Antigen tau-peptides and their application

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to immunogens based on antigenic tau-peptide, and can be used in medicine. Obtained is immunogen, which contains antigenic tau-peptide, consisting of amino acid sequence, selected from SEQ ID NO:6, 8-19, 21-26, 105 and 108-112, covalently bound with immunogenic carrier by means of linker, represented by formula (G)nC, where n equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Linker can be located either on C-terminal (peptide -(G)nC), or on N-terminal (C(G)n-peptide) of peptide. Obtained immunogens are used as base for creation of pharmaceutical compositions for treatment of tau-associated neurological disorders.

EFFECT: invention makes it possible to induce immune response against tau autoantigen in efficient way.

12 cl, 10 dwg, 5 tbl, 16 ex

 

The scope of the invention

The present group of inventions relates to immunogenum, immunogenic compositions and pharmaceutical compositions containing antigenic tau-peptide, which is linked to the immunogenic carrier, such as virus-like particle (the VLP), for the treatment of tau-associated neurological disorders or conditions such as Alzheimer's disease and mild cognitive impairment. The invention also relates to methods of producing these immunogens, immunogenic compositions and pharmaceutical compositions and their use in medicine.

Prior art

Alzheimer's disease, also called dementia Alzheimer's or BA, is a progressive neurodegenerative disorder or condition that causes memory loss and serious mental disorder. BA represents the most common form of dementia, causing more than half of all dementias. It is estimated that over 26 million people worldwide suffer from the effects of BA, and it is expected that this number will increase fourfold by 2050 as a result of ageing of the population (Brookmeyer et al., Alzheimer's & Dementia 3:186-191 (2007)). In addition to human losses and reduced quality of life, economic costs to society are enormous, considering that the average patient with BA live from 8 to 10 years after diagnosis and t is Eboue intensive daily care. Early-stage patients who complain of mild memory loss and confusion, are characterized as suffering from mild cognitive impairment (MCI), which in some cases progresses to the classic symptoms of Alzheimer's disease, leading to severe deterioration of mental and social abilities.

Alzheimer's disease (AD) is usually characterized by the accumulation Narineh plaques and tangles in the brain, leading to neuronal cell death with subsequent progressive decline in cognitive abilities. Most of the currently available treatments for BA is aimed at treating the symptoms, but not necessarily stop the progression of the disease. Accordingly, it is clear that desired are new approaches to identify treatments that can protect neurons from the debilitating effects of BA.

Most modern therapeutic approaches in the treatment of ad is based on the widely recognized "hypothesis the amyloid cascade". This concept assigns the pathophysiological role of the amyloid-β (β) as neuro - and synaptotagmin in the form from a Monomeric to oligomeric and postponed in the form of polymer in amyloid plaques, one of the characteristic features of the pathology of ad. I believe that monoclonal antibodies against a range of the and forms β are effective, because they shift the balance the brain-blood side of the blood, thus depleting the reserves β in the brain.

Pathophysiology of ad is characterized not only by the deposition β in senile plaques, but also includes the accumulation of neurofibrillary tangles (NFT). NFT are fibrils formed of paired helical filaments, which are connected together by means hyperphosphorylated Tau protein. Tau can be randomly phosphorylated by different kinases in more than 30 different residues serine and threonine (Hanger et al., J. Neurochem. 71:2465-2476 (1998)), and several tyrosine residues (Lebouvier et al., JAD 18: 1-9 (2009)). Obviously, in ad there is an imbalance kinase and fosfatnykh activities, leading to hyperphosphorylated forms of the Tau protein, which aggregate and accumulate in the form of NFT.

Mild cognitive impairment (MCI) is most often defined as the presence of a measurable worsening of memory in excess of the normally expected with aging, but still not displaying other symptoms of dementia or AD. Apparently, MCI is a transitional state between the cognitive changes associated with normal aging and early dementia. When memory loss is the predominant symptom, then this type of MCI additionally defined as amnestic MCI. Individuals with this subtype of MCI exposed to N. the highest probability of progression to BA at a speed of about 10-15% per year (Grundman M, et al., Arch Neurol. 61, 59-66, 2004). A large study published in 2005, was the first clinical trial, which demonstrated that treatment of patients with MCI may slow the transition in BA during the first year of testing (Petersen RC et al., NEJM 352, 2379-2388, 2005), which means that these patients represent a population that is appropriate for therapeutic intervention in AD.

A recent study showed that vaccination against phosphorylated Tau peptides in the model tangles of pathological Tau in mice led to a decrease in aggregated Tau in the brain and improvements in associated with tangles behavioral disorders (Asuni et al., J. Neurosci. 27:9115-9129 (2007)). Although the influence of hyperphosphorylated Tau and NFT loss of cognitive abilities and progression of ad is not clear, modern concepts agree that targeting only the amyloid enough to see an improvement in the disease course, which makes it necessary to search for additional or alternative targets (Oddo et al., J. Biol. Chem. 281:39413 (2006)). Therefore, to obtain an effective therapeutic vaccine against ad and MCI may be necessary to approach with an active vaccine that aims to associated with the disease conformation of Tau protein.

In addition, there are a number of diseases in addition to BA and MCI, which are also associated with Tau pathological the Oia or "toptime", who could potentially benefit from Tau vaccine, specifically aimed at involved pathological forms. These diseases include, for example, fronto-temporal dementia, Parkinson's disease, a disease of the Peak, progressive supranuclear palsy and complex amyotrophic lateral sclerosis/parkinsonism-dementia (see, for example, Spires-Jones et al., TINS 32:150-9(2009)).

Summary of the invention

In the present invention proposed new immunogen, immunogenic compositions and pharmaceutical compositions that contain at least one antigenic Tau peptide, which is able to induce an immune response, in particular or antibody-based test answers, resulting in antibody titer against autoantigen Tau in its pathological hyperphosphorylation condition. Such immunogen, immunogenic compositions and pharmaceutical compositions exhibit numerous desirable properties, such as the ability to induce an immune response, in particular or antibody-based test answers, with a therapeutic effect against the occurrence and development of neurodegenerative diseases associated with hyperphosphorylated Tau, such as Alzheimer's disease and MS.

In one aspect of the invention proposed an immunogen containing at least one antigenic Tau peptide associated with immunogenic wear elem, where specified antigenic Tau peptide contains phospho-Tau epitope selected from the pSer-396 phospho-Tau epitope, pThr-231/pSer-235 phospho-Tau epitope, pThr-231 phospho-Tau epitope, pSer-235 phospho-Tau epitope, pThr-212/pSer-214 phospho-Tau epitope, pSer-202/pThr-205 phospho-Tau epitope and epitope.

In one example, the specified phospho-Tau epitope is a pSer-396 phospho-Tau epitope. In another example, the indicated phospho-Tau epitope is a pThr-231/pSer-235 phospho-Tau epitope. In another example, the indicated phospho-Tau epitope is a pThr-231 phospho-Tau epitope. In another example, the indicated phospho-Tau epitope is a pSer-235 phospho-Tau epitope. In another example, the indicated phospho-Tau epitope is a pThr-212/pSer-214 phospho-Tau epitope. In another example, the indicated phospho-Tau epitope is a pSer-202/pThr-205 phospho-Tau epitops another example, the indicated phospho-Tau epitope is a pTyr-18 phospho-Tau epitope.

In another aspect of the invention proposed an immunogen containing at least one antigenic Tau peptide associated with immunogenic carrier, where the specified antigenic Tau peptide contains an amino acid sequence selected from SEQ ID NO:4, 6-26, 105, 108-112.

In one example, the specified antigenic Tau peptide covalently associated with the specified immunogenic carrier via a linker represented by the formula (G)nC, where the specified link is R is either on the C-end (peptide-(G) nC)or M-(C(G)n-peptide) of the peptide, and where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another example, the specified linker is N-the end of the specified Tau-peptide, and n is 1 or 2. In another example, the specified linker is-the end of the specified Tau-peptide, and n is 1 or 2. In another example, the specified antigenic Tau peptide contains an amino acid sequence selected from SEQ ID NO:4 and 6-13. In another example, the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID NO:4 and 6-13. In another example, the specified antigenic Tau peptide consists of the amino acid sequence represented in SEQ ID NO:11.

In another example, the specified antigenic Tau peptide contains an amino acid sequence selected from SEQ ID NO:14-19. In another example, the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID NO:14-19. In another example, the specified antigenic Tau peptide consists of the amino acid sequence represented in SEQ ID NO:16.

In another example, the specified antigenic Tau peptide contains an amino acid sequence selected from SEQ ID nos:20-24. In another example, the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID nos:20-24. In another example, the specified antigenic Tau peptide of sostoi is from amino acid sequence, presented in SEQ ID NO:21.

In another example, the specified antigenic Tau peptide contains an amino acid sequence selected from SEQ ID NO:105 and 108 to 112. In another example, the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID NO:105 and 108 to 112. In another example, the specified antigenic Tau peptide consists of the amino acid sequence represented in SEQ ID NO:105.

In one aspect of the present invention proposed any immunogens described in this application where indicated immunogenic carrier is hemocyanin (such as KLH (hemocyanin lymph snails)), serum albumin, globulin, a protein extracted from Ascaris, or an inactivated bacterial toxin.

In one aspect of the present invention proposed any immunogens described in this application where indicated immunogenic carrier is a virus-like particle selected from the group consisting of HBcAg the VLP, the VLP HBsAg and Qbeta the VLP. In one example, the invention proposed a composition comprising at least two of the immunogen, as described in this application. In another example, the composition contains at least three immunogen, as described in this application.

In one example, the present invention proposed a composition comprising at least two of the immunogen, as described in the Anna of the application where:

a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:4 and 6-13; and

b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID NO:14-19.

In another example, the present invention proposed a composition comprising at least two of the immunogen, as described in this application, where:

a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:4 and 6-13; and

b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID nos:20-24.

In another example, the invention proposed a composition comprising at least two of the immunogen, as described in this application, where:

a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:14-19; and

b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID nos:20-24.

In another example, the present invention proposed a composition comprising at least two of the immunogen, as described in this application, where:

a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:4 and 6-13; and

b) antigenic Tau peptide of the second and the of monogene, selected from SEQ ID NO:105 and 108 to 112.

In another example, the present invention proposed a composition comprising at least two of the immunogen, as described in this application, where:

a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:14-19; and

b) antigenic Tau peptide of the second immunogen selected from SEQ ID NO:105 and 108 to 112.

In another example, the present invention proposed a composition comprising at least two of the immunogen, as described in this application, where:

a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID nos:20-24; and

b) antigenic Tau peptide of the second immunogen selected from SEQ ID NO:105 and 108 to 112.

In another example, the invention proposed a composition comprising at least three of the four immunogen, as described in this application, where:

a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:4 and 6-13;

b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID NO:14-19; and

C) antigenic Tau peptide of the third immunogen consists of an amino acid sequence selected from SEQ ID nos:20-24.

d) antigenic Tau peptide of the fourth immunogen selected from SEQ ID NO:105 and 108 to 112.

In another when the ore in the proposed invention, any of the compositions, described in this application, where each of these antigenic Tau peptides independently covalently associated with the specified immunogenic carrier via a linker represented by the formula (G)nC, where each of these linkers is independently either on-end (peptide-(C)nG)or N-(C(G)n-peptide) of the specified Tau-peptide, and where each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. In another example, in the proposed invention, any of the compositions described in this application, where each of these linkers is on the N end of Tau-peptide, and where each n independently is 1 or 2.

In another aspect of the present invention proposed a composition comprising at least three of the four immunogenic where:

a first immunogen comprises at least one antigenic Tau peptide associated with Qbeta the VLP, where specified antigenic Tau peptide consists of SEQ ID NO:11, and where the specified peptide covalently linked to the VLP specified via a linker represented by the formula (G)nC, where specified, the linker is either on the C-end (peptide-(G)nC)or N-(C(G)n-peptide) of the specified Tau-peptide, and where n is 1 or 2;

b) a second immunogen comprises at least one antigenic Tau peptide associated with Qbeta the VLP, where specified antigenic Tau peptide consists of SEQ ID NO:16, and where specified the peptide Koval the IDT is associated with the VLP specified by the linker, represented by formula (G)nC, where specified, the linker is either on the C-end (peptide-(G)nC)or N-(C(G)n-peptide) of the specified Tau-peptide, and where n is 1 or 2; and

C) the third immunogen comprises at least one antigenic Tau peptide associated with Qbeta the VLP, where specified antigenic Tau peptide consists of SEQ ID NO:21, and where the specified peptide covalently linked to the VLP specified via a linker represented by the formula (G)nC, where specified, the linker is either on the C-end (peptide-(G)nC)or N-(C(G)n-peptide) of the specified Tau-peptide, and where n is 1 or 2.

d) fourth, the immunogen comprises at least one antigenic Tau peptide associated with Qbeta the VLP, where specified antigenic Tau peptide consists of SEQ ID NO:105, and where the specified peptide covalently linked to the VLP specified via a linker represented by the formula (G)nC, where specified, the linker is either on the C-end (peptide-(G)nC)or N-end (G(C)n-peptide) of the specified Tau-peptide, and where n is 1 or 2.

In one example, each of the linkers of the first, second and third immunogens are at the N end of each of the antigenic Tau peptides and where for each of these linkers n is 2.

In another aspect of the present invention proposed a composition comprising any of immunogens or compositions described in Dan the th application additionally contains at least one adjuvant selected from alum, CpG-containing oligonucleotides and adjuvants on the basis of saponin.

In another aspect of the present invention proposed a pharmaceutical composition comprising any of immunogens or compositions described in this application, and pharmaceutically acceptable excipient. In one example, at least one adjuvant is a CpG-containing oligonucleotide selected from CpG 7909 (SEQ ID NO:27), CpG 10103 (SEQ ID NO:28) and CpG 24555 (SEQ ID NO:29).

In another aspect of the present invention proposed a pharmaceutical composition comprising any of immunogens or compositions described in this application, and pharmaceutically acceptable excipient.

In another aspect of the present invention, a method of immunization, including the introduction of any mammal(s) from immunogenum, compositions or pharmaceutical compositions described in this application. For example, in one aspect of this introduction is carried out using a pharmaceutically effective dose of any(s) from immunogenum, compositions or pharmaceutical compositions described in this application.

In another aspect of the invention, a method for treating Tau-associated neurological disorders in a mammal, comprising an introduction to the specified mammal, therapeutic the ski effective amount of any(s) from immunogenum, immunogenic compositions or pharmaceutical compositions described in this application.

In one aspect of this introduction is carried out using a pharmaceutically effective dose of any(s) from immunogenum, compositions or pharmaceutical compositions described in this application.

In another aspect of the invention, a method for treating Tau-associated neurological disorders in a mammal, comprising an introduction to the specified mammal: (a) a pharmaceutically effective dose of any(s) from immunogenum, immunogenic compositions or pharmaceutical compositions described in this application; and (b) a pharmaceutically effective dose of at least one adjuvant. In one example, at least one adjuvant selected from alum, CpG-containing oligonucleotides and adjuvants on the basis of saponin. In another example, at least one adjuvant is a CpG-containing oligonucleotide selected from CpG 7909 (SEQ ID NO:27), CpG 10103 (SEQ ID NO:28) and CpG 24555 (SEQ ID NO:29).

In another example, the specified neurological disorder is a disease of Alzheimer's. In another example, the specified neurological disorder diagnosed as mild cognitive impairment. In another example, the specified neurological disorder diagnosed as amnestic MCI.

In another example, in izaberete the AI proposed the use of any(s) from immunogenum, the compositions or pharmaceutical compositions described in this application, for the manufacture of a medicinal product. For example, in one aspect, these medicines can be used for treating Tau-associated neurological disorders in a mammal. In one example, the specified neurological disorder is a disease of Alzheimer's. In another example, the specified neurological disorder diagnosed as mild cognitive impairment (MCI). In another example, the specified neurological disorder diagnosed as amnestic MCI.

In another aspect of the invention proposed by the selected antibody that is produced in response to any of the methods of immunization, as described in this application, where the specified antibody specifically binds to hyperphosphorylation form of human Tau.

In another aspect of the invention, a method for treating Tau-associated neurological disorders in a mammal, comprising an introduction to the specified mammal antibody that specifically binds to hyperphosphorylation form of human Tau, and where the aforementioned antibody is produced in response to any of the methods of immunization, as described in this application.

In another aspect of the invention it is proposed to use any of the antibodies is, described in this application, for the manufacture of a medicinal product for treating Tau-associated neurological disorders in a mammal. In one example, the specified neurological disorder is a disease of Alzheimer's. In another example, the specified neurological disorder diagnosed as mild cognitive impairment (MCI). In another example, the specified neurological disorder diagnosed as amnestic MCI.

In another aspect of the present invention proposed a selected peptide comprising or essentially consisting of the amino acid sequence selected from SEQ ID NO:4, 6-26, 31-76 and 105-122. In another aspect of the present invention proposed a selected nucleic acid that encodes any of these selected peptides. In another aspect of the present invention is proposed expressing a vector containing any of the foregoing nucleic acids. In another aspect of the present invention proposed a host cell containing any of these expression vectors.

A brief description of graphic materials

On Figa and 1B presents a description of the groups of Balb/c mice, which were immunized subcutaneously, and the results of the titles and selectivity, as described in Example 5. Balb/c mice were immunized subcutaneously using 300 μg of peptide, 100 µg peptide is IDA-KLH or 100 μg of peptide-the VLP. Where indicated, 50 μl TiterMax Gold (Alexis Biochemicals) was used as adjuvants. Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13)was varied from 1:30 to 1:7290.

Figure 2 presents a description of the groups of Balb/c mice, which were immunized, and the results of the credits, as described in Example 5. Balb/c mice were immunized subcutaneously. Where indicated, 50 μl TiterMax Gold was used as adjuvant. Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:900 to 1:1968300.

Figure 3 presents a description of Balb/c mice, which were immunized subcutaneously as described below in Example 6. 100 μg of peptide was used for the primary (prime) immunization and 100 µg peptide-the VLP used for secondary (boosts) immunization (reimmunization). Where specified, 750 μg of alum (Al(OH)3used as adjuvants. Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:800 to 1:1750000. n/a means not determined.

On Figa, 4B and 4C show the results on mice TG4510++, which were immunized intramuscularly as described in Example 7. On Figa shows the results of titers for Groups 1-7, while Figb shows the results of titers for Groups 8-17. On FIGU shows the results of selectivity for the group is 1-6. CPG is a CpG 24555. Alum are Al(OH)3. Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:5000 to 1:15800000. n/a means not determined.

Figure 5 presents a description of mice that were immunized as described in Example 8. Balb/c mice were immunized either intramuscular (I/m)or subcutaneous (s/C) by. Where specified, used 90 μg of peptide-the VLP. Where specified, used 1,595 μg of alum (Al(OH)3), 20 μg CpG 24555 and 12 µg ABISCO-100. Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:5000 to 1:15800000. Lower limit of detection of the standard curve was 0,0025 mg/ml NA means not applicable.

Figure 6 presents a description of mice that were immunized as described in Example 11. Balb/c mice were immunized intramuscularly. Used 100 µg peptide-the VLP. Where specified, used 252 (750) μg of alum (Al(OH)3). Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:500 to 1:2720000. n/a means not determined.

Figure 7 presents a description of mice that were immunized as described in Example 11. Balb/c mice were immunized intramuscularly. 750 μg of alum (Al(OH)3used as adjuvanticity serum tested in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:500 to 1:15800000.

On Fig describes the mice, which were immunized as described in Example 12. Mice TG4510-/- (odnomomentnye animals wild-type) were immunized intramuscularly. 100 μg of each peptide is the VLP used for 0-day (primary immunization) and 14 days (reimmunization), as indicated. Used the specified amount of alum (Al(OH)3). Serum from a group of 'No treatment' together. Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:5000 to 1:15800000.

Figure 9 presents a description of mice that were immunized as described in Example 12. Mice TG4510-/- (odnomomentnye animals wild-type) were immunized intramuscularly. 100 μg of each peptide is the VLP used for 0-day (primary immunization) and 14 days (reimmunization). Used without alum or 504 μg of alum (Al(OH)3). Spleen was collected on the 21st day. Shows the number of points on 5×105the spleen cells as measured by ELIspot analysis of T cells secreting interferon-gamma (see Example 14). The results obtained from the pool 3 spleens. The HBV peptide-1 (SEQ ID NO:77) was an unrelated peptide. BSA was an unrelated protein, n/a means not defined the Lee. * means p<0,05 against an unrelated peptide or protein, depending on the situation.

Figure 10 shows the amino acid sequence of Tau isoform 2 of human, Genbank no access NP_005901 (SEQ ID NO:30).

Detailed description

Definitions and General methods

If not otherwise specified in this application of scientific and technical terms used in connection with the present invention, must have values that are generally understandable to the average experts in this field. In General, the nomenclature and methods of cell and tissue culture, molecular biology, immunology, Microbiology, genetics and chemistry of proteins and nucleic acids, hybridization, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described in this application represent the nomenclature and methods are well known and commonly used in this field.

Ways and methods of the present invention is usually carried out in accordance with conventional methods, well known in this field, and as described in various General and more specific sources that are cited and discussed in the present description, unless otherwise specified. See, for example, J. Sambrook & Russell D. Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2000); Ausubel et al., Short Protocols in Molecular Biology: A Compendium of Methods from Current Protocols inMolecular Biology, Wiley, John & Sons, Inc. (2002); Harlow and Lane, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1998); and Coligan et al., Short Protocols in Protein Science, Wiley, John & Sons, Inc. (2003). Enzymatic reactions and purification methods carried out in accordance with the manufacturer's recommendations, as usually done in this area, or as described in this application.

The term "mild cognitive impairment (MCI)", as used in this application belongs to the category of deterioration of memory and cognitive ability, which is usually characterized by a clinical dementia rating (CDR) of 0.5 (see, for example, Hughes et al., Brit. J. Psychiat. 140: 566-572, 1982) and are further characterized by memory impairment, but not function impairment in other cognitive areas. Memory impairment is preferably measured using tests such as the test's ability to understand the main idea of the passage" ("paragraph test"). The patient diagnosed with moderate cognitive impairment often has degraded the ability of delayed recall. Mild cognitive impairment in most cases related to the aging process and usually occurs in patients aged 45 years or older.

