RU2798972C2 - Tau-peptide immunogenic constructs - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to new therapeutic agents for the treatment of neurodegenerative diseases. The invention discloses peptide immunogen constructs targeting regions of the Tau protein.

EFFECT: invention can be used for the treatment or prevention of tauopathy.

12 cl, 8 dwg, 16 tbl, 13 ex

Description

This application is an international PCT application claiming priority over US provisional application, serial number 62/578124, filed October 27, 2017, which is incorporated herein by reference in its entirety.

Field of invention

This invention relates to peptide immunogenic constructs based on parts of the Tau protein and their compositions for the prevention and treatment of tauopathies.

Technical field

Tau proteins (or τ proteins, after the Greek letter of that name) are proteins that stabilize microtubules (see website: en.wikipedia.org/wiki/Tau_protein). They are abundant in neurons of the central nervous system and less abundant elsewhere, but are also expressed at very low levels in astrocytes and CNS oligodendrocytes. Pathologies and dementias of the nervous system, such as Alzheimer's disease and Parkinson's disease, are associated with Tau proteins that have become defective and no longer stabilize microtubules properly.

Tau proteins are the product of alternative splicing from a single gene, which in humans is designated as MAPT (microtubule-associated protein Tau) and is located on chromosome 17. Tau proteins were identified in 1975 as thermostable proteins, necessary for microtubule assembly and then were characterized as intrinsically disordered proteins.

Tau protein is a highly soluble microtubule-associated protein (MAP). In humans, these proteins are found primarily in neurons compared to non-neuronal cells. One of the main functions of Tau is to modulate the stability of axonal microtubules. Other MAPs of the nervous system may perform similar functions, as has been suggested in Tau knockout mice, which have not been shown to have abnormal brain development - possibly due to Tau deficiency being compensated by other MAPs. Tau is not present in dendrites and is active mainly in the distal axons, where it provides microtubule stabilization but also flexibility when needed. This is in contrast to the MAP6 (STOP) proteins in the proximal axons, which essentially block microtubules, and MAP2, which stabilize microtubules in dendrites.

Tau proteins interact with tubulin to stabilize microtubules and promote assembly of tubulin into microtubules. Tau has two ways of controlling microtubule stability: isoforms and phosphorylation.

In the adult brain, six isoforms of Tau are expressed, ranging in length from 352 to 441 amino acids; they were obtained by alternative splicing of mRNA transcripts from MAPT (Fitzpatrick, et al., 2017; UniProtKB - P10636).

Isoforms differ in the number of binding domains. Three isoforms have three binding domains and three others have four binding domains. The binding domains are located at the C-terminus of the protein and are positively charged (allowing it to bind to a negatively charged microtubule). Isoforms with four binding domains stabilize microtubules better than those with three binding domains. The isoforms are distinguished by the presence or absence of inserts of 29 or 58 amino acids in the N-terminal half, and the inclusion or absence of the 31-amino acid repeat of microtubule binding encoded by MAPT exon 10 in the C-terminal half. The inclusion of exon 10 results in three Tau isoforms with four repeats each (4R), while its exclusion results in an additional three isoforms with three repeats each (3R). Four repeats (P1 - P4) contain residues 244-368 in the Tau isoform of 441 amino acids.

Tau is a phosphoprotein with 79 potential serine (Ser) and threonine (Thr) phosphorylation sites on the longest Tau isoform. Phosphorylation has been reported at about 30 of these sites in normal Tau proteins. Tau phosphorylation is regulated by a variety of kinases, including PKN, serine/threonine kinase. When PKN is activated, it phosphorylates Tau, resulting in disruption of microtubule organization. Tau phosphorylation is also regulated during development. For example, fetal Tau is more highly phosphorylated in the fetal CNS than adult Tau. The degree of phosphorylation in all six isoforms decreases with age due to the activation of phosphatases. Like kinases, phosphatases also play a role in the regulation of Tau phosphorylation. For example, both PP2A and PP2B are present in human brain tissue and have the ability to dephosphorylate Ser396. The binding of these phosphatases to Tau affects the association of Tau with MT.

Hyperphosphorylation of Tau protein (Tau inclusions, fTau) can lead to self-assembly of paired helical filament and straight filament coils that are involved in the pathogenesis of Alzheimer's disease, frontotemporal dementia, and other tauopathies. All six Tau isoforms are present in a normally hyperphosphorylated state in paired helical filaments in the Alzheimer's brain. In other neurodegenerative diseases, the deposition of aggregates enriched in some Tau isoforms is noted. When misfolded, this highly soluble protein can form highly insoluble aggregates that contribute to a number of neurodegenerative diseases.

Recent studies indicate that Tau may be released extracellularly via an exosomal mechanism in Alzheimer's disease. Gender-specific expression of the Tau gene in different regions of the human brain has recently been associated with gender differences in the manifestations and risk of tauopathy. Some aspects of how the disease progresses also suggest that it shares some similarities with prion proteins.

Neurodegenerative diseases with excessive Tau filaments are called tauopathies. In addition to Alzheimer's disease, these diseases include neurofibrillary tangle-predominant dementia, chronic traumatic encephalopathy (CTE), argyrophilic granule disease, progressive supranuclear palsy, corticobasal degeneration, globular glial tauopathy, and Pick's disease. Unlike Alzheimer's disease, these other diseases lack β-amyloid plaques. In Alzheimer's disease, tangle-predominant dementia, and chronic traumatic encephalopathy, all six Tau isoforms (3P and 4P) are present in disease filaments. In argyrophilic granule disease, progressive supranuclear palsy, corticobasal degeneration, and globular glial tauopathy, only 4P Tau isoforms are found, while in Pick's disease, only 3P Tau inclusions are present. The emergence of human tauopathies with different filament morphologies has led to the idea that there are different molecular conformers of aggregated Tau. The presence of multiple molecular conformers may also explain why filaments from the brains of individuals with Alzheimer's disease are more effective than recombinant protein filaments assembled in vitro in inducing Tau pathology in the mouse brain.

Repetitive mild traumatic brain injury (TBI) is now recognized as a central component of traumatic brain injury in contact sports, especially American football, and the impact force of military explosions. This can lead to chronic traumatic encephalopathy (CTE), which is characterized by fibrillar tangles of hyperphosphorylated Tau. High levels of tau in the fluid surrounding the brain are associated with poor recovery from head injury.

The Tau hypothesis states that excessive or abnormal Tau phosphorylation leads to the transformation of normal adult Tau into PHF-Tau (paired helical filament) and NFT (neurofibrillary tangles) (neurofibrillary tangles). Tau protein is a highly soluble microtubule-associated protein (MAP). Through its isoform and phosphorylation, Tau interacts with tubulin to stabilize microtubule assembly. Tau proteins comprise a family of six isoforms ranging from 352 to 441 amino acids. The longest isoform in the CNS contains four repeats (P1, P2, P3 and P4) and two inserts (total 441 amino acids), while the shortest isoform contains three repeats (P1, P3 and P4) and no insert (total 352 amino acids) . All six Tau isoforms are often present in a hyperphosphorylated state in paired helical filaments in AD.

Mutations that alter the function and expression of the Tau isoform result in hyperphosphorylation. The process of Tau aggregation in the absence of mutations is unknown, but may result from increased phosphorylation, protease action, or exposure to polyanions such as glycosaminoglycans. Hyperphosphorylated Tau disassembles microtubules and segregates normal Tau, MAP 1 (microtubule-associated protein 1), MAP 2, and ubiquitin into PHF tangles. This insoluble structure impairs cytoplasmic functions and interferes with axonal transport, which can lead to cell death.

To date, there is still an unmet need to develop site-targeted peptide immunogens and their formulations for the cost-effective treatment of patients suffering from tauopathies.

Links:

1. "Tau protein," Wikipedia, The Free Encyclopedia, website address: en.wikipedia.org/wiki/Tau_protein (Accessed September 29, 2017).

2. Fitzpatrick, AWP, et al., “Cryo-EM structures of Tau filaments from Alzheimer's disease”, Nature , 547(7662):185-190 (2017).

Brief description of the essence of the invention

The present invention relates to specific peptide immunogen constructs targeting regions of the Tau protein for the treatment and/or prevention of tauopathies. The invention also relates to compositions containing peptide immunogen constructs, methods for making and using peptide immunogen constructs and antibodies produced by peptide immunogen constructs.

Described peptide immunogen constructs contain about 15 or more amino acids. The peptide immunogen constructs contain a B-cell epitope from portions of the longest human Tau isoform (GenBank: AGF19246.1) having the amino acid sequence from SEQ ID NO: 100 shown in Table 8 . The B cell epitope can be linked to a heterologous T helper cell (Th) epitope derived from pathogen proteins via an optional heterologous spacer. The described peptide immunogen constructs stimulate the production of highly specific antibodies directed against Tau. The described peptide immunogen constructs can be used as immunotherapy for patients suffering from tauopathies.

The B-cell epitope-based portion of the peptide immunogen constructs contains the amino acid sequences from the full-length Tau protein (SEQ ID NO: 100). In some embodiments of this invention, the B-cell epitope contains a sequence containing any of SEQ ID NOs: 1-21 and 101-124, as shown in Table 1 .

The peptide immunogen constructs of the invention may contain the amino acid sequence of a heterologous Th epitope derived from a pathogenic protein (eg, SEQ ID NOs: 22-50) as shown in Table 2 . In some embodiments of the present invention, the heterologous Th epitope is derived from natural pathogens such as diphtheria toxin (SEQ ID NO: 26), Plasmodium Falciparum (SEQ ID NO: 27), cholera toxin (SEQ ID NO: 29). In other embodiments of the present invention, the heterologous Th epitope is an idealized artificial Th epitope derived from measles virus fusion protein (MVF 1-5) or hepatitis B surface antigen (HBsAg 1-3) in the form of a single sequence or combinatorial sequences ( for example, SEQ ID NOS: 33, 32 and 34).

In some embodiments of the invention, the peptide immunogen constructs comprise a Tau B cell epitope linked to a heterologous T helper cell (Th) epitope via an optional heterologous spacer. In some embodiments of the invention, the peptide immunogen constructs comprise a B cell antigenic determinant having an amino acid sequence from Tau (SEQ ID NOs: 1-21 and 101-124) linked to a heterologous Th epitope derived from a pathogenic protein (e.g., SEQ ID NO: 22-50) via an optional heterologous spacer. In some embodiments of this invention, an optional heterologous spacer is a molecule or chemical structure capable of linking two amino acids and/or peptides together. In some embodiments of this invention, the spacer is a naturally occurring amino acid, a non-naturally occurring amino acid, or a combination thereof. In specific embodiments of this invention, the peptide immunogen constructs have the amino acid sequence of SEQ ID NOs: 51-71 and 125-155 shown in Table 3 .

This invention also relates to compositions containing a Tau peptide immunogen construct. In some embodiments of the present invention, the disclosed compositions comprise more than one Tau peptide immunogen construct to encompass multiple B epitopes from Tau. In some embodiments of the present invention, the compositions comprise a mixture of Tau peptide immunogen constructs (e.g., any combination of SEQ ID NOs: 51-71 and 125-155) with more than one heterologous Th epitope derived from pathogenic proteins to cover a wide range of the genetic background of the patients. Compositions containing a mixture of peptide immunogen constructs can result in a higher percentage of positive clinical response when immunized for the treatment of tauopathy compared to compositions containing only one Th peptide immunogen construct.

This invention is also directed to pharmaceutical compositions for the treatment and/or prevention of tauopathies. In some embodiments of the invention, the pharmaceutical compositions comprise the described peptide immunogen constructs in the form of a stabilized immunostimulatory complex formed by electrostatic association by mixing a CpG oligomer with a composition containing the peptide immunogen complex. Such stabilized immunostimulatory complexes can further enhance the immunogenicity of peptide immunogen constructs. In some embodiments of this invention, the pharmaceutical compositions contain adjuvants such as mineral salts, including alum gel (ALHYDROGEL), aluminum phosphate (ADJUPHOS), or water-in-oil emulsions, including MONTANIDE ISA 51 or 720.

The present invention also relates to antibodies directed against the described Tau peptide immunogen constructs. In particular, the peptide immunogen constructs of the invention are capable of stimulating the production of highly specific antibodies that cross-react with Tau amino acid sequences (SEQ ID NOS: 1-21 and 101-124) when administered to a subject. Highly specific antibodies produced by peptide immunogen constructs cross-react with recombinant Tau-containing proteins. Used to enhance immunogenicity, the described antibodies bind to Tau with high specificity, without much specificity, and with any specificity directed to heterologous Th epitopes, in stark contrast to the conventional protein or other biological carriers used to such amplify peptide antigenicity.

The invention also includes methods for the treatment and/or prevention of tauopathies using the described peptide immunogen constructs and/or antibodies directed against the peptide immunogen constructs. In some embodiments of this invention, methods for treating and/or preventing tauopathies include administering to the host a composition containing the described peptide immunogen construct. In some embodiments of the present invention, the compositions used in the methods comprise the described peptide immunogen construct in the form of a stable immunostimulatory complex with negatively charged oligonucleotides, such as CpG oligomers, via electrostatic association, these complexes being further supplemented, optionally, with mineral salts or oil. as an adjuvant, for administration to patients with tauopathies. The methods described also include regimens, dosage forms, and methods for administering the peptide immunogen constructs to a host at risk for or having tauopathy.

BRIEF DESCRIPTION OF GRAPHICS

Fig. 1A - ELISA results expressed in Log ₁₀ titers for immune sera derived from multiple Tau immunogen constructs 6 weeks after initial injection (wpi)(npi) against non-phosphorylated Tau peptides assayed at Tau antigenic sites.

Fig. 1B - ELISA results expressed as Log ₁₀ titers for immune sera derived from multiple Tau immunogen constructs 6 weeks after the initial injection (IPI) against phosphorylated Tau peptides assayed at Tau antigenic sites.

Fig. 2 - Western blot analysis of cell lysates containing monomers, dimers, trimers and oligomers of Tau protein. Different lanes indicate IgG antibodies generated in response to the non-phosphorylated Tau peptide immunogen constructs shown above each lane (left side). These peptide immunogen constructs correspond to those shown in Table 3 . Binding profiles for each antibody were analyzed and summarized in a histogram (right side). Representative immune sera to Tau immunogen constructs demonstrated binding reactivity to Tau multispecies (monomers, dimers, trimers, oligomers and polymers).

Fig. 3A - Graphs showing the level of aggregation of full-length Tau (Tau441 amino acids) after exposure to various antibodies directed against phosphorylated Tau peptide immunogens at concentrations from 0 to 5 μg/ml are shown above each graph compared to antibodies from pre-immune sera. The level of Tau aggregation was measured by staining the aggregates with Thioflavin-T (ThT).

Fig. 3B - Histograms showing the level of Tau aggregation in vitro in the presence of 5 μg/ml antibodies directed against phosphorylated Tau peptide immunogen constructs and a methylene glue known to deaggregate Tau in vitro, used as a positive control.

Fig. 4 - Graphs showing the level of Tau aggregation after exposure to antibodies directed against either phospho- or non-phospho-Tau-peptide immunogens, as indicated above in each graph. The level of Tau aggregation was measured by staining the aggregates with Thioflavin-T (ThT). Varying degrees of inhibition have been found for antibodies directed against either phospho- or non-phospho-Tau immunogens.

Fig. 5 - Plots showing the rate of Tau disaggregation of preformed artificial Tau fibrils when exposed to antibodies directed against representative Tau peptide immunogens, either phospho or non-phospho as shown below each plot. The level of Tau aggregation was measured by staining the aggregates with Thioflavin-T (ThT). Disaggregation to varying degrees was detected with antibodies from immune sera obtained from representative phospho- or non-phospho-Tau immunogens.

Fig. 6 - Western blot analysis (under denaturing conditions) of anti-phospho-Tau and anti-non-phospho-Tau IgG produced by the Tau peptide immunogens of this invention, as shown on the left. Dot blot analysis (under non-denaturing conditions) of anti-phospho-Tau and anti-non-phospho-Tau IgG produced by typical Tau peptide immunogens of the present invention, as shown in the right inset. Antibodies raised in response to phospho-Tau peptide immunogens showed preferential binding to Tau fibrils and oligomers.

Fig. 7 - Immunohistochemical staining with representative antibodies generated by both phospho and non-phospho Tau peptide immunogens of the invention. The left inset shows binding profiles in Alzheimer's disease-specific sections compared to normal brain sections; while the right inset demonstrates that there is no cross-reactivity in normal tissues.

Fig. 8A - Tissue staining demonstrating protection of neurons from Tau fibrils with representative antibodies generated by both phospho and non-phospho-Tau peptide immunogens of the invention.

Fig. 8B Graph of the quantification of protection of neurons from Tau fibrils with representative antibodies generated by both phospho and non-phospho Tau peptide immunogens of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to single peptide immunogen constructs targeting regions of the Tau protein for the treatment and/or prevention of Alzheimer's disease and/or tauopathies. The invention also relates to compositions containing peptide immunogen constructs, methods for making and using peptide immunogen constructs and antibodies produced by peptide immunogen constructs.

Described peptide immunogen constructs contain about 15 or more amino acids. The peptide immunogen constructs contain a B-cell epitope from portions of the longest human Tau isoform (GenBank: AGF19246.1) having the amino acid sequence from SEQ ID NO: 100 shown in Table 8 . The B cell epitope can be linked to a heterologous T helper cell (Th) epitope derived from pathogen proteins via an optional heterologous spacer. The described peptide immunogen constructs stimulate the production of highly specific antibodies directed against Tau. The described peptide immunogen constructs can be used as immunotherapy for patients suffering from Alzheimer's disease and/or tauopathies.

The B-cell epitope-based portion of the peptide immunogen constructs contains the amino acid sequences from the full-length Tau protein (SEQ ID NO: 100). In some embodiments of this invention, the B-cell epitope contains a sequence containing any of SEQ ID NOs: 1-21 and 101-124, as shown in Table 1 .

The peptide immunogen constructs of the invention may contain the amino acid sequence of a heterologous Th epitope derived from a pathogenic protein (eg, SEQ ID NOs: 22-50) as shown in Table 2 . In some embodiments of the present invention, the heterologous Th epitope is derived from natural pathogens such as diphtheria toxin (SEQ ID NO: 26), Plasmodium Falciparum (SEQ ID NO: 27), cholera toxin (SEQ ID NO: 29). In other embodiments of the present invention, the heterologous Th epitope is an idealized artificial Th epitope derived from measles virus fusion protein (MVF 1-5) or hepatitis B surface antigen (HBsAg 1-3) in the form of a single sequence or combinatorial sequences ( for example, SEQ ID NOS: 33, 32 and 34).

In some embodiments of the invention, the peptide immunogen constructs comprise a Tau B cell epitope linked to a heterologous T helper cell (Th) epitope via an optional heterologous spacer. In some embodiments of the invention, the peptide immunogen constructs comprise a B cell antigenic determinant having an amino acid sequence from Tau (SEQ ID NOs: 1-21 and 101-124) linked to a heterologous Th epitope derived from a pathogenic protein (e.g., SEQ ID NO: 22-50) via an optional heterologous spacer. In some embodiments of this invention, an optional heterologous spacer is a molecule or chemical structure capable of linking two amino acids and/or peptides together. In some embodiments of this invention, the spacer is a naturally occurring amino acid, a non-naturally occurring amino acid, or a combination thereof. In specific embodiments of this invention, the peptide immunogen constructs have the amino acid sequence of SEQ ID NOs: 51-71 and 125-155 shown in Table 4 .

This invention also relates to compositions containing a Tau peptide immunogen construct. In some embodiments of the present invention, the described compositions contain more than one Tau peptide immunogen construct to cover multiple B epitopes from Tau. In some embodiments of the present invention, the compositions comprise a mixture of Tau peptide immunogen constructs (e.g., any combination of SEQ ID NOs: 51-71 and 125-155) with more than one heterologous Th epitope derived from a pathogenic protein to span a broad genetic background in patients. Compositions containing a mixture of peptide immunogen constructs can result in a higher percentage of positive clinical response when immunized for the treatment of Alzheimer's disease and/or tauopathies compared to compositions containing only one peptide immunogen construct.

This invention also relates to pharmaceutical compositions for the treatment and/or prevention of Alzheimer's disease and/or tauopathies. In some embodiments of the invention, the pharmaceutical compositions comprise the described peptide immunogen constructs in the form of a stabilized immunostimulatory complex formed by electrostatic association by mixing a CpG oligomer with a composition containing the peptide immunogen complex. Such stabilized immunostimulatory complexes can further enhance the immunogenicity of peptide immunogen constructs. In some embodiments of this invention, the pharmaceutical compositions contain adjuvants such as mineral salts, including alum gel (ALHYDROGEL), aluminum phosphate (ADJUPHOS), or water-in-oil emulsions, including MONTANIDE ISA 51 or 720.

This invention also relates to antibodies directed against the described Tau peptide immunogen constructs. In particular, the peptide immunogen constructs of the present invention are capable of stimulating the production of highly specific antibodies that cross-react with Tau amino acid sequences (SEQ ID NOS: 1-21 and 101-124) when administered to a subject. Highly specific antibodies produced by peptide immunogen constructs cross-react with recombinant Tau-containing proteins. Used to enhance immunogenicity, the described antibodies bind to Tau with high specificity, without much specificity, and with any specificity directed to heterologous Th epitopes, in stark contrast to the conventional protein or other biological carriers used to such amplify peptide antigenicity.

The invention also includes methods for the treatment and/or prevention of Alzheimer's disease and/or tauopathies using the described peptide immunogen constructs and/or antibodies directed against the peptide immunogen constructs. In some embodiments of this invention, methods for treating and/or preventing Alzheimer's disease and/or tauopathies include administering to a host a composition comprising the described peptide immunogen construct. In some embodiments of the invention, the compositions used in the methods comprise the described peptide immunogen construct in the form of a stable immunostimulatory complex with negatively charged oligonucleotides, such as CpG oligomers, via electrostatic association, optionally mineral salts or oil as an adjuvant, for administration to patients with Alzheimer's disease and/or tauopathies. The methods described also include regimens, dosage forms, and methods for administering the peptide immunogen constructs to a host at risk for or suffering from Alzheimer's disease and/or tauopathy.

The section headings used in this document are for organizational purposes only and should not be construed as limiting the subject matter of the description. All references or parts of references cited in this application are expressly incorporated herein by reference in their entirety for any purpose.

Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as generally understood by one of ordinary skill in the art to which this invention pertains. Singular forms also include plural forms, unless the context clearly dictates otherwise. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Therefore, "comprising A or B" means including A, or B, or A and B. In addition, it should be understood that all amino acid sizes and all molecular weight or molecular weight values given for polypeptides are approximate and are provided for description. While methods and materials similar or equivalent to those described herein may be used in the practice or testing of the described method, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in this document are incorporated by reference in their entirety. In the event of a conflict, this specification, including explanations of terms, shall prevail. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Tau peptide immunogen construct

The present invention relates to peptide immunogen constructs comprising a B cell epitope with an amino acid sequence from Tau covalently linked to a heterologous T helper cell (Th) epitope either directly or through an optional heterologous spacer.

The phrase "Tau peptide immunogen construct" or "peptide immunogen construct" as used herein refers to a peptide containing (a) a B cell epitope having about 15 or more amino acid residues from the full-length sequence of the longest Tau isoform (SEQ ID NO: 100); (b) a heterologous Th epitope; and (c) an optional heterologous spacer.

In some embodiments of this invention, the Tau peptide immunogen construct may be represented by the formulas:

(Th) _m -(A) _n -(Tau fragment)-X

or

(fragment Tau)-(A) _n -(Th) _m -X

Where

Th is a heterologous T-helper epitope;

A is a heterologous spacer;

(fragment Tau) is a B-cell epitope having about 15 to about 40 amino acid residues from SEQ ID NO: 100;

X is α-COOH or α-CONH ₂ amino acids;

m is from 1 to about 4; And

n is from 0 to about 10.

The various components of the described Tau peptide immunogen construct are described below.

A. Fragments of Tau

Described peptide immunogen constructs contain about 15 or more amino acids. The peptide immunogen constructs contain a B-cell epitope from portions of the longest human Tau isoform (GenBank: AGF19246.1) having the amino acid sequence from SEQ ID NO: 100 shown in Table 8 . The B cell epitope can be linked to a heterologous T helper cell (Th) epitope derived from pathogen proteins via an optional heterologous spacer. The described peptide immunogen constructs stimulate the production of highly specific antibodies directed against Tau. The described peptide immunogen constructs can be used as immunotherapy for patients suffering from Alzheimer's disease and/or tauopathies.

The B-cell epitope-based portion of the peptide immunogen constructs contains the amino acid sequences from the full-length Tau protein (SEQ ID NO: 100). In some embodiments of this invention, the B-cell epitope contains a sequence containing any of SEQ ID NOs: 1-21 and 101-124, as shown in Table 1 .

In some embodiments of this invention, the Tau fragments contain non-phosphorylated amino acids (eg, SEQ ID NOs: 2, 4, 6, 8, 11, 13, 15, 17, 19, and 21) as shown in Table 1 . In other embodiments of the present invention, the Tau fragments contain phosphorylated serine and/or threonine amino acids (e.g., SEQ ID NOs: 1, 3, 5, 7, 9, 10, 12, 14, 16, 18, and 20) as demonstrated in Table 1 . The Tau fragments as well as the phosphorylation sites shown in the peptides of Table 1 are illustrative and the invention includes any other fragment and/or phosphorylation site of the full length Tau protein of SEQ ID NO: 100.

b. Heterologous T helper epitopes (Th epitopes)

The present invention relates to peptide immunogen constructs comprising a B cell epitope from Tau covalently linked to a heterologous T helper cell (Th) epitope either directly or via an optional heterologous spacer.

The heterologous Th epitope in the Tau peptide immunogen construct enhances the immunogenicity of Tau fragments, which facilitates the production of high titer specific antibodies directed against the optimized target B cell epitope (ie, Tau fragment) through rational design.

The term "heterologous" as used herein refers to an amino acid sequence that is derived from an amino acid sequence that is not part of or homologous to the wild-type Tau sequence. Thus, a heterologous Th epitope is a Th epitope derived from an amino acid sequence that is not naturally found in Tau (ie, the Th epitope is not autologous for Tau). Because the Th epitope is heterologous to Tau, the natural amino acid sequence of Tau is not extended in either the N-terminal or C-terminal directions when the heterologous Th epitope is covalently linked to the Tau moiety.

The heterologous Th epitope of this invention may be any Th epitope that does not contain the amino acid sequence found naturally in Tau. The Th epitope may have an amino acid sequence derived from any species (eg, human, pig, bovine, dog, rat, mouse, guinea pig, etc.). The Th epitope can also have heterogeneous binding motifs to MHC class II molecules of several species. In some embodiments of the invention, the Th epitope contains a plurality of heterogeneous MHC class II binding motifs to maximize T helper cell activation leading to the initiation and regulation of immune responses. The Th epitope is preferably immunosilent in itself, i.e., few, if any, antibodies generated by the Tau peptide immunogen constructs will be directed towards the Th epitope, thus providing a highly focused immune response directed at the B cell epitope. -Tau fragment target.

The epitopes of this invention include, but are not limited to, amino acid sequences derived from foreign pathogens as shown in Table 2 (SEQ ID NO: 22-50). In addition, Th epitopes include idealized artificial Th epitopes and combinatorial idealized artificial Th epitopes (eg, SEQ ID NOS: 23 and 30-36). Heterologous Th epitope peptides are presented as a combinatorial sequence (eg, SEQ ID NOs: 31-34), contain a mixture of amino acid residues presented at specific positions within the peptide structure based on the homologue variable residues for that particular peptide. An assembly of combinatorial peptides can be synthesized in one process by adding a mixture of these protected amino acids instead of one specific amino acid at a specific position during the synthesis. Such combinatorial peptides of heterologous Th epitopes can provide a broad coverage of Th epitopes for animals with diverse genetic backgrounds. Exemplary combinatorial peptide sequences for heterologous Th epitopes include SEQ ID NOs: 31-34, which are shown in Table 2 . The Th epitope peptides of this invention provide broad reactivity and immunogenicity in animals and patients from genetically diverse populations.

Tau peptide immunogen constructs containing Th epitopes are produced simultaneously in a single solid phase peptide synthesis in tandem with a Tau moiety. Th epitopes also include immunological analogues of Th epitopes. Th immunological analogs include immunomodulatory analogs, cross-reactive analogs, and segments of any of these Th epitopes that are sufficient to enhance or stimulate an immune response to Tau fragments.

Functional immunological analogues of the Th epitope peptides are also effective and are included as part of this invention. Functional immunological analogs of Th may include conservative substitutions, additions, deletions, and insertions of one to about five amino acid residues in a Th epitope that do not substantially modify the Th-stimulatory function of the Th epitope. Conservative substitutions, additions and insertions can be made with natural or non-natural amino acids as described above for Tau fragments. Table 2 identifies another functional analog variant for the Th epitope peptide. In particular, SEQ ID NOs: 23 and 30 MvF1 and MvF2 Th are functional analogs of SEQ ID NOs: 33 and 35 MvF4 and MvF5 in that they differ in the amino acid framework by deletion (SEQ ID NOs: 23 and 30) or insertion (SEQ ID NO: 33 and 35) two amino acids, each at the N- and C-termini. Differences between these two series of similar sequences do not affect the function of the Th epitopes contained within these sequences. Therefore, functional immunological analogs of Th include several versions of the Th epitope derived from the MvF1-4 Th measles virus fusion protein (SEQ ID NOs: 23, 30, 31, 33 and 35) and from the hepatitis HBsAg 1-3 Th surface protein (SEQ ID NOs: 32, 34 and 36).

