggregation. Figure 10A shows that lysates from the brains of rats vaccinated with VXX-301 showed evidence of enhanced inhibition of tau aggregation in the tau biosensor line. Figure 10B shows that antibodies from rats vaccinated with p5555kb, p5187kb, and VXX-301 were equally able to inhibit tau aggregation when compared with bepranemab. Figures 11A-11B show the results of a tau uptake assay demonstrating that anti-tau antibodies from vaccinated rats inhibited tau uptake. The data showed that B103 cells treated with pHrodo-PFF tau showed significant tau reuptake, and that bepranemab (Bep), semolinemab (Sem), or antibodies from rats vaccinated with p5555kb, p5187kb, or VXX-301 were equally able to inhibit tau reuptake. These results support the efficacy of the combination vaccine VXX-301 in inhibiting tau reuptake and aggregation.

[0257] To assess in vivo protection against tau-associated pathology and death, female P301L tau mutant mice were vaccinated with saline control, p5555kb, p6187kb, or VXX-301 for a total of six injections and monitored for 210 days. Vaccination with p5555kb, p6187kb, or VXX-301 delayed the onset of ataxia (Figure 12A) and prolonged survival (Figure 12B) in a greater percentage of mice. Vaccination with p5555kb, p6187kb, or VXX-301 also resulted in high titers of antibodies against both monomeric (Figure 12C) and fibrillar PFF (Figure 12D) tau in P301L mice.

[0258] Example 4 The epitope specificity of anti-tau antibodies generated with p5555kb, p5187kb, or VXX-301 was determined by epitope mapping in ELISA. ELISA assay plates were coated with 2 mg / mL of each of the target peptides listed in Table B, immobilized to the plate via a biotin linker in 1x TBS, and incubated overnight at 2–8°C. The target peptides "N-terminal tau (1–22)" and "p5555kb target sequence" resemble the epitope used to generate the p5555kb-derived antibody. The target peptides "p5187kb target sequence," "P301S p5187kb," "P301L p5187kb," and "3R tau p5187kb" resemble the epitope used to generate the p5187kb-derived antibody. The coated plates were blocked in PBS SuperBlock™ buffer (SBB, Thermo Fisher Scientific) for 2 hours at 28°C with shaking at 500 rpm. Serum samples from animals vaccinated with p5555kb, p5187kb, or VXX-301 were serially diluted in half-logarithmic replicates from an initial factor of 20 in 1X PBS containing 2% BSA across a 12-point series, then arrayed in duplicate on assay plates and incubated for 1 hour at 28°C. HRP-conjugated anti-rat IgG was used for titer detection at a dilution of 1:10,000 in SBB. After incubation at 28°C, 3,3',5,5'-tetramethylbenzidine (TMB) Ultra ELISA substrate was applied to each plate, and the reaction was stopped after 20 minutes by adding 2N H2SO4 acid. Absorbance was measured at 450 nm on a SpectraMax iD5 plate reader (Molecular Devices) within 10 min of stopping the reaction. A nonlinear four-parameter (4-PL) curve fit was applied to each dilution series to obtain EC 50 Titers were calculated (SoftMax Pro, ver. 7.1 or GraphPad Prism, ver. 9.2). [Table B]

[0259] As shown in Table B, p555kb-induced anti-tau antibodies induce robust EC 50 Although the p5187kb-derived anti-tau antibodies showed potency and binding ability to a target peptide similar to the p555kb B cell epitope, they did not show robust EC 50 The antibodies generated by VXX-301 showed high EC2 activity against all target peptides used. 50 These results support that the p5555kb- and p5187kb-induced antibodies bind to different target epitopes and that the combination vaccine VXX-301 against tau is effective.

[0260] Example 5 A combination vaccine was designed by combining an Aβ-targeting peptide vaccine and a tau-targeting peptide vaccine into a single vaccine formulation. The immunogenicity of the vaccine was evaluated in guinea pigs, rats, and mice. Vaccine-derived antibodies were characterized by Western blot, dot blot, and immunohistochemistry. The efficacy of the vaccine was evaluated in double transgenic mice (P301L) expressing both human Aβ and tau, focusing on Aβ and tau brain pathology by immunohistochemistry and various immunoassays in brain homogenates.