The term "dementia"as used in this application refers to a mental disorder in its most broad sense, as defined in the American Psychiatric Association: Diagnostic and Statistical Manual of Mental Disorders (American Assoc the situation of psychiatrists: the diagnostic and statistical manual of mental diseases), Fourth Edition, Washington, D.., 1994 ("DSM-IV"). DSM-IV defines "dementia" as characterized by multiple cognitive impairments, which include memory loss, and enumerates various dementia according to presumed etiology. The DSM-IV outlines the generally accepted standard for this diagnosis, categorization and treatment of dementia and associated psychiatric disorders.

The term "Tau" or "Tau protein" refer to the Tau protein, which is associated with stabilization of microtubules in nerve cells and is a component of a wide spectrum of Tau aggregates, such as neurofibrillary tangles. In particular, the term "Tau protein", as used in this application encompasses any polypeptide containing or consisting of human Tau with SEQ ID NO:30, or other human isoforms with or without modification, modifications, or the corresponding orthologues from any other animal. The term "Tau protein", as used in this application also covers post-translational modifications, including, but not limited to, glycosylation, acetylation and phosphorylation of the Tau protein, as defined above.

The term "tapatia" refers to Tau-associated disorders or conditions such as Alzheimer's disease, a progressive supranuclear palsy (PSP), kortiko-basal degeneration (CBD), diseases of the Peak, the forehead is about-temporal dementia and parkinsonism, associated with chromosome 17 (FTDP-17), Parkinson's disease, stroke, traumatic brain injury, mild cognitive impairment, and the like.

The terms "antigen" and "immunogen", which, as implied in this application are used interchangeably, refer to a molecule capable of contacting an antibody, b-cell receptor (BCR) or T-cell receptor (TCR), if he is represented by molecules Mnster "antigen" and "immunogen"as used in this application also cover T-cell epitopes. In addition, the antigen can be recognized by the immune system and/or capable of inducing a humoral immune response and/or cellular immune response leading to the activation and/or T-lymphocytes. However, this may result, at least in some cases, to the antigen contained or was associated with T-helper cell epitope and was provided by the adjuvant. The antigen may have one or more epitopes (e.g., b - and T-epitopes). Assume that the specific reaction, discussed above, indicates that the antigen is preferable to react, usually highly selective manner, with its corresponding antibody or TCR, and not with the multitude of other antibodies or TCR, which can be induced by other antigens. Antigens, as used in this application can also before the presentation of a mixture of several individual antigens. Both terms "antigen" and "immunogen" includes, but is not limited to polypeptides.

The terms "antigenic site" and the term "antigenic epitope"as used in this application interchangeably, refer to a continuous or discontinuous parts of the polypeptide, which can be immunospecificity bound by the antibody or T-cell receptor in the context of MHC molecules. Immunospecificity linking eliminates nonspecific binding, but not always exclude cross-reactivity. Antigenic sites usually contain from 5 to 10 amino acids in a spatial conformation which is unique to the antigenic site.

As used in this application, the term "phosphorylated" in relation to amino acid residue refers to the presence of phosphate groups on the side chain of this residue, where otherwise there is normally a hydroxyl group. This phosphorylation is usually done as a replacement of the hydrogen atom from the hydroxyl group on the phosphate group (RHO3H2). As is well known to specialists in this field, depending on the pH of the local environment, this phosphate group can exist in the form of uncharged, neutral group (RHO3H2), or single (RHO3N-) or double (RHO32-) a negative charge. Amino acid mod and, which can be usually phosphorylated include side chains of serine, threonine and tyrosine. In the present invention phosphorylated amino acid residue is shown in bold and underlined.

As used in this application, reference to amino acid residues denoted by a single-letter or three-letter code (see, e.g., Lehninger, Biochemistry, 2ndedition, Worth Publishers, New York, 1975, p.72).

The singular is used in this application with reference to one or more than one (i.e. at least one) of the grammatical object. As an example, "an element" means one element or more than one element. In addition, unless otherwise required by context, the terms in the singular shall include the plural, and the terms in the plural shall include uniqueness, unless the context clearly requires otherwise.

The term "peptide" or "polypeptide" refers to a polymer of amino acids, regardless of length of the polymer; thus, protein fragments, oligopeptides and proteins are included in the determination of the peptide or polypeptide. This term also does not define or exclude postexplosion modification of polypeptides, such as polypeptides, which include covalent joining helicoiling groups, acetyl groups, phosphate groups, lipid groups and the like, clearly covered by the term the "polypeptide". This definition also includes polypeptides that contain one or more analogs of an amino acid (including, for example, naturally occurring amino acids, amino acids that occur in nature only in not unrelated biological system, the modified amino acids of the systems of mammals and so on), polypeptides with substituted linkages, as well as other modifications known in this area, both natural and not natural.

The term "Tau fragment", as used in this application encompasses any polypeptide containing or consisting of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 consecutive amino acids of the Tau protein, as defined in this application.

The term "pSer-396 phospho-Tau epitope", as used in this application refers to a peptide containing the amino acid sequence of KSP (i.e. Lys-Ser 395-396 Pro-397 of human Tau sequence), where the serine residue is phosphorylated, and where the sequence numbering based on the human Tau isoform 2, which is proposed in the form of SEQ ID NO:30. pSer-396 phospho-Tau epitope typically has a length of from about 3 to about 25 amino acids.

The term "pThr-231/pSer-235 phospho-Tau epitope", as used in this application refers to a peptide containing the amino acid sequence TPPKS (SEQ ID NO:1) (that is. Thr-231 Pro-Pro 232-233 Lys-Ser 234-235 from human Tau sequence), where each of the residues of threonine and serine is phosphorylated, and where the sequence numbering based on the human Tau isoform 2, which is proposed in the form of SEQ ID NO:30. Such epitopes typically have a length of from about 5 to about 25 amino acids. pThr-231/pSer-235 phospho-Tau epitope can also refer to a form of this epitope, which contains the phosphorylated residue Thr-231, but does not include the phosphorylated residue Ser-235, or contains phosphorylated residue Ser-235, but does not include a phosphorylated epitope Thr-231. Such variants of this epitope typically have a length of from about 3 to about 20 amino acids.

The term "pThr-212/pSer-214 phospho-Tau epitope", as used in this application refers to a peptide containing the amino acid sequence of TPS (i.e. Thr-212 Pro-Ser 213-214 from human Tau sequence), where each of the residues of threonine and serine is phosphorylated, and where the sequence numbering based on the human Tau isoform 2, which is proposed in the form of SEQ ID NO:30. pThr-212/pSer-214 phospho-Tau epitope typically has a length of from about 3 to about 25 amino acids.

The term "pSer-202/pThr-205 phospho-Tau epitope", as used in this application refers to a peptide containing the amino acid sequence SPGT (SEQ ID NO:3) (i.e., Ser-202 Pro-203 Gly-Thr 204-is human Tau sequence), where each of the residues of threonine and serine is phosphorylated, and where the sequence numbering based on the human Tau isoform 2, which is proposed in the form of SEQ ID NO:30. pSer-202/pThr-205 phospho-Tau epitope typically has a length of from about 4 to about 25 amino acids.

The terms "purified" and "isolated", as used in this application are synonyms. For example, the terms "isolated" or "purified" in respect of a polypeptide refers to the polypeptide, which by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) is essentially free of other proteins from the same species, (3) is expressed by a cell from a different species, or (4) does not occur in nature. Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it occurs in nature, will be "separated" from its naturally associated components. The polypeptide can also be converted essentially free of naturally associated components by selection using the methods of protein purification well known in this field. A polypeptide is "essentially pure", "essentially homogeneous" or "essentially purified"when at least about 60 is about 75% of the sample indicate a single type of polypeptide. The polypeptide may be Monomeric or multimeric. Essentially pure polypeptide can typically contain about 50%, 60%, 70%, 80% or 90% wt./mass. polypeptide sample, more typically about 95%, and preferably may have a purity greater than 99%. The purity or homogeneity of the protein can be shown in several ways, well izvestnymi in this area, such as electrophoresis of the sample protein in polyacrylamide gel followed by visualization of a single polypeptide band upon staining the gel dye, well known in this field. For some purposes, higher resolution can be achieved using HPLC (high performance liquid chromatography) or other means well known in the field for cleaning.

The term "Tau-associated neurological disorder", as used in this application, means any disease or other condition in which Tau (especially hyperphosphorylated forms Tau), as I believe, plays a role. Such disorders, diseases and/or conditions typically correlate with the presence of neurofibrillary tangles (usually including hyperphosphorylated forms of Tau) and include, without limitation, Alzheimer's disease, MCI, fronto-temporal dementia, a disease Peak, progressive nuclear palsy, kortiko-basal degeneration, it is the complex of parkinsonism-dementia of GUAM island and other chaupati.

The term "antigenic Tau peptide", as used in this application encompasses all Tau-occurring polypeptides, such as originating from mammals, for example humans, and variants, analogs, orthologues, homologues and derivatives and fragments that exhibit the biological activity of the antigenic Tau peptide". For example, the term "antigenic Tau peptide" refers to polypeptides containing, comprising or essentially consisting of the amino acid sequence selected from SEQ ID NO:1-26, 31-76 and 105-122, and their variants, homologues and derivatives, showing essentially the same biological activity.

The term "biological activity antigenic Tau peptide", as used in this application refers to the ability of the antigenic Tau peptides according to the invention to induce auto-Tau antibodies in the subject with antagonistic profile, where such auto-antibodies can reduce the level hyperphosphorylation, pathological forms of Tau, but not being able to communicate with normal megaperfezione, non-pathological forms of Tau. In addition, antigenic Tau peptide, which has the biological activity of the antigenic Tau peptide, can be designed to minimize Tau-specific T-cell response when administered to a patient. Specialists in given the Oh region will be obvious methods which can be used to determine hits or misses the particular design in the scope of the present invention. Such methods include, but are not limited to, the methods described in the Examples section of the present invention, as well as the following. Peptide with expected biological activity of the antigenic Tau peptide can be analyzed to determine the immunogenicity of the peptide (for example, to determine whether the antisera induced putative peptide to bind hyperphosphorylated forms of Tau, but essentially does not bind megaperfezione, non-pathological forms of Tau). In addition, the peptide with the intended biological activity of the antigenic Tau peptide can be analyzed to determine induces whether the peptide or essentially no induces Tau-specific-mediated T-cell response.

The term "hyperphosphorilated" or "abnormally phosphorylated," as used in this application refers to Tau, which contains at least about 7 (i.e. about 7 or more phosphate groups in the molecule of Tau (see, for example, Kopke et al., J Biol Chem 268:24374-84 (1993)). Hyperphosphorilated Tau is a major component of neurofibrillary tangles (NFT) and paired helical filaments (PHF), detected in patients shall now with BA, and hyperphosphorylation is responsible for the loss of normal Tau biological activity and self-aggregation. Some remnants of the Tau usually detected phosphorylated only in its pathological hyperphosphorylation forms, such as PHF and NFT. These residues include Ser-202, Thr-205, Thr-212, Ser-214, Thr-231, Ser-235, Ser-396 and/or Ser-404, Tyr-18. Therefore, Tau protein phosphorylated at multiple sites, usually not involved in the binding of Tau to microtubules, in particular on sites found in the Proline-rich sites flanking the site of binding of Tau to microtubules and containing the main component of PHF and NFT are also included in the term "hyperphosphorilated Tau" or "abnormally phosphorylated Tau.

Antigenic Tau peptides

Tau-protein of man is associated with microtubule protein, which is relatively widely distributed in neurons of the Central nervous system, but less common in other areas. In brain tissue Tau exists in the form of six different isoforms resulting from alternative splicing in exons 2, 3, and 10 of the Tau gene. Human Tau-isoform 2 (SEQ ID NO:30) is used in this application as compare to the numbering of the amino acids of all Tau peptides of the present invention. Tau normally interacts with tubulin to stabilize microtubules and the tis oireet the Assembly of tubulin into microtubules, and also provides axonal transport of proteins. Tau is a regulated during development fastbloc, typically containing from 2 to 3 phosphate groups on the molecule in its normal state in the brain of an adult. However, Tau can be randomly phosphorylated by different kinases in more than 30 different residues, mainly motivated by Ser/Thr-Pro (Hanger et al., J. Neurochem. 71:2465-2476 (1998)).

Antigenic Tau peptides of the present invention will typically have a small size so that they mimic the plot of the whole of the Tau protein, which is detected epitope in the pathological form of Tau. As described previously, such pathological forms of Tau are usually characterized by fosforilirovanii for some amino acids in the Tau protein. Therefore, antigenic Tau peptides according to the invention typically have a length of less than 100 amino acids, for example less than 75 amino acids, for example less than 50 amino acids. Antigenic Tau peptides according to the invention typically have a length of approximately 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or about 30 amino acids. Specific examples of antigenic Tau peptides according to the invention, presented in the sequence listing, include peptides, the length of which varies from 4 to 31 amino acids. As will be obvious to experts in this field, such antigenic peptides are usually its odny N-end and can have either carboxypropanoyl, or aminirovanie-end.

Antigenic peptides according to the invention contain amino acid sequence derived from an area of the human Tau in his hyperphosphorylation or pathological form. In particular, such antigenic Tau peptides will typically contain specific phospho-Tau epitopes, which can be described in the literature with reference to antibodies that bind to these epitopes (such as PHF1, TG3, AT and/or AT; see, for example, Hanger et al., J. Biol. Chem. 282 (32):23645-23654 (2007); Pennanen et al., Biochem. Biophys. Res. Comm. 337:1097-1101 (2005); Porzig et al., Biochem. Biophys. Res. Comm. 358:644-649 (2007)).

In the present invention were identified by specific antigenic sites of human Tau protein, which when used alone or in combination with each other can be usefully used for the induction of immune responses against pathological forms hyperphosphorylated Tau. For example, pSer-396 phospho-Tau epitope is usually a fragment of a human Tau, which includes phosphorylated serine residue Ser-396. Such fragments typically have a length of from about 3 to about 20 amino acids (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and include at least one amino acid of the native human Tau sequence as N-terminal and C-terminal side of Ser-396. For example, pSer-396 phospho-Tau epitope will be ordinary is to contain the remains 395, 396 and 397 human Tau sequence as shown in SEQ ID NO:30 (i.e. Lys-Ser 395-396 Pro-397, where Ser-396 phosphorylated). Such pSer-396 epitopes can also optionally contain phosphorylated serine residue Ser-404 native human sequence. Examples of Tau-peptides containing a pSer-396 phospho-Tau epitope presented as SEQ ID NO:4 and 6-13.

In addition, for example, pThr-231/pSer-235 phospho-Tau epitope is typically a fragment of a human Tau, which includes both the phosphorylated threonine residue TBC-231 and phosphorylated serine residue Ser-235. Alternatively, pThr-231/pSer-235 phospho-Tau epitope includes only one of Thr-231 or Ser-235. Such epitopes typically have a length of from about 3 to about 20 amino acids (for example 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and include at least one amino acid of the native human Tau sequence at the N-terminal side of Thr-231 (i.e. Arg-230) and/or at least one amino acid at the C-terminal side of the Ser-235 (i.e. Pro-236). Examples of Tau-peptides containing pThr-231/pSer-235 epitope presented as SEQ ID NO:14-19.

In addition, for example, pThr-212/pSer-214 phospho-Tau epitope is typically a fragment of a human Tau, which includes the phosphorylated threonine residue Thr-212 and phosphorylated serine residue Ser-214. Such epitopes typically have a length of from about 3 to about 20 is of minislot (such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and include at least one amino acid of the native human Tau sequence at the N-terminal side of Thr-212 (i.e. Arg-211) and at least one amino acid at the C-terminal side of Ser-214 (i.e. Leu-215). Examples of Tau-peptides containing pThr-212/pSer-214 epitope presented as SEQ ID NO:20-24.

In addition, for example, pSer-202/pThr-205 phospho-Tau epitope is typically a fragment of a human Tau, which includes the phosphorylated threonine residue Ser-202 and the phosphorylated threonine residue Thr-205. Such epitopes typically have a length of from about 6 to about 20 amino acids (for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and typically include at least one amino acid of the native human Tau sequence at the N-terminal side of the Ser-202 (i.e. Gly-201) and at least one amino acid at the C-terminal side of Thr-205 (i.e. Pro-206). An example of Tau-peptide containing a pSer-202/pThr-205 epitope presented as SEQ ID NO:25.

In addition, for example, pTyr-18 phospho-Tau epitope is typically a fragment of a human Tau, which includes phosphorylated tyrosine residue Tyr-18. Such epitopes typically have a length of from about 6 to about 20 amino acids (for example 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20) and typically include at least one amino acid of the native human Tau sequence n the N-terminal side of the Tyr-18 (i.e. Thr-17) and at least one amino acid at the C-terminal side of the Tyr-18 (i.e. Gly-19). An example of Tau-peptide containing pTyr-18 epitope presented as SEQ ID NO:112.

Antigenic Tau peptides of the present invention can also include Tau-peptides containing phospho-Tau epitopes described above, including peptides, where a small number of amino acids has been substituted, added or deleterule, but which retain essentially the same immunological properties. In addition, such derived antigenic Tau peptides can be further modified by amino acids, especially N - and C-ends, to ensure conformationally restricted antigenic Tau peptide and/or to provide a combination of antigenic Tau peptide to the immunogenic carrier after the implementation of suitable chemical reactions.

Antigenic Tau peptides of the invention also encompass functionally active variant peptides derived from the amino acid sequence of Tau, in which amino acids have been delegated, inserted or substituted without significant reduction in their immunological properties, i.e. functionally variant peptides retain substantial biological activity of the antigenic Tau peptide. Typically, these functional variant peptides have an amino acid sequence homologous, site is preferably vysokogomogennogo, amino acid sequence described in any one of SEQ ID nos:1-26, 31-76 and 105-122.

In one aspect such functionally active variant peptides exhibit at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%identity of amino acid sequence selected from the group consisting of SEQ ID NO:1-26, 31-76 and 105-122.

The identity of amino acid sequences of polypeptides can be determined traditionally by using known computer programs such as the Bestfit, FASTA or BLAST (see, for example, Pearson, Methods Enzymol. 183:63-98 (1990); Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol. Biol. 215:403-410 (1990); Altschul et al., Nucelic Acids Res. 25:3389-3402 (1997)). When using Bestfit or any other program sequence alignment to determine whether a particular sequence is, for example, 95% identical to the comparative amino acid sequences, the parameters are set so that the percentage of identity is calculated over the entire length of the comparative amino acid sequences, and to have a valid gaps in homology of up to 5% of the total number of amino acid residues in the comparative sequence. This above method of determining the percent identity between polypeptides applies to all proteins, their fragments or variants described in this application.

Functionally active the e variants include naturally occurring functionally active variants, such as allelic variants and species variants, and not naturally occurring functionally active variants which can be obtained, for example, methods of mutagenesis or direct synthesis.

Functionally active variant differs, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid residues from any of the peptides presented in SEQ ID nos:1-26 and 31-76, and still retains the biological activity of the antigenic Tau. If this comparison requires alignment the sequences are aligned for maximum homology. Site variations can be anywhere in the peptide, provided that the biological activity is substantially similar to any of the peptides presented in SEQ ID NO:1-26, 31-76 and 105-122.

Guidance on the implementation of phenotypically silent amino acid substitutions proposed in Bowie et al., Science, 247:1306-1310 (1990), which States that there are two main research strategies of tolerance amino acid sequence to change.

The first strategy exploits the tolerance of amino acid substitutions by natural selection in evolution. By comparing amino acid sequences in different species can be identified amino acid positions that were conservative among species. These conserved amino acids, apparently, t is Auda important for the function of the protein. In contrast, amino acid positions where substitutions have been made by natural selection, indicate provisions which are not critical for the function of the protein. Thus, the position may be replaced with amino acids, can be modified while maintaining the specific immunogenic activity of the modified peptide.

The second strategy was uses genetic engineering to introduce amino acid changes at specific pagenum cloned gene to identify areas that are critical to the function of the protein. For example, can be used site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham et al., Science, 244: 1081-1085 (1989)). The obtained variant peptides can then be tested against specific antigenic biological activity of Tau.

According to Bowie et al., these two strategies have shown that proteins are unexpectedly tolerant to amino acid substitutions. The authors additionally indicate which amino acid substitutions, apparently, to be admissible in certain amino acid positions in the protein. For example, most private or internal (in the tertiary structure of the protein) amino acid residues require nonpolar side chains, while some signs of surface or exterior side chains are usually conservative.

The ways m which the relevant amino acids in protein are well known to specialists in this field (see, for example, Ausubel (ed.), Current Protocols in Molecular Biology, John Wiley and Sons, Inc. (1994); .Maniatis, E.F.Fritsch and J.Sambrook, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor laboratory, Cold Spring Harbor, N.Y. (1989)).

Mutations can also be introduced using commercially available kits, such a kit for site-directed mutagenesis "QuikChange™" (Stratagene). Creating a functionally active variant antigenic Tau peptide by replacement of amino acids, which essentially does not affect the function specified antigenic Tau peptide, can be carried out by a person skilled in the art. One type of amino acid substitutions that can be made in one of the peptides according to the present invention is a conservative amino acid substitution. "Conservative amino acid substitution" is a substitution in which the amino acid residue is replaced with another amino acid residue having a group R in the side chain with similar chemical properties (e.g. charge or hydrophobicity). Usually conservative amino acid replacement in essence will not change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other conservative substitutions, the percent sequence identity or similarity can be adjusted upwards to correct conse vatives nature of the replacement. Ways of osushestvleniya this adjustment are well known to specialists in this field (see, for example, Pearson, Methods Mol. Biol. 243:307-31 (1994)).