The Th epitope in the Tau peptide immunogen construct may be covalently linked to the N- or C-terminus of the Tau peptide. In some embodiments of this invention, the Th epitope is covalently linked to the N-terminal end of the Tau peptide. In some embodiments of this invention, the Th epitope is covalently linked to the C-terminal end of the Tau peptide. In some embodiments of the invention, more than one Th epitope is covalently linked to the Tau moiety. When more than one Th epitope is associated with a Tau fragment, each Th epitope may have the same amino acid sequence or different amino acid sequences. In addition, when more than one Th epitope is associated with a Tau fragment, the Th epitopes may be in any order. For example, Th epitopes may be sequentially linked to the N-terminal end of a Tau fragment, or may be sequentially linked to the C-terminal end of a Tau fragment, or a Th epitope may be covalently linked to the N-terminal end of a Tau fragment, while a single Th- the epitope is covalently linked to the C-terminal end of the Tau fragment. There are no restrictions on the location of the Th epitopes with respect to the Tau fragment.

In some embodiments of the present invention, the Th epitope is covalently linked to the Tau moiety directly. In other embodiments of the present invention, the Th epitope is covalently linked to the Tau moiety via a heterologous spacer, described in more detail below.

With. Heterologous spacer

The described Tau peptide immunogen constructs optionally comprise a heterologous spacer that covalently links a B cell epitope from Tau to a heterologous T helper cell (Th) epitope.

As discussed above, the term "heterologous" refers to an amino acid sequence that is derived from an amino acid sequence that is not part of or homologous to the wild-type Tau sequence. Thus, the native amino acid sequence of Tau does not extend in either the N-terminal or C-terminal directions when the heterologous spacer is covalently linked to a B-cell epitope from Tau, since the spacer is heterologous to the Tau sequence.

A spacer is any molecule or chemical structure capable of linking two amino acids and/or peptides together. The spacer may vary in length or polarity depending on the application. The attachment of the spacer can be via an amide or carboxyl bond, but other functionalities are possible. The spacer may include a chemical compound, a naturally occurring amino acid, or a non-naturally occurring amino acid.

The spacer may provide structural features for the Tau peptide immunogen construct. Structurally, the spacer provides a physical separation of the Th-epitope from the B-cell epitope of the Tau fragment. Physical separation with a spacer can disrupt any artificial secondary structures created by attaching a Th epitope to a B cell epitope. In addition, physical separation of epitopes by a spacer can eliminate interferences between Th-cell and/or B-cell responses. In addition, the spacer may be designed to create or modify the secondary structure of the peptide immunogen construct. For example, a spacer can be designed to act as a flexible hinge to enhance the separation of a Th epitope and a B cell epitope. The flexible hinge spacer can also allow more efficient interactions between the presented peptide immunogen and the corresponding Th and B cells to enhance immune responses to the Th epitope and B cell epitope. Examples of sequences encoding flexible hinges are found in the immunoglobulin heavy chain hinge region, which are often rich in proline. 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: 101), where Xaa is any amino acid and preferably aspartic acid.

The spacer may also provide functional features to the Tau peptide immunogen construct. For example, the spacer may be designed to change the overall charge of the Tau peptide immunogen construct, which may affect the solubility of the peptide immunogen construct. In addition, changing the overall charge of the Tau peptide immunogen construct can affect the ability of the peptide immunogen construct to bind to other compounds and reagents. As discussed in more detail below, the Tau peptide immunogen construct can be formed into a stable immunostimulatory complex with a highly charged oligonucleotide, such as CpG oligomers, via electrostatic association. The overall charge of the Tau peptide immunogen construct is important for the formation of these stable immunostimulatory complexes.

Chemical compounds that can be used as a spacer 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-tetraoxapenta-decanoic acid (mini-PEG3 ), trioxatridecan-succinic acid (Ttds), 12-amino-dodecanoic acid, Fmoc-5-amino-3-oxapentanoic acid (O1Pen), and the like.

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.

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

The spacer in the Tau peptide immunogen construct may be covalently linked at the N- or C-terminal end of the Th epitope and the Tau peptide. In some embodiments of this invention, the spacer is covalently linked to the C-terminal end of the Th epitope and to the N-terminal end of the Tau peptide. In other embodiments of this invention, the spacer is covalently linked to the C-terminal end of the Tau peptide and to the N-terminal end of the Th epitope. In certain embodiments of the present invention, more than one spacer may be used, for example, when more than one Th epitope is present in the peptide immunogen construct. When more than one spacer is used, each spacer may be the same as or different from the other. In addition, when more than one Th epitope is present in the peptide immunogen construct, the Th epitopes may be separated by a spacer that may be the same or different from the spacer used to separate the Th epitope from the B cell epitope. There are no restrictions on the location of the spacer relative to the Th epitope or Tau fragment.

In some embodiments of this invention, the heterologous spacer is a naturally occurring amino acid or a non-naturally occurring amino acid. In other embodiments of this invention, the spacer contains more than one naturally occurring or non-naturally occurring amino acid. In specific embodiments of this invention, the spacer is a Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 102) .

d. Specific embodiments of the Tau peptide immunogen construct

The tau peptide immunogen construct can be represented by the formulas:

(Th) _m -(A) _n -(Tau fragment)-X

or

(fragment Tau)-(A) _n -(Th) _m -X

Where

Th is a heterologous T-helper epitope;

A is a heterologous spacer;

(fragment Tau) is a B-cell epitope having from about 15 to about 40 amino acid residues from the full-length Tau protein of SEQ ID NO: 100;

X is α-COOH or α-CONH ₂ amino acids;

m is from 1 to about 4; And

n is from 0 to about 10.

In some embodiments of the invention, the heterologous Th epitope in the Tau peptide immunogen construct comprises an amino acid sequence selected from any of SEQ ID NOs: 51-71 and 125-155, and combinations thereof, shown in Table 3 . In specific embodiments of this invention, the Th epitope contains an amino acid sequence selected from any of SEQ ID NOs: 30-36. In some embodiments of the invention, the Tau peptide immunogen construct contains more than one Th epitope.

In some embodiments of this invention, the optional heterologous spacer is selected from any of Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 102) and combinations thereof. In specific embodiments of this invention, the heterologous spacer is ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 102).

In some embodiments of this invention, the Tau fragment contains from about 15 to about 40 amino acid residues from the full-length Tau protein of SEQ ID NO: 100. In specific embodiments of this invention, the Tau fragment contains the amino acid sequence shown as SEQ ID NO: 1- 21 and 101-124 as shown in Table 1 .

In some embodiments of the invention, the Tau peptide immunogen construct comprises an amino acid sequence selected from any of SEQ ID NOs: 51-71 and 125-155 as shown in Table 3 .

A. Variants, homologues and functional analogues

Variants and analogs of the above immunogenic peptides that induce and/or cross-react with antibodies to preferred Tau epitopes can also be used. Analogues, including allelic, species, and induced variants, typically differ from naturally occurring peptides at one, two, or more positions, often due to conservative substitutions. Analogues typically show at least 80% or 90% sequence identity with natural peptides. Some analogs also include non-natural amino acids or modifications of the N- or C-terminal amino acids at one, two or more positions.

Variants that are functional analogs may have a conservative substitution at the amino acid position; change in total charge; a covalent bond to another fragment; or additions, insertions or deletions of amino acids; and/or any combination thereof.

Conservative substitutions are when one amino acid residue is replaced by another amino acid residue with similar chemical properties. For example, non-polar (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.

In a specific embodiment of this invention, the functional analog has at least 50% identity with the original amino acid sequence. In another embodiment of the present invention, the functional analog has at least 80% identity with the original amino acid sequence. In yet another embodiment of the present invention, the functional analog has at least 85% identity with the original amino acid sequence. In yet another embodiment of the present invention, the functional analog has at least 90% identity with the original amino acid sequence.

Variants also include variations in phosphorylated residues. For example, variants may include various residues within peptides that are phosphorylated. The immunogenic Tau peptide variant may also include pseudophosphorylated peptides. Pseudophosphorylated peptides are prepared by replacing one or more of the phosphorylated serine, threonine and tyrosine residues of Tau peptides with acidic amino acid residues such as glutamic acid and aspartic acid.

Compositions

This invention also relates to compositions containing the described immunogenic Tau construct.

A. Peptide compositions

Compositions containing the described Tau peptide immunogen construct may be in liquid or solid form. Liquid compositions may contain water, buffers, diluents, salts, and/or any other suitable reagent that does not alter the structural or functional properties of the Tau peptide immunogen construct. The peptide compositions may contain one or more of the Tau peptide immunogen constructs described.

b. Pharmaceutical compositions

This invention also relates to pharmaceutical compositions containing the described Tau peptide immunogen construct.

Pharmaceutical compositions may contain carriers and/or other additives in a pharmaceutically acceptable delivery system. Accordingly, the pharmaceutical compositions may contain a pharmaceutically effective amount of the Tau peptide immunogen construct together with a pharmaceutically acceptable carrier, adjuvant and/or other excipients such as diluents, additives, stabilizing agents, preservatives, solubilizing agents, buffers, and the like.

Pharmaceutical compositions may contain one or more adjuvants that act to accelerate, prolong or enhance the immune response to the Tau peptide immunogen construct without any specific antigenic effect. Adjuvants used in the pharmaceutical composition may include oils, aluminum salts, virosomes, aluminum phosphate (eg ADJU-PHOS®), aluminum hydroxide (eg ALHYDROGEL®), liposin, saponin, squalene, L121, Emulsigen®, monophosphoryl lipid A (MPL), QS21, ISA 35, ISA 206, ISA50V, ISA51, ISA 720, as well as other adjuvants and emulsifiers.

In some embodiments of this invention, the pharmaceutical composition contains MONTANIDE™ ISA 51 (an oil adjuvant composition consisting of vegetable oil and oleate mannide for water-in-oil emulsions), Tween® 80 (also known as: Polysorbate 80 or Polyoxyethylene (20 ) sorbitan monooleate), CpG oligonucleotide and/or any combination thereof. In other embodiments of this invention, the pharmaceutical composition is a water-in-oil-in-water (i.e. w/o/w) emulsion with Emulsigen or Emulsigen D as an adjuvant.

Pharmaceutical compositions may be formulated as immediate release formulations or as sustained release formulations. In addition, pharmaceutical compositions can be formulated to induce systemic or localized mucosal immunity by uptake of the immunogen and co-administration with microparticles. Such delivery systems are readily identified by one of ordinary skill in the art.

Pharmaceutical compositions may be prepared as injectables, either as liquid solutions or suspensions. Liquid carriers containing the Tau peptide immunogen construct can also be prepared prior to injection. The pharmaceutical composition may be administered by any suitable route of administration, eg, s.c., iv, p.o., IM, intranasal, oral, subcutaneous, etc., and in any suitable delivery device. In certain embodiments of this option, the pharmaceutical composition is formulated for intravenous, subcutaneous, intradermal or intramuscular administration. Pharmaceutical compositions suitable for other routes of administration may also be prepared, including oral and intranasal administration.

Pharmaceutical compositions may be formulated as immediate release formulations or as sustained release formulations. In addition, pharmaceutical compositions can be formulated to induce systemic or localized mucosal immunity by uptake of the immunogen and co-administration with microparticles. Such delivery systems are readily identified by one of ordinary skill in the art.

Pharmaceutical compositions may also be formulated in suitable unit dosage form. In some embodiments of this invention, the pharmaceutical composition contains from about 0.5 μg to about 1 mg of Tau peptide immunogen constructs per kg of body weight. Effective doses of pharmaceutical compositions vary depending on many different factors, including routes of administration, target site, physiological state of the patient, whether the patient is human or animal, other drugs being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated. When delivered in multiple doses, the pharmaceutical compositions may conveniently be divided into suitable amounts into unit dosage form. The dose administered will depend on the age, weight and general health of the subject, as is well known in the art of therapy.

In some embodiments of this invention, the pharmaceutical composition contains more than one Tau peptide immunogen construct. A pharmaceutical composition containing a mixture of more than one Tau-peptide immunogenic construct to provide a synergistic enhancement of the immunogenicity of the constructs. Pharmaceutical compositions containing more than one Tau peptide immunogen construct may be more effective in a larger genetic population due to the broad coverage of MHC class II, thus providing an improved immune response to Tau peptide immunogen constructs.

In some embodiments of this invention, the pharmaceutical composition comprises a Tau peptide immunogen construct selected from SEQ ID NOs: 51-71 and 125-155, as well as their homologues, analogs and/or combinations. In particular embodiments of this invention, the pharmaceutical compositions comprise a Tau peptide immunogen construct selected from SEQ ID NOs: 51-71 and 125-155, and any combination thereof.

Pharmaceutical compositions containing a Tau peptide immunogen construct can be used to induce an immune response and produce antibodies in a host after administration.

With. Immunostimulating complexes

This invention also relates to pharmaceutical compositions containing a Tau peptide immunogen construct in the form of an immunostimulatory complex with a CpG oligonucleotide. Such immunostimulatory complexes are specifically adapted to act as an adjuvant and stabilizer of the peptide immunogen. The immunostimulatory complexes are in the form of particles that can effectively present the Tau peptide immunogen to the cells of the immune system, causing an immune response. Immunostimulatory complexes can be formulated as a suspension for parenteral administration. Immunostimulatory complexes can also be formulated as IM emulsions, as a suspension in combination with a mineral salt, or with a gelling polymer in situ to effectively deliver the Tau peptide immunogen to host immune cells after parenteral administration.

A stabilized immunostimulatory complex can be formed by complexing a Tau peptide immunogen construct with an anionic molecule, oligonucleotide, polynucleotide, or combinations thereof via electrostatic association. The stabilized immunostimulatory complex may be included in a pharmaceutical composition as an immunogen delivery system.

In some embodiments of this invention, the Tau peptide immunogen construct is designed to contain a cationic moiety that is positively charged at a pH in the range of 5.0 to 8.0. The total charge of the cationic portion of a Tau peptide immunogen construct or mixture of constructs is calculated by assigning a +1 charge to each lysine (K), arginine (R) or histidine (H), a -1 charge to each aspartic acid (D) or glutamic acid (E ) and a charge of 0 for another amino acid within the sequence. The charges are summed within the cationic portion of the Tau peptide immunogen construct and are expressed as the total average charge. A suitable peptide immunogen contains a cationic moiety with an overall average positive charge of +1. Preferably the peptide immunogen has an overall positive charge in the range greater than +2. In some embodiments of this invention, the cationic portion of the Tau peptide immunogen construct is a heterologous spacer. In some embodiments of the present invention, the cationic portion of the Tau peptide immunogen construct has a +4 charge when the spacer sequence is (α, ε-N)Lys, ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 102 ).

"Anionic molecule", as described herein, refers to any molecule that is negatively charged at a pH in the range of 5.0-8.0. In certain embodiments of this invention, the anionic molecule is an oligomer or polymer. The overall negative charge on an oligomer or polymer is calculated by assigning a charge of -1 to each phosphodiester or phosphorothioate group in the oligomer. A suitable anionic oligonucleotide is a single stranded DNA molecule of 8-64 nucleotide bases with a CpG motif repeat number ranging from 1 to 10. Preferably CpG immunostimulatory single-stranded DNA molecules contain 18-48 nucleotide bases with a CpG motif repeat number ranging from 3 to 8.

More preferably the anionic oligonucleotide is represented by the formula: 5'X ¹ CGX ² 3', where C and G are unmethylated; and X ¹ is selected from the group consisting of A (adenine), G (guanine) and T (thymine); and X ² is C (cytosine) or T (thymine). Or an anionic oligonucleotide is represented by the formula: 5'(X ³ ) ₂ CG(X ⁴ ) ₂ 3', where C and G are unmethylated; and X ³ is selected from the group consisting of A, T or G; and X ⁴ is C or T.

The resulting immunostimulatory complex is in the form of particles with a size typically in the range of 1-50 microns and is a function of many factors including the relative charge stoichiometry and the molecular weight of the interacting particles. The particulate immunostimulatory complex has the advantage of providing an adjuvant effect and activation of specific immune responses in vivo. In addition, the stabilized immunostimulatory complex is suitable for preparing pharmaceutical compositions in a variety of ways, including water-in-oil emulsions, suspensions of mineral salts, and polymer gels.

Antibodies

The invention also relates to antibodies generated in response to a Tau peptide immunogen construct.

The disclosed Tau peptide immunogen constructs containing a Tau fragment, a heterologous Th epitope, and an optional heterologous spacer are capable of inducing an immune response and antibody production when administered to a host. The design of Tau peptide immunogen constructs can break tolerance to Tau itself and induce the production of site-specific antibodies that recognize conformational rather than linear epitopes.

Antibodies produced by the Tau peptide immunogen constructs recognize and bind to Tau in the form of monomers, dimers, trimers and oligomers.

Antibodies formed in response to Tau peptide immunogen constructs (anti-aggregation activity) and can dissociate previously formed Tau aggregates (de-aggregation activity).

The resulting immune responses from animals immunized with the Tau peptide immunogen constructs of this invention demonstrated the ability of the constructs to produce potent site-directed antibodies that react with Tau in the form of monomers, dimers, trimers and oligomers.

Ways

The present invention is also directed to methods for the preparation and use of Tau peptide immunogen constructs, compositions and pharmaceutical compositions.

A. Methods for making a Tau peptide immunogen construct

The tau peptide immunogen constructs of this disclosure may be prepared by chemical synthesis methods well known to those skilled in the art (see, e.g., Fields et al., Chapter 3 in Synthetic Peptides: A User's Guide, ed. Grant, WH Freeman & Co. ., New York, New York, 1992, p. 77). Tau peptide immunogen constructs can be synthesized using Merrifield's automated solid phase synthesis methods with α-NH ₂ protected by t-Boc or F-moc chemistry using side chain amino acids, for example, on an Applied Biosystems 430A or 431 peptide synthesizer model. The preparation of Tau peptide immunogen constructs containing combinatorial library peptides for Th epitopes can be achieved by providing a mixture of alternative amino acids for binding at a given variable position.

Once the desired Tau peptide immunogen construct is fully assembled, the resin can be processed according to standard procedures to cleave the peptide from the resin and the functional groups on the amino acid side chains can be deblocked. 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 of ordinary skill in the art.

The quality of the peptides produced by this chemical process can be controlled and determined, and as a result, the reproducibility of Tau peptide immunogen constructs, immunogenicity and yield can be guaranteed. A detailed description of the production of a Tau peptide immunogen construct by solid phase peptide synthesis is provided in Example 1 .

It has been found that the range of structural variability that allows retention of intended immunological activity is much more acceptable than the range of structural variability allowed for retention of specific drug activity by a small molecule drug, or the desired activities and undesirable toxicities found in large molecules that produced in conjunction with biologically derived drugs. Thus, peptide analogs, either intentionally designed or inevitably resulting from synthetic process errors as a mixture of sequence deletion by-products, that have similar chromatographic and immunological properties to the intended peptide, are often as effective as a purified preparation of the desired peptide. Designed analogs and unintentional analog blends are effective as long as a clear quality control procedure is developed to control both the manufacturing process and the product evaluation process to ensure the reproducibility and potency of the final product using these peptides.

Tau peptide immunogen constructs can also be produced using recombinant DNA technology, including nucleic acid molecules, vectors and/or host cells. As such, nucleic acid molecules encoding the Tau peptide immunogen construct and their immunologically functional counterparts are also included in this invention as part of this invention. Similarly, vectors, including expression vectors containing nucleic acid molecules, as well as host cells containing vectors, are also included in this invention as part of this invention.

Various exemplary embodiments of the present invention also encompass methods for preparing a Tau peptide immunogen construct and its immunologically functional analogs. For example, the methods may include the step of incubating a host cell containing an expression vector containing a nucleic acid molecule encoding a Tau peptide immunogen construct and/or an immunologically functional analogue thereof, under conditions where the peptide and/or analogue is expressed. Longer synthetic peptide immunogens can be synthesized by well known recombinant DNA techniques. Such methods are presented 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 produce a nucleic acid sequence encoding the amino acid sequence, preferably with codons that are optimal for the organism in which the gene is to be expressed. The synthetic gene is then obtained, usually by synthesizing oligonucleotides that encode the peptide and any regulatory elements, if necessary. The synthetic gene is inserted into a suitable cloning vector and transfected into a host cell. The peptide is then expressed under suitable conditions suitable for the chosen expression system and host. The peptide is purified and characterized by standard methods.

b. Methods for the manufacture of immunostimulating complexes

Various exemplary embodiments of the present invention also cover methods for preparing immunostimulatory complexes comprising Tau peptide immunogen constructs and a CpG oligodeoxynucleotide (ODN) molecule (oligodeoxynucleotide). Stabilized immunostimulatory complexes (ISCs) are derived from the cationic portion of the Tau peptide immunogen construct and the polyanionic CpG ODN molecule. The self-assembling system is powered by electrostatic charge neutralization. The stoichiometry of the molar charge ratio of the cationic moiety of the Tau peptide immunogen construct to the anionic oligomer determines the degree of association. The non-covalent electrostatic association of the Tau peptide immunogen construct and the CpG ODN is a completely reproducible process. Aggregates of the immunostimulatory peptide/CpG ODN complex, which facilitate presentation to "professional" antigen-presenting cells (APCs) of the immune system, thus further enhancing the immunogenicity of the complexes. These complexes are easily characterized for quality control during production. The ISC peptide/CpG is well tolerated in vivo. This new particle system containing CpG ODNs and Tau-peptide immunogen constructs derived from a Tau fragment was designed to exploit the generalized B-cell mitogenicity associated with the use of CpG ODNs while at the same time promoting balanced Th-1 type responses. Th-2.

The CpG ODN in the disclosed pharmaceutical compositions is 100% bound to the immunogen in a process mediated by electrostatic neutralization of the opposite charge, resulting in the formation of micron sized particles. The particle shape allows for a significant reduction in CpG dosage compared to conventional use of CpG adjuvants, reduces the likelihood of adverse innate immune responses, and facilitates alternative immunogen processing pathways, including antigen presenting cells (APCs). Therefore, such formulations are conceptually novel and offer potential benefits by promoting stimulation of immune responses by alternative mechanisms.

With. Methods for making pharmaceutical compositions

Various exemplary embodiments of the present invention also encompass pharmaceutical compositions containing Tau peptide immunogen constructs. In some embodiments of this invention, water-in-oil emulsions and suspensions with mineral salts are used in pharmaceutical compositions.

In order for a pharmaceutical composition to be used by a large population and the prevention of Tau aggregation is also part of the purpose of administration, safety becomes another important factor to consider. Despite the use of water-in-oil emulsions in humans for many formulations in clinical trials, alum remains the main adjuvant for formulation use due to its safety. Therefore, alum or its mineral aluminum phosphate salts (ADJUPHOS) are often used as adjuvants in preparation for clinical use.

Other adjuvants and immunostimulatory agents include 3-De-O-acylated monophosphoryl lipid A (MPL) or 3-DMP, polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Such adjuvants may be used with or without other specific immunostimulatory agents such as muramyl peptides (e.g. N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetylnormuramyl-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-acetylgluxaminyl- N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) Theramide™) or other components of the bacterial cell wall. Oil-in-water emulsions include MF59 (see WO 90/14837, Van Nest et al., which is hereby incorporated by reference in its entirety) containing 5% squalene, 0.5% Tween 80 and 0, 5% Span 85 (optionally containing varying amounts of MTP-PE) formulated into submicron particles using a microfluidizer; SAF containing 10% squalene, 0.4% Tween 80, 5% pluronic-blocked polymer L121 and thr-MDP, either microfluidized into a submicron emulsion or vortexed to form a coarser particle size emulsion; and Ribi™ Adjuvant System (RAS) (Ribi ImmunoChem, Hamilton, Montana) containing 2% squalene, 0.2% Tween 80, and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (Detox™). Other adjuvants include complete Freund's Adjuvant (CFA) (Complete Freund's Adjuvant), incomplete Freund's Adjuvant (IFA) (Incomplete Freund's Adjuvant) and cytokines such as interleukins (IL-1, IL-2 and IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF) (granulocyte-macrophage colony stimulating factor), and tumor necrosis factor (TNF).

The choice of adjuvant depends on the stability of the immunogenic formulation containing the adjuvant, route of administration, schedule of use, effectiveness of the adjuvant in the species being vaccinated, and in humans, a pharmaceutically acceptable adjuvant is one that has been approved or approved for human administration by the appropriate regulatory authorities. 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 all combinations thereof are suitable for administration. to a person.

The compositions may contain pharmaceutically acceptable, non-toxic carriers or diluents, which are defined as carriers commonly used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is chosen so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological sodium phosphate buffer, Ringer's solutions, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or composition may also contain other carriers, adjuvants, or non-toxic, non-therapeutic, non-immunogenic stabilizers, and the like.

Pharmaceutical compositions may also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids, and copolymers (e.g., latex-functionalized Sepharose, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (eg oil drops or liposomes). In addition, these carriers can function as immunostimulatory agents (ie, adjuvants).

The pharmaceutical compositions of this invention may further include a suitable delivery vehicle. Suitable delivery vehicles include, but are not limited to, viruses, bacteria, biodegradable microspheres, microparticles, nanoparticles, liposomes, collagen minipellets, and cochleates.

d. Methods Using Pharmaceutical Compositions

This invention also includes methods of using pharmaceutical compositions containing Tau peptide immunogen constructs.

In some embodiments of this invention, pharmaceutical compositions containing Tau peptide immunogen constructs can be used to:

(a) inhibition of Tau aggregation in the host;

(b) inducing disaggregation of preformed Tau aggregates in the host;

(c) reducing neurodegeneration caused by exogenous Tau aggregates in the host;

(d) reducing neurodegeneration in cells overexpressing Tau;

(e) reducing the level of Tau in the serum of the host;

(f) reducing the level of oligomeric Tau in the host brain;

(g) reducing neuropathology and restoring motor activity in the host; etc.

The above methods include administering a pharmaceutical composition containing a pharmacologically effective amount of a Tau peptide immunogen construct to a host in need thereof.

As used herein, the term "tauopathy" encompasses any neurodegenerative disease that involves abnormal aggregation of Tau protein microtubules within the brain. Accordingly, in addition to both familial and sporadic Alzheimer's disease, other tauopathies that can be treated using the methods of this invention include, without limitation, frontotemporal dementia, chromosome 17-associated parkinsonism (FTDP-17), progressive supranuclear palsy , corticobasal degeneration, Pick's disease, progressive subcortical gliosis, dementia with a predominance of neurofibrillary tangles, diffuse neurofibrillary tangles with calcification, argyrophilic granular dementia, parkinsonism-dementia amyotrophic lateral sclerosis complex, boxer's dementia, Down syndrome, Gerstmann-Strausslen-Schleinker disease, hallervorden disease -Shpatza, myositis with Taurus turned on, Creytzfeld-Jacob disease, multiple system atropy, nimann-pike type C, cerebral amyloid angiopathy of Priona protein, subacute panerencephalitis, myotonic dystrophy, Negouine neurofibrillar disease, and post-Encapalitic pouns kinsoonism and chronic traumatic encephalopathy.

Another aspect of this invention relates to a method of promoting the clearance of Tau aggregates from the brain of a subject. The method comprises administering to a subject any one or more immunogenic Tau peptides having an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-21 and 101-124, or one or more antibodies recognizing an immunogenic Tau epitope comprising the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-21 and 101-124 and 51-71 and 125-155 under conditions effective to accelerate the clearance of Tau aggregates from the subject's brain.

The clearance of Tau aggregates includes the 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 granular dementia, boxer's dementia, chronic traumatic encephalopathy, diffuse neurofibrillary tangles with calcification, Down's syndrome, Gerstmann-Straussler-Scheinker's disease, Hallervorden-Spatz, hereditary frontotemporal dementia, chromosome 17-associated parkinsonism (FTDP-17), inclusion body myositis, Creutsfeldt-Jakob disease, multiple system atrophy, Niemann-Pick type C disease, Pick's disease, cerebral amyloid protein angiopathy prions, sporadic corticobasal degeneration, progressive supranuclear palsy, subacute sclerosing panencephalitis, myotonic dystrophy, motor neuron disease with neurofibrillary tangles, dementia with a predominance of neurofibrillary tangles, and progressive subcortical gliosis.

Another aspect of the present invention relates to a method for slowing down the development of a behavioral phenotype associated with Tau pathology in a subject. The method comprises administering to a subject any one or more immunogenic Tau peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-21 and 101-124 and 51-71 and 125-155, or one or more antibodies, recognizing an immunogenic Tau epitope comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-21 and 101-124 and 51-71 and 125-155, under conditions effective to slow down the behavioral phenotype associated with Tau pathology, at the subject.