[0261] Figures 13A and 13B show Aβ-tau combination vaccine antibody titers against tau and Aβ. Three different tau peptide immunogen constructs (p5555kb, p5404kb, and p5137kb) were used in combination with an Aβ peptide immunogen construct (p3102kb). p3102kb is the construct of SEQ ID NO: 70 shown in Table 4. The results in Figures 13A-13B show that the combination vaccine induced high titers and robust immunogenicity against both oligomeric Aβ and a wide range of tau forms (monomers, oligomers, and fibrils).

[0262] table [Table 1-1] Table 1-2 Table 2-1 Table 2-2 Table 3 Table 4 Table 5-1 Table 5-2 Table 6-1 Table 6-2 Table 7 Table 8 Table 9 Table 10 Table 11

Claims

1. A composition or kit comprising: a first tau peptide immunogen construct comprising a first tau B-cell epitope comprising or consisting of 6 to about 40 amino acid residues from a full-length tau protein of SEQ ID NO: 60; and (i) a second tau peptide immunogen construct comprising a second tau B-cell epitope comprising or consisting of 6 to about 40 amino acid residues from the full-length tau protein of SEQ ID NO: 60, wherein the second B-cell epitope is not the same as the first B-cell epitope; and / or (ii) the composition or kit, comprising an amyloid beta (Aβ) peptide immunogen construct comprising an amyloid beta B-cell epitope comprising or consisting of 6 to about 42 amino acid residues derived from the full-length amyloid beta protein of SEQ ID NO:

68.

2. 10. The composition or kit of claim 1, comprising the second tau peptide immunogen construct.

3. The composition or kit of claim 1 comprising the amyloid beta peptide immunogen construct.

4. 2. The composition or kit of claim 1, comprising the second tau peptide immunogenic construct and the amyloid beta peptide immunogenic construct.

5. 5. The composition or kit of claim 1, 2, or 4, wherein the first tau B-cell epitope comprises or consists of 6 to about 40 amino acid residues from the N-terminal amino acids 1 to 44 of SEQ ID NO: 60, and the second tau B-cell epitope comprises or consists of 6 to about 40 amino acid residues from the repeat domain amino acids 244 to 372 of SEQ ID NO:

60.

6. 6. The composition or kit of claim 1, 3, 4, or 5, wherein the amyloid beta B-cell epitope comprises at least six amino acid residues from amino acids 1 to 16 of SEQ ID NO:

68.

7. 7. The composition or kit of claim 1, wherein the first tau B-cell epitope, the second tau B-cell epitope, and the amyloid beta B-cell epitope are each about 8 to 15 amino acids in length, or 10 to 14 amino acids in length.

8. 7. The composition or kit of any one of claims 1 to 6, wherein the first tau B-cell epitope and, if present, the second tau B-cell epitope are independently selected from SEQ ID NOs: 1-8, 17-30, 63-67, 72-76, 96, and 202.

9. 9. The composition or kit of claim 8, wherein the first tau B-cell epitope and, if present, the second tau B-cell epitope are independently selected from SEQ ID NOs: 2, 4, 7, and 72-76.

10. The composition or kit of any one of claims 1 to 6, wherein the first tau B-cell epitope comprises or consists of SEQ ID NO:

2.

11. The composition or kit of any one of claims 1 to 6, wherein the first tau B-cell epitope comprises or consists of SEQ ID NO:

7.

12. 7. The composition or kit of claim 1, wherein the first tau B-cell epitope is selected from SEQ ID NO: 1 or SEQ ID NO: 2, and the second tau B-cell epitope is selected from SEQ ID NOs: 7 and 72-76.

13. 7. The composition or kit of any one of claims 1 to 6, wherein the first tau B-cell epitope comprises or consists of SEQ ID NO:2 and the second tau B-cell epitope comprises or consists of SEQ ID NO:

7.

14. The composition or kit according to any one of claims 1 to 13, wherein the amyloid beta B-cell epitope is selected from SEQ ID NOs: 69 and 82-88.