Examples of groups of amino acids that have side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; 2) aliphatic hydroxyl side chain; serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine and tryptophan; 5) the alkaline side chains: lysine, arginine and histidine; 6) acidic side chains: aspartic acid and glutamic acid; and (7) gray-containing side chains: cysteine and methionine. Preferred groups for conservative amino acid substitutions are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate and asparagine-glutamine.

Alternatively, a conservative replacement is any change having a positive value in the matrix of the logarithmic likelihood function RAM described in gonnet on et al., Science 256:1443-45 (1992). "Moderately conservative" replacement is any change having a positive value in the matrix of the logarithmic likelihood function RAM.

Functionally active variant peptide can also be selected with the use of the Finance method of hybridization. Briefly, to obtain functionally active peptide using DNA having high homology with a whole or part of a nucleic acid sequence that encodes a desired peptide, polypeptide or protein, for example, SEQ ID NO:1-26, 31-76 and 105-122. Therefore, antigenic Tau peptide of the invention also includes peptides which are functionally equivalent to any of SEQ ID nos:1-26 and 31-76 and can be encoded by a nucleic acid molecule, which hybridizes with nucleic acid that encodes any of SEQ ID nos:1-26, 31-76 and 105-122, or its complement. The person skilled in the art can easily determine the sequence of nucleic acids that encode the peptides described in this application, using easily accessible table of codons. As such, these nucleic acid sequences are not represented in this application.

The rigidity of hybridization to a nucleic acid that encodes a peptide, polypeptide or protein, which is a functionally active variant is, for example, 10% formamide, 5×SSPE, 1×denhardt's solution and 1×DNA salmon sperm (low stiffness). More preferred conditions are 25% formamide, 5×SSPE, 1×denhardt's solution and 1×DNA salmon sperm (conditions of moderate stringency), even more preferred conditions are 50% formamide, 5×SSPE,1×denhardt's solution and 1×DNA salmon sperm (high stiffness). However, some factors other than the above concentration of formamide, affect the stiffness of hybridization, and the person skilled in the art will be able, respectively, to select these factors to achieve the same stiffness.

The nucleic acid molecule encoding a functionally active variant, can also be selected using the method of gene amplification, such as PCR (polymerase chain reaction), using the portion of the molecule of nucleic acid, DNA that encodes a desired peptide, polypeptide or protein, for example, any of the peptides presented in SEQ ID NO:1-26, 31-76 and 105-122, in the form of a probe.

Obtaining peptides/proteins

The polypeptides of the present invention can be obtained from natural sources and isolated from a mammal, such as, for example, human, Primate, cat, dog, horse, mouse or rat. Thus, the polypeptides according to the invention can be isolated from sources of cells or tissues using standard methods of protein purification.

Alternatively, polypeptides can be synthesized chemically or produced using methods of recombinant DNA. For example, the polypeptide of the invention (for example, Tau fragment) can be synthesized by solid phase procedures well known in the field. Suitable syntheses can be carried out using what procedures W "T-boc or F-moc". Cyclic peptides can be synthesized by solid-phase methods using well-known procedures F-moc" and polyamide resin in a fully automated apparatus. Alternatively, the professionals in this field know the necessary laboratory procedures for carrying out the process manually. Methods and procedures for solid-phase synthesis is described in Solid Phase Peptide Synthesis: A Practical Approach by E.Atherton and R.C.Sheppard, published by IRL at Oxford University Press (1989), and Methods in Molecular Biology. Vol.35: Peptide Synthesis Protocols (ed. M.W.Pennington and B.M.Dunn), chapter 7, pp.91-171 by D. Andreau et al.

Alternatively, polynucleotide encoding the polypeptide according to the invention can be put into expressing vector, which can be expressed in a suitable expression system using methods well known in the field, followed by separation or purification of interest expressed polypeptide. Many bacterial, yeast, plant expression systems expressing systems of mammals and insects available in this area, and can be used any such expression system. Perhaps polynucleotide encoding the polypeptide according to the invention, can be translated in a cell-free system broadcast.

Antigenic Tau peptides according to the invention can also include peptides that arise from the existence of multiple who's genes alternative transcription events, alternative event RNA splicing and alternative translational and post-translational events. The polypeptide can be expressed in systems, e.g., cultured cells, which leads essentially to the same post-translational modifications present when the polypeptide is expressed in a native cell, or in systems which result in the modification or omission of post-translational modifications, such as glycosylation or cleavage, present when the expression of molecules in the cell.

The polypeptide according to the invention, such as antigenic Tau polypeptide can be obtained in the form of a fused protein that contains other non-Tau or Tau-occurring amino acid sequence, such as amino acid linkers or signal sequences, or immunogenic carriers, as defined in this application as well as ligands useful for protein purification, such as glutathione-3-transferase, his-tag tag, and staphylococcal protein A. In fused protein may be present in more than one antigenic Tau polypeptide according to the invention. The heterologous polypeptide can be fused, for example, N-end, or the end of the polypeptide according to the invention. The polypeptide according to the invention can also be obtained in the form of a fused polypeptide containing geologicalindustrial sequence, i.e. other Tau or Tau-occurring sequence.

Conformationally restricted peptides

Antigenic Tau peptides according to the invention can be linear or conformationally constrained (constrained). As used in this application with respect to the molecule, the term "conformationally constrained" means a molecule such as a polypeptide, in which the three-dimensional structure is maintained for the time being in the same spatial location. Conformationally restricted molecules can have improved properties such as increased affinity, immunogenicity, metabolic stability, membrane permeability or solubility. In addition, such conformationally restricted molecules expected to be present antigenic Tau epitope in a conformation similar to the native conformation inducyruya thus anti-Tau antibodies, are more likely to recognize their own Tau molecules. How conformational constraints is well known in this field and include, without limitation, the formation of bridges and cyclization.

For the introduction of conformational constraints in the linear peptide or polypeptide chain there are several approaches known from the prior art. For example, the formation of bridges between two adjacent amino acids in the peptide PR which leads to local conformational modifications flexibility limited in comparison with the flexibility of regular peptides. Some possibilities for the formation of such bridges include the introduction of lactams and piperazinone (see, for example, Giannis and Kolter, Angew. Chem. Int. Ed., 32:1244 (1993)).

As used in this application in respect of the peptide, the term "cyclic" refers to a structure including an intramolecular bond between two non-adjacent amino acids or analogs of amino acids. Cyclization can be achieved through covalent or non-covalent bond. Intramolecular communication include, but are not limited to, communications of the skeleton from the skeleton, the side chain skeleton, the side chain to side chain, the side chain with a terminal group and end with end. Methods of cyclization include, without limitation, the formation of a disulfide bond between the side chains of the non-contiguous amino acids or amino acid analogues; the formation of amide bond between the side chains of residues Lys and Asp/Glu; the formation of ester bonds between serine residues and residues Asp/Glu; the formation of lactam communication, for example, between a group in the side chain of one amino acid or its analog with N-terminal amine aminoanisole residue; and education lysinoalanine and dityrosine links. Can also be used carbon options disulfide bond, for example, Attila or Atil the flanged connection (J. Peptide Sc. 14:898-902 (2008)), and alkylation reaction with a suitable polyamidine electrophilic reagent, such as di-, tri - or tetrachloroethane (PNAS, 105 (40), 15293-15298 (2008); ChemBioChem, 6:821-824 (2005)). To enable conformational constraints in peptides can also be used in a variety of modified analogues of Proline (Zhang et al., J. Med Chem., 39:2738-2744 (1996); Pfeifer and Robinson, Chem. Comm., 1977-1978 (1998)). Chemicals that can be used for the cyclization of peptides according to the invention, result in peptides, cyklinowanie through communication, including, but not limited to, the following: lactam, hydrazono, oximo, thiazolidinone, thioester or sulfonate connection.

Another approach to the design of conformationally restricted peptides, which are described in patent publication U.S. No. 2004-0176283, is to attach a short amino acid sequence of interest, to the matrix to obtain the cyclic conformationally constrained peptide. Such cyclic peptides are not only structurally stable their matrices and therefore exist in three-dimensional conformations that can mimic conformational epitopes on the virus and parasites, but they are also more stable than linear peptides to proteolytic degradation in serum. In patent publication U.S. No. 2004-0176283 so the e describes the synthesis of conformationally restricted cross-linked peptides by synthetic amino acids, the combination of a skeleton with appropriately spaced amino acids to stabilize supervoice patterns the peptides. Crosslinking can be achieved through amide combination of primary amino groups orthogonally protected (2S,3R)-3-aminopropanoic balance with appropriately located carboncillo group in the side chain of glutamate. This approach was adopted upon receipt of conformationally restricted tetrapeptide repeats CS protein, where at least one Proline was replaced with (2S,3R)-3-aminopropyl, and to introduce the carboxyl group in the side chain, glutamate was included as a substitution of alanine.

Strategy stitching also include the application of metathesis reaction with the closing of the cycle of verification with the formation of cross-linked ("stapled") peptides designed to mimic alpha-helical conformations (Angew. Int. Ed. Engl. 37:3281 (1998); JACS 122:5891 (2000)); the use of polyfunctionalized saccharides; application tryptamines communication (Chemistry Eu. J. 24:3404-3409 (2008)); and the application of "click"("click")reaction of azides and alkynes, which can be included either in the form of amino acid residue side chain, either located in the skeleton of the peptide sequence (Drug Disc. Today 8 (24):1128-1137 (2003)). From the literature it is also known that metal ions can stabilize the restricted conformation of linear peptides by sequestration of certain residues (e.g., histidine), which are coordinated with metal cations (Angew. Int. Ed. Engl. 42:421 (003)). Similarly, the functionalization of linear peptide sequence unnatural acid and amine functional group, or polyamines and polyacidic functional group can be used to gain access to collisional structures in the activation and formation of amide bonds.

According to one of embodiments of the antigenic Tau peptide is conformationally restricted due to the formation of intermolecular covalent bonds between two non-contiguous amino acids of the antigenic Tau peptide, for example, N - and C-terminal amino acids. According to another embodiment of the antigenic Tau peptide of the invention is conformationally restricted due to covalent binding framework (scaffold) molecule. According to another embodiment of the antigenic Tau peptide is simply limited, i.e. related or at one end (the C - or N-end), or through another amino acid that is not located on any end, with the skeleton molecule. According to another embodiment of the antigenic Tau peptide is doubly limited, i.e. as related to P-and N-ends with the skeleton molecule.

Frame (also nazyvaemy "substrate") can be any molecule that is able to reduce, through the formation of covalent bonds, the number of conformations that can make the antigenic Tau peptide. Examples of restricting the conformation of the frames include proteins and peptides, for example, lipocalin-related molecules, such as thioredoxin and thioredoxin-like proteins containing beta-sculacciata structure, nucleases (such as Mcasa A), protease (e.g. trypsin), protease inhibitors (e.g., Eglin C), antibodies or their structurally rigid fragments, fluorescent proteins such as GFP (green fluorescent protein) or YFP (yellow fluorescent protein), conotoxins, loop sections fibronectin domain type III, CTLA-4 and virus-like particles (the VLP).

Other suitable frame molecules include carbohydrates, such as sepharose. The frame may be a linear or circular molecule, for example, closed-loop with the loop. Usually the frame is heterologous in relation to antigenic Tau peptide. Such conformationally restricted peptides associated with the frame are considered to be more resistant to proteolytic degradation than the linear peptide.

According to one embodiments the frame is an immunogenic carrier, as defined in the present invention, such as a heterologous protein carrier or the VLP. In another embodiment the antigenic Tau peptide is simply mounted on the immunogenic carrier. In another embodiment the antigenic Tau peptide is the tsya doubly secured to the immunogenic carrier. Thus, the antigenic Tau peptide forms a conformationally restricted loop structure, which, as has been proven, is a particularly suitable structure as an intracellular molecule recognition.

Antigenic Tau peptides according to the invention can be modified for ease of conjugation with the substrate, for example, by adding a terminal cysteine at one or both ends and/or by adding a linker sequence, such as a double glycine "head" (head or tail, linker, ending with a lysine residue, or any other linker for carrying out this function, known to specialists in this field. To link the full peptide sequence with the carrier can also be used biorthogonol chemistry (such as "click"-reaction described above), thus, it is possible to avoid any regiochemistry problems and problems of chemoselectivity. It is known that rigid linkers, such as linkers, as described by Jones et al. (Angew. Chem. Int. Ed. 2002, 41:4241-4244), cause improved immunological response and can also be used.

In another embodiment the antigenic Tau peptide attached to the polyvalent matrix, which itself is connected with the carrier, thus increasing the density of the antigen (see below). Polyvalent matrix can represent going the way functionalized polymer or oligomer, such as (but not limited to) oligopotent or oligochitosan.

The specified linker can be located on the N-end of the peptide or the C-end of the peptide, or at both ends of the peptide. Specified, the linker may have a length of from 0 to 10 amino acids, for example, the length from 0 to 6 amino acids. Alternatively, the addition or substitution of D-stereoisomeric form of one or more amino acids can be made to produce useful derived, for example, to increase the stability of the peptide.

A typical combination of conjugatively, all of which are within the scope of the present invention and represent various embodiments, using different linkers below.

Peptide-GGGGGC (SEQ ID NO:79)-frame; Peptide-GGGGC (SEQ ID NO:80)-frame; Peptide-GGGC (SEQ ID NO:81)-frame; Peptide-CCC-frame; Peptide-GC-frame; a Peptide With the frame; Peptide-GGGGGK (SEQ ID NO:82); Peptide-GGGGK (SEQ ID NO:83); Peptide-GGGK (SEQ ID NO:84); Peptide-GGK; Peptide-GK; Peptide-K; Peptide-GGGGSC (SEQ ID NO:85); Peptide-GGGSC (SEQ ID NO:86); Peptide-GGSC (SEQ ID NO:87); Peptide-GSC; Peptide-SC; Peptide-GGGGC (SEQ ID NO:80); Peptide-GGGC (SEQ ID NO:81); Peptide-GGC; Peptide-GC; CSGGGG (SEQ ID NO:88)-Peptide; CSGGG (SEQ ID NO:89)-Peptide; CSGG (SEQ ID NO:90)-Peptide; CSG-Peptide; CS-Peptide; CGGGG (SEQ ID NO:91)-Peptide; CGGG (SEQ ID NO:92)-Peptide; CGG-Peptide; CG-Peptide.

A typical combination of conjugatively using different linkers and doubly constrained peptides p is estaline below, where the media can be a monomer, is identical to the media or non-media monomer. In the example below, GC-linker can be substituted for any GK-linker or GSC-linker, illustrated above, or any other well-known experts in this field:

Media-CGGGGG (SEQ ID NO:93)-Peptide-GGGGGC (SEQ ID NO:79)-carrier; Carrier-CGGGG (SEQ ID NO:91)-Peptide-GGGGC (SEQ ID NO:80)-carrier; Carrier-CGGG (SEQ ID NO:92)-Peptide-GGGC (SEQ ID NO:81)-carrier; Carrier-CG-Peptide-GC-carrier; the Carrier-s-Peptide-s-media.

In one of the embodiments of the terminal cysteine residue, if it is not present in the amino acid sequence of the antigenic Tau peptide, is added to one or both ends of the antigenic Tau peptide containing or consisting of any of the sequences depicted in SEQ ID NO:1-26, to create a conformationally restricted peptide.

In another embodiment of the GC-linker containing varying the number of glycine residues and one terminal cysteine residue, is added to one or both ends of the antigenic Tau peptide containing or consisting of any of the sequences depicted in SEQ ID NO:1-26, to create a conformationally restricted peptide. Preferably, GC-linker contains from 1 to 10 glycine residues, more preferably 1, 2, 3, 4 or 5 residues of glycine.

In yet another embodiment of the GC-linker containing varying the number of residues glycine and one terminal cysteine residue, add to one end of antigenic Tau peptide containing or consisting of any of the sequences depicted in SEQ ID NO:1-26, and the terminal cysteine residue, if it is not present on the other end of the antigenic Tau peptide, add to the other end of the antigenic peptide. Preferably, GC-linker contains from 1 to 10 glycine residues, more preferably 1, 2, 3, 4 or 5 residues of glycine.

Immunogenic carriers

In one of the embodiments of the present invention antigenic Tau peptide or polypeptide according to the invention is associated with a molecule immunogenic carrier with the formation of immunogens for vaccination protocols. The term "immunogenic carrier" in this application includes the following materials, which have the ability to independently cause an immunogenic response in an animal host and which may be associated (e.g., covalently linked) to the peptide, polypeptide or protein either directly through the formation of peptide or ester links between free carboxyl, amino or hydroxyl groups in the peptide, polypeptide or protein and the corresponding groups on the immunogenic material, or, alternatively, by education relations through traditional bifunctional linking group, or in the form of a fused protein.

The types of media used in immunogenic for us is oasea the invention, will be well known to experts in this field. Examples of such immunogenic carriers include: virus-like particles (the VLP); serum albumin such as bovine serum albumin (BSA); globulins; thyroglobulin; hemoglobins; hemocyanine (especially hemocyanin lymph snails (KLH)); proteins extracted from Ascaris, inactive bacterial toxins or toxoids, such as tetanus or diphtheria toxin (TT and DT) or CRM197, purified protein derivative of tuberculin (PPD); or protein D from Haemophilus influenzae (PCT publication no WO 91/18926) or recombinant fragments (for example, domain 1 fragment of TT, or the translocation domain of DT, or 1/3 protein D, containing N-terminal amino acids with 100 110 of the protein D of Haemophilus influenzae (GB 9717953.5)); polylysine; polyglutamine acid; copolymers of lysine and glutamic acid; copolymers containing lysine or ornithine; liposomal carriers, etc.

In one of the embodiments of the immunogenic carrier is a KLH. In another embodiment the immunogenic carrier is a virus-like particle (the VLP), preferably recombinant virus-like particle.

The term "viral particle", as used in this application refers to the morphological form of the virus. In some types of the virus it contains genome surrounded by a protein capsid; others have will complement the global structure, such as shells, tails, etc.

The term "virus-like particle" (the VLP)as used in this application refers to dereplication and/or non-infectious viral particle, or refers to dereplication and/or non-communicable structure, similar to a viral particle, such as the capsid of the virus. The term "dereplication", as used in this application refers to the inability to replicate the genome, which is part of the VLP. The term "non-infectious", as used in this application refers to the inability to penetrate the cell host. In one example, the virus-like particle is dereplication and/or non-infectious, because it does not contain all or part of the viral genome or genomic functions. For example, virus-like particle is a virus particle, in which the viral genome was physically or chemically inactivated. In addition, for example, virus-like particle does not contain all or part of replicative and infectious components of the viral genome. Virus-like particle can contain a nucleic acid that is different from the genome of the virus. One example of a virus-like particle is a viral capsid, such as viral capsid of the corresponding virus, such as bacteriophage, such as a RNA-phage. The terms "viral capsid" or "capsid" refers to a macromolecular Assembly, consisting of the subunits of the viral protein. For example, it may be 60, 120, 180, 240, 300, 360 and more than 360 subunits of the viral protein. The interaction of these subunits can lead to the formation of viral capsid or structure that is similar to the viral capsid, with the repeating organization, where this structure is, for example, spherical or tubular.

As used in this application, the term "virus-like particle of a RNA-phage" refers to a virus-like particle comprising, or consisting essentially of, or consisting of envelope proteins of RNA-phage, variants or fragments. For example, virus-like particle of a RNA-phage may resemble the structure of a RNA-phage, as dereplication and/or non-communicable and not containing at least a gene or genes encoding the replication apparatus RNA-phage, and may not contain a gene or genes encoding the protein or proteins responsible for the attachment of the virus to the host or the penetration of the virus into the host. However, this definition should also include virus-like particles of RNA bacteriophages, in which the aforementioned gene or genes still exist, but are inactive, and therefore also lead to dereplication and/or non-infectious virus-like particles of RNA-phage. In the present invention, the term "subunit" and "monomer" are used interchangeably and are equivalent in this context. Chrome is also in the present invention, the term "RNA-phage" and the term "RNA-bacteriophage" are used interchangeably.

In the present invention proposed compositions and methods of inducing and/or enhancing immune responses against phosphorylated Tau in a mammal. The composition of the invention may contain virus-like particle (the VLP)associated with at least one antigenic Tau peptide. For example, antigenic Tau peptide can be linked to the VLP with the formation of an ordered and repetitive matrix antigen-the VLP. For example, in one case, at least 20, at least 30, at least 60, at least 120, at least 180, at least 360, or at least 540 peptides, as described in this application, associated with the VLP.

Capsid structure, which is formed through the self-Assembly 180 subunits envelope protein of RNA phage and possibly containing RNA master is called in this application "the VLP of the envelope protein of RNA phage". A concrete example is the VLP of the envelope protein Qbeta. In this particular case, the VLP of the envelope protein Qbeta can either be collected exclusively from subunits WED Qbeta (produced by gene expression CF Qbeta containing, for example, the stop codon TAA, preventing the expression of the longer protein A1 through suppression, see Kozlovska, T.M., et al., Intervirology 39: 9-15 (1996)), or optionally to contain subje is initi protein A1 in the Assembly of the capsid. Usually the percentage of protein Qbeta A1 relative to CF Qbeta in the Assembly of the capsid will be limited to ensure the formation of the capsid.

Examples of the VLP, suitable as immunogenic carriers in the context of the present invention include, but are not limited to, capsid proteins of hepatitis b virus (Ulrich, et al., Virus Res. 50: 141-182 (1998)), measles virus (Warnes, et al., Gene 160: 173-178 (1995)), virus Sindbis, rotavirus (U.S. patent No. 5071651 and 5374426), FMD virus (Twomey, et al., Vaccine 13: 1603-1610, (1995)), virus Norwalk (Norwalk) (Jiang, X., et al., Science 250: 1580-1583 (1990); Matsui, S. M., et al., J Clin. Invest. 87: 1456-1461 (1991)), the retroviral GAG protein (PCT publication no WO 96/30523), protein pI retrotransposon That, the surface protein of hepatitis b virus (PCT publication no WO 92/11291), human papilloma virus (PCT publication no WO 98/15631), virus polyoma person (Sasnauskas K., et al., Biol. Chem. 380 (3): 381-386 (1999); Sasnauskas K., et al., Generation of recombinant virus-like particles of different polyomaviruses in yeast, 3rd International Workshop "Virus-like particles as vaccines", Berlin, September 26-29 (2001)), RNA phages, Ty, fr-phage, GA-phage, AP 205-phage and, in particular, Qbeta-phage.