As used herein, the behavioral phenotype associated with Tau pathology includes, without limitation, cognitive impairment, early personality change and disinhibition, apathy, aboulia, mutism, apraxia, perseveration, stereotypic movements/behaviors, hyperoralism, disorganization, inability to plan or organize tasks sequentially, selfishness/callingness, antisocial traits, lack of empathy, stammering, illiterate speech with frequent paraphasic errors but relatively retained comprehension, impaired comprehension and deficits in word search, slowly progressive gait instability, retropulsation, stiffness, frequent falls, not levodopa-responsive axial rigidity, supranuclear gaze palsy, quadrate oscillations, slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia, cortical sensory loss, and tremor.

According to the methods of the invention, in one embodiment of the invention, an immunogenic Tau peptide or a combination of immunogenic Tau peptides is administered to a subject in need. Suitable immunogenic Tau peptide fragments of the Tau protein contain one or more antigenic epitopes that mimic the pathological form of the Tau protein. Exemplary Tau immunogenic epitopes are phosphorylated at one or more amino acids that are phosphorylated in the pathological form of Tau but not phosphorylated in the normal or non-pathological form of Tau.

In some embodiments of the present invention, the administration of the immunogenic Tau peptide induces an active immune response in the subject to the immunogenic Tau peptide and to the pathological form of Tau, thereby facilitating the clearance of associated Tau aggregates, slowing the progression of behavior associated with Tau pathology, and treating the underlying tauopathy. In accordance with this aspect of the present invention, the immune response includes the development of an effective humoral (mediated by antibodies) and/or cellular (mediated by antigen-specific T cells or their secretion products) response directed against the immunogenic Tau peptide.

The presence of a humoral immunological response can be determined and monitored by testing a biological sample (eg, blood, plasma, serum, urine, saliva, CSF, or lymphatic fluid) from the subject for the presence of antibodies directed to the immunogenic Tau peptide. Methods for detecting antibodies in a biological sample are well known in the art, such as ELISA, dot blotting, SDS-PAGE gels, or ELISPOT. The presence of a cell-mediated immunological response can be determined using proliferation assays (CD4+ T cells) or CTL (cytotoxic T lymphocytes) (cytotoxic T lymphocyte), which are well known in the art.

The isolated immunogenic Tau peptides of this invention comprise any one of the amino acid sequences from SEQ ID NOs: 1-21 and 101-124 and 51-71 and 125-155 are shown in Tables 1 and 3 below. The amino acid residues of each sequence that are phosphorylated are shown in bold and underlined. The names of the peptides in Table 1 correspond to the amino acid position of these peptides within the longest isoform of the human Tau protein having the amino acid sequence of SEQ ID NO: 100 as shown in Table 8 .

Specific embodiments of the present invention

(1) The tau peptide immunogen construct may be represented by the formulas:

(Th) _m -(A) _n -(Tau fragment)-X

or

(fragment Tau)-(A) _n -(Th) _m -X

Where

Th is a heterologous T-helper epitope;

A is a heterologous spacer;

(fragment Tau) is a B-cell epitope having from about 15 to about 40 amino acid residues from the full-length Tau protein of SEQ ID NO: 100;

X is α-COOH or α-CONH ₂ amino acids;

m is from 1 to about 4; And

n is from 0 to about 10.

(2) The tau peptide immunogen construct according to (1), wherein the Tau fragment is selected from the group consisting of SEQ ID NOs: 1-21 and 101-124.

(3) The tau peptide immunogen construct according to any of (1) or (2), wherein the Th epitope is selected from the group consisting of SEQ ID NOs: 22-50.

(4) The tau peptide immunogen construct according to (1), wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 51-71 and 125-155.

(5) A tau peptide immunogen construct comprising:

A B-cell epitope containing from about 15 to about 40 amino acid residues from the full-length Tau protein sequence of SEQ ID NO: 100;

T-helper epitope containing an amino acid sequence selected from the group consisting of SEQ ID NO: 22-50; And

an optional heterologous spacer selected from the group consisting of amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε-N)Lys, and ε-N-Lys-Lys-Lys-Lys (SEQ ID NO : 102),

wherein the B-cell epitope is covalently linked to the T-helper epitope either directly or through an optional heterologous spacer.

(6) The tau peptide immunogen construct according to (5), wherein the B cell epitope is selected from the group consisting of SEQ ID NOs: 1-21 and 101-124.

(7) The tau peptide immunogen construct according to (5), wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 22-50.

(8) The tau peptide immunogen construct of (5), wherein the optional heterologous spacer is (α, ε-N)Lys or ε-N-Lys-Lys-Lys-Lys (SEQ ID NO: 102).

(9) The tau peptide immunogen construct of (5), wherein the T helper epitope is covalently linked to the N terminus of the B cell epitope.

(10) The tau peptide immunogen construct of (5), wherein the T helper epitope is covalently linked to the N-terminus of the B cell epitope via an optional heterologous spacer.

(11) A composition containing a peptide immunogen construct according to any of (1) to (4).

(12) Pharmaceutical composition containing:

A. a peptide immunogen construct according to any one of (1) - (4); And

b. and a pharmaceutically acceptable delivery carrier and/or adjuvant.

(13) The pharmaceutical composition according to (12), wherein

A. The tau peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 51-71 and 125-155; And

b. The tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

(14) An isolated antibody or epitope-binding fragment thereof that specifically binds to a B cell epitope of a Tau peptide immunogen construct according to any of (1) to (10).

(15) An isolated antibody or epitope-binding fragment thereof according to (14) associated with a Tau peptide immunogen construct.

(16) An isolated antibody, or epitope-binding fragment thereof, that specifically binds to a B-cell epitope of a Tau peptide immunogen construct according to any of (1) to (10).

(17) A composition comprising an isolated antibody or epitope-binding fragment thereof according to any of (14) - (16).

Example 1

ANTIBODIES PRODUCED IN RESPONSE TO UNPHOSPHORYLATED TAU-PEPTIDE IMMUNOGENIC CONSTRUCTS AND BINDING PROFILES

A. Synthesis of Tau fragments and immunization

Methods for synthesizing engineered Tau fragments that have been included in the development of non-phosphorylated and phosphorylated Tau peptide immunogen constructs are described in the Detailed Description. Typically, peptides have been synthesized on a small scale for serological assays, on a laboratory experimental scale for field or clinical studies, and on a large scale (kilogram) for industrial/commercial production of pharmaceutical compositions. A large repertoire of Tau-linked B-cell epitope antigenic peptides having sequences ranging in length from about 10 to about 40 amino acids have been developed for extensive serological screening and selection as a B-cell epitope candidate for inclusion in the design of Tau peptide immunogen constructs for immunogenicity. and functional testing for use in Tau vaccines.

Full size Tau (Table 8) used as the recombinant protein in these serological studies was purchased from rPeptide (Tau-441, T-1001-2). Tau peptide segments used for epitope mapping in various serological assays are listed inTable 1 (SEQ ID NOs: 1-21 and 101-124). Selected Tau peptide segments were converted into Tau peptide immunogen constructs by synthetically linking Tau peptides to a carefully engineered T-helper cell (Th) epitope derived from pathogenic proteins, including measles virus fusion protein (MVF), hepatitis B surface antigen protein (HBsAg), influenza, Clostridum tetani and Epstein-Barr virus (EBV). Constructed Th epitopes are shown inTable 2 (SEQ ID NO: 22-50).

Formulations using water-in-oil emulsions and suspensions with mineral salts were prepared as described in the Detailed Description. When developing a suitable pharmaceutical composition to be used by a large population in order to prevent disease, it is important to consider the safety of the composition. Despite the use of water-in-oil emulsions in humans for many pharmaceutical compositions in clinical trials, alum remains the main adjuvant for use in pharmaceutical composition due to its safety. Alum or its mineral salts such as ADJUPHOS (aluminum phosphate) are often used as adjuvants in preparation for clinical use. The non-phosphorylated Tau peptide immunogen constructs were prepared (i) in a water-in-oil emulsion with Seppic MONTANIDE™ ISA 51 as the approved oil for human use, or (ii) mixed with mineral salts ADJUPHOS (aluminum phosphate) or ALHYDROGEL (alum ) in varying amounts of peptide constructs as indicated. The formulations were typically prepared by dissolving the Tau peptide immunogen constructs in water at a concentration of about 400 µg/mL and mixed with MONTANIDE™ ISA 51 in a water-in-oil emulsion (1:1 v/v) or mineral salts (1:1 v/v). ). The compositions were kept at room temperature for about 30 minutes and stirred with a stirrer for about 10-15 seconds prior to immunization. Some animals were immunized with more than one dose of a particular composition, which was administered at time 0 (prime) and 3 weeks after initial immunization (npi) (booster) and optionally at 6 npi for intramuscular re-stimulation. These immunized animals were then tested with the selected B cell epitope peptide(s) to evaluate the immunogenicity of the various Tau peptide immunogen constructs present in the formulation, as well as their cross-reactivity with the respective target peptides or proteins. These Tau-peptide immunogenic constructs with strong immunogenicity in initial screening in guinea pigs were then further tested in both water-in-oil emulsion, mineral salts and alum-based formulations in primates for regimens over a period as prescribed. immunization protocols.

Non-phosphorylated Tau peptide immunogen constructs (SEQ ID NOs: 52, 54, 56, 58, 61, 63, 65, 67, 69 and 71) dissolved in vehicle (ISA 51 VG, CpG3 50 μg/ml, 0.2% Tween®-80) were injected to guinea pigs according to the indicated schedule with immune sera collected for extensive serological analyses.

b. ELISA analyzes and results

Serum from immunized animals was collected at various time points after initial immunization and binding profiles were initially analyzed by ELISA to determine the immunogenicity of the respective construct against its own target B-cell epitope. Assessment of serological cross-reactivity among Tau peptides from other functional regions was carefully performed by ELISA with plates coated with various peptides selected from those shown in Tables 9a-9e .

ELISA protocols have been developed for the evaluation of immune serum samples and are described in the following Examples. Briefly, wells of 96-well plates were coated individually for 1 hour at 37°C with 100 µl of Tau peptide target fragments A145-P160, P172-P189, P182-P200, S195-P213, R209-L224, V228-L243, K257-K274, R379-L408, V275-K311 and V393-L425 peptides (SEQ ID NOs: 2, 4, 6, 8, 11, 13, 15, 17, 19 and 21, respectively) at a concentration of 2 μg/ml ( unless otherwise indicated), in 10 mM NaHCO ₃ buffer, pH 9.5 (unless otherwise indicated). Peptide-coated wells were incubated with 250 µl of 3% by weight gelatin in PBS at 37°C for 1 hour to block non-specific protein binding sites, after which they were washed three times with PBS containing 0.05% by volume Tween® 20, and then dried. Assay sera were diluted 1:20 (unless otherwise noted) with PBS containing 20% by volume normal goat serum, 1% by weight gelatin, and 0.05% by volume Tween® 20. One hundred microliters (100 µl) of diluted samples (e.g. , serum, plasma) were added to each well and left to react for 60 minutes at 37°C. The wells were then washed six times with 0.05% v/v Tween® 20 in PBS to remove unbound antibodies. Horseradish peroxidase (HRP) conjugated species-specific (eg, mouse, guinea pig or human) goat anti-IgG was used as a labeled indicator for binding to the antibody/peptide antigen complex formed in the positive wells. One hundred microliters (100 µl) of peroxidase-labeled goat anti-IgG at optimal pre-titrated dilution and in 1% v/v normal goat serum with 0.05% v/v Tween® 20 in PBS was added to each well and incubated at 37°C for an additional 30 minutes. The wells were washed six times with 0.05% v/v Tween® 20 in PBS to remove unbound antibody and reacted with 100 µl substrate mixture containing 0.04% w/w 3', 3', 5', 5'-tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for an additional 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. The reactions were stopped by adding 100 μl of 1.0 M H ₂ SO ₄ and the optical density was determined at 450 nm (A ₄₅₀ ). To determine antibody titers, immunized animals that received various Tau-derived peptide immunogens were tested with 10-fold serial dilutions of sera from 1:100 to 1:10,000, and the test serum titer, expressed as Log ₁₀ , was calculated by linear regression analysis. A ₄₅₀ with A ₄₅₀ cutoff set to 0.5. The ELISA results shown in Tables 9a-9e were further analyzed by plotting histograms as shown in FIG. 1A .

In general, high immunogenicity was found to be associated with each of the selected Tau peptide constructs when tested against its own B-cell epitope ( Tables 9a-9e and Fig. 1A ). High cross-reactivity was found with antibodies generated in response to the peptide immunogen constructs of SEQ ID NOs: 52, 54, 56, 58 and 61 ( see Fig. 1A ).

Interestingly, SEQ ID NO: 65, which contains the B cell epitope K257-K274 (SEQ ID NO: 15), was found to be a highly immunogenic sequence among the engineered Tau peptide constructs, however, antibodies generated in response to this construct were the most specific without significant cross-reactivity with any other B-cell epitope (see Fig. 1A). In contrast, the Tau peptide immunogen construct of SEQ ID NO: 67, which contains the B cell epitope R379-L408 (SEQ ID NO: 17), was the most immunogenic, had high reactivity with its own B epitope, but also had acceptable cross-reactivity with other tau B-cell epitopes (see Fig. 1A ).

Example 2

ANTIBODIES PRODUCED IN RESPONSE TO PHOSPHORYLATED TAU-PEPTIDE IMMUNOGENIC CONSTRUCTS AND BINDING PROFILES

Antibodies raised against the phosphorylated Tau peptide immunogen constructs were assessed for their ability to bind to their phosphorylated B-cell epitopes by ELISA 6 weeks after initial immunization (npi).

A. Immunizations

Phosphorylated Tau peptide immunogens (SEQ ID NOs: 51, 53, 55, 57, 59, 60, 62, 64, 66, 68 and 70) were formulated and administered to guinea pigs under the same conditions as discussed in Example 1 for non-phosphorylated Tau-peptide immunogens. Formulations containing phosphorylated Tau peptide immunogens were administered at 0, 3 and 6 npi.

b. ELISA analyzes and results

Briefly, the wells of 96-well plates were coated individually for 1 hour at 37°C with 100 µl of target tau fragments A145-P160, P172-P189, P182-P200, S195-P213, R209-L224, V228-L243, K257-K274, R379-L408, V275-K311 and V393-L425 peptides (SEQ ID NOs: 1, 3, 5, 7, 9, 10, 12, 14, 16, 18 and 20, respectively) at a concentration of 2 µg/ ml (unless otherwise indicated) in 10 mM NaHCO ₃ buffer, pH 9.5 (unless otherwise indicated). Peptide-coated wells were incubated with 250 µl of 3% by weight gelatin in PBS at 37°C for 1 hour to block non-specific protein binding sites, after which they were washed three times with PBS containing 0.05% by volume Tween® 20, and then dried. Assay sera were diluted 1:20 (unless otherwise noted) with PBS containing 20% by volume normal goat serum, 1% by weight gelatin, and 0.05% by volume Tween® 20. One hundred microliters (100 µl) of diluted samples (e.g. , serum, plasma) were added to each well and left to react for 60 minutes at 37°C. The wells were then washed six times with 0.05% v/v Tween® 20 in PBS to remove unbound antibodies. Horseradish peroxidase (HRP) conjugated species-specific (eg, mouse, guinea pig or human) goat anti-IgG was used as a labeled indicator for binding to the antibody/peptide antigen complex formed in the positive wells. One hundred microliters (100 μl) of peroxidase-labeled goat anti-IgG at optimal pre-titrated dilution and in 1% v/v normal goat serum with 0.05% v/v Tween® 20 in PBS was added to each well and incubated at 37°C for an additional 30 minutes. The wells were washed six times with 0.05% v/v Tween® 20 in PBS to remove unbound antibody and reacted with 100 µl substrate mixture containing 0.04% w/w 3', 3', 5', 5'-tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for an additional 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. The reactions were stopped by adding 100 μl of 1.0 M H ₂ SO ₄ and the optical density was determined at 450 nm (A ₄₅₀ ). To determine antibody titers, immunized animals that received various Tau-derived peptide immunogens were tested with 10-fold serial dilutions of sera from 1:100 to 1:10,000, and the test serum titer, expressed as Log ₁₀ , was calculated by linear regression analysis. A ₄₅₀ with A ₄₅₀ cutoff set to 0.5. Serum from immunized animals was collected at various time points after injection and binding profiles were analyzed by ELISA. ELISA plates were coated with various Tau fragments and the results are shown in Tables 10a-10d .

The ELISA results shown in Tables 10a-10d were further analyzed by plotting histograms as shown in FIG. 1B . Similar immunogenic and serological profiles, including cross-reactivity among B cell epitopes, have been found using phosphorylated Tau peptide constructs as immunogens.

Example 3

ANTIBODIES PRODUCED IN RESPONSE TO PHOSPHORYLATED TAU-PEPTIDE IMMUNOGENIC CONSTRUCTS AND BINDING PROFILES AGAINST THEIR NON-PHOSPHORYLATED PEPTIDE ANALOGUES

Antibodies raised against phosphorylated Tau peptides were assessed for their ability to bind to their respective non-phosphorylated analog peptide by ELISA 6 weeks post-injection (npi), as described in more detail below.

A. Immunizations

Phosphorylated Tau peptide immunogens (SEQ ID NOs: 51, 53, 55, 57, 59, 60, 62, 64, 66, 68 and 70) were formulated and administered to guinea pigs under the same conditions as discussed in Example 1 for non-phosphorylated Tau-peptide immunogens.

b. ELISA analyzes and results

Briefly, wells of 96-well plates were coated individually for 1 hour at 37°C with 100 µl of Tau peptide target fragments A145-P160, P172-P189, P182-P200, S195-P213, R209-L224, V228-L243, K257-K274, R379-L408, V275-K311 and V393-L425 peptides (SEQ ID NOs: 2, 4, 6, 8, 11, 13, 15, 17, 19 and 21, respectively) at a concentration of 2 μg/ml ( unless otherwise indicated), in 10 mM NaHCO ₃ buffer, pH 9.5 (unless otherwise indicated). Peptide-coated wells were incubated with 250 µl of 3% by weight gelatin in PBS at 37°C for 1 hour to block non-specific protein binding sites, after which they were washed three times with PBS containing 0.05% by volume Tween® 20, and then dried. Assay sera were diluted 1:20 (unless otherwise noted) with PBS containing 20% by volume normal goat serum, 1% by weight gelatin, and 0.05% by volume Tween® 20. One hundred microliters (100 µl) of diluted samples (e.g. , serum, plasma) were added to each well and left to react for 60 minutes at 37°C. The wells were then washed six times with 0.05% v/v Tween® 20 in PBS to remove unbound antibodies. Horseradish peroxidase (HRP) conjugated species-specific (eg, mouse, guinea pig or human) goat anti-IgG was used as a labeled indicator for binding to the antibody/peptide antigen complex formed in the positive wells. One hundred microliters (100 µl) of peroxidase-labeled goat anti-IgG at optimal pre-titrated dilution and in 1% v/v normal goat serum with 0.05% v/v Tween® 20 in PBS was added to each well and incubated at 37°C for additional 30 minutes. The wells were washed six times with 0.05% v/v Tween® 20 in PBS to remove unbound antibody and reacted with 100 µl substrate mixture containing 0.04% w/w 3', 3', 5', 5'-tetramethylbenzidine (TMB) and 0.12% by volume hydrogen peroxide in sodium citrate buffer for an additional 15 minutes. This substrate mixture was used to detect the peroxidase label by forming a colored product. The reactions were stopped by adding 100 μl of 1.0 M H ₂ SO ₄ and the optical density was determined at 450 nm (A ₄₅₀ ). To determine antibody titers, immunized animals that received various Tau-derived peptide immunogens were tested with 10-fold serial dilutions of sera from 1:100 to 1:10,000, and the test serum titer, expressed as Log ₁₀ , was calculated by linear regression analysis. A ₄₅₀ with A ₄₅₀ cutoff set to 0.5. Serum from immunized animals was collected at various time points after injection and binding profiles were analyzed by ELISA.

ELISA plates were coated with various non-phosphorylated Tau fragments and the results are shown in Tables 11a-11e . Similar serological binding profiles were obtained for all antibodies raised against phosphorylated Tau peptide constructs when immune sera were tested against the corresponding unphosphorylated Tau peptides of the B cell epitope. From the extensive immunogenicity and serology studies conducted in Examples 1, 2 and 3, it is clear that antibodies raised in response to Tau peptide immunogen constructs exhibit similar properties. Most of these properties can be attributed to amino acid sequences. Conformation assessment using cross-reactivity studies indicates that there were only minor differences caused by peptide phosphorylation, suggesting that these immune sera will have similar functional properties induced by their respective phosphorylated versus non-phosphorylated peptide immunogen constructs.

Example 4

BINDING PROFILE OF ANTIBODIES DIRECTED AGAINST NON-PHOSPHORYLATED TAU-PEPTIDE IMMUNOGENS

A. Specificity of anti-non-phosphorylated Tau antibodies purified from guinea pig antisera immunized with different size Tau peptide immunogen constructs

Guinea pigs were immunized with non-phosphorylated Tau peptide immunogen constructs as described in Example 1. Western blotting was used to screen for antibodies against non-phosphorylated Tau (anti-non-p-Tau antibodies) purified from guinea pig antisera immunized with various Tau peptide immunogenic constructs, on the specificity of binding to the Tau molecular complex of different sizes. Various forms (monomers, dimers, trimers, oligomers and polymers) Tau (20 μM) were separated on 12% Tris-glycine SDS-PAGE and transferred to a nitrocellulose (NC) (nitrocellulose) membrane prior to photoinduced crosslinking (PICUP) treatment. The membrane was incubated with anti-non-fTau antibodies purified from guinea pig antiserum at a concentration of 1 μg/ml, and then incubated with HRP-conjugated donkey anti-guinea pig immunoglobulin (706-035-148, Jackson). The blot was visualized with Western Lightning ECL Pro chemiluminescent reagent (PerkinElmer).

b. results

Western blot analysis of lysates containing Tau monomers, dimers, trimers and oligomers are shown in FIG. 2 (left panel). Monomeric Tau appeared in the region of about 60 kDa, and the dimer, trimer, and also oligomers appeared at their respective molecular weights. The commercial monoclonal antibodies mAb Tau 5 and mAb Tau 46 were able to detect the monomeric and, to a small extent, the dimeric form of Tau ( FIG. 2 , lanes 1 and 2). mAb Tau5 was used as a general marker for Tau and Tau46 as a marker for Tau 404-441.

IgG antibodies generated in response to non-phosphorylated peptide immunogen constructs (SEQ ID NOs: 52, 54, 56, 58, 61, 63, 65, 67, 69 and 71) detected Tau monomers, dimers, trimers and oligomers with varying efficiencies ( Fig. 2, tracks 4-13). Binding profiles for each antibody were analyzed and quantified in a histogram ( FIG. 2, right panel). In general, antibodies derived from C-terminal Tau peptide immunogen constructs were able to induce antibodies with higher preferential binding profiles compared to those from the N-terminal region (data not shown).

Example 5

ANTIBODIES DIRECTED AGAINST NON-PHOSPHORYLATED TAU-PEPTIDE IMMUNOGEN INHIBITE TAU AGGREGATION

A. Immunization and exposure to Tau

Animals were immunized with either non-phosphorylated Tau immunogen constructs (SEQ ID NOs: 52, 54, 56, 58, 61, 63, 65, 67, 69 and 71) as described in Example 1 or phosphorylated Tau immunogen constructs (SEQ ID NOs: 51, 53, 55, 57, 60, 62, 64, 66, 68 and 70) as described in Example 2.

Antibodies generated by immunization were then exposed to samples containing commercial full-length recombinant Tau441 protein under conditions that promote Tau aggregation. Tau441 β-sheet fibrils were formed using 60 μg Tau aggregated in 100 μl 1xPBS with 10 U/ml heparin at 37°C for 7 days, and then transferred to filters with a cutoff of 300 kDa (Pall) at 4° C to isolate β-sheet fibrils. These aggregations were confirmed by Thioflavin-T (ThT, Sigma) fluorescence.

Tau was aggregated in 200 μl of PBS buffer with 5 μM heparin for 3 days with or without IgG antibodies derived from Tau peptide immunogen constructs. After centrifugation (13,000 x g, 4°C, 30 minutes), Tau aggregates were collected and confirmed by ThT assays.

b. results

Fig. 3A contains graphs showing the level of aggregation of full-length Tau (Tau441) in the presence of antibodies directed against Tau-phosphorylated peptide immunogen constructs shown above each graph. The dotted line shown in Fig. 3A is a control sample that assessed Tau aggregation using pre-immune IgG. The results show that all antibodies directed against phosphorylated Tau peptide immunogens inhibited Tau aggregation compared to preimmune serum control. The antibodies were able to inhibit Tau aggregation at different rates.

On FIG. 3B quantifies the level of aggregation of full-length Tau (Tau441) by staining with ThT aggregates in the presence of antibodies directed against the non-phosphorylated Tau peptide immunogen constructs shown below the histogram. The results show that antibodies directed against non-phosphorylated Tau peptide immunogens inhibit Tau aggregation compared to a pre-immune serum control. The antibodies were able to inhibit Tau aggregation at different rates.

The results indicate that Tau polymerization exhibits a higher anti-aggregation capacity when using Tau peptide immunogen constructs having B-cell epitopes closer to the Tau C-terminal region.

Example 6

ANTIBODIES DIRECTED AGAINST TAU-PEPTIDE IMMUNOGENS INHIBITE TAU AGGREGATION

A. Immunization and exposure to Tau

Animals were immunized with non-phosphorylated Tau immunogen constructs as described in Example 1. Animals were immunized with phosphorylated Tau immunogen constructs as described in Example 2.

Antibodies generated by immunization were then exposed to cell lysates from (a) okadaic acid (OKA)-treated neuronal cells (SH-SY5Y cells) as hyperphosphorylated Tau, or (b) neuronal cells lacking TCA as non-phosphorylated Tau. . OKA was added to the medium to a final concentration of 100 nM for three hours in the presence of antisera at 6 and 9 npi to determine the dose-dependent inhibition effects of antisera on Tau aggregation.

b. results

Fig. 4 contains graphs showing the level of Tau aggregation of various lysates after exposure to antibodies directed against the Tau peptide immunogens shown above each graph. The level of Tau aggregation was measured by staining with ThT aggregates. Again, antibodies from both phosphorylated and non-phosphorylated Tau peptide immunogen constructs were able to inhibit Tau aggregation, consistent with the serological assay described in the previous Examples.

The results indicate that Tau polymerization exhibits a high anti-aggregation capacity with Tau epitopes derived from the central and C-terminal region of Tau.

Example 7

DISAGGREGATION OF REPRESENTED TAU AGGREGATES AFTER EXPOSURE TO ANTIBODIES DIRECTED AGAINST TAU-PEPTIDE IMMUNOGENES

A. Immunization and exposure to preformed Tau aggregates

Animals were immunized with non-phosphorylated Tau immunogen constructs as described in Example 1. Animals were immunized with phosphorylated Tau immunogen constructs as described in Example 2.

Antibodies resulting from immunization were then exposed to preformed Tau fibrils. The preformed Tau aggregates were then incubated with or without anti-f-Tau antibodies or anti-p-Tau antibodies purified from guinea pig antisera for 2 and 4 days. After incubation, aggregates were collected after centrifugation at 13,000×g at 4°C for 30 minutes and then quantified by ThT analysis.

b. results

Fig. 5 contains graphs showing the level of Tau disaggregation after exposure to antibodies directed against Tau peptide immunogens (representative peptide constructs from SEQ ID NOs: 66 and 67) as shown above in FIG. 5 (right panel). The level of Tau aggregation was measured by ThT staining and Western blotting.

Data from both Western blotting and ThT analysis show that anti-non-f/f-Tau 379-408 IgG induced from SEQ ID NOs: 67 and 66, respectively, have the ability to disrupt the aggregation of recombinant Tau fibrils.

Example 8

DOT BLOT ANALYSIS FOR SPECIFIC BINDING OF NON-DENATURED TAU FIBRILES, OLIGOMERS AND TAU MONOMERS USING REPRESENTATIVE ANTIBODIES FORMED BY BOTH PHOSPHORYLATED TAU AND NON-PHOSPHORYLATED TAU PEPTIDE IMMUNOGENIC CONSTRUCTS.

A. Immunization, Western blot and dot blot analysis of anti-Tau antibody binding profiles

Animals were immunized with non-phosphorylated Tau immunogen constructs as described in Example 1. Animals were immunized with phosphorylated Tau immunogen constructs as described in Example 2.

Various types of Tau protein (i.e. α-helical monomers, β-sheet monomers, β-sheet oligomers and β-sheet fibrils) were prepared as described in Example 5. Western blot and dot blot analyzes with different types of Tau protein was performed using anti-f-Tau antibodies or anti-non-f-Tau antibodies purified from guinea pig antisera immunized with various Tau peptide immunogen constructs as primary antibodies. Various forms of β-sheet fibrils, oligomers, monomers, α-helical monomers, and other controls were plotted on the blot for precise, specific control. Detailed procedures for the preparation of these reagents for serological testing and evaluation are described further in Example 11.

b. results

Fig. 6 (left panel) contains Western blot analysis (under denaturing conditions) of anti-f-Tau IgG produced by Tau peptide immunogens (SEQ ID NOS: 59, 66 and 70) and anti-f-Tau IgG produced by Tau β-peptide immunogen (SEQ ID NO: 67). Western blotting results show that anti-f-Tau IgG directed against the C-terminal end of Tau specifically recognizes the high molecular weight Tau aggregate but not the Tau monomer.