15. The composition or kit of any one of claims 1 to 13, wherein the amyloid beta B-cell epitope comprises or consists of SEQ ID NO: 69 or SEQ ID NO:

82.

16. 16. The composition or kit of any one of claims 1 to 15, wherein the first tau peptide immunogen construct comprises a first heterologous T helper (Th) epitope, the second tau peptide immunogen construct, if present, comprises a second heterologous Th epitope, and the amyloid beta peptide immunogen construct, if present, comprises a third heterologous Th epitope.

17. 17. The composition or kit of claim 16, wherein the first heterologous Th epitope, the second heterologous Th epitope, and the third heterologous Th epitope are each independently selected from SEQ ID NOs: 31-59 and 97.

18. 17. The composition or kit of claim 16, wherein the first heterologous Th epitope, the second heterologous Th epitope, and the third heterologous Th epitope are each independently selected from SEQ ID NOs: 42, 44, 45, and 97.

19. 17. The composition or kit of claim 16, wherein the first heterologous Th epitope, the second heterologous Th epitope, and the third heterologous Th epitope each have SEQ ID NO:

44.

20. 20. The composition or kit of any one of claims 1 to 19, wherein the first heterologous Th epitope and the first tau B-cell epitope, the second heterologous Th epitope and the second tau B-cell epitope, and the third heterologous Th epitope and the amyloid beta B-cell epitope are covalently linked, respectively, either directly or via a heterologous spacer.

21. 21. The composition or kit of claim 20, comprising the heterologous spacer linking the first heterologous Th epitope and the first tau B cell epitope, the second heterologous Th epitope and the second tau B cell epitope, and / or the third heterologous Th epitope and the amyloid beta B cell epitope.

22. 22. The composition or kit of claim 21, wherein the heterologous spacer comprises one or more amino acids.

23. 23. The composition or kit of claim 22, wherein the one or more amino acids are Lys-, Gly-, Lys-Lys-Lys-, (α,ε-N)Lys, Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95), or ε-Lys-Lys-Lys-Lys (SEQ ID NO: 62).

24. 23. The composition or kit of claim 22, wherein the one or more amino acids are Lys-Lys-Lys-ε-Lys (SEQ ID NO: 95) or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 62).

25. The first tau peptide immunogen construct, and, if present, the second tau peptide immunogen construct, have the formula: (Th) ｍ -(A) ｎ -(tau fragment)-X or (Tau fragment)-(A) ｎ - (Th) ｍ -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer; (tau fragment) is a B-cell epitope having 6 to about 40 amino acid residues from said full-length tau protein of SEQ ID NO: 60, optionally said B-cell epitope having about 10 to about 40 amino acid residues from said full-length tau protein of SEQ ID NO: 60; X is α-COOH or α-CONH of an amino acid ２ and m is from 1 to about 4; 25. The composition or kit of any one of claims 1 to 24, wherein n is 0 to about 10.

26. The amyloid beta peptide immunogen construct has the formula: (Th) ｍ - (A) ｎ - (Aβ fragment) - X or (Aβ fragment) - (A) ｎ — (Th) ｍ -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer; (Aβ fragment) is a B-cell epitope having 6 to about 42 amino acid residues from the full-length Aβ protein of SEQ ID NO: 68, optionally wherein the B-cell epitope has about 10 to about 42 amino acid residues from the full-length Aβ protein of SEQ ID NO: 68; X is α-COOH or α-CONH of an amino acid ２ and m is from 1 to about 4; 26. The composition or kit of any one of claims 1 to 25, wherein n is 0 to about 10.

27. 27. The composition or kit of any one of claims 1 to 26, wherein the first tau peptide immunogenic construct comprises the first heterologous Th epitope covalently linked to the carboxyl terminus of the first tau B-cell epitope, the second tau peptide immunogenic construct comprises the second heterologous Th epitope covalently linked to the amino terminus of the second tau B-cell epitope, and / or the amyloid beta peptide immunogenic construct comprises the third heterologous Th epitope covalently linked to the carboxyl terminus of the amyloid beta B-cell epitope.