As will be obvious to experts in this field, the VLP used as immunogenic carrier according to the invention is not limited to any specific form. The particle can be synthesized chemically or through a biological process, which may be natural or unnatural. As an example, this type of embodiment the Oia includes virus-like particle or a recombinant form. In a more specific embodiment of the VLP may contain, or alternatively consists of recombinant polypeptides of any of the viruses, which are known to form the VLP. The VLP may further comprise, or alternatively consist of one or more fragments of such polypeptides, and variants of such polypeptides. Variants of the polypeptide can have, for example, at least 80%, 85%, 90%, 95%, 97% or 99%identity at the amino acid level with their prototypes wild type. Variant the VLP, suitable for use in the present invention may be from any organism, provided that they can form a "virus-like particle" and can be used as an "immunogenic carrier," as defined in this application.

Preferred the VLP according to the invention include the capsid protein or a core and surface antigen of HBV (HBcAg and HBsAg, respectively) or recombinant proteins, or fragments thereof, and envelope proteins of RNA-phages or recombinant proteins, or fragments thereof, more preferably the envelope protein Qbeta or recombinant proteins, or fragments thereof.

In one of the embodiments of the immunogenic carrier used in combination with the antigenic Tau peptide according to the invention, is NSAD protein. Examples NSAD proteins that can be used in the context of this image is to be placed, can be easily determined by the person skilled in the art. Examples include, but are not limited to, core protein of HBV, described in Yuan et al., J. Virol. 73:10122-10128 (1999), and in PCT publications No. WO 00/198333, WO 00/177158, WO 00/214478, WO 00/32227, WO 01/85208, WO 02/056905, WO 03/024480 and WO 03/024481. HbcAg, suitable for use in the present invention may be from any organism, provided that they can form a "virus-like particle" and can be used as an "immunogenic carrier," as defined in this application.

Options HbcAg, special interest, which can be used in the context of the present invention are variants in which one or more naturally occurring cysteine residues were either delegated or replaced. In this field it is well known that free cysteine residues can be involved in various chemical reactions, including disulfide exchange reaction with chemicals or metabolites that, for example, inherits or formed in combination therapy with other substances, or direct oxidation and reaction with nucleotides under the action of UV light. Thus, can produce toxic adducts, especially considering the fact that HBcAgs have a strong tendency to bind nucleic acids. Thus, axione adducts will be distributed between many species, each of which individually may be present in low concentration, but reaches toxic levels, when present together. In light of the above, one advantage of using HBcAgs in vaccine compositions that have been modified to remove the naturally occurring cysteine residues, is that sites with which it can communicate toxic type when attaching antigens or antigenic determinants, will be numerically reduced or even eliminated.

In addition, protestirovanny form of HBcAg, not containing N-terminal leader sequence of the protein precursor of measles antigen hepatitis b, can also be used in the context of the invention, especially when HBcAg is produced under conditions in which the processing will not occur (for example, expression in bacterial systems).

Other options HBcAg according to the invention include: 1) a polypeptide sequence that is at least 80%, 85%, 90%, 95%, 97% or 99% identical to one of the HBcAg amino acid sequence of wild-type or its sub-regions, using standard computer algorithms to compare sequences, 3) mutants, truncated at the C-end, including mutants, where at least 1, 5, 10, 15, 20, 25, 30, 34 or 35 amino acids were deleted from the C-end, 4) mutants, truncated N-con is, including mutants, where at least 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the N-Terminus, 3) mutants, abbreviated as N-end and C-end, including HBcAgs, where at least 1, 2, 5, 7, 9, 10, 12, 14, 15 or 17 amino acids have been deleted from the N-Terminus and at least 1, 5, 10, 15, 20, 25, 30, 34 or 35 amino acids were deleted from the C-end.

Another variant proteins NSAD within the scope of the invention are variants, modified to enhance immunogenic presentation of a foreign epitope, where one or more of the four arginine repeats was deleterows, but in which the C-terminal cysteine saved (see, for example, PCT publication no WO 01/98333), and chimeric, shortened at the C-end of HBcAg, such as described in PCT publications No. WO 02/14478, WO 03/102165 and WO 04/053091.

In another embodiment the immunogenic carrier used in combination with the antigenic Tau peptide according to the invention, represents the HBsAg protein. HBsAg proteins that can be used in the context of the present invention, can be easily determined by the person skilled in the art. Examples include, but are not limited to, HBV surface proteins, are described in U.S. patent No. 5792463 and PCT publications No. WO 02/10416 and WO 08/020331. HBsAgs, suitable for use in the present invention may be from any organism, provided that they are capable of images is to "virus-like particle" and can be used as an "immunogenic carrier", as defined in this application.

In yet another embodiment the immunogenic carrier used in combination with the antigenic Tau peptide according to the invention, is a protein shell Qbeta. Found that the protein shell Qbeta organized around the capsid during expression in E. coli (Kozlovska T.M. et al., GENE 137: 133-137 (1993)). The resulting capsid or virus-like particles demonstrated icosahedral agopton capsid structure with a diameter of 25 nm and quasi-symmetry T=3. In addition, it has been resolved crystal structure of bacteriophage Qbeta. The capsid contains 180 copies of a protein shell, which are connected with covalent pentamers and hexamers disulfide bridges (Golmohammadi, R. et al., Structure 4: 5435554 (1996)), leading to the remarkable stability of the capsid shell protein Qbeta. Capsid protein Qbeta also shows unusual resistance to organic solvents and denaturing agents. The high stability of the capsid shell protein Qbeta is a preferred characteristic, in particular, for use in immunization and vaccination of mammals and humans in the context of the present invention

Examples of envelope proteins Qbeta, which can be used in the context of the present invention can be easily determined by the person skilled in the art. Examples have been widely described in the PCT publications No. WO 02056905, WO 03/024480, WO 03/024481 and include, but are not limited to, amino acid sequences described in the database PIR (Protein information resource), no access VCBPQbeta against CF Qbeta; no access AAA against Qbeta A1 protein; and their variants, including variant protein in which the N-terminal methionine derived; C-terminal truncated forms of Qbeta A1, in which there are no up to 100, 150 or 180 amino acids; variant proteins that have been modified by removal of the lysine residue with deletions or substitutions, or by adding lysine residue by substitution or insertion (see, for example, Qbeta-240, Qbeta-243, Qbeta-250, Qbeta-251 and Qbeta-259, described in PCT publication NoWO 03/024481), and variants showing at least 80%, 85%, 90%, 95%, 97% or 99%identity with any of crustal proteins Qbeta described in this application. Variant envelope proteins Qbeta, suitable for use in the present invention may be from any organism, provided that they can form a "virus-like particle" and can be used as immunogenic carriers"as defined in this application.

Communication

Antigenic Tau peptides according to the invention can be subjected to a combination of immunogenic carriers by chemical conjugation or by the expression of genetically engineered partners with whom iania. The combination need not be direct, but may occur through linker sequences. More generally, in the case where the antigenic peptides fused, conjugated or otherwise attached to an immunogenic carrier, spacer elements or linker sequence usually add on one or both ends of the antigenic peptides. Such linker sequences usually contain sequences recognized by the proteasome, proteases endosomes or other vesicular compartment cells.

In one of the embodiments of the peptides of the present invention are expressed in the form of fused proteins with immunogenic carrier. The fusion peptide may be exercised by inserting in the primary sequence of the immunogenic carrier, or by merging with either N-or C-end immunogenic carrier. Further, when referring to the slit proteins peptide to the immunogenic carrier, covered by a merger or ends the sequence of the a subunit, or insert peptide within the sequence of media. The merger, as described later, can be performed by inserting the antigenic peptide in the sequence of media, by replacing parts of the sequence of the carrier of antigenic peptide, or through a combination of deletion, substitution or insertion.

When immunogenic what the media represents the VLP, then the subunit chimeric antigenic peptide-the VLP will, in General, capable of self-Assembly in the VLP. The VLP-displayed epitopes, merged with their subunits, also referred to in this application chimeric the VLP. For example, in EP 0421635 described IN the application of chimeric crustal antigenic particles hepadnavirus for the presentation of foreign peptide sequences in the virus-like particle.

Flanking amino acid residues can be added to any end of the sequence of the antigenic peptide, subject to the merger, to any end of the sequence of the VLP subunit, or for the insertion of this peptide sequence in the sequence of the VLP subunit. Residues of glycine and serine are especially preferred amino acids to be used in flanking sequences, added to the peptide to be merged. Residues of glycine provide additional flexibility, which can reduce the potentially destabilizing effect of the merger alien sequence on the sequence of the VLP subunit.

In a particular embodiment of the invention the immunogenic carrier is a HBcAg the VLP. Slit proteins are antigenic peptide or N-end HBcAg (Neyrinck, S. et al., Nature Med. 5:11571163 (1999)), or insert in the so-called main immunodominant site (MIR, major immunodominant region) have been described (Pumpes et al., Intervirology 44:98-114 (2001), PCT publication no WO 01/98333), and represent specific embodiments of the invention. Naturally occurring variants of HBcAg with divisions in MIR have also been described (Pumpens et al., Intervirology 44:98-114 (2001)), and fusion with the N - or C-end, and insert in position MIR corresponding to the site of deletions compared with HBcAg wt (wild type), represent an additional embodiment of the invention. Merge with C-end have also been described (Pumpens et al., Intervirology 44:98-114 (2001)). The person skilled in the art will easily find a guide on how to design fused proteins using classical methods of molecular biology. Vectors and plasmids encoding HBcAg and fused proteins HBcAg and useful for the expression of HBcAg and merged HBcAg protein, have been described (Pumpens et al., Intervirology 44:98-114 (2001), Neyrinck, S. et al., Nature Med. 5:1157-1163 (1999)) and can be used to implement the present invention. An important factor to optimize the effectiveness of self-Assembly and display of epitopes to be included in the MIR HBcAg is the choice of a site for insertion, as well as the number of amino acids to be deleted from a sequence of HBcAg within MIR (European patent number EP 0421635; U.S. patent No. 6231864) resulting from the insertion, or, in other words, what are the amino acids that form HBcAg, to be replaced by a new epitope. For example, was described replacement HBcAg amino acids 76-80, 79-81, 79-80, 75-85 80-81 or courtniebyrne (Pumpens et al., Inte/virology 44:98-114 (2001); European patent # EP 0421635; U.S. patent No. 6231864, patent PCT publication no WO 00/26385). HBcAg arginine contains a long tail, which is not essential for the Assembly of the capsid and are able to bind nucleic acids. HBcAg, either containing or not containing the arginine tail, both represent an embodiment of the present invention.

In another specific embodiment of the invention the immunogenic carrier is a the VLP of RNA phage, preferably Qbeta. The major envelope proteins of RNA-phages spontaneously gather in the VLP upon expression in bacteria and in particular in E. coli. Were described fused protein structure, where the antigenic peptides were fused with the C-end of the truncated form of the protein A1 of Qbeta, or inserted into a protein A1 (Kozlovska et al., Intervirology, 39:9-15 (1996)). Protein A1 is formed by suppression in the stop codon UGA and has a length of 329 amino acids, or 328 amino acids, taking into account the removal of N-terminal methionine. Cleavage of N-terminal methionine alanine before (the second amino acid of the encoded gene CF Qbeta) usually occurs in E. coli, and this is the case for N-ends of the envelope proteins Qbeta. Part of the A1 gene, 3' from amber-codon UGA encodes elongation CF, which has a length of 195 aminokislotami antigenic peptide between position 72 and 73 extend CF leads to additional embodiments from which retene (Kozlovska et al., Intervirology 39:9-15 (1996)). Merge antigenic peptide on the C-end shortened at the C-end of protein A1 Qbeta leads to additional preferred embodiments of the invention. For example, in Kozlovska et al., Intervirology, 39:9-15 (1996) described the slit proteins A1 Qbeta, where the epitope fused at the C-end elongation WED Qbeta, shortened position 19.

As described in Kozlovska et al., Intervirology, 39:9-15 (1996), Assembly of particles, exposing the slit epitopes, usually requires the presence of both the fusion protein A1-antigen, and CF wild type with the formation of the mosaic particles. However, embodiments containing virus-like particles, and thus, in particular, the VLP of the envelope protein of RNA phage Qbeta, which consist only of the VLP subunits, with antigenic peptide, attached to them, are also within the scope of the present invention.

Products mosaic particles can be done in different ways. In Kozlovska et al., Intervirology 39:9-15 (1996) described three ways, all of which can be used to carry out the invention. In the first approach, the effective exposure fused epitope on the VLP-mediated expression plasmid that encodes a protein A1 Qbeta with the stop codon UGA between CF and extension CF in E. coli strain that carries a plasmid encoding the cloned suppressor tRNA UGA, which leads to the translation of the UGA codon in TGR (plasmid pISM3001 (Smiley et al., Gene 134:33-40 (1993)). In another p the diode stop codon of the gene modified WED at UAA, and a second plasmid expressing the fusion protein A1-antigen, is subjected to cotransformation. The second plasmid encodes resistance to another antibiotic, and the replication origin is compatible with the first plasmid. In the third approach, CF and the fusion protein A1-antigen encode in bicistronic manner, functionally associating with a promoter, such as Thr promoter, as described in figure 1 in Kozlovska et al., Intervirology, 39:9-15 (1996).

In addition, the VLP, suitable for fusion of antigens or antigenic determinants, are described in PCT publication no WO 03/024481 and include the bacteriophage fr, RNA phage MS-2 capsid protein of human papillomavirus, The retrotransposon, yeast, and retrovirology particles, HIV2 Gag, mosaic virus of cow pea (Cowpea Mosaic Virus), the VLP VP2 of parvovirus, HBsAg (U.S. patent No. 4722840 and European patent number EP 0020416 B1). Examples of the VLP chimeric suitable for carrying out the invention are also examples described in Intervirology 39:1 (1996). Other examples of the VLP considered for use in the present invention, are: HPV-1, HPV-6, HPV-11, HPV-16, HPV-18, HPV-33, HPV-45, CRPV, CPOV, HIV GAG and tobacco mosaic virus. Additional examples include the VLP of SV-40, polyomavirus, adenovirus, herpes simplex virus, rotavirus, and Norwalk virus.

For any recombinante expressed peptide or protein, which forms part of the present invention, including what I antigenic Tau peptide according to the invention, associated or not associated with immunogenic carrier, nucleic acid, which encodes the specified peptide or protein that also forms an aspect of the present invention, as well as expressing a vector containing a nucleic acid, a host cell containing the specified expressing vector (stand-alone or integrated in the chromosome). The recombinant method of obtaining a peptide or protein through its expression in the above-mentioned cell host and the selection of the immunogen from it is another aspect of the invention.

In another embodiment, the peptide according to the invention is subjected to chemical combination with an immunogenic carrier, using methods well known in the field. Conjugation can occur with ensuring the free movement of peptides through a single point of conjugation (e.g., either N-terminal or C-terminal position) or in the form of a fixed (locked down) structure where both ends of the peptides conjugated to either immunogenic protein carrier or frame structure, such as the VLP. Such conjugation may be carried out by means of the conjugation chemistry, known to specialists in this field, for example, through cysteine residues, lysine residues or other carboxy group, commonly known as points of conjugation, such as glutamic acid sludge is aspartic acid. Thus, for example, for direct covalent binding can be used carbodiimide, glutaraldehyde or (N-[y-maleimidomethyl]operations ester using conventional commercially available heterobifunctional linkers, such as CDAP and SPDP (using the manufacturer's instructions). Examples of conjugation of peptides, especially collisionally peptides with protein carrier through acylhydrazines peptide derivatives described in PCT publication no WO 03/092714. After the reaction, the combination of the immunogen can be easily isolated and purified by dialysis method, method, gel filtration, fractionation method, etc. Peptides ending at residue cysteine (preferably outside linker collisioning plot), can be easily conjugated to a protein carrier through maleimide chemistry.

When the immunogenic carrier is a the VLP, then multiple antigenic peptides having either identical amino acid sequence, or another amino acid sequence, can be subjected to a combination of one molecule of the VLP, which preferably leads to a repetitive and ordered structure representing several antigenic determinants in a certain order, as described in PCT publications No. WO 00/32227, WO 03/024481, WO 02/056905 and WO 04/007538.

In one aspect of the invention, the antigenic peptide is linked to the VLP by forming chemical cross-linkages, typically and preferably by using heterobifunctional cross-linker. In this area there are several hetero-bifunctional cross-linkers. In some embodiments of the hetero-bifunctional cross-linker contains a functional group that can react with the first sites of accession, i.e. with the amino group of the side chain lysine residues the VLP or the VLP subunit, and another functional group that can react with a second preferred site of accession, i.e. a cysteine residue fused with antigenic peptide and may also be available for response through recovery. The first stage of this procedure, commonly called derivatization, is a reaction of the VLP with the cross-linker. The product of this reaction is activated the VLP, also called activated media. In the second stage, unreacted cross-linker is removed using standard methods, such as gel filtration or dialysis. In the third stage, the antigenic peptide is subjected to interaction with activated the VLP, and this stage is usually referred to as the stage combinations. Unreacted antigenic peptide can be removed in the fourth stage, for example by dialysis. In this area there are several hetero-bifunctional cross-linkers. They include the preferred cross Inkeri SMPH (Pierce), Sulfo-MBS, Sulfo-EMCS, Sulfo-GMBS, Sulfo-fairs are forthcoming-Siab, Sulfo-SMPB, Sulfo-SMCC, SVSB, SIA and other cross-linkers, such as those available from Pierce Chemical Company (Rockford, IL, USA) and having one functional group reactive towards amino groups and one functional group reactive against cysteine residues. All of the above cross-linkers lead to the formation of thioester linkages.

Another class of cross-linkers suitable for carrying out the invention is characterized by introduction of a disulfide bond between the antigenic peptide and the VLP when combined. Preferred cross-linkers belonging to this class include, for example, SPDP and Sulfo-LC-SPDP (Pierce). The degree of derivatization the VLP cross-linker may be affected by changing the experimental conditions such as concentration of each of the partners of the reaction, an excess of one reagent over the other, pH, temperature and ionic strength. The degree of combination, i.e. the number of antigenic peptide to the VLP subunit, can be adjusted by varying the experimental conditions described above, to meet the requirements of the vaccine.

Another way of binding antigenic peptide to the VLP is a binding lysine residue on the surface of the VLP with a cysteine residue on antigenic peptide. In some embodiments for combining with the VLP may be necessary to merge amino acid linker, is holding a cysteine residue, as the second site of accession or in part, with the antigenic peptide. In General, flexible amino acid linkers are preferred. Examples of the amino acid linker is selected from the group consisting of: (a) CGG; (b) N-terminal gamma 1-linker; (C) N-terminal gamma 3-linker; (d) Ig hinge areas; (d) N-terminal glycine of linkers; (f) (G)kC(G)nwith n=0 to 12 and k=0-5; (g) N-terminal glycine-serine linkers; (h) (G)kC(G)m(S)i(GGGGS)nwith n = 0 to 3, k = 0-5, m = from 0 to 10, i = from 0 to 2; (I) GGC; (K) GGC-NH2; (l) C-terminal gamma 1-linker; (m) C-terminal gamma 3-linker; (n) C-terminal glycine of linkers; (o) (G)nC(G)kwith n = 0-12 and k = 0-5; (p) C-terminal glycine-serine linkers; (R) (G)m(S)t(GGGGS)n(G)oC(G)kwith n = 0 to 3, k = 0-5, m = from 0 to 10, t = 0 to 2, and o=0 to 8. Additional examples of amino acid linkers are hinged section of immunoglobulins, glycine-serine linkers (GGGGS)nand glycine linkers (G)n, all optionally containing a cysteine residue as second site connection and possibly additional glycine residues. Generally preferable examples of the above amino acid linkers represent the N-terminal gamma 1: CGDKTHTSPP (SEQ ID NO:94); C-terminal gamma 1: DKTHTSPPCG (SEQ ID NO:95); N-terminal gamma 3: CGGPKPSTPPGSSGGAP (SEQ ID NO:96);-to Navoi gamma 3: PKPSTPPGSSGGAPGGCG (SEQ ID NO:97); N-terminal glycine linker: GCGGGG (SEQ ID NO:98) and C-terminal glycine linker: GGGGCG (SEQ ID NO:99).

Other amino acid linkers, especially suitable for carrying out the invention, when a hydrophobic antigenic peptide is associated with the VLP, are CGKKGG (SEQ ID NO:100) or CGDEGG (SEQ ID NO:101) for N-terminal linkers, or GGKKGC (SEQ ID NO:102) and GGEDGC (SEQ ID NO:103) for C-terminal linkers. For C-terminal linkers, terminal cysteine may amitirova on the C-end.

In some embodiments of the present invention linkers GGCG (SEQ ID NO:104), GGC or GGC-NH2 ("NH2" means the amidation) on the s-end of the peptide or CGG at its N-end are preferred as amino acid linkers. In General, the residues of glycine would be built between the bulky amino acids and cysteine is used as the second site of accession, in order to avoid possible steric inconsistencies more bulky amino acids in the reaction combinations. In another embodiment of the invention, the amino acid linker GGC-NH2 are merging with the end of the antigenic peptide.