Fig. 6 (right panel) contains dot blot analysis (under non-denaturing conditions) of anti-f-Tau and anti-non-f-Tau IgG produced by the Tau peptide immunogens of SEQ ID NOS: 66 and 67, respectively. The results show that both anti-non-p-/p-Tau379-408 IgGs have specific binding to various forms of Tau, but not to other amyloidogenic proteins (ie, Aβ1-42 and alpha-synuclein). In addition, compared to the mAb Tau 5 monoclonal antibody and other IgGs against the non-C-terminal region of Tau, the anti-p/anti-non-p-Tau379-408 IgGs prepared according to the invention (SEQ ID NO: 66 and 67, respectively) have a higher affinity for fibrils and oligomers than for monomers. In addition, anti-f-Tau379-408 IgG does not bind to β-sheet monomers.

Example 9

TISSUE BINDING PROFILES OF ANITEL AGAINST TAU-PEPTIDE IMMUNOGENS

A. Immunization and tissue staining

Animals were immunized with non-phosphorylated Tau immunogen constructs as described in Example 1. Animals were immunized with phosphorylated Tau immunogen constructs as described in Example 2.

The antibodies formed as a result of immunization were then used to stain healthy (normal) and Alzheimer's disease (AD) brain tissues. A human tissue panel (Pantomics) was deparaffinized with xylene, rehydrated in ethanol and then treated with 0.25% trypsin solution with 0.5% CaCl ₂ in PBS for 30 minutes and incubated in 1% hydrogen peroxide in methanol to block endogenous peroxidase activity, and then incubated with 10% Block Ace (Sigma) in PBS before applying either anti-Tau 349-408 (SEQ ID NO: 66 or 67) or preimmune tissue sera. Sections were designed using 3-3'-diaminobenzidine (DAB) (diaminobenzidine) and stained with hematoxylin prior to microscopic examination.

b. results

Fig. 7 shows the staining of images from this experiment. Antibodies directed against unphosphorylated Tau 379-408 (SEQ ID NO: 67) and phosphorylated Tau 379-408 (SEQ ID NO: 66) did not cross-react with normal tissue sections, but did cross-react with Alzheimer's brain tissue. An antibody directed against a phosphorylated peptide (SEQ ID NO: 66) showed stronger binding in the hippocampal region of the AD brain.

Example 10

ANTIBODIES FORMED IN RESPONSE TO TAU-PEPTIDE IMMUNOGENIC CONSTRUCTS AND THEIR COMPOSITIONS: NEUROPROTECTIVE EFFECT ON THE REDUCTION OF NEURODEGENERATION PROVOKED BY EXOGENOUS TAU FIBRILES

To evaluate the neuroprotective effect of anti-Tau antibodies purified from guinea pig antisera immunized with various Tau peptide immunogen constructs, an in vitro model of neurodegeneration with exogenous, preformed Tau aggregates on NGF-treated, neuron-differentiated PC12 cells was used.

PC12 cells were treated with NGF (100 ng/ml) for 6 days to induce neuronal differentiation. The morphology of neuronally differentiated cells was confirmed and analyzed using a high-performance cell ³ iMager duos imaging system (Screen). Fluorescein dye was used to mark cell death and cells were counted using cell counting software. Antibodies generated in response to the Tau peptide immunogen constructs were able to enter cells and bind to Tau aggregates (data not shown). The neurotrophic effects of NGF were reflected in neurite outgrowth, and the number of neuronal differentiated cells was quantified. Levels of neurite outgrowth and the number of neuron differentiated cells were shown as a percentage (mean ± SEM) after normalization. The neurite length of PC12 cells with and without NGF treatment was taken as 100% and 0%, respectively. The number of neuron-differentiated PC12 cells after 6 days of NGF treatment was normalized to 100%.

Neurodegeneration was observed by adding exogenous, pre-formed heparin-treated Tau aggregates for one week at 37° C. on neuron differentiated PC12 cells. In the presence of preformed Tau aggregates, the length of the neurite was shortened, and the number of cells in neuronally differentiated PC12 cells decreased. This Tau fibril-induced neurodegeneration was proportional to the amount of exogenous Tau aggregates added in a concentration dependent manner. Commercially available anti-Tau antibodies (eg mAb Tau 5 and mAb Tau46), but not antibodies purified from naive guinea pigs, attenuated Tau-fibril-induced neurodegeneration. This model was adopted as a screening platform to determine which anti-Tau antibodies purified from guinea pig antisera immunized with various Tau peptide immunogen constructs had neuroprotective effects in restoring neuronal survival in a concentration-dependent manner ( Fig. 8A and B ).

As a result, anti-Tau antibodies purified from guinea pig antisera immunized with more than half of the different Tau peptide immunogen constructs restored neurite outgrowth. The tau fibril was provided at a concentration of 5 μg/ml, previously optimized to cause injury ( FIG. 8A ). Anti-Tau antibodies purified from guinea pig antisera immunized with phosphorylated or non-phosphorylated Tau peptide constructs protected neuron-differentiated PC12 cells from Tau fibril-induced neuronal death. On FIG. 8B shows anti-non-f-Tau antibody data obtained by immunization from SEQ ID NOS: 65, 67 and 69.

The results in FIG. 8A and 8B show that anti-Tau antibodies (eg, SEQ ID NOs: 65, 67 and 69) are able to protect neurons from Tau fibril neurotoxicity. The anti-neurodegenerative effects of anti-Tau antibodies generated in response to Tau peptide immunogen constructs (SEQ ID NOS: 65, 67 and 69) were observed and quantified by calculation using Cell ³ Imager duos software (Miteklab) for the number of surviving cells in culture.

With respect to neuroprotection by anti-Tau antibodies by evaluating in vitro functional assays, it has been shown that in vitro Tau nucleation can markedly reduce pathology and improve cognitive function in vivo, as reported by Yanamandra, K, et al. ( Neuron 80:402-414, 2013). The Tau monomer is considered to be natively unstructured and may not have a major functional role in Tau aggregation and associated cell cytotoxicity. Recent results indicate that the onset of pathological aggregation may begin with the conversion of the Tau monomer from an inert form to a nucleatable form (Mirbaha, H., et al., ELife 2018; 7: e36584; doi: 10.7554/eLife.36584). Based on these results, we tested anti-Tau antibodies generated in response to Tau peptide immunogen constructs for their ability to reduce nucleation activity. In particular, anti-Tau antibodies directed against Tau peptide immunogen constructs were incubated with Alzheimer's disease (AD) patient brain homogenates and found to have the ability to reduce such nucleation activity (data not shown). Our extensive Tau-based immunogen design, immunogenicity and serological assays have contributed to the development of neuroprotective intervention as vaccines at the molecular and serological levels for the treatment of severe tauopathies.

Example 11

ADDITIONAL SERO TESTS AND REAGENTS

Serological assays and reagents for evaluating the functional immunogenicity of synthetic peptide constructs and their formulations are detailed below.

A. Accurate specificity analysis and epitope mapping to Tau fragments using peptide-based ELISA tests with a cluster of B-cell epitopes

Analysis of the precise specificity of anti-Tau antibodies in immunized hosts was determined by epitope mapping. Briefly, wells of 96-well plates were coated with individual Tau fragment peptides (SEQ ID NO: 115 and 156-171) from Tau 275-311 plot (SEQ ID NO: 19) for mapping, 0.5 μg per 0.1 ml per well and then 100 µl of serum samples (1:100 dilution in PBS) were incubated in duplicate in wells of the plate, following the steps of the antibody ELISA method described above. The B-cell epitope of the Tau peptide immunogen construct and its associated analysis of the precise specificity of anti-Tau antibody immune sera in immunized hosts was also tested with the corresponding Tau peptides (e.g., SEQ ID NOS: 1-21, 101-124, as shown in Table 1, SEQ ID NO: 72-99, as shown in Tables 4-7, and SEQ ID NO: 100, as shown in Table 8) without spacer and Th sequences, for additional reactivity and confirmation of specificity.

b. Assessment of immunogenicity

Samples of preimmune and immune sera from animals were collected according to experimental immunization protocols and heated at 56°C for 30 minutes to inactivate serum complement factors. Following administration of the Tau peptide immunogen constructs, blood samples were obtained according to the protocols and their immunogenicity for the specific target site(s) was assessed. Serially diluted sera were tested and positive titers were expressed as Log ₁₀ times the dilution. The immunogenicity of a particular Tau peptide immunogen construct was assessed by its ability to elicit a high titer B cell response directed against the desired epitope specificity within the target antigen while maintaining low or negligible antibody reactivity to the "T helper cell epitopes" used to provide enhancement of desired B-cell responses.

With. Preparation of Tau aggregates (dimers, trimers, oligomers and fibrils) with recombinant Tau protein

To prepare aggregated Tau, purified recombinant Tau protein (0.1 µg/µl) in 100 µl PBS/KCl aggregation buffer (2.5 mM MgCl ₂ , 50 mM HEPES and 150 mM KCl in 1 x PBS, pH 7.4 ) were incubated at 37°C in 1.5 ml Eppendorf tubes for 7 days in a thermomixer (Eppendorf) without shaking. Aggregated Tau was immediately frozen at -80°C for later use.

d. Purification of anti-Tau antibodies

Anti-Tau antibodies were purified from sera collected 3-15 weeks after initial immunization (npi) of guinea pigs immunized with Tau peptide immunogen constructs containing peptides of different sequences (SEQ ID NOS: 51-71 and 125-155) using affinity columns (Thermo Scientific, Rockford). Briefly, after equilibrating the buffer (0.1 M phosphate and 0.15 M sodium chloride, pH 7.2), 400 µl of serum was added to the Nab Protein G Spin column, followed by mixing for 10 minutes and centrifugation at 5800 xg for 1 minute . The column was washed three times with binding buffer (400 μl). Subsequently, an elution buffer (400 μl, 0.1 M glycine, pH 2.0) was added to the spin column to elute the antibodies after centrifugation at 5800×g for 1 minute. The eluted antibodies were mixed with neutralizing buffer (400 μl, 0.1 M Tris, pH 8.0) and the concentrations of these purified antibodies were measured using Nan-Drop at OD ₂₈₀ with BSA (bovine serum albumin) as standard.

e. Specificity of Anti-Tau Antibodies Purified from Guinea Pig Antisera Immunized with Various Tau Peptide Immunogenic Constructs of Different Sizes

Western blotting was used to screen anti-Tau antibodies purified from guinea pig antisera immunized with various Tau peptide immunogen constructs for binding specificity to Tau molecular complex of various sizes. Various forms of Tau (20 μM) were separated on 12% Tris-glycine SDS-PAGE and transferred to a nitrocellulose (NC) membrane prior to photoinduced crosslinking (PICUP) treatment. The membrane was incubated with anti-Tau antibodies purified from guinea pig antiserum at 1 μg/ml and then incubated with HRP-conjugated donkey anti-guinea pig immunoglobulin (706-035-148, Jackson). The blot was visualized with Western Lightning ECL Pro chemiluminescent reagent (PerkinElmer). Monomeric Tau was determined by blotting to be about 60 kDa while the dimer, trimer or oligomers had corresponding molecular weights several times greater than monomeric Tau. A commercial antibody that is capable of detecting various oligomeric species such as dimers, trimers and larger oligomers, eg mAb Tau 5 or mAb Tau 46, was used as a positive control.

f. Dot blot analysis with different types of amyloidogenic proteins

The preparation of α-helical monomers, β-sheet monomers, β-sheet oligomers and β-sheet fibrils of Aβ _1-42 , Tau and α-Syn is described as follows.

1.Aβ _1-42 α- helical monomers: 20 µg β-sheet Aβ monomers_1-42 (50 μl) was added to 1xPBS containing 20% trifluoroacetic acid and 20% hexafluoroisopropanol (10 μl) and incubated at 4° C. for 24 hours to form α-helical monomers.

2.Aβ _1-42 β-sheet monomers: 60 mcg Aβ_1-42 in 120 μl of 1xPBS containing 5% TFA aggregated at 37° C. for 24 hours was transferred to a 10 kDa cut-off filter (Millipore) to recover β-sheet monomers.

3.Aβ _1-42 β- sheet oligomers: 60 mcg Aβ_1-42 in 120 μl of 1xPBS aggregated at 37° C. for 3 days, sonicated on ice and transferred to 10 and 30 kDa cut-off filters (Millipore) to isolate <35 kDa β-sheet oligomeric fibrils.

4.Aβ _1-42 β- sheet fibrils: 60 μg Aβ_1-42 in 120 μl of 1xPBS aggregated at 37° C. for 3 days, sonicated on ice and transferred to 30 kDa cut-off filters (Millipore) to isolate β-sheet fibrils.

5. α -Syn α -helical monomers: 40 μg of freshly prepared α-Syn was dissolved in 100 μl of cold 1xPBS at 4°C and immediately transferred to a 10 kDa filter (Millipore) to isolate the α-helical monomer.

6. α -Syn β -sheet monomers: 40 μg of α-Syn incubated in 100 μl of PBS/KCl buffer at 37°C for 24 hours was transferred to a 10 kDa filter (Millpore) to isolate β-sheet monomers .

7. α -Syn β -sheet oligomers: 40 μg of α-Syn aggregated in 100 μl of PBS/KCl buffer at 37° C. for 8 days was sonicated on ice and then transferred to filters with a cutoff of 30 and 100 kDa for isolation of β-sheet oligomers.

8. α -Syn β -sheet fibrils: 40 µg of α-Syn aggregated in 100 µl of PBS/KCl buffer at 37° C. for 8 days was sonicated on ice and then transferred to 30 and 100 kDa cut-off filters for isolation of β-sheet fibrils.

9. Tau441 α -helical monomers: 60 μg of Tau, prepared in 100 μl of 1xPBS at 4°C, was transferred to a 100 kDa filter to isolate α-helical monomers.

10. Tau441 β -sheet monomers: 60 µg Tau aggregated in 100 µl 1xPBS with 10 U/ml heparin at 25°C for 48 hours was transferred to a 100 kDa filter at 4°C to isolate β-sheets. monomers.

11. Tau441 β -sheet oligomers: 60 µg Tau aggregated in 100 µl 1xPBS with 10 U/ml heparin at 37°C for 48 hours, transferred to a filter with a cutoff of 100 and 300 kDa (Pall) at 4°C to isolate β-sheet oligomers.

12. Tau441 β -sheet fibrils: 60 μg Tau aggregated in 100 μl 1xPBS with 10 U/ml heparin at 37°C for 6 hours was transferred to a 300 kDa cut-off filter (Pall) at 4°C for isolation β-sheet fibrils.

These monomers and oligomers were confirmed by fluorescence of Thioflavin-T (ThT, Sigma) or PAAG (polyacrylamide gel electrophoresis). Amyloidogenic protein concentrations were measured using Nano-Drop with a commercial amyloidogenic Aβ _1-42 stock solution as standard. These monomers and oligomers were applied separately to PVDF membranes in an amount of 3 μg for Aβ _1-42 , 4 μg for α-Syn and 7 μg for Tau. The membranes were incubated with anti-Tau antibodies purified from guinea pig antisera (1:1000 dilution) as primary antibody, followed by hybridization with an HRP-conjugated secondary antibody against guinea pig immunoglobulin (1:5000; Vector Laboratories). The membranes were treated with Luminata Western HRP substrates (Bio-Rad, Hercules, CA, USA) and signals were recorded using a ChemiDoc-It 810 digital imaging system (UVP Inc., Upland, CA, USA).

Example 12

ANIMALS USED IN IMMUNOGENICITY AND EFFICACY STUDIES

a. Guinea pigs:

Immunogenicity studies were performed on mature, naive, adult male and female Duncan-Hartley guinea pigs (300-350 g/body weight). A minimum of 3 guinea pigs per group were used in the experiments. Protocols involving Duncan-Hartley guinea pigs (8-12 weeks of age; Covance Research Laboratories, Denver, PA, USA) were performed according to IACUC approved applications in contract vivariums, as well as in United Biomedical, Inc. (UBI), as a sponsor.

Example 13

IDENTIFICATION OF A FOCUSED ANTIBODY RESPONSE CAUSED BY A TAU PEPTIDE IMMUNOGENIC CONSTRUCT TO A B-CELL TARGET EPITOPE: A REPRESENTATIVE STUDY

In a representative accurate epitope mapping study ( Table 16 ) to determine the binding site(s) of an antibody with specific residues formed in response to a Tau peptide immunogen construct directed to its 275-311 Tau region (SEQ ID NO: 19), overlapping peptides with increasing length from this region (SEQ ID NOS: 115 and 156-171) were synthesized and individually applied to the wells of a 96-well microtiter plate as solid phase immunoabsorbents. Peptides (0.5 μg per 0.1 ml per well) were applied to the plates, and then 100 μl of serum samples (1:100 dilution in PBS) were incubated in duplicate in the wells of the plates, following the steps of the antibody ELISA method described above.

6 NPI pooled porcine antiserum directed against the Tau peptide immunogen construct SEQ ID NO: 68 was added at a 1:100 dilution in sample diluent buffer to plate wells coated with overlapping Tau peptides from this site at 2.0 μg/mL , and then incubated for one hour at 37°C. After washing the wells of the plate with wash buffer, protein A/G conjugated with horseradish peroxidase is added and incubated for 30 min. After washing again with PBS, the substrate is added to the wells to measure the optical density at 450 nm using an ELISA plate reader, the samples of which are analyzed in duplicate. Binding of the antisera to the corresponding long Tau peptide of the B epitope immunogen construct represents the maximum binding.

As shown in Table 16 , pooled 6 bp guinea pig immune sera exhibited a high fidelity binding profile in which two Tau peptides of SEQ ID NOs: 164 and 115 having only two amino acids differing in length but the longer SEQ ID peptide NO: 115 was able to present a strong Tau epitope from this site of hyperphosphorylation and aggregation. However, the pooled immune sera were unable to recognize the peptide of SEQ ID NO: 164 due to the deletion of two N-terminal amino acids (I297 and K298), indicating that these two amino acids are critical to present this site as an immunodominant epitope.

This region has been the subject of further rigorous development of Tau peptide constructs for immunogenicity studies, as shown in Tables 12, 13a, 13b, and 15 , including peptide immunogen constructs comprising the trans conformation of proline at position 301 to evaluate immunogenicity. In general, high immunogenicity was found with carefully designed Tau peptide immunogen constructs SEQ ID NOS: 126-132, 135, 136, 140 and 141 except for SEQ ID NOS: 128 at this site of hyperphosphorylation and aggregation.

Additional Tau peptide immunogen constructs were used for the Tau "fuzzy shell" region, which is located at the N-terminal region, which is often removed from Tau isomers. Tau peptide immunogen constructs from this region (SEQ ID NO: 137-139) as shown in Table 3 were also constructed and evaluated for their respective immunogenicity.

Table 14 shows that the Tau peptide immunogen constructs from the fuzzy envelope region were highly immunogenic. These results suggest that Tau peptide immunogen constructs from the N-terminal region of the fuzzy shell may be useful for inclusion in an antibody detection formulation to prevent Tau aggregation in addition to potentially reducing monomeric Tau nucleation initiation leading to Tau aggregation.

Table 1

Amino acid sequences of Tau and phospho-Tau (phTau) (pTau) fragments

SEQ ID Code Description Sequence ¹ 1 p4578 fTau - ak145-160
(pT149/pT153) ADGKTKIATPGAAPP 2 p4579 Tau - ak145-160 ADGKTKIATPGAAPP 3 p4580 fTau - ak172-189
(pT175/pT181/pS185) PAKTPPPAPKTPPSSGEPP 4 p4581 Tau - ak172-189 PAKTPPPAPKTPPSSGEPP 5 p4582 fTau - ak182-200
(pS184/pY197) PPSSGEPPKSGDRSGYSSP 6 p4583 Tau - ak182-200 PPSSGEPPKSGDRSGYSSP 7 p4586 fTau - ak195-213
(pS198/pS208/pS210) SGYSSPGSPGTPGSSRRTP 8 p4585 Tau - ak195-213 SGYSSPGSPGTPGSSRRTP 9 p4584 fTau - ak195-213
(pS199/pS202/pT205) SGYSSPGSPGTPGSSRRTP 10 p4587 fTau - ak209-224
(pT212/pS214/pT217) RSRTPSLPTPPTREPK eleven p4588 Tau - ak209-224 RSRTPSLPTPPTREPK 12 p4589 fTau - ak228-243
(pT231/pS235/pS238) VVRTPPPKSPSSAKSRL 13 p4598 Tau - ak228-243 VVRTPPPKSPSSAKSRL 14 p4599 fTau - ak257-274
(ps262) KSKIGSTENLKHQPGGGK 15 p4600 Tau - ak257-274 KSKIGSTENLKHQPGGGK 16 p4601 fTau - ak379-408
(pS396/pS404) RENAKAKKTDHGAEIVYKSPVVSGDTSPRHL 17 p4602 Tau - ak379-408 RENAKAKKTDHGAEIVYKSPVVSGDTSPRHL 18 p4603 fTau - ak275-311
*P301->S
(pS293/pS301) ͙VQIINKKLDLSNVQSKCGSKDNIKHVSGGGSVQIVYK 19 p4604 Tau - ak275-311 VQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYK 20 p4606 fTau - ak393-425
(pS396/pS404/pS422) VYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQL 21 p4605 Tau - ak393-425 VYKSPVVSGDTSPRHLSNVSSTGSIDMVDSPQL 101 p5031a Tau - ak131-141 SKDGTGSDDKK 102 p5032a Tau - ak241-257 SRLQTAPVVPMPDLKNVK 103 p5033a Tau - ak243-259 LQTAPVVPMPDLKNVKSK 104 p5134a Tau - ak275-281 VQIINKK 105 p5135a Tau - ak281-294 KLDLSNVQSKCGSK 106 p5136a Tau - ak275-294 VQIINKKLDLSNVQSKCGSK 107 p5137a Tau - ak294-311 KDNIKHVPGGGSVQIVYK 108 p5138a Tau - ak294-311, P301→S301 KDNIKHVSGGGSVQIVYK 109 p5158a Tau - ak294-311, p301 trans KDNIKHVPGGGSVQIVYK 110 p5159a Tau - ak275-311, p301 trans VQIINKKLDLSNVQSKCGSKDNIKHVPGGGSVQIVYK 111 p5183a Tau - ak297-311, p301 trans IKHVPGGGSVQIVYK 112 p5160a Tau - ak22-34 DRKDQGGYTMHQD 113 p5161a Tau - ak18-34 YGLGDRKDQGGYTMHQD 114 p5162a Tau - ak22-38 DRKDQGGYTMHQDQEGD 115 p5186a Tau - ak297-311 IKHVPGGGSVQIVYK 116 p5404a Tau - ak359-370 NITHVPGGGNKK 117 p5405a Tau - ak349-370 RVQSKIGSLDNITHVPGGGNKK 118 p5406a Tau - ak339-370 VKSEKLDFKDRVQSKIGSLDNITHVPGGGNKK 119 p5407a Tau - ak329-370 HHKPGGGQVEVKSEKLDFKDRVQSKIGSLDNITHVPGGGNKK 120 p5408a Tau - ak329-349 HHKPGGGQVEVKSEKLDFKDR 121 p5456a Tau - ak1-40 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTD 122 p5457a Tau - ak11-50 MEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQT 123 p5458a Tau - ak1-34 MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQD 124 p5459a Tau - ak1-22 MAEPRQEFEVMEDHAGTYGLGD

¹ Phosphorylated residues are in bold and underlined.

table 2

Amino acid sequences of Th epitopes derived from pathogenic proteins, including idealized artificial Th epitopes for use in the development of Tau peptide immunogen constructs

Description Subsequence SEQID NO Th Clostridium tetani1 KKQYIKANSKFIGITEL 22 Th MvF1 LSEIKGVIVHRLEGV 23 Th Bordetella pertussis GAYARCPNGTRALTVAELRGNAEL 24 Th Clostridium tetani2 WVRDIIDDFTNESSQKT 25 Th Diphtheria DSETADNLEKTVAALSILPGHGC 26 Th Plasmodium falciparum DHEKKHAKMEKASSVFNVVNS 27 Th Schistosoma mansoni KWFKTNAPNGVDEKHRH 28 Th cholera toxin ALNIWDRFDVFCTLGATTGYLKGNS 29 Th MvF2 ISEIKGVIVHKIEGI thirty Th KKKMvF3 KKKISISEIKGVIVHKIEGILF
T RT TR T 31 Th HBsAg1 KKKLFLLTKLLTLPQSLD
RRRIKII RII IL IR
VRVV VV VIV
F FF FF FVF
F 32 Th MvF4 (UBITh®3) ISISEIKGVIVHKIETILF
T RT TR 33 HBsAg2 Th KKKIITITRIITIPQSLD
FFLL L ITTI 34 Th MvF5 (UBITh®1) ISITEIKGVIVHRIETILF 35 Th HBsAg3 (UBITh®2) KKKIITITRIITIITTID 36 Th Influenza MP1_1 FVFTTLTVPSER 37 Th Influenza MP1_2 SGPLKAEIAQRLEDV 38 Th Influenza NSP1 DRLRRDQKS 39 Th EBV BHRF1 AGLTLSLLVICSYLFISRG 40 Th Clostridium tetani TT1 QYIKANSKFIGITEL 41 Th EBV EBNA-1 PGPLRESIVCYFMVFLQTHI 42 Th Clostridium tetani TT2 FNNFTVSFWLRVPKVSASHLE 43 Th Clostridium tetani TT3 KFIIKRYTPNNEIDSF 44 Th Clostridium tetani TT4 VSIDKFRIFCKALNPK 45 Th EBV CP VPGLYSPCRAFFNKEELL 46 Th HCMV IE1 DKREMWMACIKELH 47 Th EBV GP340 TGHGARTSTEPTTDY 48 Th EBV BPLF1 KELKRQYEKKLRQ 49 Th EBV EBNA-2 TVFYNIPPMPL 50

Table 8

Tau protein sequence, full-length (441 aa) isoform with 4R and 2N (GenBank: AGF19246.1)

Sequence(SEQ ID NO: 100) MAEPRQEFEVMEDHAGTYGLGDRKDQGGYTMHQDQEGDTDAGLKESPLQTPTEDGSEEPGSETSDAKSTP 70
TAEDVTAPLVDEGAPGKQAAAQPHTEIPEGTTAEEAGIGDTPSLEDEAAGHVTQARMVSKSKDGTGSDDK 140
KAKGADGKTKIATPRGAAPPGQKGQANATRIPAKTPPAPKTPPSSGEPPKSGDRSGYSSPGSPGTPGSRS 210
RTPSLPTPPTREPKKVAVVRTPPKSPSSAKSRLQTAPVVPMPDLKNVKSKIGSTENLKHQPGGGKVQIINK 280
KLDLSNVQSKCGSKDNIKHVPGGGSVQIVYKPVDLSKVTSKCGSLGNIHHKPGGGQVEVKSEKLDFKDRV 350
QSKIGSLDNITHVPGGGNKKIETHKLTFRENAKAKAKTDHGAEIVYKSPVVSGDTSPRHLSNVSSTGSIDMV 420
DSPQLATLADEVSASLAKQGL