28. 28. The composition or kit of any one of claims 1 to 27, wherein the first tau peptide immunogenic construct and the second tau peptide immunogenic construct are selected from SEQ ID NOs: 9-16 and 77-81, or are constructs in which the positions of the peptide cellular epitope and the Th epitope are reversed.

29. 29. The composition or kit of any one of claims 1 to 28, wherein the first tau peptide immunogen construct comprises or consists of the amino acid sequence of SEQ ID NO: 10 or SEQ ID NO:

15.

30. 30. The composition or kit of any one of claims 1 to 29, wherein the first tau peptide immunogenic construct comprises or consists of the amino acid sequence of SEQ ID NO: 10 and the second tau peptide immunogenic construct comprises or consists of the amino acid sequence of SEQ ID NO:

15.

31. 29. The composition or kit of any one of claims 1 to 28, wherein the first tau peptide immunogenic construct and / or the second tau peptide immunogenic construct is selected from SEQ ID NOs: 77-81, and optionally the first tau peptide immunogenic construct has the amino acid sequence of SEQ ID NO: 10 and the second tau peptide immunogenic construct is selected from SEQ ID NOs: 77-81.

32. 32. The composition or kit of any one of claims 1 to 31, wherein the amyloid beta peptide immunogen construct is selected from SEQ ID NOs: 70, 71, and 89, or a construct in which the positions of the B cell epitope and the Th epitope are reversed.

33. 33. The composition or kit of claim 32, wherein the amyloid beta peptide immunogen construct comprises or consists of SEQ ID NO:

70.

34. 33. The composition or kit of claim 32, wherein the amyloid beta peptide immunogen construct comprises or consists of SEQ ID NO:

89.

35. A tau peptide immunogen construct comprising a B-cell epitope comprising an amino acid sequence selected from any one of SEQ ID NOs: 72-76.

36. formula: (Th) ｍ -(A) ｎ -(B cell epitope)-X or (B cell epitope)-(A) ｎ - (Th) ｍ -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer; (B-cell epitope) comprises, consists essentially of, or consists of an amino acid sequence selected from any one of SEQ ID NOs: 72-76; X is α-COOH or α-CONH of an amino acid ２ and m is from 1 to about 4; 36. The tau peptide immunogen construct of claim 35, wherein n is from 0 to about 10.

37. 36. The tau peptide immunogen construct of claim 35, comprising an amino acid sequence selected from any one of SEQ ID NOs: 77-81.

38. An amyloid beta peptide immunogen construct comprising a B-cell epitope comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:

82.

39. formula: (Th) ｍ -(A) ｎ -(B cell epitope)-X or (B cell epitope)-(A) ｎ - (Th) ｍ -X wherein: Th is a heterologous T helper epitope; A is a heterologous spacer; (B cell epitope) comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 82; X is α-COOH or α-CONH of an amino acid ２ and m is from 1 to about 4; 39. The amyloid beta peptide immunogen construct of claim 38, wherein n is 0 to about 10.

40. 39. The amyloid beta peptide immunogen construct of claim 38, comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:

89.

41. A composition or kit comprising the tau peptide immunogenic construct of any one of claims 35 to 37.

42. A composition or kit comprising the amyloid beta peptide immunogenic construct of any one of claims 38 to 40.

43. 43. The composition or kit of any one of claims 1 to 34, 41, and 42, which is a single composition comprising the first tau peptide immunogenic construct and at least one of the second tau peptide immunogenic construct and the amyloid beta peptide immunogenic construct.

44. 43. The composition or kit of any one of claims 1 to 34, 41, and 42, which is a kit comprising the first tau peptide immunogenic construct and at least one of the second tau peptide immunogenic construct and the amyloid beta peptide immunogenic construct.

45. 45. The composition or kit of claim 44, wherein prior to administration to a subject, the components of the kit are mixed to create a single composition comprising the first tau peptide immunogenic construct and at least one of the second tau peptide immunogenic construct and the amyloid beta peptide immunogenic construct.