The cysteine residue present on the antigenic peptide, preferably is in its reduced state to interact with the hetero-bifunctional cross-linker for activated the VLP, i.e. should be available free cysteine or a cysteine residue with a free sulfhydryl group In the case when the cysteine residue acts as a binding site in the oxidized form, for example, if it forms a disulfide bridge, is preferred restoration of this disulfide bridge, for example, using DTT, TCER or p-mercaptoethanol. Low concentrations of reducing agent is comparable to the combination, as described in PCT publication no WO 02/05690, whereas higher concentrations inhibit the reaction mix, as will be known to the specialist, and in this case, the reducing agent must be removed or its concentration is reduced before the combination, for example, by dialysis, gel filtration or HPLC with reversed phase.

The binding of the antigenic peptide to the VLP using a hetero-bifunctional cross-linker according to the methods described above, makes possible the combination of antigenic peptide to the VLP oriented way. Other methods of binding antigenic peptide to the VLP include the ways in which the antigenic peptide cross-linked to the VLP using the carbodiimide EDC, and NHS.

In other ways antigenic peptide is attached to the VLP using a Homo-bifunctional cross-linker such as glutaraldehyde, DSGBM [REO] 4, BS3, (Pierce Chemical Company, Rockford, IL, USA) or other known Homo-bifunctional cross-linkers with functional groups reactive towards amine groups or carboxy who inim groups of the VLP.

Other methods of binding the VLP with antigenic peptide include methods where the VLP biotinylation and antigenic peptide is expressed in the form of streptavidin-fused protein, or methods, where the antigenic peptide, and the VLP biotinylated, for example as described in PCT publication no WO 00/23955. In this case, the antigenic peptide can first be linked with streptavidin or Avidya by adjusting the ratio of the antigenic peptide to streptavidin, so that free binding sites are still available for binding the VLP, which is added at the next stage. Alternatively, all components can be mixed in dorectory" ("one-pot"reaction. Other ligand-receptor pairs, where a soluble form of the receptor and ligand is available, and capable transversely to contact the VLP or antigenic peptide can be used as binding agents for binding antigenic peptide to the VLP. Alternatively, either the ligand or the receptor can be merged with the antigenic peptide, and thus to mediate binding the VLP, chemically modified or merged respectively with either receptor or ligand. Fusion can also be affected by the insertion or replacement.

One subunit of the capsid or the VLP of the envelope proteins of the RNA-phage can be attached one or more antigenic molecules, predpochtitel what about the over exposed lysine residues of the VLP RNA-phages, if this steric valid. Thus, the specific feature of the VLP of the envelope protein of RNA phage and, in particular, the VLP of the envelope protein Qbeta, is the ability to bind multiple antigens for subunit. This makes possible the formation of a dense antigenic matrix.

In one of the embodiments of the invention the linking and joining, respectively, at least one antigen or antigenic determinants for virus-like particle is carried out by means of interaction and Association, respectively, between at least one first site joining virus-like particle and at least one second website join antigenic peptide.

The VLP or capsid of the envelope protein Qbeta exhibit a certain number of lysine residues on its surface, with a certain topology with three lysine residues, which are oriented to the inside of the capsid and interacting with RNA, and the other four lysine residues exposed on the outer surface of the capsid. These specific properties contribute to attach antigens to the outer surface of the particles and not to the inner surface of particles, where lysine residues interact with RNA. The VLP from other envelope proteins of RNA-phage also have a certain number of lysine residues on its surface and a certain topology e is their lysine residues.

In another embodiment of the present invention, the first site of accession is a lysine residue and/or the second site of accession contains a sulfhydryl group or a cysteine residue. In yet another embodiment of the present invention, the first site of accession is a lysine residue, and a second site accession represents a cysteine residue. In other embodiments, the antigen or antigenic determinant is bound via a cysteine residue with lysine residues of the VLP of shell protein RNA-phage, and, in particular, the VLP of the envelope protein Qbeta.

Another advantage of the VLP derived from RNA-phages is their high yield expression in bacteria, which makes possible the production of large quantities of material at an affordable price. Furthermore, the use of the VLP as the media makes possible the formation of a reliable antigenic matrices and conjugates, respectively, with adjustable antigenic density. In particular, the use of the VLP of RNA phage and, in particular, the use of the VLP of the envelope protein of RNA phage Qbeta allows to achieve very high epitope density.

In some embodiments of the immunogenic composition can contain a mixture of immunogenic conjugates, i.e. immunogenic carriers subjected combination with one or more antigenic Tau peptides. Thus, these them is Unesennye composition can comprise immunogenic carriers, which differ in amino acid sequence. For example, can be obtained vaccine compositions containing the VLP "wild-type" and modified the VLP protein in which one or more amino acid residues have been modified (for example, delegated, inserted, or replaced). Alternatively, it may be used the same immunogenic carrier, but subjected to combination with the antigenic Tau peptides with different amino acid sequences.

Therefore, the present invention also relates to methods of producing immunogen, comprising: 1) providing antigenic Tau peptide according to the invention, 2) providing immunogenic carrier according to the invention, preferably the VLP, and 3) the Union of the specified antigenic Tau peptide and the immunogenic carrier. In one of the embodiments of the specified stage of the Association shall be implemented by the formation of chemical cross-links, preferably using heterobifunctional cross-linker.

A composition containing antigenic Tau peptide

The present invention also relates to compositions, especially to immunogenic compositions, also referred to as "the immunogenic compositions containing antigenic Tau peptide according to the invention, preferably associated with immunogenic carrier, more than before occhialino with the VLP, even more preferably, HBsAg, HBcAg or Qbeta the VLP, and possibly at least one adjuvant. Such immunogenic compositions, especially prepared in the form of pharmaceutical compositions, are useful for the prevention, cure or relief of Tau-associated disorders such as Alzheimer's disease.

Immunogenic compositions

In some embodiments considered immunogenic composition according to the invention contains antigenic Tau peptide containing amino acid sequence selected from SEQ ID NO:1-26, 31-76 and 105-122. In some embodiments specified antigenic Tau peptide linked to immunogenic carrier, preferably with the VLP, preferably with HBsAg, HBcAg or Qbeta the VLP.

Consider immunogenic composition comprising antigenic Tau peptide according to the invention, can be prepared in various ways, as described in more detail below.

In some embodiments considered immunogenic composition comprises one type of antigenic Tau peptide, for example, the immunogenic composition comprises a population of antigenic Tau peptides, which are essentially all have the same amino acid sequence. In other embodiments considered immunogenic composition comprises two or more different antigenic Tau peptides, for example, the immunogenic composition of the ash is separated by the population of antigenic Tau peptides the members of which may vary amino acid sequence.

For example, in some embodiments considered immunogenic composition comprises a first antigenic Tau peptide, preferably associated with immunogenic carrier, more preferably with the VLP, even more preferably with HBsAg, HBcAg or Qbeta the VLP, and containing the first amino acid sequence selected from SEQ ID NO:1-26, 31-76 and 105-122; and at least a second antigenic Tau peptide, preferably associated with immunogenic carrier, more preferably with the VLP, even more preferably with HBsAg, HBcAg or Qbeta the VLP, and containing the second amino acid sequence, preferably selected from SEQ ID NO:1-26, 31-76 and 105-122, where the second amino acid sequence differs from the first amino acid sequence at least 1, 2, 3, 4, 5, 6-10 or 15 amino acids.

As another example, consider immunogenic composition comprises a first antigenic Tau peptide, preferably associated with immunogenic carrier, more preferably with the VLP, even more preferably with HBsAg, HBcAg or Qbeta the VLP, and containing the first amino acid sequence selected from SEQ ID NO:1-26, 31-76 and 105-122; second antigenic Tau peptide, preferably associated with immunogenic carrier, more preferably with the VLP, even more preferably with HBsAg, HBcA or Qbeta the VLP, and containing the second amino acid sequence, preferably selected from SEQ ID NO:1-26, 31-76 and 105-122, where the second amino acid sequence differs from the first amino acid sequence at least 1, 2, 3, 4, 5, 6 up to 10 or 15 amino acids; and at least a third antigenic Tau peptide, preferably associated with immunogenic carrier, more preferably with the VLP, even more preferably with HBsAg, HBcAg or Qbeta the VLP, and containing a third amino acid sequence, preferably selected from SEQ ID NO:1-26, 31-76 and 105-122, where third amino acid sequence different from the first and second amino acid sequence at least 1, 2, 3, 4, 5, 6 up to 10 or 15 amino acids.

In other embodiments considered immunogenic composition comprises multimediabay antigenic Tau peptide, as described above. As used in this application, the terms "immunogenic composition comprising antigenic Tau peptide", or "immunogenic composition according to the invention" or "the immunogenic composition" refers to an immunogenic composition containing any one kind (multimediabay or not) or repetitive type of antigen(s) Tau-peptide(s), subjected to combined or not subjected to combination with immunogenic carrier.

Adjuvants

In some embodied what s considered immunogenic composition comprises at least one adjuvanticity adjuvants include adjuvants, suitable for use in mammals, preferably in humans. Examples of known suitable adjuvants that can be used in humans, include but are not necessarily limited to, alum, aluminum phosphate, aluminum hydroxide, MF59™ (4,3% wt./about. squalene, 0.5% wt./about. Polysorbate 80 (Tween 80), 0.5% wt./about. sorbitrate (Span 85)), CpG-containing nucleic acid (where the cytosine is demetilirovanny), QS21 (adjuvant based on saponin), MPL (monophosphorylated A), 3DMPL (3-0-describeany MPL), extracts from Aquilla, ISCOMS (see, e.g., Sjolander et al" J. Leukocyte Biol. 64:713 (1998)); PCT publication nos WO 90/03184, WO 96/11711, WO 00/48630, WO 98/36772, WO 00/41720, WO 06/134423 and WO 07/026190), LT/CT mutants, poly(D,L-lactide-co-glycolide)nye (PLG) microparticles, Quil A, interleukins, and the like. For veterinary applications, including, but not limited to experiments on animals, you can use beta-blockers, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyrisperidone)-ethylamine (CGP 19835A, referred to MTP-PE) and RIBI, which contains three components extracted from bacteria, monophosphorylated, dimycolate trehalose and cell wall skeleton (MPL+TDM+CWS) in the emulsion 2% squalene/Tween 80.

Other exemplary and juvante to increase the effectiveness of the composition include, but are not limited to: (1) drugs emulsion of the type oil-in-water (with other specific immunostimulating agents such as muramylpeptide (see below) or components of bacterial cell walls, or without them), such as for example (a) MF59™ (PCT publication no WO 90/14837; Chapter 10 in Vaccine design: the subunit and adjuvant approach, eds. Powell &Newman, Plenum Press 1995), containing 5% squalene, 0.5% of Tween 80 (polyoxyethylenesorbitan monooleate) and 0.5% Span 85 (sorbitan trioleate) (possibly containing muramyldipeptide, covalently associated with dipalmitoylphosphatidylethanolamine (MTP-PE)), prepared in the form of submicron particles using microfluidizer, (b) SAF, containing 10% squalene, 0.4% of Tween 80, 5% pluronic-blocked polymer L121, and thr-MDP, either subjected microfluidizer in submicron emulsion or subjected to shaken to form an emulsion with particles of larger size, and (C) adjuvant system RIBI™ (RAS), (Ribi Immunochem, Hamilton, MT)containing 2% squalene, 0.2% of Tween 80, and one or more components of the bacterial cell wall, such as monophosphorylated A (MPL), dimycolate trehalose (TDM) and cell wall skeleton (CWS), preferably MPL+CWS (DETOX™); (2) adjuvants on the basis of saponins such as QS21, STIMULON™ (Cambridge Bioscience, Worcester, MA), Abisco® (Isconova, Sweden) or Iscomatrix® (Commonwealth Serum Laboratories, Australia), may be used or particles formed from them, that is their as ISCOM (immunostimulating complexes), which ISCOMS may be free of additional detergent, for example, PCT publication no WO 00/07621; (3) complete adjuvant's adjuvant (CFA) and incomplete adjuvant's adjuvant (IFA); (4) cytokines, such as interleukins (such as IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12 (PCT publication no WO 99/44636) etc.), interferons (e.g. gamma interferon), colony stimulating factor, macrophage (M-CSF)the tumor necrosis factor (TNF), etc; (5) monophosphorylated A (MPL) or 3-0-describeany MPL (3dMPL), for example, UK patent No. GB-2220221 and European patent number EP-A-0689454 possibly at substantial absence of alum when used with pneumococcal saccharides, for example, PCT publication no WO 00/56358; (6) combinations of 3dMPL with, for example, QS21 and/or emulsions of the type oil-in-water, for example, EP-A-0835318, EP-A-0735898, EP And 0761231; (7) oligonucleotides containing CpG-motifs [Krieg, Vaccine (2000) 19:618-622; Krieg, CurrOpin Mol Ther(2001) 3:15-24; Roman et al., Nat. Med. (1997) 3:849-854; Weiner et al., PNAS USA (1997) 94:10833-10837; Davis et al., J. Immunol (1998) 160:870-876; Chu et al., J. Exp. Med (1997) 186:1623-1631; Lipford et al., Ear. J. Immunol. (1997) 27:2340-2344; Moldoveami et al., Vaccine (1988) 16:1216-1224, Krieg et al., Nature (1995) 374:546-549; Klinman et al., PNAS USA (1996) 93:2879-2883; Ballas et al., J. Immunol, (1996) 157:1840-1845; Cowdery et al., J. Immunol (1996) 156:4570-4575; Halpern et al., Cell Immunol. (1996) 167:72-78; Yamamoto et al., Jpn. J. Cancer Res., (1988) 79:866-873; Stacey et al., J. Immunol., (1996) 157:2116-2122; Messina et al., J. Immunol, (1991) 147:1759-1764; Yi et al., J. Immunol (1991) 157:4918-4925; Yi et al., J. Immunol (1996) 157:5394-5402; Yi et al., J. Immunol, (1998) 160:4755-4761; and Yi et al., J. Immunol, (1998) 160:5898-5906; PCT publication No. what is WO 96/02555, WO 98/16247, WO 98/18810, WO 98/40100, WO 98/55495, WO 98/37919 and WO 98/52581], i.e. containing at least one CG dinucleotide, where the cytosine is demetilirovanny; (8) simple polyoxyethylene ether or complex polyoxyethylene ether, for example, PCT publication no WO 99/52549; (9) surface-active substance based on a complex ester of polyoxyethylenesorbitan in combination with octoxynol (PCT publication no WO 01/21207), or surfactant-based polyoxyethyleneglycol simple ester or complex ester in combination with at least one additional non-ionic surface-the active substance, such as an octoxynol (PCT publication no WO 01/21152); (10) a saponin and an immunostimulating oligonucleotide (e.g. a CpG-oligonucleotide) (PCT publication no WO 00/62800); (11) an immunostimulant and a particle of metal salt, for example, PCT publication no WO 00/23105; (12) a saponin and an emulsion of oil-in-water, for example, PCT publication no WO 99/11241; (13) a saponin (e.g. QS21)+3dMPL+IM2 (possibly+a Sterol), for example, PCT publication no WO 98/57659; (14) other substances that act as immunostimulating agents to enhance the effectiveness of the composition, such as muramylpeptide, including N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-25 acetyl-norbornyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamine-L-alanine-2-(1'-2'-dipalmitoyl-sn-glycero-3-hydroxyprop is Biloxi)-ethylamine MTP-D), (15) ligands for toll-like receptors (TLR), natural or synthesized (e.g., as described in the Kanzler et al., Nature Med. 13:1552-1559 (2007)), including TLR3 ligands, such as poly(1:C) and similar compounds such as Hiltonol and Ampligen.

In one of the embodiments of the immunogenic composition of the present invention contains at least one adjuvant specific embodiment of the specified adjuvant is an immune-stimulating oligonucleotide and more preferably CpG oligonucleotide. In one of the embodiments of the CpG oligonucleotide has a nucleic acid sequence 5' TCGTCGTTTTGTCGTTTTGTCGTT 3' (CpG 7909; SEQ ID NO:27). In another embodiment of the CpG oligonucleotide has a nucleic acid sequence 5' TCGTCGTTTTTCGGTGCTTTT 3' (CpG 24555; SEQ ID NO:29). The sequence of the nucleic acid immunostimulating oligonucleotide a SEQ ID NO:29 differs from the previously described immunostimulating oligonucleotide (CpG 10103) 5' TCGTCGTTTTTCGGTCGTTTT3' (SEQ ID NO:28) contacting the nearest to the 3'-end of dinucleotide CG. The similarity in activity between these two immunostimulating oligonucleotide is unexpected because earlier it was reported that immunostimulirutuyu activity of CpG-oligonucleotides does not depend on the number of CpG motifs, sequences flanking dinucleotide CG, localization CDs-motif(s) and the distance between CpG-motifs (Ballas et al., 1996, J. Immunol.; Hartmann et al., 2000, J. Immunol.; Klinman et al. 2003, Clin. Exp. Immunol.). Remove the one closest to the 3'-end of dinucleotide CG in immunostimuliruyushhim the oligonucleotide CpG 24555 did not lead to a negative impact on the ability of this immunostimulating oligonucleotide to enhance antigen-specific immune responses, as expected from previous descriptions. CpG 24555 demonstrated similar and in some cases increased immunostimulirutuyu activity when compared with CpG 10103.

Immune-stimulating oligonucleotide may be double-stranded or single-stranded. Usually double-stranded molecules are more stable in vivo, whereas single-stranded molecules have an increased immune activity. Thus, in some aspects of the invention it is preferable that the nucleic acid was stranded, and in other aspects it is preferable that the nucleic acid was double-stranded.

For any of CpG sequences described in this application (e.g., CpG 24555, CpG 10103 and CpG 7909), any mezhnukleotidnyh ties can be phosphorothioate or phosphodiester bond.

The terms "nucleic acid" and "oligonucleotide" are used in this application interchangeably to denote multiple nucleotides (i.e. molecules containing sugar (e.g. ribose or deoxyribose)linked to a phosphate group and the substituted organic based is eat, which is either a substituted pyrimidine (e.g. cytosine (C), thymidine (T) or uracil (U))or a substituted purine (e.g. adenine (a) or guanine (G)). As used in this application, these terms apply to oligoribonucleotides (i.e. polynucleotide minus the phosphate) and any other polymer containing organic base. Molecules of nucleic acids can be obtained from existing sources of nucleic acids (e.g., genomic or cDNA), but are preferably synthetic (for example, obtained by means of the synthesis of nucleic acids).

In one embodiments the immunostimulatory oligonucleotides can encompass various chemical modifications and substitutions, in comparison to natural RNA and DNA, including fosfodiesterzy magnolioideae bridge, β-D-ribose (deoxyribose) link and/or natural nucleoside base (adenine, guanine, cytosine, thymine, uracil). Examples of chemical modifications are known to the expert and are described, for example, in Uhlmann, E. et al. (1990), Chem. Rev. 90:543; "Protocols for Oligonucleotides and Analogs", Synthesis and Properties &Synthesis and Analytical Techniques, S. Agrawal, Ed., Humana Press, Totowa, USA 1993; Crooke, S.T. et al. (1996) Annu. Rev. Pharmacol. Toxicol. 36:107-129; and J. Hunziker et al., (1995), Mod. Synth. Methods 7:331-417. Oligonucleotide according to the invention can have one or more modifications, where each modification is located at a specific fosfomifira minuk easydna the bridge and/or in specific β-D-(deoxy)ribose link and/or in a specific position of the natural nucleoside bases compared with the oligonucleotide of the same sequence, which consists of natural DNA or RNA.

For example, the oligonucleotides may contain one or more modifications. Such modifications can be selected from: a) replacement phosphodieterase magnolioideae bridge located at the 3'- and/or 5'-end nucleoside modified magnoliopsida bridge, b) replacement phosphodieterase bridge located at the 3'- and/or 5'-end nucleoside, geostatistical, in) replacement of sugar-phosphate units from the sugar-phosphate skeleton of another link, g) replacement of β-D-ribosome link modified sugar level, and d) replacement of the natural nucleoside bases.

Nucleic acids also include substituted purines and pyrimidines such as 5 propylpyrimidine and 7-deaza-7-substituted purine modified bases (Wagner et al., 1996, Nat. Biotechnol. 14:840-4). Purines and pyrimidines include, but are not limited to, adenine, cytosine, guanine, thymidine, 5-methylcytosine, 2-aminopurine, 2-amino-6-globulin, 2,6-diaminopurine, gipoksantin and other natural and man-made nucleobase, substituted and unsubstituted aromatic groups. Other such modifications are well known to specialists in this field.

Modified base is any base that is chemically different from the naturally occurring bases, usually found in DNA and RNA, such as T, C, G, a and U, but which has the General chemical structure of these naturally occurring bases. A modified nucleoside base can be selected, for example, from gipoksantina, uracil, dihydrouracil, pseudorutile, 2-thiouracil, 4-thiouracil, 5-aminouracil, 5-(C1-C6)-alkylaryl, 5-(C2-C6)-alkenylacyl, 5-(C2-C6)-alkynylaryl, 5-(hydroxymethyl)uracil, 5-florouracil, 5-fluorouracil, 5-bromouracil, 5-hydroxycytidine, 5-(C1-C6)-allylcysteine, 5-(C2-C6)-alkenylsilanes, 5-(C2-C6)-alkenylsilanes, 5-chlorcyclizine, 5-fertilizin, 5-brazilain, N2-dimethylguanosine, 2,4-diamino-purine, 8-sapurina, substituted 7-deazapurine, preferably 7-deaza-7-substituted and/or 7-deaza-8-substituted purine, 5-hydroxymethylcytosine, N4-allylcysteine, such as N4-Utilitaire, 5-hydroxydesloratadine, 5-hydroxymethylcytosine, N4-alkylmethacrylamide, such as N4-ethyldiethanolamine, 6-didesoxymannitol and deoxyribonucleosides of nitropyrrole, C5-propylpyrimidine and diaminopurine, for example 2,6-diaminopurine, inosine, 5-methylcytosine, 2-aminopurine, 2-amino-6-chloropurine, gipoksantina or other modifications of the natural nucleoside bases. This list is exemplary and should not be interpreted as limiting.