Table 9a

Analysis of the immunogenicity of Tau-peptide constructs

(Data correspond to Fig. 1A)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4579a (A145-P160)
Log ₁₀ titer ELISA
(SEQ ID NO: 2) Anti-p4581a (P172-P189)
Log ₁₀ titer ELISA
(SEQ ID NO: 4) Group No. Immunogen MS no. (MS-guinea pig) 0npi 3npi 6npi 9npi 0npi 3npi 6npi 9npi 1 p4579kb: Tau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 52 5811 0.15 5.07 5.12 5.42 0.11 8.05 6.70 6.03 5812 0.11 5.11 5.05 5.15 0.08 4.81 4.76 4.99 5813 0.11 9.40 7.49 5.58 0.08 5.06 5.33 5.24 Avg. 0.12 6.52 5.89 5.38 0.09 5.97 5.60 5.42 2 p4581kb: Tau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 55 5814 0.11 5.89 5.46 5.26 0.08 12.54 9.44 6.81 5815 0.09 4.52 4.96 5.05 0.07 4.99 5.89 5.45 5816 0.11 5.18 5.33 5.12 0.08 4.51 5.07 5.17 Avg. 0.10 5.20 5.25 5.14 0.08 7.35 6.80 5.81 3 p4583kb: Tau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 56 5817 0.11 4.60 4.97 5.36 0.08 4.40 4.52 4.72 5818 0.12 4.57 4.67 4.95 0.10 4.55 4.86 5.06 5819 0.11 4.76 4.85 4.82 0.11 4.72 4.91 4.94 Avg. 0.11 4.64 4.83 5.04 0.10 4.56 4.76 4.91 4 p4585kb: Tau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 58 5820 0.09 3.09 4.33 4.20 0.09 3.32 4.67 4.66 5821 0.10 4.21 4.95 4.92 0.09 4.51 5.05 4.93 5822 0.09 4.81 4.64 4.85 0.08 4.79 4.54 4.58 Avg. 0.09 4.04 4.64 4.66 0.08 4.21 4.75 4.72 5 p4588kb: Tau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 61 5823 0.08 2.66 4.42 4.63 0.08 2.96 4.78 4.90 5824 0.09 2.81 3.81 4.35 0.08 3.29 4.56 4.83 5845 0.09 3.03 3.10 3.85 0.08 2.93 3.59 4.50 Avg. 0.09 2.83 3.78 4.28 0.08 3.06 4.31 4.74 6 p4598kb: Tau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 63 5826 0.10 1.67 2.99 3.30 0.12 2.66 4.31 4.37 5827 0.11 1.86 3.18 3.66 0.12 2.95 4.62 4.52 5828 0.09 3.31 4.06 4.38 0.08 3.87 4.61 4.77 Avg. 0.10 2.28 3.41 3.78 0.11 3.16 4.51 4.55 7 p4600kb: Tau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 65 5829 0.08 3.02 4.39 4.72 0.08 2.99 4.60 4.77 5830 0.08 3.93 4.83 4.75 0.08 4.46 5.03 4.93 5831 0.08 3.02 4.02 3.86 0.07 3.64 4.52 3.41 Avg. 0.08 3.32 4.42 4.44 0.08 3.70 4.72 4.37 8 p4602kb: Tau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 67 5832 0.08 3.08 4.27 4.29 0.08 3.26 4.62 4.64 5833 0.08 2.99 4.04 3.54 0.08 3.15 4.37 4.28 5834 0.10 2.86 3.63 4.29 0.10 2.87 4.10 4.45 Avg. 0.09 2.98 3.98 4.04 0.09 3.09 4.36 4.46 9 p4604kb: Tau construct, UBITh1-εk-kkk-(V275-K311) SEQ ID NO: 69 5835 0.12 3.85 4.55 4.33 0.12 4.87 5.08 4.90 5836 0.08 3.98 4.64 4.44 0.09 4.26 4.95 4.77 5837 0.10 4.83 4.83 4.85 0.08 4.44 4.85 4.89 Avg. 0.10 4.22 4.67 4.54 0.09 4.52 4.96 4.85 10 p4605kb: Tau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 71 5838 0.08 3.05 4.36 4.37 0.08 3.75 4.62 4.63 5839 0.08 3.18 4.52 4.56 0.08 3.40 4.71 4.76 5840 0.10 3.16 4.52 4.59 0.09 3.76 4.85 4.90 Avg. 0.09 3.13 4.46 4.51 0.08 3.64 4.73 4.76

Table 9b

Analysis of the immunogenicity of Tau-peptide constructs

(Data correspond to Fig. 1A)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4583a (P182-P200)
Log ₁₀ titer ELISA
(SEQ ID NO: 6) Anti-p4585a (S195-P213)
Log10 titer ELISA
(SEQ ID NO: 8) Group # Immunogen MS No. 0npi 3npi 6npi 9npi 0npi 3npi 6npi 9npi 1 p4579kb : Tau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 52 5811 0.12 3.15 3.59 4.62 0.12 3.02 3.29 4.72 5812 0.09 1.20 0.66 2.91 0.12 2.06 2.96 4.24 5813 0.08 3.10 3.28 3.76 0.10 3.65 4.23 4.38 Avg. 0.10 2.48 2.51 3.76 0.11 2.91 3.49 4.45 2 p4581kb: Tau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 55 5814 0.08 4.65 4.83 4.65 0.10 4.42 4.78 4.53 5815 0.08 2.36 4.25 4.52 0.10 2.23 4.31 4.52 5816 0.09 0.00 0.65 1.75 0.09 2.64 3.05 2.90 Avg. 0.08 2.34 3.24 3.64 0.10 3.09 4.05 3.98 3 p4583kb: Tau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 56 5817 0.09 4.83 5.53 6.59 0.08 3.08 4.40 4.60 5818 0.11 4.69 5.19 5.57 0.09 3.87 4.83 5.02 5819 0.12 5.04 5.59 5.34 0.10 3.81 4.90 4.93 Avg. 0.10 4.85 5.44 5.83 0.09 3.58 4.71 4.85 4 p4585kb: Tau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 58 5820 0.09 2.15 2.82 3.11 0.10 4.02 4.91 4.93 5821 0.08 3.12 4.60 4.69 0.09 4.88 5.17 5.08 5822 0.08 4.31 4.29 4.36 0.08 5.80 7.07 5.62 Avg. 0.08 3.20 3.90 4.05 0.09 4.90 5.71 5.21 5 p4588kb: Tau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 61 5823 0.08 0.00 3.13 3.06 0.08 0.21 3.26 3.10 5824 0.08 0.00 2.14 2.92 0.07 0.00 1.74 2.79 5845 0.09 0.00 0.00 0.00 0.08 0.00 1.39 2.62 Avg. 0.08 0.00 1.76 1.99 0.08 0.07 2.13 2.84 6 p4598kb: Tau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 63 5826 0.10 0.00 0.00 0.00 0.09 0.00 0.00 0.85 5827 0.12 0.00 0.00 0.00 0.10 0.00 0.00 0.00 5828 0.09 0.00 0.00 0.52 0.09 0.00 0.00 0.39 Avg. 0.10 0.00 0.00 0.17 0.09 0.00 0.00 0.41 7 p4600kb: Tau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 65 5829 0.08 0.00 2.68 3.14 0.08 0.00 2.52 3.13 5830 0.08 0.00 2.93 2.70 0.07 0.00 2.37 1.96 5831 0.08 0.00 0.28 0.00 0.07 0.00 0.00 0.00 Avg. 0.08 0.00 1.96 1.95 0.07 0.00 1.63 1.70 8 p4602kb: Tau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 67 5832 0.09 0.00 0.24 1.24 0.07 0.00 0.00 0.35 5833 0.09 0.00 1.35 0.00 0.07 0.78 2.53 0.33 5834 0.10 0.00 0.00 0.00 0.08 1.52 0.00 0.00 Avg. 0.09 0.00 0.53 0.41 0.07 0.77 0.84 0.23 9 p4604kb: Tau construct, UBITh1-εk-kkk-(V275-K311) SEQ ID NO: 69 5835 0.11 0.00 0.73 0.00 0.10 1.00 3.03 2.28 5836 0.08 2.57 2.97 2.59 0.09 0.47 0.00 0.00 5837 0.08 1.78 2.99 2.79 0.08 1.02 2.58 1.94 Avg. 0.09 1.45 2.23 1.79 0.09 0.83 1.87 1.41 10 p4605kb: Tau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 71 5838 0.08 0.00 3.08 2.91 0.08 0.00 3.02 2.66 5839 0.08 0.00 3.00 3.12 0.07 0.00 2.75 2.96 5840 0.09 0.66 3.04 3.18 0.08 0.00 2.92 3.10 Avg. 0.08 0.22 3.04 3.07 0.08 0.00 2.90 2.90

Table 9c

Analysis of the immunogenicity of Tau-peptide constructs

(Data correspond to Fig. 1A)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4588a (R209-L224)
Log10 titer ELISA
(SEQ ID NO: 11) Anti-p4598a (V228-L243)
Log ₁₀ titer ELISA
(SEQ ID NO: 13) Group # Immunogen MS No. 0npi 3npi 6npi 9npi 0npi 3npi 6npi 9npi 1 p4579kb : Tau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 52 5811 0.105 0.000 0.000 0.000 0.093 0.000 0.000 0.000 5812 0.095 0.000 0.000 0.000 0.081 0.000 0.000 0.000 5813 0.074 0.752 0.000 0.000 0.078 0.000 0.000 0.000 Avg. 0.091 0.251 0.000 0.000 0.084 0.000 0.000 0.000 2 p4581kb: Tau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 55 5814 0.071 0.000 0.000 0.000 0.074 0.000 0.000 0.000 5815 0.070 0.000 0.000 0.000 0.075 0.000 0.000 0.000 5816 0.067 0.000 0.000 0.000 0.076 0.000 0.000 0.000 Avg. 0.069 0.000 0.000 0.000 0.075 0.000 0.000 0.000 3 p4583kb: Tau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 56 5817 0.080 2.922 2.659 1.231 0.084 0.000 0.000 0.000 5818 0.084 0.843 0.689 0.000 0.084 0.000 0.000 0.000 5819 0.092 2.483 1.984 0.033 0.097 0.000 0.000 0.000 Avg. 0.085 2.083 1.777 0.421 0.088 0.000 0.000 0.000 4 p4585kb: Tau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 58 5820 0.077 3.288 3.340 3.061 0.077 0.000 2.956 0.000 5821 0.076 1.958 0.627 0.000 0.088 0.000 0.000 0.000 5822 0.073 3.106 3.455 3.144 0.074 0.000 0.000 0.000 Avg. 0.075 2.784 2.474 2.068 0.080 0.000 0.985 0.000 5 p4588kb: Tau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 61 5823 0.109 6.026 6.512 6.387 0.080 0.000 0.000 0.000 5824 0.070 4,858 5.132 5.159 0.081 0.000 0.000 0.000 5845 0.142 5.287 5.314 7.040 0.082 0.000 0.000 0.000 Avg. 0.107 5,390 5.653 6.195 0.081 0.000 0.000 0.000 6 p4598kb: Tau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 63 5826 0.083 0.000 1.053 1.759 0.086 4.514 4.778 4.929 5827 0.094 0.000 0.218 2.044 0.098 4.966 5.146 5.138 5828 0.080 0.000 0.000 0.736 0.100 4,563 4,660 4.962 Avg. 0.086 0.000 0.424 1.513 0.095 4.681 4,861 5.010 7 p4600kb: Tau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 65 5829 0.089 0.000 0.000 0.000 0.084 0.000 0.000 0.000 5830 0.076 0.000 0.000 0.000 0.082 0.000 0.000 0.000 5831 0.073 0.000 0.000 0.000 0.082 0.000 0.000 0.000 Avg. 0.079 0.000 0.000 0.000 0.083 0.000 0.000 0.000 8 p4602kb: Tau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 67 5832 0.069 2.468 2.504 1.654 0.079 0.000 0.000 0.000 5833 0.093 1.952 2.677 2.274 0.083 0.000 0.000 0.000 5834 0.083 2.847 1.839 0.648 0.091 0.000 0.000 0.000 Avg. 0.082 2.422 2.340 1.525 0.084 0.000 0.000 0.000 9 p4604kb: Tau construct, UBITh1-εk-kkk-(V275-K311) SEQ ID NO: 69 5835 0.099 0.000 0.000 0.000 0.089 0.000 0.000 0.000 5836 0.086 0.000 0.000 0.000 0.077 0.000 0.000 0.000 5837 0.080 0.000 0.000 0.000 0.082 0.000 0.000 0.000 Avg. 0.088 0.000 0.000 0.000 0.083 0.000 0.000 0.000 10 p4605kb: Tau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 71 5838 0.077 2.752 3.484 4.146 0.076 0.000 0.000 0.000 5839 0.070 2.538 4.371 4.716 0.079 0.000 0.000 0.000 5840 0.075 2.951 4.301 4,570 0.080 0.000 0.000 0.000 Avg. 0.074 2.747 4.052 4.477 0.078 0.000 0.000 0.000

Table 9d

Analysis of the immunogenicity of Tau-peptide constructs

(Data correspond to Fig. 1A)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4600a (K257-K274)
Log ₁₀ titer ELISA
(SEQ ID NO: 15) Anti-p4602a (R379-L408)
Log ₁₀ titer ELISA
(SEQ ID NO: 17) Group # Immunogen MS No. 0npi 3npi 6npi 9npi 0npi 3npi 6npi 9npi 1 p4579kb : Tau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 52 5811 0.102 0.000 0.000 0.000 0.076 3.314 2.855 2.809 5812 0.102 0.000 0.000 0.000 0.065 2,881 1.857 1.207 5813 0.083 0.000 0.000 0.000 0.064 3.113 2.407 2.778 Avg. 0.096 0.000 0.000 0.000 0.068 3.103 2.373 2.265 2 p4581kb: Tau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 55 5814 0.093 0.000 0.000 0.000 0.069 3.036 2.075 0.330 5815 0.086 0.000 0.000 0.000 0.068 3.020 2.942 2.471 5816 0.103 0.000 0.000 0.000 0.073 2.791 2.711 2.393 Avg. 0.094 0.000 0.000 0.000 0.070 2.949 2.576 1.731 3 p4583kb: Tau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 56 5817 0.090 0.000 0.000 0.000 0.074 0.000 0.000 0.679 5818 0.087 0.000 0.000 0.000 0.083 0.000 0.000 0.000 5819 0.083 0.000 0.000 0.000 0.080 0.000 0.642 2.602 Avg. 0.087 0.000 0.000 0.000 0.079 0.000 0.214 1.094 4 p4585kb: Tau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 58 5820 0.078 0.000 0.000 0.000 0.073 1.094 2.307 2.171 5821 0.076 0.000 0.000 0.000 0.073 2.935 2.798 2.205 5822 0.080 0.000 0.000 0.000 0.071 3.865 3.386 3.227 Avg. 0.078 0.000 0.000 0.000 0.072 2.631 2,830 2.534 5 p4588kb: Tau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 61 5823 0.083 0.000 0.000 0.000 0.069 3.035 4.551 4,580 5824 0.084 0.000 0.000 0.000 0.079 3.032 2,980 3,100 5845 0.076 0.000 0.000 0.000 0.074 2,846 3.654 4.357 Avg. 0.081 0.000 0.000 0.000 0.074 2.971 3.728 4.012 6 p4598kb: Tau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 63 5826 0.091 0.000 0.000 0.000 0.082 3.187 3.971 4,583 5827 0.074 0.000 0.000 0.000 0.085 3.224 4.256 4.648 5828 0.073 0.000 0.000 0.000 0.075 1.762 2.510 3.068 Avg. 0.079 0.000 0.000 0.000 0.081 2.724 3.579 4,100 7 p4600kb: Tau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 65 5829 0.063 4.619 5.104 5.879 0.065 0.000 0.877 2.010 5830 0.067 5.125 9.486 8.478 0.069 0.000 0.000 0.000 5831 0.067 5.454 10,840 14.170 0.066 0.000 0.000 0.185 Avg. 0.066 5.066 8.477 9.509 0.067 0.000 0.292 0.732 8 p4602kb: Tau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 67 5832 0.071 0.000 0.000 1.879 0.205 8.520 8.112 6.557 5833 0.076 0.000 0.000 0.160 0.074 12.920 11,790 11,200 5834 0.080 0.000 0.000 2.851 0.085 9.382 10,350 8.024 Avg. 0.076 0.000 0.000 1.630 0.121 10.274 10.084 8.594 9 p4604kb: Tau construct, UBITh1-εk-kkk-(V275-K311) SEQ ID NO: 69 5835 0.086 0.000 0.000 0.000 0.083 4,050 5.398 4.995 5836 0.076 0.000 0.000 0.000 0.067 4.503 4.974 4,876 5837 0.074 0.000 0.000 0.000 0.068 5.233 4,940 4,574 Avg. 0.079 0.000 0.000 0.000 0.073 4.595 5.104 4.815 10 p4605kb: Tau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 71 5838 0.075 0.000 0.000 0.000 0.069 4.676 4.511 4,574 5839 0.070 0.000 0.000 0.000 0.064 4.722 4.686 4,868 5840 0.077 0.000 0.000 0.000 0.070 5.202 4.954 4,846 Avg. 0.074 0.000 0.000 0.000 0.068 4,867 4.717 4,763

Table 9e

Analysis of the immunogenicity of Tau-peptide constructs

(Data correspond to Fig. 1A)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4604a (V275-K311)
Log ₁₀ titer ELISA
(SEQ ID NO: 19) Anti-p4605a (V393-L425)
Log ₁₀ titer ELISA
(SEQ ID NO: 21) Group # Immunogen MS No. 0npi 3npi 6npi 9npi 0npi 3npi 6npi 9npi 1 p4579kb : Tau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 52 5811 0.084 0.000 0.000 0.000 0.088 0.000 0.000 0.000 5812 0.072 0.000 0.000 0.000 0.088 0.000 0.000 0.000 5813 0.075 0.000 0.000 0.000 0.083 0.000 0.000 0.000 Avg. 0.077 0.000 0.000 0.000 0.086 0.000 0.000 0.000 2 p4581kb: Tau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 55 5814 0.075 0.000 0.000 0.000 0.078 0.000 0.000 0.000 5815 0.072 0.000 0.000 1.342 0.070 0.000 0.000 0.000 5816 0.078 0.000 0.000 0.000 0.075 0.000 0.000 0.234 Avg. 0.075 0.000 0.000 0.447 0.074 0.000 0.000 0.078 3 p4583kb: Tau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 56 5817 0.090 0.000 0.000 0.000 0.083 0.000 0.000 0.000 5818 0.088 0.000 0.000 0.000 0.101 0.000 0.000 0.000 5819 0.086 0.000 0.000 0.000 0.090 0.000 0.000 0.301 Avg. 0.088 0.000 0.000 0.000 0.091 0.000 0.000 0.100 4 p4585kb: Tau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 58 5820 0.077 0.000 0.000 0.000 0.084 0.000 0.000 0.000 5821 0.074 0.000 0.000 0.000 0.080 0.000 0.578 0.182 5822 0.075 0.000 0.000 0.000 0.072 0.000 0.000 1.969 Avg. 0.075 0.000 0.000 0.000 0.079 0.000 0.193 0.717 5 p4588kb: Tau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 61 5823 0.074 0.000 0.000 0.000 0.074 4.755 5.129 5.495 5824 0.076 0.000 0.000 0.000 0.068 4.658 4,882 5.120 5845 0.081 0.000 0.000 0.000 0.082 4,583 4.943 5.727 Avg. 0.077 0.000 0.000 0.000 0.075 4.665 4.985 5.447 6 p4598kb: Tau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 63 5826 0.120 0.000 0.000 0.000 0.112 2,883 2.688 1,790 5827 0.097 0.000 0.000 0.000 0.090 2.910 2.322 1.864 5828 0.080 0.000 0.000 0.000 0.085 0.000 0.000 0.000 Avg. 0.099 0.000 0.000 0.000 0.096 1.931 1.670 1.218 7 p4600kb: Tau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 65 5829 0.082 0.000 0.000 0.000 0.078 0.000 0.000 0.000 5830 0.084 0.000 0.000 0.000 0.079 0.000 0.000 0.000 5831 0.084 0.000 0.000 0.000 0.069 0.000 0.000 0.000 Avg. 0.083 0.000 0.000 0.000 0.075 0.000 0.000 0.000 8 p4602kb: Tau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 67 5832 0.085 5.034 4.818 4,554 0.071 2.955 2.752 2.808 5833 0.095 5.333 4.969 4.957 0.083 3.885 4.090 4.557 5834 0.104 5.102 4.827 4.607 0.095 2.931 2,880 3.032 Avg. 0.095 5.156 4,871 4.706 0.083 3.257 3.241 3.466 9 p4604kb: Tau construct, UBITh1-εk-kkk-(V275-K311) SEQ ID NO: 69 5835 0.085 4.967 10.060 7.244 0.100 0.000 0.000 0.000 5836 0.079 4,896 7.084 6.091 0.090 0.000 0.000 0.000 5837 0.073 5.492 5,930 5.425 0.086 0.000 0.000 1.137 Avg. 0.079 5.118 7.691 6.253 0.092 0.000 0.000 0.379 10 p4605kb: Tau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 71 5838 0.079 0.000 0.000 0.000 0.090 5.709 5.234 5.387 5839 0.086 0.000 0.000 0.000 0.069 7.296 6.288 6.562 5840 0.084 0.000 0.000 0.000 0.113 5.513 5.446 5.673 Avg. 0.083 0.000 0.000 0.000 0.091 6.173 5.656 5,874

Table 10a

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

(Data correspond to Fig. 1B)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4578a (A145-P160)
Log ₁₀ titer ELISA
(SEQ ID NO: 1) Anti-p4580a (P172-P189)
Log ₁₀ titer ELISA
(SEQ ID NO: 3) Anti-p4582a (P182-P200)
Log ₁₀ titer ELISA
(SEQ ID NO: 5) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.110 5.936 5.193 0.087 4.949 4,856 0.083 3.147 3.229 5779 0.089 6.520 5.559 0.090 5.069 5.189 0.080 3.141 3.043 5780 0.096 6.844 5.642 0.083 4,920 5.013 0.065 2,887 2.904 Avg. 0.098 6.433 5.465 0.087 4.979 5.019 0.076 3.058 3.059 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.092 5.127 5.277 0.063 5,430 5.732 0.082 3.292 3.754 5782 0.097 5.035 5.003 0.083 5.401 5.331 0.088 4.421 4,394 5783 0.086 5.293 5,594 0.070 5.782 6.406 0.076 1.897 2.395 Avg. 0.092 5.152 5.291 0.072 5.538 5.823 0.082 3.203 3.514 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.164 4.056 4.521 0.095 3.932 4.344 0.089 4,650 4.929 5785 0.094 3.135 3.108 0.076 0.725 1.047 0.090 4,817 4,900 5786 0.089 5.049 4.981 0.090 4.988 4,920 0.083 5.512 5,843 Avg. 0.116 4.080 4.203 0.087 3.215 3.437 0.087 4.993 5.224 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.081 4.938 4.758 0.089 4.736 4.644 0.071 3.614 4.508 5788 0.091 4,694 4.414 0.085 4.071 4.276 0.067 3.009 3.446 5789 0.074 4.958 4.729 0.091 4.659 4.557 0.089 3.985 4.383 Avg. 0.082 4,863 4.634 0.088 4.489 4.492 0.076 3.536 4.112 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.085 4,670 4.442 0.092 4.714 4.382 0.084 4,350 4.064 5791 0.078 4.956 4,876 0.077 4.908 4,803 0.088 4.813 4,816 5792 0.078 5.534 6.162 0.095 5.489 6.171 0.083 5.139 6.607 Avg. 0.080 5.053 5.160 0.088 5.037 5.119 0.085 4.767 5.162 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.090 0.000 0.000 0.090 0.000 0.000 0.093 0.000 0.998 5794 0.090 0.000 0.000 0.087 0.000 0.000 0.098 2.279 1.536 5795 0.096 0.939 0.165 0.095 0.000 0.000 0.094 0.000 0.000 Avg. 0.092 0.313 0.055 0.091 0.000 0.000 0.095 0.760 0.845 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.102 0.000 0.000 0.092 0.000 0.000 0.086 0.000 0.000 5797 0.076 0.000 0.000 0.078 0.000 0.000 0.083 0.000 0.000 5798 0.082 0.000 0.000 0.081 0.000 3.108 0.080 0.000 0.000 Avg. 0.087 0.000 0.000 0.084 0.000 1.036 0.083 0.000 0.000 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.078 0.000 0.000 0.080 0.000 0.000 0.075 0.000 0.000 5800 0.080 0.000 0.000 0.084 0.000 0.000 0.095 0.000 0.000 5801 0.092 0.000 0.000 0.079 0.000 0.000 0.096 0.000 0.000 Avg. 0.083 0.000 0.000 0.081 0.000 0.000 0.089 0.000 0.000 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.086 2.493 1.345 0.087 2,680 1.766 0.093 0.000 0.000 5803 0.102 3.097 2.330 0.099 3.699 2.294 0.080 0.000 0.000 5804 0.089 3.213 2.922 0.085 2,930 1.135 0.076 0.000 0.000 Avg. 0.092 2.934 2.199 0.090 3.103 1.732 0.083 0.000 0.000 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.075 0.000 0.000 0.087 0.000 0.000 0.085 0.000 0.000 5806 0.085 0.000 0.000 0.084 0.000 0.000 0.081 0.000 0.000 5807 0.074 0.000 0.000 0.084 0.000 0.000 0.088 0.000 0.000 Avg. 0.078 0.000 0.000 0.085 0.000 0.000 0.085 0.000 0.000 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.080 0.000 0.000 0.092 0.000 0.000 0.103 0.000 0.000 5809 0.089 0.000 0.000 0.081 0.000 0.000 0.109 0.000 0.000 5810 0.081 0.000 0.000 0.069 0.000 0.000 0.096 0.000 0.000 Avg. 0.083 0.000 0.000 0.081 0.000 0.000 0.103 0.000 0.000

Table 10b

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

(Data correspond to Fig. 1B)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4584a (S195-P213)
Log ₁₀ titer ELISA
(SEQ ID NO: 9) Anti-p4586a (S195-P213)
Log ₁₀ titer ELISA
(SEQ ID NO: 7) Anti-p4587a (R209-L224)
Log ₁₀ titer ELISA
(SEQ ID NO: 10) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.075 4.442 4.006 0.070 2.921 3.053 0.069 0.000 0.000 5779 0.079 3,820 3.724 0.069 2.993 2.826 0.074 0.000 0.000 5780 0.094 4.014 4.077 0.066 2.961 3,050 0.066 0.000 0.000 Avg. 0.083 4.092 3.936 0.068 2.958 2.976 0.070 0.000 0.000 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.087 4.063 4.016 0.073 3.106 4.017 0.078 0.000 0.000 5782 0.085 4,398 4.372 0.072 4.221 4.168 0.075 0.000 0.000 5783 0.084 3.091 3.087 0.068 0.793 1.362 0.080 0.000 0.000 Avg. 0.085 3.851 3.825 0.071 2.707 3.182 0.078 0.000 0.000 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.069 3.216 4.245 0.063 2.992 3.818 0.086 0.000 0.000 5785 0.073 2.995 2.808 0.069 0.642 1.791 0.074 1.030 0.000 5786 0.076 4.758 5.304 0.078 4.538 5.010 0.081 2.568 2,820 Avg. 0.073 3.656 4.119 0.070 2.724 3,540 0.080 1.199 0.940 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.069 5.280 5.320 0.071 4,890 5.056 0.085 2.979 3.035 5788 0.071 6.587 7.196 0.069 5.232 6.139 0.073 4.435 4.492 5789 0.071 5.045 5.312 0.075 4.744 5.013 0.083 2.925 2,940 Avg. 0.070 5.637 5.943 0.072 4.955 5.403 0.080 3.446 3.489 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.076 5.185 5.545 0.083 5.475 5.314 0.086 3.082 2.865 5791 0.075 5.345 5.635 0.079 5.163 5.357 0.079 3.249 3.093 5792 0.066 5.701 8.875 0.073 5.547 9.583 0.091 4,832 4,670 Avg. 0.072 5.410 6.685 0.078 5.395 6.751 0.085 3.721 3.543 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.069 2.902 2.787 0.075 0.000 0.000 0.087 4,763 4,857 5794 0.078 2.983 3.088 0.069 2.326 2.836 0.074 4.725 4,864 5795 0.069 3.108 2.993 0.065 2.637 2.079 0.074 5,050 4.968 Avg. 0.072 2.998 2.956 0.070 1.654 1.638 0.078 4,846 4,896 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.082 0.000 0.000 0.071 0.000 0.000 0.078 0.685 3.032 5797 0.074 0.000 0.000 0.068 0.000 0.000 0.076 2.436 2,700 5798 0.080 0.000 0.000 0.066 0.000 0.000 0.081 0.000 0.000 Avg. 0.079 0.000 0.000 0.068 0.000 0.000 0.078 1.040 1.911 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.069 0.000 0.000 0.068 0.000 0.000 0.081 0.000 0.000 5800 0.071 0.000 0.000 0.081 0.000 0.000 0.086 0.000 0.000 5801 0.071 0.000 0.000 0.073 0.000 0.000 0.082 0.000 0.000 Avg. 0.070 0.000 0.000 0.074 0.000 0.000 0.083 0.000 0.000 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.068 0.000 0.000 0.071 0.424 0.000 0.088 0.764 2.819 5803 0.062 0.000 0.000 0.070 0.000 0.000 0.096 3.152 2,660 5804 0.058 2.905 1.915 0.066 2.973 1.427 0.104 2.802 0.621 Avg. 0.063 0.968 0.638 0.069 1.132 0.476 0.096 2.239 2.033 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.062 0.000 0.000 0.073 0.000 0.000 0.108 0.000 0.000 5806 0.061 0.000 0.000 0.068 0.000 0.000 0.104 0.000 0.000 5807 0.062 0.000 0.000 0.078 0.000 0.000 0.096 0.000 0.000 Avg. 0.062 0.000 0.000 0.073 0.000 0.000 0.103 0.000 0.000 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.074 0.000 0.000 0.102 0.000 0.000 0.111 3.418 4,861 5809 0.064 0.000 0.000 0.079 0.000 0.000 0.106 3.321 4,861 5810 0.061 0.000 0.000 0.079 0.000 0.000 0.071 3.925 3.423 Avg. 0.066 0.000 0.000 0.087 0.000 0.000 0.096 3.555 4.382