46. 45. The composition or kit of claim 44, wherein the components of the kit are used to create a first composition comprising the first tau peptide immunogenic construct, and a second composition comprising at least one of the second tau peptide immunogenic construct and the amyloid beta peptide immunogenic construct.

47. 47. A pharmaceutical composition comprising the composition of any one of claims 43, 45, or 46 and a pharmaceutically acceptable delivery vehicle and / or adjuvant.

48. 48. The pharmaceutical composition of claim 47, wherein the first tau peptide immunogen construct, the second tau peptide immunogen construct, and / or the amyloid beta peptide immunogen construct are mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immune stimulatory complex.

49. 49. The pharmaceutical composition of claim 47 or 48, comprising three or four of the tau peptide immunogen constructs.

50. 50. The pharmaceutical composition of claim 47, 48, or 49, comprising two or more amyloid beta peptide immunogen constructs.

51. An isolated antibody or epitope-binding fragment thereof which specifically binds to said B-cell epitope of said tau peptide immunogenic construct or said amyloid beta peptide immunogenic construct, obtainable by administering to a mammal a composition(s) of any one of claims 43, 45, and 46-50, or a peptide immunogenic construct of any one of claims 35-40.

52. 52. The isolated antibody or epitope-binding fragment thereof of claim 51, bound to said tau peptide immunogenic construct or said amyloid beta peptide immunogenic construct.

53. 53. A composition comprising the isolated antibody or epitope-binding fragment thereof of claim 51 or 52.

54. A method for preventing, inhibiting, reducing the severity of, delaying or treating a tauopathy in a subject, said method comprising administering to said subject a tau peptide immunogenic construct, amyloid beta peptide immunogenic construct, composition(s) according to any one of the preceding claims.

55. 55. The method of claim 54, comprising administering the composition, wherein the first tau peptide immunogen construct and at least one of the second tau peptide immunogen construct and the amyloid beta peptide immunogen construct are contained within a single composition.

56. 1. A method of preventing, inhibiting, reducing the severity of, delaying, or treating a tauopathy in a subject, said method comprising administering to said subject a first tau peptide immunogen construct comprising a first tau B-cell epitope comprising, or consisting of, from 6 to about 40 amino acid residues from a full-length tau protein of SEQ ID NO: 60; (i) a second tau peptide immunogen construct comprising a second tau B-cell epitope comprising or consisting of 6 to about 40 amino acid residues from the full-length tau protein of SEQ ID NO: 60, wherein the second B-cell epitope is not the same as the first B-cell epitope; and / or (ii) administering an amyloid beta (Aβ) peptide immunogen construct comprising an amyloid beta B-cell epitope comprising, or consisting of, 6 to about 42 amino acid residues from the full-length amyloid beta protein of SEQ ID NO: 68; The method, wherein the first tau peptide immunogenic construct and the second tau peptide immunogenic construct, and / or the amyloid beta peptide immunogenic construct are contained within separate compositions that are administered separately.

57. 57. The method of any one of claims 54 to 56, wherein the tauopathy is selected from Alzheimer's disease, Lewy body disease, frontotemporal dementia, Parkinsonism linked to chromosome 17 (FTDP-17), progressive supranuclear palsy, corticobasal degeneration, Pick's disease, progressive subcortical gliosis, neurofibrillary tangle dementia, diffuse neurofibrillary tangles with calcifications, argyrophilic grain dementia, amyotrophic lateral sclerosis / Parkinsonism / dementia combined, dementia pugilistica, Down syndrome, Gerstmann-Sträussler-Scheinker disease, Hallervorden-Spatz disease, inclusion body myositis, Creutzfeldt-Jakob disease, multiple system atrophy, Niemann-Pick disease type C, prion protein cerebral amyloid angiopathy, subacute sclerosing panencephalitis, myotonic dystrophy, non-Guam motor neuron disease with neurofibrillary tangles, postencephalitic parkinsonism, and chronic traumatic encephalopathy.

58. 58. The method of any one of claims 54 to 57, wherein the tauopathy is Alzheimer's disease.

59. 59. The method of any one of claims 54 to 58, wherein the subject is a human.

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