In some aspects of the invention CpG dinucleotide of immunostimulatory oligonucleotide is s, described in this application, is preferably demetilirovanny. Demetilirovanny CpG-motif is a sequence demetilirovannogo cytosine-gurinovka dinucleotide (i.e. demetilirovanny 5'-cytosine, then 3'-guanosine associated phosphate bond). In other aspects of the CpG motifs are methylated. Methylated CpG motif is a sequence of dinucleotide methylated cytosine-guanine (i.e. methylated 5'-cytosine, then 3'-guanosine associated phosphate bond).

In some aspects of the invention immunostimulating oligonucleotide may contain a modified cytosine. Modified cytosine is a naturally occurring or not naturally occurring analogue of the pyrimidine bases cytosine, which can replace this base without compromising immunostimulatory activity of a given oligonucleotide. Modified cytosine include, but are not limited to, 5-substituted cytosine (for example 5-methyl-cytosine, 5-fluoro-cytosine, 5-chloro-cytosine, 5-bromo-cytosine, 5-iodine-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, 5-deformity-cytosine, and unsubstituted or substituted 5-quinil-cytosine), 6-substituted cytosine, M4 is substituted cytosine (e.g. N4-ethyl-cytosine), 5-Aza-cytosine, 2-mercapto-cytosine, isocytosine, pseudo-isocytosine, and alogi cytosine with condensed ring systems (for example N,N'-propylenimine or phenoxazine), and uracil and its derivatives (such as 5-fluoro-uracil, 5-bromo-uracil, 5-bromovinyl-uracil, 4-thio-uracil, 5-hydroxy-uracil, 5-PROPYNYL-uracil). Some of the preferred cytosine include 5-methyl-cytosine, 5-fluoro-cytosine, 5-hydroxy-cytosine, 5-hydroxymethyl-cytosine, and N4-ethyl-cytosine. In another embodiment of the invention, the base cytosine is substituted by a universal base (e.g. 3-nitropyrrole, R-base), an aromatic ring system (e.g florasulam or differentlal) or hydrogen atom (dSpacer).

In some aspects of the invention immunostimulating oligonucleotide may contain a modified guanine. Modified guanine is a naturally occurring or not naturally occurring analogue of the purine bases guanine, which can replace this base without compromising immunostimulatory activity of a given oligonucleotide. Modified guanine include, but are not limited to, 7-deazaguanine, 7 deaza-7-substituted guanine, gipoksantin, N2-substituted guanine (for example N2-methyl-guanine), 5-amino-3-methyl-3H,6N-thiazolo[4,5-d]pyrimidine-2,7-dione, 2,6-diaminopurine, 2-aminopurine, purine, indole, adenine, substituted adenine (for example MB-methyl-adenine, 8-oxo-adenine), 8-substituted guanine (for example 8-hydroxyguanine and 8-bromoguanine) and 6-tioguanin. In another embodiment of izopet is of Guaynabo base is substituted by a universal base (e.g. 4-methyl-indole, 5-nitro-indole, and K-base), an aromatic ring system (such as benzimidazole or dichloro-benzimidazole, amidon 1-methyl-1H-[1,2,4]triazole-3-carboxylic acid) or hydrogen atom (dSpacer).

In some aspects, the oligonucleotides may include modified mezhnukleotidnyh communication. These modified links can be partially resistant to degradation (e.g., stable). "Stabilized nucleic acid molecule" means a molecule of nucleic acid, which is relatively resistant to degradation in vivo (for example by Exo - or endo-nuclease). Stabilization may depend on the length or secondary structure. Nucleic acids have a length of from tens to hundreds of thousands of base pairs, relatively resistant to degradation in vivo. As for shorter nucleic acids, secondary structure can stabilize and increase their effect. Education patterns "stem-loop" can stabilize the molecule of nucleic acid. For example, if the 3'end of the nucleic acid is semicompetent with the upper section, so that it can be folded to form the structure of the "stem-loop", the nucleic acid can be stabilized and show a lot of activity.

For use in vivo, nucleic acids are preferably Rel is relatively resistant to degradation (e.g., by endo - and Exo-nucleases). It has been demonstrated that modification of the skeleton nucleic acid provides increased activity of the nucleic acid when introduced in vivo. Secondary structures such as stem-loop can stabilize nucleic acids against degradation. Alternatively, the stabilization of nucleic acids can be performed by modifications of the phosphate backbone. Preferred stabilized nucleic acid has at least partial phosphorothioate modified selectortimeout can be synthesized using automated methods that use either phosphoramidate or H-phosphonate chemistry. Aryl - and alkyl-phosphonates can be obtained, for example, as described in U.S. patent No. 4469863; and alkylphosphocholine (in which the charged oxygen group is alkylated, as described in U.S. patent No. 5023243 and European patent No. 092574) can be obtained by automated solid-phase synthesis using commercially available reagents. Ways to implement other modifications and substitutions in the skeleton's DNA have been described (Uhlmann, E. and Peyman, A. (1990) Chem. Rev. 90:544; Goodchild, J. (1990) Bioconjugate Chem. 1:165). 2'-O-methyl nucleic acids with CpG motifs also cause immune activation, as ethoxy-modified CpG nucleic KIS is the notes. In fact, there was no skeletal modifications that completely eliminate the CpG effect, although it is greatly reduced by substitution With 5-methyl-N-Structures with phosphorothioate communication, provide maximum activity and protect the nucleic acid from degradation of intracellular Exo - and endo-nucleases. Other modified nucleic acids include phosphodiester-modified nucleic acids, combinations fosfomifira and phosphorothioate nucleic acid, methylphosphonate, methylphosphonothioate, phosphorodithioate, p-ethoxy and combinations thereof. Each of these combinations and their particular effects on immune cells is discussed in more detail in relation to CpG nucleic acids in PCT publications No. WO 96/02555 and WO 98/18810 and in U.S. Pat. U.S. No. 6194388 and 6239116. I believe that these modified nucleic acid may exhibit a greater stimulating activity due to increased resistance to nucleases, increased cellular uptake, increased protein binding and/or altered intracellular localization.

For administration in vivo, the nucleic acid can be associated with a molecule that results in higher affinity binding to the surface of target cells (such as dendritic cells, b cells, macrophage cells cells and natural-killer (NK)) and/or isonomy cellular uptake by target cells with the formation of the complex for delivery of nucleic acids". Nucleic acids may be ionic or covalently associated with suitable molecules using methods that are well known in the field. Can be used many agents combining or stitching, for example, protein a, carbodiimide and N-Succinimidyl-3-(2-pyridyldithio)propionate (SPDP). Alternatively, nucleic acids can be encapsulated in liposomes or virosome using well known methods.

Other stabilized nucleic acids include, but are not limited to, non-ionic analogs of DNA, such as alkyl - and arylphosphate (in which the charged phosphonate oxygen is replaced by an alkyl or aryl group), phosphodiester and alkylphosphocholine, in which the charged oxygen group is alkilirovanny. Nucleic acids that contain a diol, such as tetraethylene glycol or hexamethyleneimine, either on one or on both ends, also has been shown to be essentially resistant to degradation by nucleases. In some embodiments immune-stimulating oligonucleotide of the invention may include at least one lipophilic substituted nucleotide analog and/or pyrimidine-purine dinucleotide.

The oligonucleotides can have one or two accessible 5'-end. You can create a modified oligonucleotides having two t the fir 5'-end, for example, by attaching two oligonucleotides via a 3'-3' linkage to create the oligonucleotide having one or two accessible 5'-end. 3'-3'-connection can be fosfodiesterzy, phosphorothioates or any other modified magnolioideae bridge. How to create such linkages are known in this field. For example, these relationships were described in Seliger, N. et al., Oligonucleotide analogs with terminal 3'-3'- and 5'-5'-internucleotidic linkages as antisense inhibitors of viral gene expression, Nucleosides &Nucleotides (1991), 10 (1-3), 469-77 and Jiang, et al., Pseudo-cyclic oligonucleotides: in vitro and in vivo properties, Bioorganic & Medicinal Chemistry (1999), 7 (12), 2727-2735.

In addition, 3'-3'-linked oligonucleotides, where the relationship between the 3'-terminal nucleoside is not fosfomifira, phosphorothioate or other modified bridge, can be obtained using an additional spacer, such as tri - or Tetra-etilenglikolevye grouping (Durand, M. et al., Triple-helix formation by an oligonucleotide containing one (dA)12 and two (dT)12 sequences bridged by two hexaethylene glycol chains, Biochemistry (1992), 31 (38), 9197-204, Pat. U.S. No. 5658738 and 5668265). Alternatively, neucleotides the linker may be derived from ethanediol, propane diol or devoid of nitrogenous bases desoxyribose (dSpacer) link (Fontanel, Marie Laurence et al., Nucleic Acids Research (1994), 22 (11), 2022-7) using standard phosphoramidite chemistry. Dinucleotide linkers may be included one or more times, or combined with each other is ω, providing any desirable distance between the 3'ends of the two oligonucleotides to be binding.

Fosfodiesterzy magnolioideae bridge located at the 3'- and/or 5'-end nucleoside, may be replaced by a modified magnoliopsida bridge, where the modified magnolioideae bridge selected, for example, from phosphorothioate, phosphorodithioate, NR1R2-phosphoramidates, boranophosphate, α-hydroxyethylphosphonate, phosphate-(C1-C21)-O-acylcarnitine, phosphate-[(C6-C12)aryl-(C1-C21)-O-alkyl]ester, (C1-C8)alkylphosphonate and/or (C6-C12)arylphosphonate bridges, (C7-C12)-α-hydroxymethyl-aryl (for example, described in PCT publication no WO 95/01363), where (C6-C12)aryl, (C6-C20)aryl and (C6-C14)aryl possibly substituted with halogen, alkyl, alkoxy, nitro, cyano, and where R1and R2independently of one another represent hydrogen, (C1-C18)-alkyl, (C6-C20)-aryl, (C6-C14)-aryl, (C1-C8)-alkyl, preferably hydrogen, (C1-C8)-alkyl, preferably (C1-C4)-alkyl and/or methoxyethyl, or R1and R2form together with the nitrogen atom carrying them, a 5-6-membered heterocyclic ring, which is additional to contain an additional heteroatom, selected from the group O, S and N.

Replacement phosphodieterase bridge located at the 3'- and/or 5'end of a nucleoside by geostatistical (geostatistic described, for example, in Uhlmann, E. and Peyman, A., In "Methods in Molecular Biology", Vol.20, "Protocols for Oligonucleotides and Analogs", S.Agrawal, Ed., Humana Press, Totowa 1993, Chapter 16, pp.355ff), where geostatistic selected, for example, from geostatistical formatline, 3'-toformation, methylhydroxylamine, oxomnik, methylindenyl-hydrazo, dimethylsulfone and/or silyl groups.

Immunostimulatory oligonucleotides of the invention may be chimeric skeletons. Chimera skeleton is a skeleton, which contains more than one type of communication. In one of the embodiments of the chimeric skeleton can be represented by the formula: 5'-Y1N1ZN2Y2 3'. Y1 and Y2 represent a molecule of nucleic acid, having from 1 to 10 nucleotides. Each of Y1 and Y2 includes at least one modified mezhnukleotidnyh communication. Since at least 2 nucleotides of the chimeric oligonucleotides include modifications of the skeleton, these nucleic acids are an example of one type of "stabilized immunostimulatory nucleic acids".

As for the chimeric oligonucleotides, then Y1 and Y2 are considered independently from each other. This means that each of Y1 and Y2 may or may not be different from each sequence is different skeletal relationships in the same molecule. In some embodiments, Y1 and/or Y2 are from 3 to 8 nucleotides. N1 and N2 are nucleic acid molecules having from 0 to 5 nucleotides, provided that N1ZN2 is at least 6 nucleotides in General. Nucleotides N1ZN2 have fosfodiesterzy skeleton and do not include nucleic acids having modified the skeleton. Z represents an immunostimulating motif nucleic acid, preferably selected from those listed in the application.

Central nucleotides (N1ZN2) formula Y1N1ZN2Y2 have phosphodieterase mezhnukleotidnyh communication, and Y1 and Y2 have at least one, but can have more than one, or even can have all modified mezhnukleotidnyh communication. In preferred embodiments, Y1 and/or Y2 have at least two or from two to five modified mezhnukleotidnyh ties, or Y1 has five modified mezhnukleotidnyh ties, and Y2 has two modified mezhnukleotidnyh communication. Modified mezhnukleotidnyh relationship in some embodiments is phosphorothioate modified link, phosphorodithioate communication or p-ethoxy-modified link.

Nucleic acids include nucleic acids having a skeletal sugars which are covalently attached to low molecular weight organic groups, but not to the hydroxyl group at the 2'-position the Institute and not to the phosphate group at the 5'-position. Thus, the modified nucleic acids can include 2'-O-alkilirovanny robozou group. In addition, the modified nucleic acids may include sugars such as arabinose or 2'-ferramenta, instead of ribose. Thus, the nucleic acid can be heterogeneous in composition of the skeleton, while containing any possible combination of polymer units linked together such as peptide-nucleic acids (which have amino acid skeleton with nucleic acid bases). In some embodiments, nucleic acids are homogeneous in composition of the skeleton.

Sugar-phosphate link (i.e. β-D-ribose and fosfodiesterzy magnolioideae bridge, together forming a sugar-phosphate group) of the sugar-phosphate skeleton (i.e. sugar-phosphate skeleton consists of sugar-phosphate units) can be replaced by another link another link, for example, suitable for the creation of "morpholino-derivative" oligomer (as described, for example, in Stirchak E.P. et al. (1989) Nucleic Acid Res. 17:6129-41), ie, for example, replacement of morpholino-derived; or to create polyamide nucleic acids ("PNA"; as described, for example, Nielsen P.E. et al. (1994) Bioconjug. Chem. 5:3-7), for example, replacement PNA skeletal element, for example, 2-aminoantipyrine. The oligonucleotide may have other carbohydrate skeletal modifications and substitutions, such as peptide the E. nucleic acids with phosphate groups (PHONA), closed (locked) nucleic acids (LNA) and the oligonucleotides having the skeletal segments with alkyl linkers or aminocinnamate. The alkyl linker may be branched or unbranched, substituted or unsubstituted, and chiral net, or a racemic mixture.

R-Ribose link or β-D-2' -deoxyribose link may be replaced by a modified sugar level, where the modified sugar level is selected, for example, β-D-ribose, α-D-2'-deoxyribose, L-2'-deoxyribose, 2'-F-2' -deoxyribose, 2'-F-arabinose, 2'-O-(C1-C6)alkyl-ribose, preferably 2'-O-(C1-C6)alkyl-ribose is a 2'-O-methylribose, 2'-O-(C1-C6)alkenyl-ribose, 2'-[O-(C1-C6)alkyl-O-(C1-C6)alkyl]-ribose, 2'-NH2-2'-deoxyribose, β-D-Xylo-furanose, α-arabinofuranose, 2,4-dideoxy-β-D-Erythro-hexo-pyranose, and carbocyclic sugar analogs (described, for example, in Froehler J. (1992) Am. Chem. Soc. 114:8320) and/or sugar analogues open zero (described, for example, in Vandendriessche et al. (1993) Tetrahedron 49:7223) and/or bicycloalkanes analogs (described, for example, in Tarkov M. et al. (1993) Helv. Chim. Acta. 76:481).

In some embodiments sugar is a 2'-O-methylribose, especially for one or both of the nucleotides associated fosfomifira or phosphodiester-like magnolioideae connection.

Ol is gonucleotide according to the invention can be synthesized cte novo using any of a variety of procedures, well known in this field. For example, b-cyanomethylphosphonate method (Beaucage, S.L., and Caruthers, M.N., (1981) Tet. Let. 22:1589); nucleoside H-phosphonate method (Garegg et al., (1986) Tet. Let. 27:4051-4054; Froehler et al., (1986) Nucl. Acid Res. 14:5399-5407; Garegg et al., (1986) 27:4055-4058; Gaffney et al., (1988) Tet. Let. 29:2619-2622). These chemical methods can be accomplished using numerous automated nucleic acid synthesizers commercially available. These oligonucleotides are referred to as synthetic oligonucleotides. Alternatively, T-rich and/or TG dinucleotides is possible to produce on a large scale in plasmids, (see Sambrook T. et al., "Molecular Cloning: A Laboratory Manual", Cold Spring Harbor Laboratory Press, New York, 1989), and divided into smaller segments or to enter a whole. Nucleic acids can be obtained from existing nucleic acids sequences (e.g. genomic or cDNA) using known methods, such as methods in which use restriction enzymes, ectonucleoside or endonuclease.

Modified skeletons, such as phosphorothioate, can be synthesized using automated methods, employ or phosphoramidate or H-phosphonate chemistry. Aryl - and alkyl-phosphonates can be obtained, for example, as described in U.S. Pat. U.S. No. 4469863, and alkylphosphoric (in which the charged oxygen group is alkilirovan is Noah, as described in U.S. Pat. U.S. No. 5023243) can be obtained by using an automated solid-phase synthesis using commercially available reagents. Demonstrated how to implement other modifications and substitutions in the skeleton of DNA (e.g. Uhlmann, E. and Peyman, A., Chem. Rev. 90:544, 1990; Goodchild, J., Bioconjugate Chem. 1:165, 1990).

Nucleic acid obtained in this way is called isolated nucleic acids. "Isolated nucleic acid" generally refers to nucleic acid that is separated from components with which it is secreted from the cell, the nucleus, mitochondria or from chromatin, and any other components that can be considered as pollutants.

In some embodiments CpG-containing oligonucleotides can be simply mixed with immunogenic carriers according to methods known to experts in this field (see, for example, PCT publication no WO 03/024480). In other embodiments of the invention CpG-containing oligonucleotides can be incorporated into the VLP (see, for example, PCT publication no WO 03/024481).

Examples of adjuvants in the context of the present invention include alum; CpG-containing oligonucleotides, such as CpG 7909, CpG 10103 and CpG 24555; and adjuvants on the basis of a saponin, such as Iscomatrix, which can be used separately or in combination.

Therefore, in the invention proposed immunogene the composition, containing antigenic Tau peptide, preferably containing amino acid sequence selected from the group consisting of SEQ ID NO:1-26, 31-76 and 105-122, and at least one adjuvant. Specified antigenic Tau peptide is preferably linked to immunogenic carrier, preferably with the VLP, preferably with HBsAg, HBcAg or Qbeta the VLP. In one of the specified embodiments the adjuvant is an adjuvant based on saponin, preferably Iscomatrix. In another embodiment of the specified adjuvant is an alum. In yet another embodiment of the specified adjuvant is a CpG-containing oligonucleotide. Preferably, the specified adjuvant is a CpG 7909 or CpG 10103. More preferably, the specified adjuvant is a CpG 24555.

In yet another embodiment of the specified at least one adjuvant contains two adjuvant, preferably selected from the group consisting of alum adjuvants on the basis of saponin and CpG-containing oligonucleotides. In the preferred embodiment these adjuvants are alum and CpG-containing oligonucleotide, preferably CpG 7909, preferably CpG 10103, more preferably CpG 24555. In another preferred embodiment of the above adjuvants are adjuvant based on saponin, preferably Iscomatrix, and CpG-containing oligonucleotide, before occhialino CpG 7909, preferably CpG 10103, more preferably CpG 24555. In another preferred embodiment of the above adjuvants are alum and adjuvant on the basis of saponin, preferably Iscomatrix.

In yet another embodiment of the specified at least one adjuvant contains three adjuvant, preferably selected from the group consisting of alum adjuvant on the basis of saponin, preferably Iscomatrix, and CpG-containing oligonucleotides, such as CpG 7909, CpG 10103 and CpG 24555.

Drugs

The present invention also proposed a pharmaceutical composition containing antigenic Tau peptide of the invention or immunogenic composition, the drug together with one or more pharmaceutically acceptable(and) excipients(AMI). The term "excipient" is used in this application to describe any ingredient other than the active ingredient, i.e. the antigenic Tau peptide according to the invention, subsequently subjected to combination with immunogenic carrier and possibly combined with one or more adjuvants. The choice of excipient(s) to a large extent will depend on such factors as the specific route of administration, the effect of excipient on solubility and stability, and the nature of the dosage form. As used in this application, a "pharmaceutically acceptable excipient" includes any and all solvents, dispersion the physical environment, coatings, antibacterial and antifungal agents, isotonic and slow absorption agents and the like that are physiologically compatible. Some examples of pharmaceutically acceptable excipients are water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In many cases it will be preferable to include isotonic agents, for example, sugar, polyalcohol, such as mannitol, sorbitol, or sodium chloride in the composition. Additional examples of pharmaceutically acceptable substances are moisturizing agents or small amounts of auxiliary substances such as moisturizing or emulsifying agents, preservatives or buffers, which enhance the shelf life or effectiveness of the active ingredient.

The pharmaceutical compositions of the present invention and methods for their preparation will be obvious to experts in this field. Such compositions and methods for their preparation can be found, for example, in Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company, 1995). The pharmaceutical compositions preferably are manufactured under GMP (Good Manufacturing Practice, good manufacturing practices).

The pharmaceutical composition according to the invention can prigoda ivalsa, be Packed or sold no, as a single dose or in multiple single doses. As used in this application, "single dose" is the amount of pharmaceutical composition containing a specified amount of the active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient that is administered to a subject, or an appropriate fraction of such a dosage such as, for example, half or one third of such a dosage.

The pharmaceutical compositions according to the invention is generally suitable for parenteral administration. As used in this application, "parenteral pharmaceutical composition includes any route of administration characterized by physical penetration into the tissue of the subject and the introduction of the pharmaceutical composition through a puncture in the tissue, usually resulting in the direct introduction into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, the introduction of a pharmaceutical composition by injection of the composition, by introducing the composition through a surgical incision, by introducing the composition through the tissue-penetrating non-surgical wound, and the like. In particular, believe that parenteral administration including the et in itself, but not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intra-articular injection or infusion; and methods of dialysis infusion kidney. Preferred embodiments include intravenous, subcutaneous, intradermal and intramuscular route.