Table 10c

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

(Data correspond to Fig. 1B)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Anti-p4589a (V228-L243)
Log ₁₀ titer ELISA
(SEQ ID NO: 12) Anti-p4599a (K257-K274)
Log ₁₀ titer ELISA
(SEQ ID NO: 14) Anti-p4601a (R379-L408)
Log ₁₀ titer ELISA
(SEQ ID NO: 16) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.078 0.000 0.000 0.066 0.000 0.000 0.074 3.717 3.152 5779 0.100 0.000 0.000 0.071 0.000 0.000 0.077 3.186 3.033 5780 0.107 0.000 0.000 0.074 0.000 0.000 0.086 3.242 2.855 Avg. 0.095 0.000 0.000 0.070 0.000 0.000 0.079 3.382 3.013 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.102 0.000 0.000 0.069 0.000 0.000 0.078 3.139 3.211 5782 0.083 0.000 0.000 0.070 0.000 0.000 0.076 2.869 2,760 5783 0.093 0.000 0.000 0.067 0.000 0.000 0.082 2.823 3.188 Avg. 0.093 0.000 0.000 0.069 0.000 0.000 0.079 2.944 3.053 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.092 0.000 0.000 0.096 0.000 0.000 0.122 0.000 0.000 5785 0.097 0.000 0.000 0.082 0.000 0.000 0.118 0.000 0.000 5786 0.070 0.000 0.000 0.080 0.000 0.000 0.073 0.000 0.000 Avg. 0.086 0.000 0.000 0.086 0.000 0.000 0.104 0.000 0.000 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.090 0.000 0.000 0.084 0.000 0.000 0.071 4.639 4.505 5788 0.079 0.000 0.000 0.090 0.000 0.000 0.080 4.220 4.681 5789 0.079 0.000 0.000 0.085 0.000 0.000 0.076 3.268 3.687 Avg. 0.083 0.000 0.000 0.086 0.000 0.000 0.076 4.042 4.291 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.078 0.000 0.000 0.080 0.000 0.000 0.077 4,890 4.715 5791 0.078 0.000 0.000 0.079 0.000 0.000 0.078 4.375 4,300 5792 0.085 0.000 0.000 0.080 0.000 0.000 0.078 4.236 3.342 Avg. 0.080 0.000 0.000 0.080 0.000 0.000 0.078 4,500 4.119 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.095 3.155 1.806 0.115 0.000 0.000 0.108 4,500 4,740 5794 0.062 0.639 0.000 0.072 0.000 0.000 0.075 3.827 3.816 5795 0.062 2.892 2.388 0.070 0.000 0.000 0.079 4.045 4.326 Avg. 0.073 2.229 1.398 0.086 0.000 0.000 0.087 4.124 4.294 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.077 4.738 5.004 0.089 0.000 0.000 0.096 4.608 4,879 5797 0.063 4,970 5.077 0.073 0.000 0.000 0.076 4.752 4.739 5798 0.067 4.341 4,684 0.071 0.000 0.000 0.072 3.476 4,450 Avg. 0.069 4,683 4.922 0.078 0.000 0.000 0.081 4.279 4.689 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.083 0.000 0.000 0.073 10,440 >10 0.078 0.000 0.539 5800 0.242 0.000 0.000 0.095 6.378 5.732 0.085 0.000 0.000 5801 0.085 0.000 0.000 0.090 7.182 >10 0.097 0.000 0.000 Avg. 0.137 0.000 0.000 0.086 8,000 5.732 0.087 0.000 0.180 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.060 0.000 0.000 0.098 0.000 0.000 0.097 9.906 >10 5803 0.069 2,844 2.839 0.082 0.000 0.000 0.121 >10 >10 5804 0.071 2.693 2.027 0.098 0.000 0.000 0.100 >10 >10 Avg. 0.067 1.846 1.622 0.093 0.000 0.000 0.106 9.906 >10 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.068 0.000 0.000 0.081 0.000 0.000 0.094 4,664 5.271 5806 0.068 0.000 0.000 0.074 0.000 0.000 0.096 4.452 5.467 5807 0.069 0.000 0.000 0.082 0.000 0.000 0.092 4.758 5.015 Avg. 0.068 0.000 0.000 0.079 0.000 0.000 0.094 4.625 5.251 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.089 2.308 0.000 0.098 0.000 0.000 0.084 6.308 11,990 5809 0.094 4.224 3.237 0.107 0.000 0.000 0.115 8.761 9.667 5810 0.089 2.392 1.767 0.100 0.000 0.000 0.093 4,950 5.308 Avg. 0.091 2.975 1.668 0.102 0.000 0.000 0.097 6.673 8.988

Table 10d

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

(Data correspond to Fig. 1B)

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Log ₁₀ titer ELISA for anti-p4603a (V275-K311)
(SEQ ID NO: 18) Log ₁₀ titer ELISA for anti-p4606a (V393-L425)
(SEQ ID NO: 20) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.116 0.000 0.000 0.069 0.000 1.797 5779 0.111 0.000 0.000 0.070 0.000 2.797 5780 0.105 0.000 0.000 0.057 0.000 1.854 Avg. 0.111 0.000 0.000 0.065 0.000 2.149 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.107 0.000 0.000 0.055 0.000 0.000 5782 0.089 0.000 0.000 0.064 0.000 0.000 5783 0.090 0.000 0.000 0.066 0.000 0.000 Avg. 0.095 0.000 0.000 0.062 0.000 0.000 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.096 0.000 0.000 0.085 0.000 0.000 5785 0.275 0.000 0.000 0.065 0.000 0.000 5786 0.084 0.000 0.000 0.075 0.000 0.000 Avg. 0.152 0.000 0.000 0.075 0.000 0.000 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.087 0.000 0.000 0.070 0.000 0.000 5788 0.082 0.159 0.000 0.061 0.000 2.433 5789 0.079 0.000 0.000 0.058 0.000 0.000 Avg. 0.083 0.053 0.000 0.063 0.000 0.811 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.075 0.000 0.000 0.065 0.000 0.000 5791 0.082 0.000 0.000 0.072 0.000 0.000 5792 0.092 0.000 0.000 0.085 0.000 2.807 Avg. 0.083 0.000 0.000 0.074 0.000 0.936 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.076 0.000 0.000 0.075 0.000 4.829 5794 0.085 0.000 0.000 0.077 0.000 4.545 5795 0.083 0.000 0.000 0.077 0.000 4,818 Avg. 0.081 0.000 0.000 0.076 0.000 4.731 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.090 0.000 0.000 0.067 0.000 4.675 5797 0.072 0.000 0.000 0.226 0.000 4,394 5798 0.076 0.000 0.000 0.072 0.000 3.176 Avg. 0.079 0.000 0.000 0.122 0.000 4.082 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.078 0.000 0.000 0.075 0.000 0.000 5800 0.096 0.000 0.000 0.096 0.000 0.000 5801 0.080 0.000 0.000 0.087 0.000 0.000 Avg. 0.085 0.000 0.000 0.086 0.000 0.000 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.082 4,683 4.463 0.070 4.947 4.788 5803 0.084 5.542 5.047 0.079 4.993 4,910 5804 0.076 4.827 4.520 0.065 4.965 4.938 Avg. 0.081 5.017 4.677 0.071 4.968 4,879 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.071 5,080 >10 0.061 0.000 0.000 5806 0.074 4,898 >10 0.069 0.000 0.000 5807 0.078 4.942 10.260 0.072 0.000 0.000 Avg. 0.074 4.973 10.260 0.067 0.000 0.000 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.111 0.000 0.678 0.091 >10 >10 5809 0.080 0.000 0.000 0.089 >10 >10 5810 0.095 0.000 0.000 0.089 7,200 8.246 Avg. 0.095 0.000 0.226 0.090 7,200 8.246

Table 11a

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3, 6 npi

Anti-p4583a (P182-P200)
Log ₁₀ titer ELISA
(SEQ ID NO: 2) Anti-p4585a (S195-P213)
Log ₁₀ titer ELISA
(SEQ ID NO: 4) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.17 5.61 5.20 0.14 4.88 4.87 5779 0.14 5.70 5.73 0.10 4.88 5.05 5780 0.11 6.26 5.81 0.09 4.76 4.95 Avg. 0.14 5.86 5.58 0.11 4.84 4.96 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.11 5.01 5.15 0.11 5.28 5.94 5782 0.11 4.94 4.97 0.09 5.25 5.33 5783 0.11 5.18 5.18 0.09 5.79 6.06 Avg. 0.11 5.04 5.10 0.10 5.44 5.78 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.14 3.73 4.34 0.09 3.67 4.16 5785 0.12 2.95 2.85 0.10 0.00 0.00 5786 0.13 5.00 4.87 0.12 4.96 4.81 Avg. 0.13 3.89 4.02 0.10 2.88 2.99 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.11 4.71 4.64 0.09 4.57 4.60 5788 0.10 4.65 4.30 0.08 3.46 3.66 5789 0.16 4.65 4.42 0.08 4.54 4.38 Avg. 0.12 4.67 4.45 0.08 4.19 4.21 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.09 4.44 3.89 0.08 4.70 4.20 5791 0.10 4.71 4.61 0.09 4.83 4.69 5792 0.10 5.04 5.33 0.10 5.31 5.57 Avg. 0.10 4.73 4.61 0.09 4.95 4.82 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.11 0.00 0.00 0.10 0.00 0.00 5794 0.14 0.00 0.00 0.12 0.00 0.00 5795 0.11 0.14 0.00 0.10 0.00 0.00 Avg. 0.12 0.05 0.00 0.11 0.00 0.00 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.10 0.00 0.00 0.09 0.00 0.00 5797 0.09 0.00 0.00 0.08 0.00 0.00 5798 0.09 0.00 0.00 0.08 0.00 0.00 Avg. 0.09 0.00 0.00 0.08 0.00 0.00 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.09 0.00 0.00 0.09 0.00 0.00 5800 0.10 0.00 0.00 0.09 0.00 0.00 5801 0.10 0.00 0.00 0.10 0.00 0.00 Avg. 0.10 0.00 0.00 0.09 0.00 0.00 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.12 2.12 0.00 0.10 2.69 0.97 5803 0.10 3.05 1.36 0.08 2.36 0.00 5804 0.09 3.08 2.43 0.09 2.89 0.53 Avg. 0.10 2.75 1.26 0.09 2.65 0.50 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.08 0.00 0.00 0.08 0.00 0.00 5806 0.09 0.00 0.00 0.09 0.00 0.00 5807 0.09 0.00 0.00 0.09 0.00 0.00 Avg. 0.09 0.00 0.00 0.09 0.00 0.00 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.09 0.00 0.00 0.09 0.00 0.00 5809 0.10 0.00 0.00 0.10 0.00 0.00 5810 0.10 0.00 0.00 0.08 0.00 0.00 Avg. 0.10 0.00 0.00 0.09 0.00 0.00

Table 11b

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3, 6 npi

Anti-p4583a (P182-P200)
Log ₁₀ titer ELISA
(SEQ ID NO: 6) Anti-p4585a (S195-P213)
Log ₁₀ titer ELISA
(SEQ ID NO: 8) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.11 2.70 3.06 0.13 2.96 3.16 5779 0.09 2.29 2.70 0.11 2.69 2.94 5780 0.09 2.56 2.53 0.11 3.02 3.29 Avg. 0.10 2.52 2.76 0.12 2.89 3.13 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.09 2.45 3.32 0.12 2.78 3.09 5782 0.09 3.41 3.72 0.11 3.28 3.59 5783 0.10 0.97 1.11 0.11 0.72 0.30 Avg. 0.09 2.28 2.72 0.11 2.26 2.33 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.10 3.23 4.60 0.09 2.78 3.64 5785 0.10 2.74 4.31 0.13 1.24 0.87 5786 0.11 4.84 5.56 0.10 4.47 4.94 Avg. 0.10 3.60 4.82 0.11 2.83 3.15 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.09 3.00 4.41 0.10 5.06 5.43 5788 0.09 2.54 3.54 0.09 5.53 9.14 5789 0.09 3.08 3.84 0.10 4.78 5.14 Avg. 0.09 2.87 3.93 0.10 5.12 6.57 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.09 3.26 3.53 0.10 4.84 5.02 5791 0.09 4.38 4.73 0.09 4.82 5.10 5792 0.10 4.85 6.71 0.08 5.18 8.62 Avg. 0.09 4.16 4.99 0.09 4.95 6.25 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.11 0.00 0.00 0.10 2.59 2.12 5794 0.10 0.00 0.00 0.10 3.04 3.03 5795 0.09 0.00 0.00 0.10 2.82 2.49 Avg. 0.10 0.00 0.00 0.10 2.82 2.55 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.09 0.00 0.00 0.10 0.00 0.00 5797 0.08 0.00 0.00 0.09 0.00 0.00 5798 0.08 0.00 0.00 0.09 0.00 0.00 Avg. 0.08 0.00 0.00 0.09 0.00 0.00 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.09 0.00 0.00 0.08 0.00 0.00 5800 0.10 0.00 0.00 0.09 0.00 0.00 5801 0.11 0.00 0.00 0.10 0.00 0.00 Avg. 0.10 0.00 0.00 0.09 0.00 0.00 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.11 0.00 0.00 0.11 0.00 0.00 5803 0.10 0.00 0.00 0.09 0.00 0.00 5804 0.09 0.00 0.00 0.09 2.90 1.43 Avg. 0.10 0.00 0.00 0.10 0.97 0.48 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.08 0.00 0.00 0.09 0.00 0.00 5806 0.09 0.00 0.00 0.08 0.00 0.00 5807 0.09 0.00 0.00 0.08 0.00 0.00 Avg. 0.09 0.00 0.00 0.08 0.00 0.00 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.15 0.00 0.00 0.08 0.00 0.00 5809 0.11 0.00 0.00 0.10 0.00 0.00 5810 0.12 0.00 0.00 0.11 0.00 0.00 Avg. 0.13 0.00 0.00 0.10 0.00 0.00

Table 11c

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3, 6 npi

Anti-p4588a (R209-L224)
Log ₁₀ titer ELISA
(SEQ ID NO: 11) Anti-p4598a (V228-L243)
Log ₁₀ titer ELISA
(SEQ ID NO: 13) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.10 0.00 0.00 0.20 0.00 0.00 5779 0.09 0.00 0.00 0.11 0.00 0.00 5780 0.09 0.00 0.00 0.10 0.00 0.00 Avg. 0.09 0.00 0.00 0.14 0.00 0.00 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.10 0.00 0.00 0.10 0.00 0.00 5782 0.08 0.00 0.00 0.10 0.00 0.00 5783 0.08 0.00 0.00 0.11 0.00 0.00 Avg. 0.09 0.00 0.00 0.10 0.00 0.00 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.08 0.00 0.00 0.10 0.00 0.00 5785 0.10 0.18 0.00 0.11 0.00 0.00 5786 0.11 2.01 2.67 0.19 0.00 0.00 Avg. 0.10 0.73 0.89 0.13 0.00 0.00 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.10 3.13 2.83 0.10 0.00 0.00 5788 0.09 4.76 4.39 0.08 0.00 0.00 5789 0.10 1.33 2.51 0.09 0.00 0.00 Avg. 0.10 3.07 3.24 0.09 0.00 0.00 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.09 2.35 2.55 0.09 0.00 0.00 5791 0.08 3.33 3.26 0.09 0.00 0.00 5792 0.08 4.82 4.82 0.09 0.00 0.00 Avg. 0.08 3.50 3.54 0.09 0.00 0.00 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.10 5.01 5.11 0.09 0.00 0.00 5794 0.10 4.98 5.05 0.19 0.00 0.00 5795 0.09 5.27 5.22 0.09 2.62 1.56 Avg. 0.10 5.09 5.13 0.12 0.87 0.52 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.09 0.00 2.74 0.08 4.40 4.98 5797 0.09 0.65 1.98 0.09 4.77 5.16 5798 0.08 0.00 0.00 0.09 3.75 4.68 Avg. 0.09 0.22 1.57 0.09 4.31 4.94 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.08 0.00 0.00 0.08 0.00 0.00 5800 0.08 0.00 0.00 0.10 0.00 0.00 5801 0.10 0.00 0.00 0.10 0.00 0.00 Avg. 0.09 0.00 0.00 0.09 0.00 0.00 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.09 0.00 2.28 0.18 0.00 0.00 5803 0.08 2.95 1.69 0.09 0.00 1.47 5804 0.08 2.26 0.00 0.08 0.00 0.83 Avg. 0.08 1.74 1.32 0.12 0.00 0.77 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.09 0.00 0.00 0.08 0.00 0.00 5806 0.08 0.00 0.00 0.09 0.00 0.00 5807 0.09 0.00 0.00 0.09 0.00 0.00 Avg. 0.09 0.00 0.00 0.09 0.00 0.00 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.08 3.29 4.85 0.09 0.00 0.00 5809 0.11 3.24 4.90 0.10 2.60 0.85 5810 0.09 3.16 3.17 0.10 0.00 0.00 Avg. 0.09 3.23 4.31 0.10 0.87 0.28

Table 11d

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3, 6 npi

Anti-p4600a (K257-K274)
Log ₁₀ titer ELISA
(SEQ ID NO: 15) Anti-p4602a (R379-L408)
Log ₁₀ titer ELISA
(SEQ ID NO: 17) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.17 0.00 0.00 0.20 3.48 2.87 5779 0.09 0.00 0.00 0.10 3.10 2.93 5780 0.08 0.00 0.00 0.08 3.23 2.62 Avg. 0.11 0.00 0.00 0.13 3.27 2.81 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.08 0.00 0.00 0.08 2.88 2.67 5782 0.08 0.00 0.00 0.08 0.32 0.00 5783 0.08 0.00 0.00 0.08 2.23 2.04 Avg. 0.08 0.00 0.00 0.08 1.81 1.57 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.12 0.00 0.00 0.08 0.00 0.00 5785 0.10 0.00 0.00 0.09 0.00 0.00 5786 0.18 0.00 0.00 0.19 0.00 0.00 Avg. 0.13 0.00 0.00 0.12 0.00 0.00 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.09 0.00 0.00 0.09 2.91 2.84 5788 0.08 0.00 0.00 0.09 2.86 3.42 5789 0.08 0.00 0.00 0.08 1.88 0.93 Avg. 0.08 0.00 0.00 0.09 2.55 2.40 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.09 0.00 0.00 0.08 2.99 2.83 5791 0.08 0.00 0.00 0.08 4.20 4.03 5792 0.09 0.00 0.00 0.09 3.23 2.98 Avg. 0.09 0.00 0.00 0.08 3.47 3.28 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.10 0.00 0.00 0.10 3.52 4.45 5794 0.18 0.00 0.00 0.15 2.71 2.85 5795 0.09 0.00 0.00 0.08 3.08 3.43 Avg. 0.12 0.00 0.00 0.11 3.10 3.58 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.10 0.00 0.00 0.08 3.62 4.56 5797 0.08 0.00 0.00 0.09 4.48 4.34 5798 0.08 0.00 0.00 0.07 3.11 3.76 Avg. 0.09 0.00 0.00 0.08 3.74 4.22 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.09 5.62 14.11 0.07 0.00 0.00 5800 0.10 5.84 9.55 0.09 0.00 0.00 5801 0.10 5.51 11.39 0.09 0.00 0.00 Avg. 0.10 5.66 11.68 0.08 0.00 0.00 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.18 0.00 0.00 0.15 7.75 6.71 5803 0.10 0.00 0.00 0.09 13.34 12.63 5804 0.08 0.00 0.00 0.09 9.08 9.59 Avg. 0.12 0.00 0.00 0.11 10.06 9.64 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.08 0.00 0.00 0.09 4.71 5.55 5806 0.08 0.00 0.00 0.08 4.53 5.34 5807 0.09 0.00 0.00 0.09 4.60 4.91 Avg. 0.08 0.00 0.00 0.09 4.61 5.27 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.09 0.00 0.00 0.10 3.94 4.40 5809 0.10 0.00 0.00 0.10 2.81 3.41 5810 0.11 0.00 0.00 0.09 3.36 3.44 Avg. 0.10 0.00 0.00 0.10 3.37 3.75

Table 11e

Analysis of the immunogenicity of phosphorylated Tau peptide constructs

Adjuvant: ISA 51VG; CpG3 50 µg/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/im (four injection sites, 0.25 ml/site); Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3, 6 npi

Anti-p4604a (V275-K311)
Log ₁₀ titer ELISA
(SEQ ID NO: 19) Anti-p4605a (V393-L425)
Log ₁₀ titer ELISA
(SEQ ID NO: 21) Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 1 p4578kb: fTau construct, UBITh1-εk-kkk-(A145-P160) SEQ ID NO: 51 5778 0.10 0.00 0.00 0.16 0.00 0.31 5779 0.09 0.00 0.00 0.09 0.00 0.00 5780 0.08 0.00 0.00 0.09 0.00 1.89 Avg. 0.09 0.00 0.00 0.11 0.00 0.73 2 p4580kb: fTau construct, UBITh1-εk-kkk-(P172-P189) SEQ ID NO: 53 5781 0.09 0.00 0.00 0.10 0.00 0.00 5782 0.08 0.00 0.00 0.09 0.00 0.00 5783 0.10 0.00 0.00 0.09 0.00 0.00 Avg. 0.09 0.00 0.00 0.09 0.00 0.00 3 p4582kb: fTau construct, UBITh1-εk-kkk-(P182-P200) SEQ ID NO: 55 5784 0.09 0.00 0.00 0.11 0.00 0.00 5785 0.11 0.00 0.00 0.10 0.00 0.00 5786 0.10 0.00 0.00 0.09 0.00 0.00 Avg. 0.10 0.00 0.00 0.10 0.00 0.00 4 p4584kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 57 5787 0.09 0.00 0.00 0.09 0.00 0.00 5788 0.08 0.00 0.00 0.10 0.00 0.00 5789 0.09 0.00 0.00 0.10 0.00 0.00 Avg. 0.09 0.00 0.00 0.10 0.00 0.00 5 p4586kb: fTau construct, UBITh1-εk-kkk-(S195-P213) SEQ ID NO: 59 5790 0.09 0.00 0.00 0.10 0.00 0.00 5791 0.09 0.00 0.00 0.11 0.00 0.00 5792 0.09 0.00 0.00 0.13 0.13 2.11 Avg. 0.09 0.00 0.00 0.11 0.04 0.70 6 p4587kb: fTau construct, UBITh1-εk-kkk-(R209-L224) SEQ ID NO: 60 5793 0.11 0.00 0.00 0.12 4.38 4.78 5794 0.10 0.00 0.00 0.12 4.38 4.47 5795 0.08 0.00 0.00 0.10 4.38 4.76 Avg. 0.10 0.00 0.00 0.11 4.38 4.67 7 p4589kb: fTau construct, UBITh1-εk-kkk-(V228-L243) SEQ ID NO: 62 5796 0.08 0.00 0.00 0.11 4.37 4.23 5797 0.09 0.00 0.00 0.10 4.33 4.51 5798 0.09 0.00 0.00 0.09 2.58 1.70 Avg. 0.09 0.00 0.00 0.10 3.76 3.48 8 p4599kb: fTau construct, UBITh1-εk-kkk-(K257-K274) SEQ ID NO: 64 5799 0.09 0.00 0.00 0.10 0.00 0.00 5800 0.10 0.00 0.00 0.11 0.00 0.00 5801 0.11 1.57 0.00 0.11 0.00 0.00 Avg. 0.10 0.52 0.00 0.11 0.00 0.00 9 p4601kb: fTau construct, UBITh1-εk-kkk-(R379-L408) SEQ ID NO: 66 5802 0.10 4.40 4.00 0.08 1.48 2.92 5803 0.09 5.10 4.90 0.09 3.03 3.17 5804 0.09 4.60 4.20 0.09 2.96 3.00 Avg. 0.09 4.70 4.37 0.09 2.49 3.03 10 p4603kb: fTau construct, UBITh1-εk-kkk-(V275-K311, P301→S301) SEQ ID NO: 68 5805 0.10 4.90 12.50 0.10 0.00 0.00 5806 0.10 4.80 13.10 0.09 0.00 0.00 5807 0.10 4.80 7.40 0.10 0.00 0.00 Avg. 0.10 4.83 11.00 0.10 0.00 0.00 eleven p4606kb: fTau construct, UBITh1-εk-kkk-(V393-L425) SEQ ID NO: 70 5808 0.10 0.00 0.00 0.11 10.14 13.15 5809 0.13 0.00 0.00 0.12 10.14 12.89 5810 0.09 0.00 0.00 0.09 5.87 6.92 Avg. 0.11 0.00 0.00 0.11 8.72 10.99

Table 12

Analysis of the immunogenicity of Tau-peptide constructs containing a B-epitope from the site of Tau hyperphosphorylation and aggregation

Adjuvant: ISA 51VG; CpG3: 50 μm/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/m four injection sites, 0.25 ml/site; Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Log ₁₀ ELISA titer for anti-p5031a (131-141)
SEQ ID NO: 101 Log ₁₀ titer ELISA for anti-p5032a (241-257)
SEQ ID NO: 102 Log ₁₀ ELISA titer for anti-p5033a (243-259)
SEQ ID NO: 103 Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 1 p5031kb
UBITh1-εk-kkk-Tau 131-141
SEQ ID NO: 125 6670 0.245 3.110 4.985 0.072 0.000 3.373 0.073 0.000 0.000 6671 0.291 4.538 5.765 0.083 2.309 4.448 0.069 0.988 3.366 6672 0.420 3.094 4,640 0.079 0.728 3.971 0.068 1.741 4,446 Avg. 0.319 3.581 5.130 0.078 1.012 3.931 0.070 0.910 2.604 2 p5032kb
UBITh1-εk-kkk-Tau 241-257
SEQ ID NO: 126 6673 0.565 2.622 0.000 0.089 4,960 5.276 0.091 4.415 4.737 6674 0.336 2.691 0.000 0.084 >10 10.66 0.104 5.228 4.904 6675 0.174 2.595 0.000 0.076 >10 >10 0.071 5.488 5.266 Avg. 0.358 2.636 0.000 0.083 4,960 7.966 0.089 5.044 4.969 3 p5033kb
UBITh1-εk-kkk-Tau 243-259
SEQ ID NO: 127 6676 0.162 0.095 0.000 0.065 5.063 5.085 0.060 4.979 5.225 6677 0.202 1.374 0.000 0.065 4,694 4,784 0.065 4.644 4,893 6678 0.306 2.219 0.000 0.075 5.088 5.055 0.063 5.557 5.968 Avg. 0.223 1.229 0.000 0.068 4.948 4.975 0.063 5.060 5.362

Table 13a

Analysis of the immunogenicity of Tau peptide constructs derived from the B epitope from the Tau region 275-311

Adjuvant: ISA 51VG; CpG3: 50 μm/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/m four injection sites, 0.25 ml/site; Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Log ₁₀ titer ELISA for anti-p5134a (275-281)
SEQ ID NO: 104 Log ₁₀ titer ELISA for anti-p5135a (281-294)
SEQ ID NO: 105 Log ₁₀ titer ELISA for anti-p5136a (275-294)
SEQ ID NO: 106 Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 1 p5134kb
UBITh1-εk-kkk-Tau 275-281
SEQ ID NO: 128 6742 0.193 0.000 2,847 0.113 0.113 0.000 0.113 0.000 2.973 6743 0.192 0.284 3.255 0.102 0.102 1.240 0.102 0.648 3.160 6744 0.181 1.133 2.164 0.109 0.109 5.208 0.109 4.416 5.134 Avg. 0.189 0.472 2.755 0.108 0.108 2.149 0.108 1.688 3.756 2 p5135kb
UBITh1-εk-kkk-Tau 281-294
SEQ ID NO: 129 6745 0.160 1.466 1.327 0.110 0.110 7.273 0.110 10.235 7,100 6746 0.587 1,100 2.755 0.150 0.150 5.29 0.150 5.086 5.397 6747 0.269 1.509 3.056 0.103 0.103 7.733 0.103 9.398 8.508 Avg. 0.339 1.358 2.379 0.121 0.121 6.765 0.121 8.240 7.002 3 p5136kb
UBITh1-εk-kkk-Tau 275-294
SEQ ID NO: 130 6748 0.485 0.000 1.848 0.126 0.126 6.723 0.126 5.107 >10 6749 0.232 1.555 1.048 0.149 0.149 7.746 0.149 6,862 >10 6750 0.144 0.000 2.797 0.101 0.101 5.182 0.101 5.134 5.488 Avg. 0.287 0.518 1.898 0.125 0.125 6,550 0.125 5.701 5.488 4 p5137kb
UBITh1-εk-kkk-Tau 294-311
SEQ ID NO: 131 6751 0.339 2.714 2.356 0.125 0.125 0.377 0.125 3.116 2.954 6752 0.213 2,800 2.552 0.122 0.122 1.243 0.122 0.509 2.442 6753 0.201 2.701 2.709 0.101 0.101 0.000 0.101 3.522 3.743 Avg. 0.251 2.738 2.539 0.116 0.116 0.540 0.116 2.382 3.046 5 p5138kb
UBITh1-εk-kkk-Tau 294-311, P301 -> S301
SEQ ID NO: 132 6754 0.222 2.829 2.985 0.154 0.154 2.201 0.154 1.348 2.159 6755 0.378 2.971 3.121 0.106 0.106 1.361 0.106 0.000 0.000 6756 0.454 3.012 2.823 0.127 0.127 1.513 0.127 3.343 3.065 Avg. 0.351 2.937 2.976 0.129 0.129 1.692 0.129 1.564 1.741