Drugs pharmaceutical compositions suitable for parenteral administration, generally contain the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such preparations can be manufactured, packaged, or sold in a form suitable for bolus injection or continuous infusion. Injectable preparations can be manufactured, packaged, or sold in a standard dosage form, e.g., in ampoules or in mnogochasovykh containers containing a preservative. Preparations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous carriers, pastes and the like. These preparations may additionally contain one or more additional ingredients including, but not limited to, suspendida, stabilizing or dispergiruyushchie the agents. In one of the embodiments of the preparation for parenteral administration the active ingredient offered in dry (i.e. powder or granular) form for reconstitution with a suitable carrier (e.g. sterile pyrogen-free water) prior to parenteral introduction of the diluted composition. Parenteral preparations include aqueous solutions which may contain excipients such as salts, carbohydrates and buffering agents (preferably to a pH of from 3 to 9), but for some applications they may be more preferably prepared in the form of a sterile non-aqueous solution or as a dried form to be used in combination with a suitable carrier, such as sterile, pyrogen-free water. Model forms for parenteral administration include solutions or suspensions in sterile aqueous solutions, for example, aqueous solutions of propylene glycol or dextrose. Such dosage forms can be suitably buffered, if desired. Other drugs for parenteral administration, which are useful include preparations that contain the active ingredient in microcrystalline form or in a liposomal preparation. Preparations for parenteral administration can be prepared for immediate and/or modified release. Drugs with modified visw is the release include drugs deferred, slow, pulse, controlled, targeted and programmed release.

For example, in one aspect of sterile injectable solutions can be obtained by incorporating antigenic Tau peptide, preferably associated with immunogenic carrier, eventually in combination with one or more adjuvants, in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, if required, followed by sterilization by filtration. Typically, the dispersion is obtained by incorporating the active compound into a sterile vehicle, which contains the basic dispersion medium and other necessary ingredients from above. In the case of sterile powders for obtaining sterile injectable solutions, the preferred methods of obtaining are vacuum drying and lyophilization, which lead to the formation of a powder of the active ingredient plus any additional desired ingredient from a previously sterilized by filtration of the solution. The proper fluidity of the solution can be maintained, for example, by applying a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prolonged absorption of the compositions for injection is s can be achieved by incorporating in the composition of the agent, which slows down the absorption, for example, monostearate salts and gelatin.

Exemplary, non-limiting pharmaceutical composition according to the invention is a product in the form of a sterile aqueous solution having a pH that ranges from about 5.0 to 6.5, and containing from about 1 mg/ml to about 200 mg/ml of the peptide according to the invention, from about 1 mm to about 100 mm his-tag buffer, from about 0.01 mg/ml to about 10 mg/ml Polysorbate 80, from about 100 mm to about 400 mm trehalose, and from about 0.01 mm to about 1.0 mm disodium EDTA dihydrate.

Antigenic Tau peptides according to the invention can also be entered intranasally or by inhalation, typically in the form of a dry powder (either alone, as a mixture or in the form of mixed particles of the components, for example, mixed with a suitable pharmaceutically acceptable excipient) from a dry powder inhaler, as an aerosol spray from a pressurized container, pump, spray, atomizer (preferably atomizer using electrohydrodynamics for the production of fine mist), or a nebulizer, with or without the use of a suitable propellant, or in the form of nose drops.

The pressurized container, pump, spray, atomizer, or nebulizer typically contains a solution or suspension of the composition according to the invention, with the containing a series, for example, a suitable agent for dispersion, solubilization or extending release of the active agent, and propellant(s) as solvent.

Before use in a dry powder or suspension of the drug, drug product is usually subjected to micronization to a size suitable for delivery by inhalation (typically less than 5 microns). This can be achieved by any suitable grinding method, such as grinding in a spiral jet mill, grinding in a jet mill, fluidized bed, supercritical fluid processing is the formation of nanoparticles, high pressure homogenization, or spray drying.

Capsules, blisters and cartridges for use in an inhaler or insufflator can be cooked the same way, to contain a powder mix of the compound according to the invention, a suitable powder base and modifier efficiency.

Suitable drug solution for use in the atomizer using electrohydrodynamics for the production of fine mist may contain a suitable dose of the antigenic Tau peptide according to the invention for triggering, and the amount of operation can be varied, for example, from 1 μl to 100 μl.

In such preparations according to the invention, intended for inhaled/intranasal, may be add the ENES suitable flavorings, such as menthol and levomenthol, or sweeteners, such as saccharin or saccharin sodium.

Preparations for inhalation/intranasal can be prepared with possible immediate and/or modified release. Drugs with modified release include drugs deferred, delayed, pulsed, controlled, targeted and programmed release.

In the case of dry powder inhalers and aerosols, unit dosages are determined by valve that delivers a measured amount. Units in accordance with the invention are usually for the introduction of a metered dose or "zilch compositions of the present invention. The overall daily dose will typically enter in a single dose or more often in the form of fractional doses during the day.

Pharmaceutical composition containing antigenic Tau peptide, can be prepared for administration through the mouth. Oral administration may include swallowing, so that the connection enters the gastrointestinal tract, and/or transbukkalno, lingual or sublingual introduction, with which the connection enters the bloodstream directly from the mouth.

Preparations suitable for oral administration include solid, semi-solid and liquid systems, is such as tablets; soft or hard capsules containing multi - or nanoparticles, liquid or powder; cakes (including fluid filled); chewing gum; gels; quickly dispergirujutsja dosage form; films; ovule; sprays; and transbukkalno/mucoadhesive patches.

Liquid preparations include suspensions, solutions, syrups and elixirs. Such drugs can be used as fillers in soft or hard capsules (made, for example, from gelatin or hydroxypropylmethylcellulose) and usually contain a carrier, for example, water, ethanol, polyethylene glycol, propylene glycol, methylcellulose, or a suitable oil, and one or more emulsifying agents and/or suspendida agents. Liquid preparations can also be obtained by breeding solids, for example, from Sasha.

Dosing

The composition of the invention can be used for the treatment, mitigation or prevention of Tau-associated disorders or symptoms in a subject at risk of or suffering from such a disorder or symptom by stimulating an immune response in a specified subject by immunotherapy. Immunotherapy may include a primary immunization followed an additional, for example, one, two, three or more reimmunization.

"Immunologically effective amount" antigenic Tau-p is putida according to the invention, or its composition, is a quantity that is delivered to the subject is a mammal or in a single dose or as part of a series, which is effective to induce an immune response against pathological forms of Tau in the specified subject. This number varies depending on the health status and physical condition of the individual, which are treated, the taxonomic group of individual who are treated, the immune system of an individual to induce humoral and/or cellular immune responses, drug, vaccine, and other important factors. Expect that the amount will fall in a relatively broad range that can be determined using the appropriate test.

"Pharmaceutically effective dose" or "therapeutically effective dose" is a dose that is needed for treating or preventing, or alleviating one or more Tau-associated disorders or symptoms in the subject. Pharmaceutically effective dose may depend on the input connection, the severity of symptoms, susceptibility of the subject to side effects, type of disease, the composition, the route of administration, the type of mammal, which is being treated, the physical characteristics of a particular question of a mammal, such as the status of ZV is cited and physical condition, concomitant treatment, the activity of the immune system of the individual, the degree of desired protection, and other factors that are known to experts in the field of medicine. For preventive purposes, the amount of peptide in each dose is selected as an amount which induces immunoprotective response without significant adverse side effects in typical vaccines. After the initial vaccination, subjects may receive one or more reimmunization, through appropriate intervals.

It should be understood that the specific dose level for any particular patient will depend on many factors including the activity of the specific compound, the age, body weight, General health, sex, diet, time of administration, route of administration, rate of excretion, combination of drugs and the severity of the particular disease being treated.

For example, antigenic Tau peptides according to the invention, subjected to combination with immunogenic carrier, you can enter the subject in a dose of from about 0.1 μg to about 200 mg each, for example, from about 0.1 mcgd about 5 μg, from about 5 μg to about 10 μg, from about 10 μg to about 25 μg, from about 25 μg to about 50 μg, from about 50 μg to about 100 μg, from about 100 μg to about 500 μg, from about 500 μg to about 1 mg, about what about 1 mg to about 10 mg, from about 10 mg to about 50 mg, or from about 50 mg to about 200 mg, with possible reimmunization conducted, for example, after 1 week, 2 weeks, 3 weeks, 4 weeks, 2 month, 3 month and/or year. In some embodiments, the number of antigenic Tau peptide to determine the dose based on body weight. For example, in some embodiments the antigenic peptide is administered in an amount of from about 0.5 mg/kg to about 100 mg/kg, for example, from about 0.5 mg/kg to about 1 mg/kg, from about 1 mg/kg to about 2 mg/kg, from about 2 mg/kg to about 3 mg/kg, from about 3 mg/kg to about 5 mg/kg, from about 5 mg/kg to about 7 mg/kg, from about 7 mg/kg to about 10 mg/kg, from about 10 mg/kg to about 15 mg/kg, from about 15 mg/kg to about 20 mg/kg, from about 20 mg/kg to about 25 mg/kg, from about 25 mg/kg to about 30 mg/kg, from about 30 mg/kg to about 40 mg/kg, from about 40 mg/kg to about 50 mg/kg per dose, from about 50 mg/kg to about 60 mg/kg, from about 60 mg/kg to about 70 mg/kg, from about 70 mg/kg to about 80 mg/kg, from about 80 mg/kg to about 90 mg/kg, or from about 90 mg/kg to about 100 mg/kg, or more than about 100 mg/kg

In some embodiments injected with a single dose of the antigenic Tau peptide according to the invention. In other embodiments administered multiple doses of antigenic Tau peptide according to the invention. Cha is Thoth introduction may vary depending on any of a variety of factors, for example, the severity of symptoms, extent desired immunoprotective whether the composition for prevention or curative goals, etc. for Example, in some embodiments the antigenic Tau peptide according to the invention is administered once per month, twice per month, three times per month, every other week (qow), once per week (qw), twice per week (biw), three times per week (tiw), four times per week five times per week six times per week, in a day (qod), one once a day (qd), twice a day (qid), or three times daily (tid). When the composition according to the invention is used for preventive purposes, then it will typically enter as for Primerose (priming)and boosting (boosting) doses. Expect the booster dose will be divided adequate periods of time, or preferably be given annually or at the time when the levels of circulating antibodies fall below the desired level, the Booster dose may consist of antigenic Tau peptide in the absence of initial immunogenic molecules of the carrier. Such booster designs can include alternative immunogenic carrier or may not contain any media. Such booster compositions can be prepared with either adjuvant or without adjuvant.

The duration of antigenic Tau peptide according to the invention, for example, the time period for a cat is cerned enter antigenic Tau peptide, may vary depending on any of a variety of factors, for example, the reaction of the patient, etc. for Example, antigenic Tau peptide can be entered within a period of time ranging from about one day to about one week, from about two weeks to about four weeks, from about one month to about two months, from about two months to about four months, from about four months to about six months, from about six months to about eight months, from about eight months to about 1 year, from about 1 year to about 2 years, or from about 2 years to about 4 years or more.

Applications and methods of treatment

The present invention also covers various methods of treatment, which include the introduction of antigenic Tau peptide according to the invention. Methods of treatment include methods of inducing an immune response in an individual on auto-Tau in its pathological(s) form(s) and methods of preventing, alleviating or treating Tau-associated disorder or symptom in the individual.

In one aspect of the present invention, a method for treatment, prevention or relief of Tau-associated disorder or symptom in a subject, comprising introducing a therapeutically effective amount of the antigenic Tau peptide is about invention, or immunogenic or pharmaceutical composition, the specified entity.

In another aspect of the present invention, a method for inducing an immune response against autologous Tau in its pathological(s) form(s) for a subject, comprising introducing a therapeutically or immunogen effective amount of the antigenic Tau peptide of the invention, or immunogenic or pharmaceutical composition, the specified entity.

"Treat", "treatment" and "treatment" refer to a method of alleviating or eliminating a biological disorder and/or at least one of its related symptoms. As used in this application, "facilitate" a disease, disorder or condition means to reduce the severity and/or frequency of symptoms of the disease, disorder or condition. In addition, references in this application to "treatment" include references to curative, palliative and prophylactic treatment. The specified subject is preferably a human, and can be either a man or a woman of any age.

Other aspects the invention relates to antigenic Tau peptide according to the invention, or immunogenic compositions or pharmaceutical compositions, for use as a drug, preferably in treating Tau-associated the Sam the properties.

In another aspect of the present invention proposed the use of antigenic Tau peptide of the invention, or immunogenic compositions or pharmaceutical compositions, in the manufacture of medicines, preferably for the treatment of Tau-associated disorders.

The present invention is additionally illustrated by the following examples, which should not be construed as further limiting. The content of all graphic materials and all references, patents and published patent applications, cited in this document are expressly incorporated in this application by reference in their entirety.

EXAMPLES

Attempts have been made to ensure accuracy in terms of numbers (e.g. amounts, temperature, etc.), but should take into account some experimental errors and deviations. Unless indicated otherwise, parts are parts by weight, molecular weight is an average molecular weight, temperature is expressed in degrees Celsius, and pressure is an atmospheric or close to atmospheric. As used in the examples below, the following abbreviations have the following meaning and, unless otherwise stated, readily available from commercial suppliers: DMF: dimethylformamide; TFA: triperoxonane acid; TIS: triisopropylsilyl-triftorbyenzola; TSER: Tris(2-carboxyethyl)phosphine; mcKLH: hemocyanin lymph snails from marine culture; HBTU: O-benzotriazole-N,N,N, N'-tetramethyl-urani-hexaplar-phosphate; EDTA: ethylene-diamine-tetraoxane acid; DMSO: dimethyl sulfoxide.

Example 1: Plasmid construction Qbeta

Native envelope protein Qbeta: the Coding sequence corresponding to a protein shell Qbeta, nucleotides from 1304 to 1705 from GenBank access # AY099114, synthesized using DNA 2,0 (DNA 2.0, Menio Park, CA). Included a 5'-modification (CCatgg) for introducing an NcoI site and a 3'-modification to introduce two stop codons and a XhoI site (gtaTTAATGACTCGAG- SEQ ID NO:78).

The codon-optimized shell protein Qbeta: a Sequence encoding a protein shell Qbeta, optimized for expression using Gene Designer (Villalobos et al., BMC Bioinformatics 7:285 (2006)). Identical to the 5'- and 3'-modifications were introduced in the codon-optimized shell protein Qbeta.

Both native and codon-optimized sequence of the envelope protein Qbeta was introduced in expressing vector RET using traditional methods sublimirovanny DNA, including the digestion of restrictase and ligation reaction.

Example 2: obtain the synthetic Tau peptide

Tau-peptides (referred to as A-1 to A-11; b-1 to b-6; C-1 to C-5; D-1; F-1 and F1; and phosphorylated variants of these peptides is listed as A-1R, A-2R, IS-3P, and so on), presented as SEQ ID NO:31-76, 105-107 and shown in Table 5 below with their respective names to be used in the following examples was prepared as follows. Synthesis of phosphorylated or nefosfaurilirovanna Tau-peptides containing the linker sequence (CGG or GGC), was carried out using the technology of solid-phase synthesis of a peptide synthesizer Symphony (Protein Technologies, Inc). Mono-protected amino acid Fmoc-Ser[PO(O-Bzl)OH]-OH, Fmoc-Thr[PO(O-Bzl)OH]-OH and Fmoc-Tyr[PO(O-Bzl)OH]-OH (EMD Chemicals, Inc) was used to enable phosphoserine, posttraining and phosphotyrosine in phosphorylated variants of these sequences. The reaction was started by mixing resin NovaSyn TGA (EMD Chemicals, Inc.), containing the first amino acid, 6,25-fold excess of Fmoc-protected second amino acid (1 mmol), which was activated using 1 mmol of HBTU in the course of 1 hour. The reaction mix was repeated once for each amino acid. Removal of Fmoc group was achieved in 20%piperidine in DMF for 2×5 minutes. The synthesized peptide was released from the resin by incubation of the resin with 5 ml TFA containing 2.5% TIPS and 2.5% thioanisole, for 3 hours at room temperature. The crude peptide was isolated after filtration, mediated diethyl ether precipitation and vacuum drying. Purification of the peptide was carried out in HPLC with reversed phase (Waters 2525 Binary Gradient Modue) preparative column VEINS 130 C18. The mobile phase consisted of 0.1% TFA in water as buffer a and 0.1% of TFA in acetonitrile as buffer C. the Collected fractions containing the peptide were pooled and ifileservice under vacuum. Approximately 20 mg of the peptide was purified from a typical injection of 100 mg of the crude peptide with a purity of more than 95%. All purified peptides was checked by LC-MS.

Similarly, synthesize and purify additional Tau-peptides (SEQ ID: 108-122).

Example 3: Qbeta-the VLP: expression, purification and conjugation with Tau-peptides

Expression of Qbeta in E. coli: Plasmid RET containing cDNA Qbeta, were subjected to transformation into competent cells of E. coli BL21 (DE3). One colony was inoculable in 5 ml of medium 2×YT containing 50 μg/ml kanamycin, at 37°C over night. Night inoculum was diluted to 500 ml medium TV containing 50 μg/ml kanamycin, and grown to OD600 of 0.8 at 250 rpm at 37°C and induced with 0.4 mm IPTG (isopropyl-β-D-1-thiogalactopyranoside) during the night. The cells were collected by centrifugation at 2500 RCF (units relative centrifugal acceleration from the English. relative centrifuge force) for 15 minutes. The cell sediments were stored at -80°C.

Cleaning Qbeta the VLP from E. coli: all stages of the purification were performed at 4°C. the Cell sediment expressing Qbeta, resuspendable in lytic buffer containing 25 mm Tris pH 8.0, 150 mm NaCl, 5 mm EDTA, 0.1% Triton-100, supplemented with a cocktail of inhibitors FR is AZ (Roche). Resuspending solution was passed through microfluidizer (Microfluidics Corp.) with subsequent ultracentrifugation. Proteins were besieged by adding ammonium sulfate to 50%saturation, followed by centrifugation at 15000 RCF for 30 minutes. Sediment resuspendable and were dialyzed in buffer containing 25 mm Hepes pH 7.5, 100 mm NaCl, 1 mm EDTA, at 4°C over night. Cialisovernight the solution was centrifuged and then was applied on the column, Capto Q (GE), equilibrated in 25 mm HEPES pH 7.5, 100 mm NaCl, 1 mm EDTA. The column was washed and chased gradient from 100 mm NaCl to 1 M NaCl in buffer containing 25 mm HEPES pH 7.5, 1 mm EDTA. Protein Qbeta identified using SDS-PAGE (polyacrylamide gel electrophoresis with sodium dodecyl sulfate). The fractions containing Qbeta, were dialyzed in 10 mm potassium phosphate, pH 7.4, 150 mm KCl, during the night and was applied to a hydroxyapatite column (Type II, Bio-Rad Inc.). The column was washed and was suirable gradient from 100% buffer containing 10 mm potassium phosphate pH 7.5, 150 mm KCl, and 100% buffer containing 500 mm potassium phosphate, pH 7.5, 0.5 m KCl. The fractions containing Qbeta, combined, were dialyzed and applied on a phenyl column, equilibrated in 25 mm Tris-Cl, pH 8.0, 150 mm NaCI, 0.7 M (NH4)2SO4. Protein was suirable gradient from 100% buffer containing 25 mm Tris-Cl, pH 8.0, 150 mm NaCl, 0.7 M (NH4)2SO4up to 100% of buffer containing 25 mm Tris-Cl, pH 8.0, 50 mm Nal. The fractions containing pure Qbeta, were combined and were dialyzed in PBS at 4°C over night. The protein concentration was determined by the Bradford method.

The combination of Tau-peptides with Qbeta the VLP: a Combination of Tau-peptides with Qbeta-the VLP oposredovany via a bifunctional cross-linker SMPH (Succinimidyl-6-[p-maleimidopropionamide]hexanoate) (Thermo Scientific) (Freer et al., Virology 322 (2):360-369 (2004)). The peptide was dissolved in PBS (Invitrogen), pH 7.0 containing 5 mm EDTA, 10 mg/ml and was restored by incubation with the disulfide-revitalizing gel with immobilized TSAR in equal volume at room temperature for 1 hour. A solution of the peptide was isolated by centrifugation at 1000×g for 2 minutes. Protein Qbeta-the VLP at 2 mg/ml in PBS (Invitrogen) was activated by incubation with 7 mm SMPH in DMSO at room temperature for 1 hour. Derivatizing the VLP was absoluely by passing through a column of Zeba Desalt Spin (Thermo Scientific) at 1000×g for 2 minutres activated the VLP mixed with 10-fold molar excess of recovered peptides at room temperature for 2-3 hours. The reaction mixture was concentrated and were dialyzed either in PBS or in 25 mm histidine pH of 7.4, containing 50 mm NaCl at 4°C over night. The protein concentration was determined using Coomassie protein analysis Plus from Thermo Scientific.

Example 4: obtaining a conjugate of the peptide-KLH

Tau-peptide A-1R with CGG-linker (SEQ ID NO:31) which was sjugirov with mcKLH (Thermo Scientific, cat.№77605) for evaluation of its immunogenicity in mice. Conjugation was mediated via a bifunctional cross-linker SMPH (Succinimidyl-6-[(3-maleimidopropionamide]hexanoate) (Thermo Scientific). Peptide A-1 at 10 mg/ml in PBS, pH 7.0, containing 5 mm EDTA, first treated with a disulfide-revitalizing gel with immobilized TSAR in equal amount by stirring at room temperature for 1 hour. A solution of the peptide was isolated by centrifugation at 1000×g for 2 minuteclinic KLH was carried out by incubation KLH at 10 mg/ml in PBS with 200 μl of 100 mm SMPH in DMSO for 1 hour at room temperature. The reaction mixture was passed through a column Zeba Desalt Spin (Thermo Scientific). Collected derivationally KLH and mixed with the recovered-1P for 2 hours at room temperature. The reaction mixture were dialyzed in PBS containing 0.6 M NaCl, at 4°C over night. The protein concentration was determined using Coomassie protein analysis Plus from Thermo Scientific.