Table 13b

Log ₁₀ titer ELISA for anti-p5137a (294-311)
SEQ ID NO: 107 Anti-p5138a (294-311, P301->S301)
Log ₁₀ titer ELISA
SEQ ID NO: 108 Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 1 p5134kb
UBITh1-εk-kkk-Tau 275-281
SEQ ID NO: 128 6742 0.120 0.000 0.000 0.124 0.000 0.000 6743 0.104 0.000 0.000 0.125 0.000 0.000 6744 0.114 1.075 4.557 0.096 0.804 4.514 Avg. 0.113 0.358 1.519 0.115 0.268 1.505 2 p5135kb
UBITh1-εk-kkk-Tau 281-294
SEQ ID NO: 129 6745 0.092 0.000 0.000 0.095 0.000 0.000 6746 0.068 0.000 0.000 0.097 0.000 0.000 6747 0.102 0.000 0.000 0.146 0.000 0.000 Avg. 0.087 0.000 0.000 0.113 0.000 0.000 3 p5136kb
UBITh1-εk-kkk-Tau 275-294
SEQ ID NO: 130 6748 0.085 0.000 0.805 0.117 0.000 0.712 6749 0.086 2.787 2,896 0.096 2.892 2.715 6750 0.105 0.000 2.448 0.110 0.000 2.466 Avg. 0.092 0.929 2,050 0.108 0.964 1.964 4 p5137kb
UBITh1-εk-kkk-Tau 294-311
SEQ ID NO: 131 6751 0.097 4,910 5.151 0.130 4.927 5.122 6752 0.112 5.015 5.130 0.099 5.035 5.136 6753 0.111 4,999 5.236 0.088 5.001 5.199 Avg. 0.107 4.975 5.172 0.105 4.988 5.152 5 p5138kb
UBITh1-εk-kkk-Tau 294-311, P301 -> S301
SEQ ID NO: 132 6754 0.086 4,851 5.185 0.103 4,896 5.405 6755 0.077 4.997 11.375 0.125 5.078 11,562 6756 0.080 4.945 4.957 0.108 4,960 5.034 Avg. 0.081 4.931 7.172 0.112 4.978 7.334

Table 14

Analysis of the immunogenicity of Tau peptide constructs from the N-terminal "fuzzy coating" of Tau

Adjuvant: ISA 51VG; CpG3: 50 μm/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/m four injection sites, 0.25 ml/site; Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Log ₁₀ titer ELISA for anti-p5160a (D22-D34)
SEQ ID NO: 112 Log ₁₀ titer ELISA for anti-p5161a (Y18-D34)
SEQ ID NO: 113 Log ₁₀ ELISA titer for anti-p5162a (D22-D38)
SEQ ID NO: 114 Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 1 p5160kb
UBITh1-εK-KKK-Tau D22-D34
SEQ ID NO: 137 6898 0.190 4.913 5,564 0.080 5.943 9.261 0.075 3.303 4.186 6899 0.205 4.786 5.002 0.071 5.356 5.206 0.084 3.316 4.188 6900 0.205 4,844 5.016 0.073 5.047 5.343 0.070 0.000 3.392 Avg. 0.200 4,848 5.194 0.075 5.449 6.603 0.076 2.206 3.922 2 p5161kb
UBITh1-εK-KKK-Tau Y18-D34
SEQ ID NO: 138 6901 0.327 5.017 7.534 0.079 6,650 10.454 0.064 3.264 4,380 6902 0.135 3.461 4,870 0.057 4.783 5.189 0.056 2.164 3.982 6903 0.310 3.643 5.048 0.070 4,887 5.339 0.059 1.919 3.472 Avg. 0.257 4.040 5.817 0.069 5,440 6.994 0.060 2.449 3.945 3 p5162kb
UBITh1-εK-KKK-Tau D22-D38
SEQ ID NO: 139 6904 0.159 3.471 4.139 0.062 4,883 4,862 0.055 4,600 5.408 6905 0.264 3.092 3.683 0.076 4.663 4.696 0.071 4,694 5.228 6906 0.117 3.316 4.505 0.081 4.826 5.004 0.063 3.726 5.091 Avg. 0.180 3.293 4.109 0.073 4.791 4,854 0.063 4,340 5.242

Table 15

Immunogenicity assays for Tau peptide constructs containing P301 trans from a position prone to Tau aggregation

Adjuvant: ISA 51VG; CpG3: 50 μm/ml; 0.2% Twin 80

Dose of immunogen: Prime: 400 mcg/1.0 ml/dose/m four injection sites, 0.25 ml/site; Boost: 100 mcg/0.25 ml/dose/IM

Vaccination schedule: 0, 3 and 6 npi

Log ₁₀ ELISA titer for anti-p5159a (275-311, p301 trans)
SEQ ID NO: 110 Log ₁₀ titer ELISA for anti-p5183a (297-311, p301 trans)
SEQ ID NO: 111 Log ₁₀ titer ELISA for anti-p5137a (294-311)
SEQ ID NO: 107 Log ₁₀ titer ELISA for anti-p4604a (275-311)
SEQ ID NO: 19 Group # Immunogen MS No. 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 0npi 3npi 6npi 1 p5158kb
UBITh1-εK-KKK-K294-K311, p301 trans
SEQ ID NO: 135 6919 0.094 5,080 5,866 0.064 2.813 4.759 0.075 3.727 5.083 0npi 3npi 6npi 6920 0.078 > 10 13.839 0.086 4,850 4.913 0.095 5.533 5.217 0.069 5.069 6.126 6921 2.312 5.113 6.209 0.078 2.227 4.753 0.093 3.991 5.033 0.101 > 10 11.667 Avg. 0.828 5.097 8.638 0.076 3.297 4,808 0.088 4.417 5.111 0.073 5.209 6.836 2 p5159kb
UBITh1-εK-KKK-V275-K311, p301 trans
SEQ ID NO: 136 6922 0.097 5.532 5.772 0.090 4,683 4.774 0.080 4.911 5.006 0.081 5.139 8.210 6923 0.084 7.204 > 10 0.099 4.687 5,000 0.086 4.975 5,540 0.075 5.604 5.938 6924 0.068 5.105 8.242 0.069 2.709 4.729 0.072 3.425 4.991 0.096 7.207 > 10 Avg. 0.083 5.947 7.007 0.086 4.026 4,834 0.080 4.437 5.179 0.074 5.143 9.951 3 p5183kb
UBITh1-εK-KKK-I297-K311, p301 trans
SEQ ID NO: 140 6925 0.086 5,834 7.915 0.085 4.944 5.068 0.071 4.975 5.195 0.081 5.985 7.945 6926 0.063 5.245 13.971 0.072 3.510 5.111 0.071 4,898 5.792 0.061 5.991 8.297 6927 0.077 5.446 6.285 0.069 4.525 4.792 0.063 4.829 5.118 0.059 5.307 13.885 Avg. 0.075 5.508 9.390 0.076 4.326 4,990 0.068 4,901 5.368 0.062 5.574 6.294 4 p5158kb, I297-K311, p301 trans
KKK-εK-UBITh1
SEQ ID NO: 141 6928 0.071 3.613 5.096 0.089 2.818 3.646 0.074 1.703 4.023 0.060 5.624 9.492 6929 0.084 4.787 5.038 0.079 3.364 3.898 0.107 3.287 4,563 0.063 3.536 5.039 6930 0.079 4.959 5.109 0.077 3.506 4.645 0.078 3.005 4.781 0.065 4,806 5.058 Avg. 0.078 4.453 5.081 0.082 3.229 4.063 0.086 2.665 4.456 0.070 4.787 5.091

Table 16

Localization of the immunogenic site within Tau 275-311 through precise epitope mapping

--->

SEQUENCE LIST

<110> United Neuroscience

UNS IP Holdings, LLC

Wang, Chang Yi

<120> TAU-PEPTIDE IMMUNOGENIC CONSTRUCTS

<130> 1008578.102WO2 (UNS1002-TW)

<140> TBD

<141> 26.10.2018

<150> US 62/578 124

<151> 27.10.2017

<160> 171

<170> PatentIn version 3.5

<210> 1

<211> 16

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> fTau-peptide - ak145-160

(pT149/pT153)

<220>

<221> MOD_RES

<222> (5)..(5)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222>(9)..(9)

<223> PHOSPHORYLATION

<400> 1

Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro

1 5 10 15

<210> 2

<211> 16

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> Tau-peptide - ak145-160

<400> 2

Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro

1 5 10 15

<210> 3

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> fTau-peptide - ak172-189

(pT175/pT181/pS185)

<220>

<221> MOD_RES

<222> (4)..(4)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (10)..(10)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (14)..(14)

<223> PHOSPHORYLATION

<400> 3

Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Ser Ser Ser Gly Glu

1 5 10 15

Pro Pro

<210> 4

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Tau-peptide - ak172-189

<400> 4

Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Ser Ser Ser Gly Glu

1 5 10 15

Pro Pro

<210> 5

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> fTau-peptide - ak182-200

(pS184/pY197)

<220>

<221> MOD_RES

<222> (3)..(3)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (16)..(16)

<223> PHOSPHORYLATION

<400> 5

Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr

1 5 10 15

Ser Ser Pro

<210> 6

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Tau-peptide - ak182-200

<400> 6

Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr

1 5 10 15

Ser Ser Pro

<210> 7

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> fTau-peptide - ak195-213

(pS198/pS208/pS210)

<220>

<221> MOD_RES

<222> (4)..(4)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (14)..(14)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (16)..(16)

<223> PHOSPHORYLING

<400> 7

Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser

1 5 10 15

Arg Thr Pro

<210> 8

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Tau-peptide - ak195-213

<400> 8

Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser

1 5 10 15

Arg Thr Pro

<210> 9

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> fTau-peptide - ak195-213

(pS199/pS202/pT205)

<220>

<221> MOD_RES

<222> (5)..(5)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222>(8)..(8)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (11)..(11)

<223> PHOSPHORYLATION

<400> 9

Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser

1 5 10 15

Arg Thr Pro

<210> 10

<211> 16

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> fTau-peptide - ak209-224

(pT212/pS214/pT217)

<220>

<221> MOD_RES

<222> (4)..(4)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222>(6)..(6)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222>(9)..(9)

<223> PHOSPHORYLATION

<400> 10

Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys

1 5 10 15

<210> 11

<211> 16

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> tau-peptide - ak209-224

<400> 11

Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys

1 5 10 15

<210> 12

<211> 16

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> fTau-peptide - ak228-243

(pT231/pS235/pS238)

<220>

<221> MOD_RES

<222> (4)..(4)

<223> PHOSPHORYLING

<220>

<221> MOD_RES

<222>(8)..(8)

<223> PHOSPHORYLING

<220>

<221> MOD_RES

<222> (11)..(11)

<223> PHOSPHORYLING

<400> 12

Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu

1 5 10 15

<210> 13

<211> 16

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> tau-peptide - ak228-243

<400> 13

Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg Leu

1 5 10 15

<210> 14

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> fTau-peptide - ak257-274

(ps262)

<220>

<221> MOD_RES

<222>(6)..(6)

<223> PHOSPHORYLING

<400> 14

Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly

1 5 10 15

Gly Lys

<210> 15

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Tau-peptide - ak257-274

<400> 15

Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly

1 5 10 15

Gly Lys

<210> 16

<211> 30

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(30)

<223> fTau-peptide - ak379-408

(pS396/pS404)

<220>

<221> MOD_RES

<222> (18)..(18)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (26)..(26)

<223> PHOSPHORYLATION

<400> 16

Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr

1 5 10 15

Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu

20 25 30

<210> 17

<211> 30

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(30)

<223> Tau-peptide - a379-408

<400> 17

Arg Glu Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr

1 5 10 15

Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu

20 25 30

<210> 18

<211> 37

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(37)

<223> fTau-peptide - ak275-311

*P301->S

(pS293/pS301)

<220>

<221> MOD_RES

<222> (19)..(19)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (27)..(27)

<223> PHOSPHORYLATION

<400> 18

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser Lys Asp Asn Ile Lys His Val Ser Gly Gly Gly Ser Val

20 25 30

Gln Ile Val Tyr Lys

35

<210> 19

<211> 37

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(37)

<223> Tau-peptide - ak275-311

<400> 19

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val

20 25 30

Gln Ile Val Tyr Lys

35

<210> 20

<211> 33

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(33)

<223> fTau-peptide - ak393-425

(pS396/pS404/pS422)

<220>

<221> MOD_RES

<222> (4)..(4)

<223> PHOSPHORYLING

<220>

<221> MOD_RES

<222> (12)..(12)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (30)..(30)

<223> PHOSPHORYLATION

<400> 20

Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu

1 5 10 15

Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln

20 25 30

Leu

<210> 21

<211> 33

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(33)

<223> tau-peptide - ak393-425

<400> 21

Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu

1 5 10 15

Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln

20 25 30

Leu

<210> 22

<211> 17

<212> Protein

<213> Clostridium tetani

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Th Clostridium tetani 1

<400> 22

Lys Lys Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu

1 5 10 15

Leu

<210> 23

<211> 15

<212> Protein

<213> measles virus

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Th MvF1

<400> 23

Leu Ser Glu Ile Lys Gly Val Ile Val His Arg Leu Glu Gly Val

1 5 10 15

<210> 24

<211> 24

<212> Protein

<213> Bordetella pertussis

<220>

<221> PEPTIDE

<222> (1)..(24)

<223> Th Bordetella pertussis

<400> 24

Gly Ala Tyr Ala Arg Cys Pro Asn Gly Thr Arg Ala Leu Thr Val Ala

1 5 10 15

Glu Leu Arg Gly Asn Ala Glu Leu

20

<210> 25

<211> 17

<212> Protein

<213> Clostridium tetani

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Th Clostridium tetani 2

<400> 25

Trp Val Arg Asp Ile Ile Asp Asp Phe Thr Asn Glu Ser Ser Gln Lys

1 5 10 15

Thr

<210> 26

<211> 23

<212> Protein

<213> diphtheria bacilli

<220>

<221> PEPTIDE

<222> (1)..(23)

<223> Th Diphtheria

<400> 26

Asp Ser Glu Thr Ala Asp Asn Leu Glu Lys Thr Val Ala Ala Leu Ser

1 5 10 15

Ile Leu Pro Gly His Gly Cys

20

<210> 27

<211> 21

<212> Protein

<213> Plasmodium falciparum

<220>

<221> PEPTIDE

<222> (1)..(21)

<223> Th Plasmodium falciparum

<400> 27

Asp His Glu Lys Lys His Ala Lys Met Glu Lys Ala Ser Ser Val Phe

1 5 10 15

Asn Val Val Asn Ser

20

<210> 28

<211> 17

<212> Protein

<213> Schistosoma mansoni

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Th Schistosoma mansoni

<400> 28

Lys Trp Phe Lys Thr Asn Ala Pro Asn Gly Val Asp Glu Lys His Arg

1 5 10 15

His

<210> 29

<211> 25

<212> Protein

<213> cholera toxin

<220>

<221> PEPTIDE

<222> (1)..(25)

<223> Th cholera toxin

<400> 29

Ala Leu Asn Ile Trp Asp Arg Phe Asp Val Phe Cys Thr Leu Gly Ala

1 5 10 15

Thr Thr Gly Tyr Leu Lys Gly Asn Ser

20 25

<210> 30

<211> 15

<212> Protein

<213> measles virus

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Th MvF 2

<400> 30

Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile Glu Gly Ile

1 5 10 15

<210> 31

<211> 22

<212> Protein

<213> measles virus

<220>

<221> PEPTIDE

<222> (1)..(22)

<223> Th KKKMvF 3

<220>

<221> Protein

<222>(7)..(7)

<223> S or T

<220>

<221> Protein

<222> (10)..(10)

<223> K or R

<220>

<221> Protein

<222> (11)..(11)

<223> G or T

<220>

<221> Protein

<222> (15)..(15)

<223> H or T

<220>

<221> Protein

<222> (16)..(16)

<223> K or R

<220>

<221> Protein

<222> (19)..(19)

<223> G or T

<400> 31

Lys Lys Lys Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa

1 5 10 15

Ile Glu Xaa Ile Leu Phe

20

<210> 32

<211> 18

<212> Protein

<213> hepatitis B virus

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Th HBsAg 1

<220>

<221> Protein

<222> (1)..(1)

<223> K or R

<220>

<221> Protein

<222> (2)..(2)

<223> K or R

<220>

<221> Protein

<222> (3)..(3)

<223> K or R

<220>

<221> Protein

<222> (4)..(4)

<223> L or I or V or F

<220>

<221> Protein

<222> (5)..(5)

<223> F or K or R

<220>

<221> Protein

<222>(6)..(6)

<223> L or I or V or F

<220>

<221> Protein

<222>(7)..(7)

<223> L or I or V or F

<220>

<221> Protein

<222>(9)..(9)

<223> K or R

<220>

<221> Protein

<222> (10)..(10)

<223> L or I or V or F

<220>

<221> Protein

<222> (11)..(11)

<223> L or I or V or F

<220>

<221> Protein

<222> (13)..(13)

<223> L or I or V or F

<220>

<221> Protein

<222> (15)..(15)

<223> Q or L or I or V or F

<220>

<221> Protein

<222> (17)..(17)

<223> L or I or V or F

<220>

<221> Protein

<222> (18)..(18)

<223> D or R

<400> 32

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Thr Xaa Xaa Xaa Thr Xaa Pro Xaa Ser

1 5 10 15

Xaa Xaa

<210> 33

<211> 19

<212> Protein

<213> measles virus

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 4

<220>

<221> Protein

<222> (4)..(4)

<223> S or T

<220>

<221> Protein

<222>(7)..(7)

<223> K or R

<220>

<221> Protein

<222>(8)..(8)

<223> G or T

<220>

<221> Protein

<222> (12)..(12)

<223> H or T

<220>

<221> Protein

<222> (13)..(13)

<223> K or R

<400> 33

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe

<210> 34

<211> 18

<212> Protein

<213> hepatitis B virus

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Th HBsAg 2

<220>

<221> Protein

<222> (4)..(4)

<223> I or F

<220>

<221> Protein

<222> (5)..(5)

<223> I or F

<220>

<221> Protein

<222>(6)..(6)

<223> T or L

<220>

<221> Protein

<222>(7)..(7)

<223> I or L

<220>

<221> Protein

<222> (11)..(11)

<223> I or L

<220>

<221> Protein

<222> (14)..(14)

<223> P or I

<220>

<221> Protein

<222> (15)..(15)

<223> Q or T

<220>

<221> Protein

<222> (16)..(16)

<223> S or T

<220>

<221> Protein

<222> (17)..(17)

<223> L or I

<400> 34

Lys Lys Lys Xaa Xaa Xaa Xaa Thr Arg Ile Xaa Thr Ile Xaa Xaa Xaa

1 5 10 15

Xaa Asp

<210> 35

<211> 19

<212> Protein

<213> measles virus

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 5

<400> 35

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe

<210> 36

<211> 18

<212> Protein

<213> hepatitis B virus

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Th HBsAg 3

<400> 36

Lys Lys Lys Ile Ile Thr Ile Thr Arg Ile Ile Thr Ile Ile Thr Thr

1 5 10 15

Ile Asp

<210> 37

<211> 11

<212> Protein

<213> influenza virus

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> Th matrix protein of influenza virus 1 _1

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> Th matrix protein of influenza virus 1_1

<400> 37

Phe Val Phe Thr Leu Thr Val Pro Ser Glu Arg

1 5 10

<210> 38

<211> 15

<212> Protein

<213> influenza virus

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Th matrix protein of influenza virus 1_2

<400> 38

Ser Gly Pro Leu Lys Ala Glu Ile Ala Gln Arg Leu Glu Asp Val

1 5 10 15

<210> 39

<211> 9

<212> Protein

<213> influenza virus

<220>

<221> PEPTIDE

<222> (1)..(9)

<223> Th non-structural protein of influenza virus 1

<400> 39

Asp Arg Leu Arg Arg Asp Gln Lys Ser

15

<210> 40

<211> 19

<212> Protein

<213> Epstein-Barr virus

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th EBV BHRF1

<400> 40

Ala Gly Leu Thr Leu Ser Leu Leu Val Ile Cys Ser Tyr Leu Phe Ile

1 5 10 15

Ser Arg Gly

<210> 41

<211> 15

<212> Protein

<213> Clostridium tetani

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Th Clostridium tetani TT1

<400> 41

Gln Tyr Ile Lys Ala Asn Ser Lys Phe Ile Gly Ile Thr Glu Leu

1 5 10 15

<210> 42

<211> 20

<212> Protein

<213> Epstein-Barr virus

<220>

<221> PEPTIDE

<222> (1)..(20)

<223> Th EBV EBNA-1

<400> 42

Pro Gly Pro Leu Arg Glu Ser Ile Val Cys Tyr Phe Met Val Phe Leu

1 5 10 15

Gln Thr His Ile

20

<210> 43

<211> 21

<212> Protein

<213> Clostridium tetani

<220>

<221> PEPTIDE

<222> (1)..(21)

<223> Th Clostridium tetani TT2

<400> 43

Phe Asn Asn Phe Thr Val Ser Phe Trp Leu Arg Val Pro Lys Val Ser

1 5 10 15

Ala Ser His Leu Glu

20

<210> 44

<211> 16

<212> Protein

<213> Clostridium tetani

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> Th Clostridium tetani TT3

<400> 44

Lys Phe Ile Ile Lys Arg Tyr Thr Pro Asn Asn Glu Ile Asp Ser Phe

1 5 10 15

<210> 45

<211> 16

<212> Protein

<213> Clostridium tetani

<220>

<221> PEPTIDE

<222> (1)..(16)

<223> Th Clostridium tetani TT4

<400> 45

Val Ser Ile Asp Lys Phe Arg Ile Phe Cys Lys Ala Leu Asn Pro Lys

1 5 10 15

<210> 46

<211> 18

<212> Protein

<213> Epstein-Barr virus

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Th EBV CP

<400> 46

Val Pro Gly Leu Tyr Ser Pro Cys Arg Ala Phe Phe Asn Lys Glu Glu

1 5 10 15

Leu Leu

<210> 47

<211> 14

<212> Protein

<213> human cytomegalovirus

<220>

<221> PEPTIDE

<222> (1)..(14)

<223> Th HCMV IE1

<400> 47

Asp Lys Arg Glu Met Trp Met Ala Cys Ile Lys Glu Leu His

1 5 10

<210> 48

<211> 15

<212> Protein

<213> Epstein-Barr virus

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Th EBV GP340

<400> 48

Thr Gly His Gly Ala Arg Thr Ser Thr Glu Pro Thr Thr Asp Tyr

1 5 10 15

<210> 49

<211> 13

<212> Protein

<213> Epstein-Barr virus

<220>

<221> PEPTIDE

<222> (1)..(13)

<223> Th EBV BPLF1

<400> 49

Lys Glu Leu Lys Arg Gln Tyr Glu Lys Lys Leu Arg Gln

1 5 10

<210> 50

<211> 11

<212> Protein

<213> Epstein-Barr virus

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> Th EBV EBNA-2

<400> 50

Thr Val Phe Tyr Asn Ile Pro Pro Met Pro Leu

1 5 10

<210> 51

<211> 39

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(39)

<223> fTau 145-P160 (pT149/pT153)

<220>

<221> MOD_RES

<222> (28)..(28)

<223> PHOSPHORYLING

<220>

<221> MOD_RES

<222> (32)..(32)

<223> PHOSPHORYLING

<400> 51

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ala Asp Gly Lys Thr Lys Ile Ala Thr

20 25 30

Pro Arg Gly Ala Ala Pro Pro

35

<210> 52

<211> 39

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(39)

<223> Tau 145-160

<400> 52

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ala Asp Gly Lys Thr Lys Ile Ala Thr

20 25 30

Pro Arg Gly Ala Ala Pro Pro

35

<210> 53

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> fTau 172-189 (pT175/pT181/pS185)

<220>

<221> MOD_RES

<222> (27)..(27)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (33)..(33)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (37)..(37)

<223> PHOSPHORYLATION

<400> 53

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Pro Ala Lys Thr Pro Pro Ala Pro Lys

20 25 30

Thr Pro Pro Ser Ser Gly Glu Pro Pro

35 40

<210> 54

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> Tau 172-189

<400> 54

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Pro Ala Lys Thr Pro Pro Ala Pro Lys

20 25 30

Thr Pro Pro Ser Ser Gly Glu Pro Pro

35 40

<210> 55

<211> 42

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(42)

<223> fTau 182-200 (pS184/pY197)

<220>

<221> MOD_RES

<222> (26)..(26)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (39)..(39)

<223> PHOSPHORYLATION

<400> 55

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Pro Ser Ser Gly Glu Pro Pro Lys

20 25 30

Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro

35 40

<210> 56

<211> 42

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(42)

<223> Tau 182-200

<400> 56

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Pro Ser Ser Gly Glu Pro Pro Lys

20 25 30

Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro

35 40

<210> 57

<211> 42

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(42)

<223> fTau 195-213 (pS199/pS202/pT205)

<220>

<221> MOD_RES

<222> (28)..(28)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (31)..(31)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (34)..(34)

<223> PHOSPHORYLATION

<400> 57

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ser Gly Tyr Ser Ser Pro Gly Ser Pro

20 25 30

Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro

35 40

<210> 58

<211> 42

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(42)

<223> Tau 195-213

<400> 58

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ser Gly Tyr Ser Ser Pro Gly Ser Pro

20 25 30

Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro

35 40

<210> 59

<211> 42

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(42)

<223> fTau 195-213 (pS198/pS208/pS210)

<220>

<221> MOD_RES

<222> (27)..(27)

<223> PHOSPHORYLING

<220>

<221> MOD_RES

<222> (37)..(37)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (39)..(39)

<223> PHOSPHORYLATION

<400> 59

Ile Ser Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ser Gly Tyr Ser Ser Pro Gly Ser Pro

20 25 30

Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro

35 40

<210> 60

<211> 39

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(39)

<223> fTau 209-224 (pT212/pS214/pT217)

<220>

<221> MOD_RES

<222> (27)..(27)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (29)..(29)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (32)..(32)

<223> PHOSPHORYLATION

<400> 60

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Arg Ser Arg Thr Pro Thr Pro Gly Ser

20 25 30

Arg Ser Arg Thr Pro Ser Leu

35

<210> 61

<211> 39

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(39)

<223> Tau 209-224

<400> 61

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Arg Ser Arg Thr Pro Thr Pro Gly Ser

20 25 30

Arg Ser Arg Thr Pro Ser Leu

35

<210> 62

<211> 39

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(39)

<223> fTau 228-243 (pT231/pS235/pS238)

<220>

<221> MOD_RES

<222> (27)..(27)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (31)..(31)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (34)..(34)

<223> PHOSPHORYLATION

<400> 62

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Val Arg Thr Pro Pro Lys Ser Pro

20 25 30

Ser Ser Ala Lys Ser Arg Leu

35

<210> 63

<211> 39

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(39)

<223> Tau 228-243

<400> 63

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Val Arg Thr Pro Pro Lys Ser Pro

20 25 30

Ser Ser Ala Lys Ser Arg Leu

35

<210> 64

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> fTau 257-274 (pS262)

<220>

<221> MOD_RES

<222> (29)..(29)

<223> PHOSPHORYLATION

<400> 64

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Ser Lys Ile Gly Ser Thr Glu Asn

20 25 30

Leu Lys His Gln Pro Gly Gly Gly Lys

35 40

<210> 65

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> Tau 257-274

<400> 65

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Ser Lys Ile Gly Ser Thr Glu Asn

20 25 30

Leu Lys His Gln Pro Gly Gly Gly Lys

35 40

<210> 66

<211> 53

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(53)

<223> fTau 379-408 (pS396/pS404)

<220>

<221> MOD_RES

<222> (41)..(41)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (49)..(49)

<223> PHOSPHORYLATION

<400> 66

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Arg Glu Asn Ala Lys Ala Lys Thr Asp

20 25 30

His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr

35 40 45

Ser Pro Arg His Leu

50

<210> 67

<211> 53

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(53)

<223> Tau 379-408

<400> 67

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Arg Glu Asn Ala Lys Ala Lys Thr Asp

20 25 30

His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr

35 40 45

Ser Pro Arg His Leu

50

<210> 68

<211> 60

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(60)

<223> fTau 275-311 (*P301->S301; pS293/pS301)

<220>

<221> MOD_RES

<222> (42)..(42)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (50)..(50)

<223> PHOSPHORYLATION

<400> 68

Ile Ser Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Gln Ile Ile Asn Lys Lys Leu Asp

20 25 30

Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His

35 40 45

Val Ser Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

50 55 60

<210> 69

<211> 60

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(60)

<223> Tau 275-311

<400> 69

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Gln Ile Ile Asn Lys Lys Leu Asp

20 25 30

Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His

35 40 45

Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

50 55 60

<210> 70

<211> 56

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(56)

<223> fTau 393-425 (pS396/pS404/pS422)

<220>

<221> MOD_RES

<222> (27)..(27)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (35)..(35)

<223> PHOSPHORYLATION

<220>

<221> MOD_RES

<222> (53)..(53)

<223> PHOSPHORYLATION

<400> 70

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Tyr Lys Ser Pro Val Val Ser Gly

20 25 30

Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile

35 40 45

Asp Met Val Asp Ser Pro Gln Leu

50 55

<210> 71

<211> 56

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(56)

<223> Tau 393-425

<400> 71

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Tyr Lys Ser Pro Val Val Ser Gly

20 25 30

Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile

35 40 45

Asp Met Val Asp Ser Pro Gln Leu

50 55

<210> 72

<211> 12

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(12)

<223> Tau-peptide, L215-V226

<400> 72

Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val

1 5 10

<210> 73

<211> 22

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(22)

<223> Tau-peptide, T205-V226

<400> 73

Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr

1 5 10 15

Arg Glu Pro Lys Lys Val

20

<210> 74

<211> 32

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(32)

<223> Tau-peptide, S195-V226

<400> 74

Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser

1 5 10 15

Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val

20 25 30

<210> 75

<211> 42

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(42)

<223> Tau-peptide, S185-V226

<400> 75

Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro

1 5 10 15

Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro

20 25 30

Thr Pro Pro Thr Arg Glu Pro Lys Lys Val

35 40

<210> 76

<211> 52

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(52)

<223> Tau-peptide, T175-V226

<400> 76

Thr Pro Pro Ala Pro Lys Thr Pro Ser Ser Gly Glu Pro Pro Lys

1 5 10 15

Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro

20 25 30

Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu

35 40 45

Pro Lys Lys Val

50

<210> 77

<211> 62

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(62)

<223> Tau-peptide, Q165-V226

<400> 77

Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys

1 5 10 15

Thr Pro Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly

20 25 30

Tyr Ser Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr

35 40 45

Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys Lys Val

50 55 60

<210> 78

<211> 72

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(72)

<223> Tau-peptide, R155-V226

<400> 78

Arg Gly Ala Ala Pro Pro Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg

1 5 10 15

Ile Pro Ala Lys Thr Pro Pro Ala Pro Lys Thr Pro Pro Ser Ser Gly

20 25 30

Glu Pro Pro Lys Ser Gly Asp Arg Ser Gly Tyr Ser Ser Pro Gly Ser

35 40 45

Pro Gly Thr Pro Gly Ser Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro

50 55 60

Pro Thr Arg Glu Pro Lys Lys Val

65 70

<210> 79

<211> 12

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(12)

<223> Tau-peptide, V287-K298

<400> 79

Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys

1 5 10

<210> 80

<211> 22

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(22)

<223> Tau-peptide, I277-K298

<400> 80

Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly

1 5 10 15

Ser Lys Asp Asn Ile Lys

20

<210> 81

<211> 32

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(32)

<223> Tau-peptide, K267-K298

<400> 81

Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu

1 5 10 15

Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys

20 25 30

<210> 82

<211> 42

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(42)

<223> Tau-peptide, K257-K298

<400> 82

Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly

1 5 10 15

Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln

20 25 30

Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys

35 40

<210> 83

<211> 52

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(52)

<223> Tau-peptide, P247-K298

<400> 83

Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser Lys Ile Gly Ser

1 5 10 15

Thr Glu Asn Leu Lys His Gln Pro Gly Gly Gly Lys Val Gln Ile Ile

20 25 30

Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys

35 40 45

Asp Asn Ile Lys

50

<210> 84

<211> 62

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(62)

<223> Tau-peptide, S237-K298

<400> 84

Ser Ser Ala Lys Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp

1 5 10 15

Leu Lys Asn Val Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His

20 25 30

Gln Pro Gly Gly Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu

35 40 45

Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys

50 55 60

<210> 85

<211> 72

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(72)

<223> Tau-peptide, A227-K298

<400> 85

Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys Ser Arg

1 5 10 15

Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser

20 25 30

Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly Gly Lys

35 40 45

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

50 55 60

Cys Gly Ser Lys Asp Asn Ile Lys

65 70

<210> 86

<211> 12

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(12)

<223> Tau-peptide, N359-K370

<400> 86

Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys

1 5 10

<210> 87

<211> 22

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(22)

<223> Tau-peptide, R349-K370

<400> 87

Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro

1 5 10 15

Gly Gly Gly Asn Lys Lys

20

<210> 88

<211> 32

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(32)

<223> Tau-peptide, V339-K370

<400> 88

Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile

1 5 10 15

Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys

20 25 30

<210> 89

<211> 42

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(42)

<223> Tau-peptide, H329-K370

<400> 89

His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu

1 5 10 15

Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile

20 25 30

Thr His Val Pro Gly Gly Gly Asn Lys Lys

35 40

<210> 90

<211> 52

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(52)

<223> Tau-peptide, T319-K370

<400> 90

Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly

1 5 10 15

Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val

20 25 30

Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly

35 40 45

Gly Asn Lys Lys

50

<210> 91

<211> 62

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(62)

<223> Tau-peptide, V309-K370

<400> 91

Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser Lys Cys Gly Ser

1 5 10 15

Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys

20 25 30

Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser

35 40 45

Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys

50 55 60

<210> 92

<211> 72

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(72)

<223> Tau-peptide, H299-K370

<400> 92

His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Pro Val Asp

1 5 10 15

Leu Ser Lys Val Thr Ser Lys Cys Gly Ser Leu Gly Asn Ile His His

20 25 30

Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu Asp Phe

35 40 45

Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His

50 55 60

Val Pro Gly Gly Gly Asn Lys Lys

65 70

<210> 93

<211> 11

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> Tau-peptide, E431-L441

<400> 93

Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu

1 5 10

<210> 94

<211> 21

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(21)

<223> Tau-peptide, D421-L441

<400> 94

Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu

1 5 10 15

Ala Lys Gln Gly Leu

20

<210> 95

<211> 31

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(31)

<223> Tau-peptide, V411-L441

<400> 95

Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala

1 5 10 15

Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu

20 25 30

<210> 96

<211> 41

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(41)

<223> Tau-peptide, G401-L441

<400> 96

Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser

1 5 10 15

Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val

20 25 30

Ser Ala Ser Leu Ala Lys Gln Gly Leu

35 40

<210> 97

<211> 51

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(51)

<223> Tau-peptide, E391-L441

<400> 97

Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr Ser Pro Arg

1 5 10 15

His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp Met Val Asp Ser

20 25 30

Pro Gln Leu Ala Thr Leu Ala Asp Glu Val Ser Ala Ser Leu Ala Lys

35 40 45

Gln Gly Leu

50

<210> 98

<211> 61

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(61)

<223> Tau-peptide, N381-L441

<400> 98

Asn Ala Lys Ala Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser

1 5 10 15

Pro Val Val Ser Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser

20 25 30

Ser Thr Gly Ser Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu

35 40 45

Ala Asp Glu Val Ser Ala Ser Leu Ala Lys Gln Gly Leu

50 55 60

<210> 99

<211> 71

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(71)

<223> Tau-peptide, I371-L441

<400> 99

Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala Lys Thr

1 5 10 15

Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp

20 25 30

Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser Ile Asp

35 40 45

Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val Ser Ala

50 55 60

Ser Leu Ala Lys Gln Gly Leu

65 70

<210> 100

<211> 441

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(441)

<223> Tau isoform with 4R and 2N (GenBank: AGF19246.1)

<400> 100

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His

20 25 30

Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly Leu Lys Glu Ser Pro Leu

35 40 45

Gln Thr Pro Thr Glu Asp Gly Ser Glu Glu Pro Gly Ser Glu Thr Ser

50 55 60

Asp Ala Lys Ser Thr Pro Thr Ala Glu Asp Val Thr Ala Pro Leu Val

65 70 75 80

Asp Glu Gly Ala Pro Gly Lys Gln Ala Ala Ala Gln Pro His Thr Glu

85 90 95

Ile Pro Glu Gly Thr Thr Ala Glu Glu Ala Gly Ile Gly Asp Thr Pro

100 105 110

Ser Leu Glu Asp Glu Ala Ala Gly His Val Thr Gln Ala Arg Met Val

115 120 125

Ser Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys Ala Lys Gly

130 135 140

Ala Asp Gly Lys Thr Lys Ile Ala Thr Pro Arg Gly Ala Ala Pro Pro

145 150 155 160

Gly Gln Lys Gly Gln Ala Asn Ala Thr Arg Ile Pro Ala Lys Thr Pro

165 170 175

Pro Ala Pro Lys Thr Pro Ser Ser Gly Glu Pro Pro Lys Ser Gly

180 185 190

Asp Arg Ser Gly Tyr Ser Pro Gly Ser Pro Gly Thr Pro Gly Ser

195 200 205

Arg Ser Arg Thr Pro Ser Leu Pro Thr Pro Pro Thr Arg Glu Pro Lys

210 215 220

Lys Val Ala Val Val Arg Thr Pro Pro Lys Ser Pro Ser Ser Ala Lys

225 230 235 240

Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val

245 250 255

Lys Ser Lys Ile Gly Ser Thr Glu Asn Leu Lys His Gln Pro Gly Gly

260 265 270

Gly Lys Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln

275 280 285

Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly

290 295 300

Ser Val Gln Ile Val Tyr Lys Pro Val Asp Leu Ser Lys Val Thr Ser

305 310 315 320

Lys Cys Gly Ser Leu Gly Asn Ile His His Lys Pro Gly Gly Gly Gln

325 330 335

Val Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser

340 345 350

Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn

355 360 365

Lys Lys Ile Glu Thr His Lys Leu Thr Phe Arg Glu Asn Ala Lys Ala

370 375 380

Lys Thr Asp His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser

385 390 395 400

Gly Asp Thr Ser Pro Arg His Leu Ser Asn Val Ser Ser Thr Gly Ser

405 410 415

Ile Asp Met Val Asp Ser Pro Gln Leu Ala Thr Leu Ala Asp Glu Val

420 425 430

Ser Ala Ser Leu Ala Lys Gln Gly Leu

435 440

<210> 101

<211> 11

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> Tau-peptide - ak131-141

<400> 101

Ser Lys Asp Gly Thr Gly Ser Asp Asp Lys Lys

1 5 10

<210> 102

<211> 17

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Tau-peptide - ak241-257

<400> 102

Ser Arg Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val

1 5 10 15

Lys

<210> 103

<211> 17

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Tau-peptide - ak243-259

<400> 103

Leu Gln Thr Ala Pro Val Pro Met Pro Asp Leu Lys Asn Val Lys Ser

1 5 10 15

Lys

<210> 104

<211> 7

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(7)

<223> Tau-peptide - ak275-281

<400> 104

Val Gln Ile Ile Asn Lys Lys

15

<210> 105

<211> 14

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(14)

<223> Tau-peptide - ak281-294

<400> 105

Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys

1 5 10

<210> 106

<211> 20

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(20)

<223> Tau-peptide - ak275-294

<400> 106

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser Lys

20

<210> 107

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Tau-peptide - ak294-311

<400> 107

Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val

1 5 10 15

Tyr Lys

<210> 108

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Tau peptide - ak294-311, P301 to S301

<400> 108

Lys Asp Asn Ile Lys His Val Ser Gly Gly Gly Ser Val Gln Ile Val

1 5 10 15

Tyr Lys

<210> 109

<211> 18

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(18)

<223> Tau-peptide - a294-311, p301 trans

<400> 109

Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val

1 5 10 15

Tyr Lys

<210> 110

<211> 37

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(37)

<223> Tau-peptide - ak275-311, p301 trans

<400> 110

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val

20 25 30

Gln Ile Val Tyr Lys

35

<210> 111

<211> 15

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Tau-peptide - ak297-311, p301 trans

<400> 111

Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

1 5 10 15

<210> 112

<211> 13

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(13)

<223> Tau-peptide - ak22-34

<400> 112

Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His Gln Asp

1 5 10

<210> 113

<211> 17

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Tau-peptide - ak18-34

<400> 113

Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His Gln

1 5 10 15

asp

<210> 114

<211> 17

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Tau-peptide - ak22-38

<400> 114

Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His Gln Asp Gln Glu Gly

1 5 10 15

asp

<210> 115

<211> 15

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Tau-peptide - ak297-311

<400> 115

Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

1 5 10 15

<210> 116

<211> 12

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(12)

<223> Tau-peptide - ak359-370

<400> 116

Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys

1 5 10

<210> 117

<211> 22

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(22)

<223> Tau-peptide - ak349-370

<400> 117

Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro

1 5 10 15

Gly Gly Gly Asn Lys Lys

20

<210> 118

<211> 32

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(32)

<223> Tau-peptide - a339-370

<400> 118

Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys Ile

1 5 10 15

Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys

20 25 30

<210> 119

<211> 42

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(42)

<223> Tau-peptide - a329-370

<400> 119

His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu

1 5 10 15

Asp Phe Lys Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile

20 25 30

Thr His Val Pro Gly Gly Gly Asn Lys Lys

35 40

<210> 120

<211> 21

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(21)

<223> Tau-peptide - ak329-349

<400> 120

His His Lys Pro Gly Gly Gly Gln Val Glu Val Lys Ser Glu Lys Leu

1 5 10 15

Asp Phe Lys Asp Arg

20

<210> 121

<211> 40

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(40)

<223> Tau-peptide - ak1-40

<400> 121

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His

20 25 30

Gln Asp Gln Glu Gly Asp Thr Asp

35 40

<210> 122

<211> 40

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(40)

<223> Tau-peptide - ak11-50

<400> 122

Met Glu Asp His Ala Gly Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln

1 5 10 15

Gly Gly Tyr Thr Met His Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly

20 25 30

Leu Lys Glu Ser Pro Leu Gln Thr

35 40

<210> 123

<211> 34

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(34)

<223> Tau-peptide - ak1-34

<400> 123

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His

20 25 30

Gln Asp

<210> 124

<211> 22

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(22)

<223> Tau-peptide - ak1-22

<400> 124

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp

20

<210> 125

<211> 34

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(34)

<223> Tau S131-K141

<400> 125

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ser Lys Asp Gly Thr Gly Ser Asp Asp

20 25 30

Lys Lys

<210> 126

<211> 40

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(40)

<223> Tau S241-K257

<400> 126

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ser Arg Leu Gln Thr Ala Pro Val Pro

20 25 30

Met Pro Asp Leu Lys Asn Val Lys

35 40

<210> 127

<211> 40

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(40)

<223> Tau L243-K259

<400> 127

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Leu Gln Thr Ala Pro Val Pro Met Pro

20 25 30

Asp Leu Lys Asn Val Lys Ser Lys

35 40

<210> 128

<211> 30

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(30)

<223> Tau V275-K281

<400> 128

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Gln Ile Ile Asn Lys Lys

20 25 30

<210> 129

<211> 37

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(37)

<223> Tau K281-K294

<400> 129

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Leu Asp Leu Ser Asn Val Gln Ser

20 25 30

Lys Cys Gly Ser Lys

35

<210> 130

<211> 43

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(43)

<223> Tau V275-K294

<400> 130

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Gln Ile Ile Asn Lys Lys Leu Asp

20 25 30

Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys

35 40

<210> 131

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> Tau K294-K311

<400> 131

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Asp Asn Ile Lys His Val Pro Gly

20 25 30

Gly Gly Ser Val Gln Ile Val Tyr Lys

35 40

<210> 132

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> Tau K294-K311, P301 to S301

<400> 132

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Asp Asn Ile Lys His Val Ser Gly

20 25 30

Gly Gly Ser Val Gln Ile Val Tyr Lys

35 40

<210> 133

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 4 (UBITh3)

<220>

<221> Protein

<222> (4)..(4)

<223> S or T

<220>

<221> Protein

<222>(7)..(7)

<223> K or R

<220>

<221> Protein

<222>(8)..(8)

<223> G or T

<220>

<221> Protein

<222> (12)..(12)

<223> H or T

<220>

<221> Protein

<222> (13)..(13)

<223> K or R

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> Tau K257-K274

<400> 133

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Ser Lys Ile Gly Ser Thr Glu Asn

20 25 30

Leu Lys His Gln Pro Gly Gly Gly Lys

35 40

<210> 134

<211> 53

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 4 (UBITh3)

<220>

<221> Protein

<222> (4)..(4)

<223> S or T

<220>

<221> Protein

<222>(7)..(7)

<223> K or R

<220>

<221> Protein

<222>(8)..(8)

<223> G or T

<220>

<221> Protein

<222> (12)..(12)

<223> H or T

<220>

<221> Protein

<222> (13)..(13)

<223> K or R

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(53)

<223> Tau R379-L408

<400> 134

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Arg Glu Asn Ala Lys Ala Lys Thr Asp

20 25 30

His Gly Ala Glu Ile Val Tyr Lys Ser Pro Val Val Ser Gly Asp Thr

35 40 45

Ser Pro Arg His Leu

50

<210> 135

<211> 41

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(41)

<223> Tau K294-K311, p301 trans

<400> 135

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Lys Asp Asn Ile Lys His Val Pro Gly

20 25 30

Gly Gly Ser Val Gln Ile Val Tyr Lys

35 40

<210> 136

<211> 60

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(60)

<223> Tau V275-K311, p301 trans

<400> 136

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Gln Ile Ile Asn Lys Lys Leu Asp

20 25 30

Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His

35 40 45

Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

50 55 60

<210> 137

<211> 36

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(36)

<223> Tau D22-D34

<400> 137

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Asp Arg Lys Asp Gln Gly Gly Tyr Thr

20 25 30

Met His Gln Asp

35

<210> 138

<211> 40

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(40)

<223> Tau Y18-D34

<400> 138

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Tyr Gly Leu Gly Asp Arg Lys Asp Gln

20 25 30

Gly Gly Tyr Thr Met His Gln Asp

35 40

<210> 139

<211> 40

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(40)

<223> Tau D22-D38

<400> 139

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Asp Arg Lys Asp Gln Gly Gly Tyr Thr

20 25 30

Met His Gln Asp Gln Glu Gly Asp

35 40

<210> 140

<211> 38

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(38)

<223> Tau I297-K311, p301 trans

<400> 140

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ile Lys His Val Pro Gly Gly Gly Ser

20 25 30

Val Gln Ile Val Tyr Lys

35

<210> 141

<211> 38

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Tau I297-K311

<220>

<221> Protein

<222> (16)..(16)

<223> epsilon K

<220>

<221> PEPTIDE

<222> (16)..(19)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (20)..(38)

<223> Th MvF5 (UBITh1)

<400> 141

Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys Lys

1 5 10 15

Lys Lys Lys Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg

20 25 30

Ile Glu Thr Ile Leu Phe

35

<210> 142

<211> 38

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (21)..(38)

<223> Tau K294-K311

<400> 142

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser

20 25 30

Val Gln Ile Val Tyr Lys

35

<210> 143

<211> 35

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (21)..(35)

<223> Tau I297-K311

<400> 143

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile

20 25 30

Val Tyr Lys

35

<210> 144

<211> 38

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(38)

<223> Tau I297-K311

<400> 144

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ile Lys His Val Pro Gly Gly Gly Ser

20 25 30

Val Gln Ile Val Tyr Lys

35

<210> 145

<211> 39

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 4 (UBITH3)

<220>

<221> Protein

<222> (4)..(4)

<223> S or T

<220>

<221> Protein

<222>(7)..(7)

<223> K or R

<220>

<221> Protein

<222>(8)..(8)

<223> G or T

<220>

<221> Protein

<222> (12)..(12)

<223> H or T

<220>

<221> Protein

<222> (13)..(13)

<223> K or R

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(39)

<223> Tau A145-P160

<400> 145

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Ala Asp Gly Lys Thr Lys Ile Ala Thr

20 25 30

Pro Arg Gly Ala Ala Pro Pro

35

<210> 146

<211> 60

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 4 (UBITh3)

<220>

<221> Protein

<222> (4)..(4)

<223> S or T

<220>

<221> Protein

<222>(7)..(7)

<223> K or R

<220>

<221> Protein

<222>(8)..(8)

<223> G or T

<220>

<221> Protein

<222> (12)..(12)

<223> H or T

<220>

<221> Protein

<222> (13)..(13)

<223> K or R

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(60)

<223> Tau V275-K311

<400> 146

Ile Ser Ile Xaa Glu Ile Xaa Xaa Val Ile Val Xaa Xaa Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Gln Ile Ile Asn Lys Lys Leu Asp

20 25 30

Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His

35 40 45

Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

50 55 60

<210> 147

<211> 35

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(35)

<223> Tau N359-K370

<400> 147

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Asn Ile Thr His Val Pro Gly Gly Gly

20 25 30

Asn Lys Lys

35

<210> 148

<211> 45

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF 5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(45)

<223> Tau R349-K370

<400> 148

Ile Ser Ile Ser Glu Ile Lys Gly Val Ile Val His Lys Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Arg Val Gln Ser Lys Ile Gly Ser Leu

20 25 30

Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys Lys

35 40 45

<210> 149

<211> 55

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(55)

<223> Tau V339-K370

<400> 149

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys Val Lys Ser Glu Lys Leu Asp Phe Lys

20 25 30

Asp Arg Val Gln Ser Lys Ile Gly Ser Leu Asp Asn Ile Thr His Val

35 40 45

Pro Gly Gly Gly Asn Lys Lys

50 55

<210> 150

<211> 65

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(65)

<223> Tau H329-K370

<400> 150

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys His His Lys Pro Gly Gly Gly Gln Val

20 25 30

Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg Val Gln Ser Lys

35 40 45

Ile Gly Ser Leu Asp Asn Ile Thr His Val Pro Gly Gly Gly Asn Lys

50 55 60

Lys

65

<210> 151

<211> 44

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Th MvF5 (UBITh1)

<220>

<221> Protein

<222> (20)..(20)

<223> epsilon-K

<220>

<221> PEPTIDE

<222> (20)..(23)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (24)..(44)

<223> Tau H329-R349

<400> 151

Ile Ser Ile Thr Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr

1 5 10 15

Ile Leu Phe Lys Lys Lys Lys His His Lys Pro Gly Gly Gly Gln Val

20 25 30

Glu Val Lys Ser Glu Lys Leu Asp Phe Lys Asp Arg

35 40

<210> 152

<211> 63

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(40)

<223> Tau M1-D40

<220>

<221> Protein

<222> (41)..(41)

<223> epsilon K

<220>

<221> PEPTIDE

<222> (41)..(44)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (45)..(63)

<223> Th MvF5 (UBITh1)

<400> 152

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His

20 25 30

Gln Asp Gln Glu Gly Asp Thr Asp Lys Lys Lys Lys Ile Ser Ile Thr

35 40 45

Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr Ile Leu Phe

50 55 60

<210> 153

<211> 63

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(40)

<223> Tau M11-T50

<220>

<221> Protein

<222> (41)..(41)

<223> epsilon K

<220>

<221> PEPTIDE

<222> (41)..(44)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (45)..(63)

<223> Th MvF5 (UBITh1)

<400> 153

Met Glu Asp His Ala Gly Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln

1 5 10 15

Gly Gly Tyr Thr Met His Gln Asp Gln Glu Gly Asp Thr Asp Ala Gly

20 25 30

Leu Lys Glu Ser Pro Leu Gln Thr Lys Lys Lys Lys Ile Ser Ile Thr

35 40 45

Glu Ile Lys Gly Val Ile Val His Arg Ile Glu Thr Ile Leu Phe

50 55 60

<210> 154

<211> 57

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(34)

<223> Tau M1-D34

<220>

<221> Protein

<222> (35)..(35)

<223> epsilon K

<220>

<221> PEPTIDE

<222> (35)..(38)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (39)..(57)

<223> Th MvF5 (UBITh1)

<400> 154

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp Arg Lys Asp Gln Gly Gly Tyr Thr Met His

20 25 30

Gln Asp Lys Lys Lys Lys Ile Ser Ile Thr Glu Ile Lys Gly Val Ile

35 40 45

Val His Arg Ile Glu Thr Ile Leu Phe

50 55

<210> 155

<211> 45

<212> Protein

<213> Synthetic peptide

<220>

<221> PEPTIDE

<222> (1)..(22)

<223> Tau M1-D22

<220>

<221> Protein

<222> (23)..(23)

<223> epsilon K

<220>

<221> PEPTIDE

<222> (23)..(26)

<223> epsilon K-KKK as spacer

<220>

<221> PEPTIDE

<222> (27)..(45)

<223> Th MvF5 (UBITh1)

<400> 155

Met Ala Glu Pro Arg Gln Glu Phe Glu Val Met Glu Asp His Ala Gly

1 5 10 15

Thr Tyr Gly Leu Gly Asp Lys Lys Lys Lys Ile Ser Ile Thr Glu Ile

20 25 30

Lys Gly Val Ile Val His Arg Ile Glu Thr Ile Leu Phe

35 40 45

<210> 156

<211> 31

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(31)

<223> Tau-peptide - ak281-311

<400> 156

Lys Leu Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn

1 5 10 15

Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

20 25 30

<210> 157

<211> 29

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(29)

<223> Tau-peptide - ak283-311

<400> 157

Asp Leu Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys

1 5 10 15

His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

20 25

<210> 158

<211> 27

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(27)

<223> Tau-peptide - ak285-311

<400> 158

Ser Asn Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val

1 5 10 15

Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

20 25

<210> 159

<211> 25

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(25)

<223> Tau-peptide - ak287-311

<400> 159

Val Gln Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly

1 5 10 15

Gly Gly Ser Val Gln Ile Val Tyr Lys

20 25

<210> 160

<211> 23

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(23)

<223> Tau-peptide - ak289-311

<400> 160

Ser Lys Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly

1 5 10 15

Ser Val Gln Ile Val Tyr Lys

20

<210> 161

<211> 21

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(21)

<223> Tau-peptide - ak291-311

<400> 161

Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val

1 5 10 15

Gln Ile Val Tyr Lys

20

<210> 162

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Tau-peptide - ak293-311

<400> 162

Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile

1 5 10 15

Val Tyr Lys

<210> 163

<211> 17

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(17)

<223> Tau-peptide - ak295-311

<400> 163

Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr

1 5 10 15

Lys

<210> 164

<211> 13

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(13)

<223> Tau-peptide - ak299-311

<400> 164

His Val Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

1 5 10

<210> 165

<211> 11

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> Tau-peptide - a301-311

<400> 165

Pro Gly Gly Gly Ser Val Gln Ile Val Tyr Lys

1 5 10

<210> 166

<211> 31

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(31)

<223> Tau-peptide - ak275-305

<400> 166

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro Gly Gly Gly Ser

20 25 30

<210> 167

<211> 27

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(27)

<223> Tau-peptide - ak275-301

<400> 167

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser Lys Asp Asn Ile Lys His Val Pro

20 25

<210> 168

<211> 23

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(23)

<223> Tau-peptide - ak275-297

<400> 168

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser Lys Asp Asn Ile

20

<210> 169

<211> 19

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(19)

<223> Tau-peptide - ak275-293

<400> 169

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser Lys

1 5 10 15

Cys Gly Ser

<210> 170

<211> 15

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(15)

<223> Tau-peptide - ak275-289

<400> 170

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser Asn Val Gln Ser

1 5 10 15

<210> 171

<211> 11

<212> Protein

<213> Homo sapiens

<220>

<221> PEPTIDE

<222> (1)..(11)

<223> Tau-peptide - ak275-285

<400> 171

Val Gln Ile Ile Asn Lys Lys Leu Asp Leu Ser

1 5 10

<---

Claims (31)

1. Tau-peptide immunogenic construct, which can be represented by the formulas: (Th) _m -(A) _n - Tau-X fragment or fragment Tau-(A) _n -(Th) _m -X, where Th is a heterologous T-helper epitope; A is a heterologous spacer; the Tau fragment is a B cell epitope selected from the group consisting of SEQ ID NOs: 1-21 and 101-124; X is α-COOH or α-CONH ₂ amino acids; m is from 1 to 4; And n is from 1 to 10. 2. The tau peptide immunogen construct of claim 1, wherein the Th epitope is selected from the group consisting of SEQ ID NOs: 22-50. 3. The tau peptide immunogen construct according to claim 1, wherein the peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 51-71 and 125-155. 4. Tau-peptide immunogenic construct, containing: B-cell epitope selected from the group consisting of SEQ ID NOs: 1-21 and 101-124; T-helper epitope containing an amino acid sequence selected from the group consisting of SEQ ID NO: 22-50; And an optional heterologous spacer selected from the group consisting of amino acids, Lys-, Gly-, Lys-Lys-Lys-, (α, ε- N )Lys, and ε- N -Lys-Lys-Lys-Lys of SEQ ID NO : 102 where the B cell epitope is covalently linked to the T helper epitope either directly or through an optional heterologous spacer. 5. The tau peptide immunogen construct of claim 4 wherein the T helper epitope is selected from the group consisting of SEQ ID NOs: 22-50. 6. The tau peptide immunogen construct of claim 4, wherein the optional heterologous spacer is (α, ε- N )Lys or ε- N -Lys-Lys-Lys-Lys of SEQ ID NO: 102. 7. The tau peptide immunogen construct of claim 4, wherein the T helper epitope is covalently linked to the N-terminus of the B cell epitope. 8. The tau peptide immunogen construct of claim 4, wherein the T helper epitope is covalently linked to the N-terminus of the B cell epitope via an optional heterologous spacer. 9. Immunogenic composition containing a peptide immunogenic construct according to any one of paragraphs. 1-3. 10. Pharmaceutical composition for the treatment of tauopathy, containing: A. a peptide immunogen construct according to any one of paragraphs. 1-3; And b. a pharmaceutically acceptable delivery carrier and/or adjuvant. 11. Pharmaceutical composition for the prevention of tauopathy, containing: A. a peptide immunogen construct according to any one of paragraphs. 1-3; And b. a pharmaceutically acceptable delivery carrier and/or adjuvant. 12. Pharmaceutical composition according to claim 10 or 11, where A. The tau peptide immunogen construct is selected from the group consisting of SEQ ID NOs: 51-71 and 125-155; And b. The tau peptide immunogen construct is mixed with a CpG oligodeoxynucleotide (ODN) to form a stabilized immunostimulatory complex.

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