Example 5: a Study of peptide immunization against immunogenicity and b-cell memory

The experiment was performed in order to determine whether the selected peptides are shown in Table 5, immunogenic, and to determine whether developing immunological memory. Groups of 3 Balb/c mice were primiraly peptide or peptide, conjugated to Qbeta the VLP, 0) with the TCA and re-stimulated in 14 and 101 days, while some mice only have primiraly in 101 days, as shown in Figa, 1B and 2. Serum was collected at 28, 101, 104, 108 and 115 days. Sera from selected mice were collected 94 days. Or antibody-based test responses from immunized animals was investigated using tests detect antigen-specific titers (as described in Example 13).

Results in title antigen-specific IgG, which indicate that the peptides are immunogenic when using serum samples from 28-days, are summarized in Figb. This study showed that the peptides a-1, a-1R, 1R and 1R are immunogenic when immunization using TiterMax Gold (Alexis Biochemicals) as adjuvant. Primary immunization with peptide A-1R and TiterMax Gold or A-1, conjugated to Qbeta-the VLP, followed by reimmunization A-1P-Qbeta-the VLP on the 14th day gave large titers of antibodies than A-1R TiterMax "Prime-boost" group. Primary immunization and reimmunization on the 14th day using A-1, conjugated to KLH (obtained as described in Example 4), as adjuvant also gave large titers of antibodies than A-1R TiterMax "Prime-boost" group.

Also investigated the selectivity of antibodies induced in response to phosphorylated (A-1P, 1P, D-1P-1P) or nefosfaurilirovanna peptide (a-1)used for immunization. This was carried out by comparing the titer of antibodies to the to on phosphorylated, and nefosfaurilirovanna options for each peptide used for immunization (see Figb). Calculate the ratio of the specific titer of non-specific title. In this experiment, or antibody-based test response against a-1 (Group 1) was selective (ratio <0,1) for phosphorylated state of the peptides, which were immunized animals, whereas antibodies against C-1P (Group 5), likely to be selective (title-1P/s-1>7). Group 2 (A-1R) was not selective.

Results showing secondary immune (recall) the response of memory cells on A-1R, as shown in figure 2. Group a (primary immunization AND-1R with TiterMax, reimmunization using A-1R - Qbeta-the VLP) and Group B (primary immunization and reimmunization using A-1R - Qbeta-the VLP) was compared with Group C, which was primiraly on the 101st day A-1, conjugated to Qbeta-the VLP. All three groups showed an IgM response. IgG was determined at 104 days in both groups, re-stimulated on the 101st day, but not earlier than 7 days for the group, premirovanii on the 101st day. Titers of 104 days were more than the titles 94 days. The IgG titers at 7 and 14 days were more than the group premirovany for 101 days (Group C). The IgG titers for Groups a and b at 108 and 115 days were the same, whereas the IgG titer for Group did not reach a peak until 115 days. These data indicate that the long-or antibody-based test the responses and stimulation of b-cell memory.

Example 6: a Study of primary immunization with peptide and reimmunization peptide-on the VLP immunogenicity

The experiment was performed in order to determine the selected peptides from Table 5 immunogenic when primary immunization peptide with addition of alum (Al(OH)3; Alhydrogel 2% '85' , Brenntag Biosector) adjuvant, followed by reimmunization peptide, conjugated to Qbeta-the VLP. Groups of 4 Balb/c mice were primiraly in 0 days and re-stimulated at 28 and 56 days, as shown in Figure 3. Serum was collected at 70 days. Or antibody-based test responses from immunized animals was investigated using analysis determination of antigen-specific titers (as described in Example 13).

The results are summarized in figure 3. In Groups 1-6, IgG antibodies against the peptide used for immunization was determined at the maximum tested dilution (1:1749600), showing a strong or antibody-based test responses to peptide antigen used for immunization. In the untreated group antibodies have not been identified (Group 7). Antibodies obtained by immunization with the use of peptides D-1P and 1P recognized the peptide F-1R. Peptides D-1P and 1P completely contained in E-1R.

Investigated the selectivity of antibodies induced phosphorylated (A-1P, 1P, D-1P, 1P, E-1R) or nefosfaurilirovanna peptide (a-1)used for them is unisule. This is carried out by determining the titer of antibodies to nefosfaurilirovanna options phosphorylated peptides and phosphorylated variants nefosfaurilirovanna peptides (see figure 3). Calculate the ratio of the specific titer of non-specific title. In this experiment, antibodies against D-1P (Group 4)-1R (Group 5) and F-1R (Group 6) were selective (title >10) for phosphorylated state of the peptides, which were immunized animals.

Example 7: a Study of immunization with peptide-on the VLP immunogenicity

The experiment was performed in order to determine whether the selected peptides and combinations of peptides from Table 5 immunogenic when immunization in the form of conjugates Qbeta-the VLP with different adjuvants. As shown in figure 4, groups of 4 mice TG4510+/+ (double transgenic positive, see Ramsden et al, J. Neuroscience 25 (46):10637 (2005)) or TG4510-/- (odnomomentnye control animals wild-type) primiraly in 0 days and re-stimulated for 56 days in any 28 or 29 days. Serum was collected for 63 days. Or antibody-based test responses from immunized animals was investigated using analysis determine the titer of antigen-specific IgG, as described in Example 13.

Results samples for 63 days summarized in figure 4. In each group, antibodies (IgG) against the peptide or combination of peptides used for immuniza the AI, determined with average titers ranging from E+04 to E+06. Immunization three conjugates of peptide-Qbeta-the VLP in combination with 100 μg or 10 μg of each induced titers similar to immunization with 100 μg of the conjugate peptide-Qbeta-the VLP. Titles A-1P, 1P and 1P from Groups 1 and 2 combined dosing ranges from 1.7 to 4.4 times regarding the relevant groups of single dosing (Groups 3, 4 and 5). Titles A-1P, 1P and 1P from Groups 11 and 12 combined dosage range from 0.32 to 2.8 times regarding the relevant groups of single dosing (Group 13, 14 and 15). Antibodies were determined using adjuvant (alum or CpG 24555 (provisional application for U.S. patent No. 61/121022, filed December 9, 2008) or ABISCO-100 (Isconova) with CpG-24555) or without adjuvant. In the untreated control antibodies against peptides have not been identified.

In the selected groups have investigated the selectivity of antibodies induced phosphorylated (A-1P, 1P, D-1P, 1P, E-1R)used for immunization. This is carried out by determining the titer of antibodies to nefosfaurilirovanna options phosphorylated peptides for Groups 1-7 (Figure 4). Calculate the ratio of the specific titer of non-specific title. In this experiment, antibodies were selective (>10-fold ratio of credits) for IN-1R relatively In-1 in all dosing groups. Antibodies were selective in whom Oseni WITH-1R relative to C-1 only in Group 6 (group, does not contain alum). Antibodies were selective against A-1R relative to A-1 from Groups 2, 3 and 6, but not in Group 1 (high immunization dose combinations with the addition of alum as adjuvant). In the untreated control antibodies against nefosfaurilirovanna peptides have not been identified.

Example 8: a Study of immunization with peptide-the VLP in respect of the way, adjuvant and isotype

The experiment was performed in order to compare the immunogenicity and the isotype of the antibodies induced by using different adjuvants and routes of administration. Groups of 3 Balb/c mice were primiraly in 0 days and re-stimulated 17-e day, as shown in Figure 5. Serum was collected on the 24th day. Or antibody-based test responses from immunized animals was investigated using analysis determination of antigen-specific titers as described in Example 13.

A-1, conjugated to Qbeta-the VLP, delivered balb/c mice by subcutaneous or intramuscular injection. Different combinations of antigens were also tested using the intramuscular route. The results using samples on the 27th day are summarized in figure 5. Both subcutaneous and intramuscular injection of A-1, conjugated to Qbeta-the VLP, and with the addition of alum as adjuvant caused IgG or antibody-based test response. Group intramuscular dose had a greater ratio of titer AND-1R to the -1 (70), than group subcutaneous dose (11). This shows that the route of administration may influence the selectivity of the response.

As shown in Figure 5, all used adjuvant combination induced IgG1 and IgG2a antibodies with alum-containing groups (ratio of 21 and 12 for Groups 2 and 5, respectively), with a much higher ratio of IgG1 to IgG2a than Group 3 (0,17) and 4 (0,17)that did not include alum as adjuvant. This is consistent with the known effects of alum on the immune response, shifted towards Th2 (see Lindblad, Immunol Cell Biol. 82 (5):497-505 (2004); Marrack et al., Nature Rev. 9:287-293 (2009)). These results imply that adjuvants can be used to change or antibody-based test responses to the vaccine used in this example. In the untreated control antibodies against peptides have not been identified.

Example 9: Immunization with peptide-for the VLP analysis of linkers

The experiment was performed in order to determine, whether on the immunogenicity of the position of the linker (CGG or GGC) of selected peptides from Table 5. There used peptide-1R with a linker, which was located on the N-end (i.e. SEQ ID NO:31 - a-1R) or at the C-end (i.e. SEQ ID NO:41 AND R) peptide. Groups of 4 mice TG4510+/+primiraly in 0 days and re-stimulated for 14 days, as shown below in Table 1. Mice were bled on the 20th day. Or antibody-based test answers from the immunized animal studies and, using analysis to detect antigen-specific titers as described in Example 13.

Based on the results presented in Table 1, the linker sequence for Qbeta-the VLP can be placed on N- (CGG) or late (GGC) Tau-specific sequence and still cause selective in relation to phosphorylation IgG response >10-fold respect of titles, table 1). The peptides used in this experiment (SEQ ID NO:31 and 41)have the same sequence, except that CGG-linker is N-terminal to SEQ ID NO:31, and GGC-linker is C-terminal to SEQ ID NO:41. Both induced similar titer of IgG in the samples on the 20th day. The antibodies induced by the two peptide sequences, were selective, as determined from the ratios of IgG titers phosphorylated peptide against nefosfaurilirovanna - 49 >132, as shown in Table 1. In the untreated controls on the 56th day of antibodies against peptides have not been identified (Group 7 in figure 4).

Table 1:
Mice were immunized intramuscularly. Used 100 µg peptide-the VLP and 750 μg of alum (Al(OH)3). Dilution of the serum under test in the analysis of the determination of antigen-specific titers (see Example 13), ranged from 1:5000 to 1:1800000.
VaccineAdjuvantMouseNThe titer of IgG in 20 daysSelectivity
A-1R IgG (mg/ml)Title A-1PTitle a-1A-1/a-1
A-1P-the VLPAlumTG4510++40,62E+05E+0449,0
A-R-the VLPAlumTG4510++40,42E+05<5,E+03>132

Example 10: the Binding of polyclonal antibodies with short peptides

The experiment was performed in order to determine whether contained selected peptides from Table 5 immunogenic epitopes present in A-1R, 1R or S-1R, which were induced antibodies. Serum was collected from mice vaccinated with A-1R, 1R or S-1R, as shown below in Table 2. Or antibody-based test from the Yety from immunized animals investigated, using analysis to detect antigen-specific titers (as described in Example 13) with the following modification in data analysis: a signal twice the average value of uncoated wells were considered positive, whereas the signal is below twice the average value of uncoated wells were considered negative.

In order to determine whether antibodies from animals immunized with peptide-by the VLP conjugates of any of the peptides A-1R, 1R or S-1R, contact a shorter variants of each of these peptides was performed by ELISA (enzyme-linked immunosorbent assay). Each test Tau-peptide used as an tablet antigen, and serum in dilutions 1:4×104and 1:4×105from mice immunized with A-1R --1R - or C-1P-the VLP, was tested to determine whether they can contact the relevant peptide (see Table 3). These sera, as shown earlier, have antigen-specific antibodies. Sera were from mice immunized with the relevant parent peptide (A-1R a-1R and derivatives, B-1 to B-1P and derivatives, C-1 to C-1 and C-1P/S-1R derivatives) (see Table 2). Each of the antisera used in 2 dilutions (1:4×104and 1:4×105). If defined binding peptide, resulted in a positive rezultatele signal was not identified from any is the value of serum the led negative resultats the samples tested, with the exception of A-5P AND 10P and 2P, had a positive signal that indicates that the antibodies induced full-size (parent) peptides, have also been associated with most shorter tested derivatives.

of 14.28
Table 2:
Mice were immunized intramuscularly. Used 100 µg peptide, 100 μg of peptide-the VLP and 750 μg of alum (Al(OH)3), where indicated. Dilution 1:4×104and 1:4×105tested in the analysis of the determination of antigen-specific titers (Example 13) for each serum.
Primary immunization (Day 0)Reimmunization
Saworo-TKAVaccineVaccineDayMouse
Line
Collection of serum (Day)
1A-1P-the VLP+AlumA-1P-the VLP+AlumTG4510-/-42
2A-1P-the VLP+AlumA-1P-the VLP+Alum14TG4510-/-20
3In-1PB-1P-the VLP28,56Balb/c70
4In-1PB-1 P-the VLP28,56Balb/c70
5C-1P-the VLP+AlumC-1P-the VLP+Alumof 14.28TG4510-/-42
6C-1PP-1 P-the VLP28,56Balb/c70
Table 3:
"Positive" indicates that the OD (optical square is Tosti) this hole was at least twice the average background values OD (uncovered hole). "Negative" indicates that the OD for the wells was less than twice the average background values OD (uncovered hole).
PeptideSerum AndSerum
A-1PPositivePositive
A-2PPositivePositive

PeptideSerum AndSerum
A-4PPositiveNegative
A-5PNegativeNegative
A-6PositivePositive
A-7ĐPositivePositive
A-8P PositivePositive
A-9RPositivePositive
A-10PNegativeNegative
In-1PPositivePositive
In-2PNegativeNegative
In-LAPositivePositive
In-4RPositiveNegative
In-5PPositiveNegative
In-6RPositiveNegative
C-1PPositivePositive
With-2PPositivePositive
C-SP PositivePositive
With-4RPositivePositive
With-5PPositivePositive

Example 11: a Study of immunization with short peptides in terms of immunogenicity and memory

Two experiments were conducted to determine whether the selected peptides from Table 5 immunogenic when immunization in the form of conjugates Qbeta-the VLP. One of these studies also used to determine whether developing immunological memory. In order to avoid potential binding peptide antigens with T-cell ligands of MHC class I and MHC class II, tested shorter versions "parent" peptide A-1P, 1P and 1P. Selected peptides with a length of from 7 to 11 amino acids, since the molecules of the MHC class II typically bind peptides with 13-17 amino acids, and the peptide with a length of at least 8 amino acids required for binding MHC I (Murphy et al., Janeway''s Immunobiology, Garland Science (2007)). Therefore, the peptides having 11 or fewer amino acids, should not induce CD4 T-cell response, limited MHC class II and the peptide of 7 amino acids should not induce neither CD4 T-cell or CD8 T-cell is the answer, limited MHC class I. Also tested the peptide F-1P with a length of 7 amino acids. Groups of 3 or 6 Balb/c mice were primiraly in 0 days and re-stimulated for 14 days, as shown in Fig.6. Three groups also re-stimulated for 108 days and three groups have primiraly on 108th day (see Fig.7). Serum was collected at 21 days or 28 days, or 111, 115 and 122 days or 21, 105, 111, 115 and 122 days. Or antibody-based test responses from immunized animals was investigated using analysis determination of antigen-specific titers (as described in Example 13).

The results are summarized in Fig.6. All the conjugates of peptide-Qbeta-the VLP induced antigen-specific IgG antibodies in all mice tested in ELISA, with the exception of IN-5P, in which only 2 of the 3 mice had detectable antibodies in the serum dilution 1:15800. These results show that 7-11-amino acid Tau-peptides with CGG-linker are immunogenic and can induce antibodies that are specific against the immunogen.

Investigated the selectivity of antibodies induced in response to phosphorylated peptide form used for immunization (see Fig.6). Most of these peptides were selective (>10-fold ratio of titers) against phosphorylated form of the peptide relative to nefosfaurilirovanna form. Many of truncated derivatives of A-1R, A-1R and C-1R n who had no ELISA signal, defined when using nefosfaurilirovanna variant peptide for immunization as a tablet antigen. The selectivity of many shortened derivatives of A-1R, A-1R and C-1R equal to or greater than the selectivity of the parent peptide. Active immunization with peptide A-2P without CGG-linker reportedly reduces aggregated Tau in the brain and slows down the development associated with glomeruli sensorimotor impairments in an animal model JNPL3 overexpression of Tau P301L (Asuni et al., J. Neurosci. 27:9115 (2007)). A-2P, when the conjugation with Qbeta-the VLP was immunogenic. However, the induced antibodies were not selective against phosphorylated variant peptide (A-2P) relative nefosfaurilirovanna options (a-2) ELISA (titles A-2P/A-21,7). In contrast, these antibodies were selective against A-1R relative to a-1 (titles A-1/a-1>10,0). Titles, when using A-2P and 1P as ELISA antigens were the same. This implies that the epitopes of the most apostolicity antibodies include 12 amino acids of the peptide A-2P, which are not contained in A-1R. In this experiment WITH-1R had a higher selectivity when tested without alum adjuvant than alum (Groups 14 and 10, respectively). Adjuvants, such as alum, may be used for modification of selection the STI against phosphorylated peptide compared to nefosfaurilirovanna peptide. In the untreated control antibodies against peptides have not been identified. These results show that 7-11-amino acid Tau-peptides with CGG-linker can induce antibodies that are selective for phospho-peptides.

The test results of the secondary immune response (memory recall response) a-1P, 1R and 1R are shown in Fig.7. Titers of IgG 111, 115 and 122 days (day +3, +7 and +14 from the last immunization, respectively) for mice immunized with peptide-Qbeta-the VLP, which has primiraly and re-stimulated at 0, 14 and 108 days, compared with titers of IgG from mice, premirovany for 108 days. Groups 1, 2 and 3 had higher IgG titers for 105 days, 84 days after the last reimmunization. Compared with groups premirovanii on 108th day (Groups 4, 5 and 6), these groups also had a large increase in the IgG titer between 111 and 115-mi day. These data indicate that the long-or antibody-based test response and secondary immune response.

Example 12: a Study of immunization with short peptides in terms of immunogenicity and T-cell response in combination with alum and without alum

The experiment was performed in order to determine whether peptides derived from A-1R, A-1R and C-1R (table 5), immunogenic after immunization with 100 μg Qbeta-the VLP conjugate with 0 or 504 μg of alum (Al(Oh)3or when taken in combination conjugates peptide-Qbta-the VLP with or without alum alum. Also analyzed T-cell responses in the spleen. Group of 3 odnopolnyh mice TG4510-/- wild type was primiraly in 0 days and re-stimulated for 14 days, as shown in Fig. Serum and spleen were collected at 21 days. Or antibody-based test responses from immunized animals was investigated using analysis determination of antigen-specific titers (as described in Example 13) and ELISPOT analysis of IFN-γ (as described in Example 14).

Titers of antigen-specific IgG show that all tested peptides were immunogenic when immunization in the form of a conjugate Qbeta-the VLP with 504 μg of alum (Al(OH)3or without alum (see Fig). Immunization AND-8P,-3P and 2P in combination, generally 750 μg of alum to 300 μg of conjugated peptide-Qbeta-the VLP resulted in selective or antibody-based test response for all 3 peptides.

The selectivity of antibodies induced in response to phosphorylated peptide against nefosfaurilirovanna variant of this peptide used for immunization was investigated using ELISA (Fig). Calculate the ratio of specific versus non-specific title, where a higher ratio indicates a higher selectivity. The induced antibodies were selective against phosphorylated form of the peptide regardless, were included alum during the primary immunization and reimmunization or not, and regardless of what westlakes immunization with conjugates of peptide-Qbeta-the VLP alone or in combination.

T-cell responses in the spleen after immunization by a single peptide Qbeta-the VLP were analyzed using ELISPOT analysis of IFN-y (see Fig.9). The frequency of T cells secreting IFN-y-specific to the parental Tau-peptides (A-1R, 1R, 1R) and its truncated variants were analyzed for 21 days through 7 days after the last reimmunization peptide Qbeta-the VLP. Relative to an unrelated control peptide (HBV-1), no significant amounts of B-1P, B-1, B-3, b-3, C-1, C-1, C-2R or-2-specific IFN-γ-secreting T cells were not formed after immunization using IN-3P-Qbeta-the VLP and C-2P-Qbeta-the VLP either in the presence or in the absence of alum. Significant levels (p<0.05) AND-3P-specific IFN-γ T-cell responses were induced after immunization with the use of A-3P-Qbeta-the VLP. Peptide A-3 contains the predicted murine epitope binding MHC class I Kb(IVYKSPW, see Lundegaard et al. Bioinfonnatics 24:1397-1398 (2008)), and this epitope may contribute to the T-cell response, which is observed in A-3P-immunized animals. This epitope is also present in A-1R, a-1, a-2R, a-2 and a-3. When the peptide A-1R was shortened to a peptide with a length of 7 amino acids (A-8P Qbeta-the VLP), then IFN-γ-specific T-cell responses in a-8P Qbeta-the VLP-immunized mice was reduced to background levels.

CD4 T-helper cells are required for education or antibody-based test answers with areclosely the isotype and education In-memory cells (see Murphy et al., Janway's Immunobiology, Garland Science, (2007)). Thus, the discovery that the IgG or antibody-based test answers were formed on their corresponding peptide epitopes after immunization shortened phospho-Tau peptide Qbeta-the VLP, implies that CD4 T-helper responses are induced against vaccines. Because no significant levels of Tau-peptide-specific T cells were not formed after immunization shortened peptide conjugates were tested T-cell response to another component of the vaccine. Analysis of T-cell responses to the VLP protein demonstrates that IFN-y-specific T cells were generated against the VLP epitopes (4-15-ratio relative to an unrelated protein control (BSA, Sigma Aldrich A9418).

Example 13: Determination of the titer of antigen-specific antibodies

The following analysis used to determine or antibody-based test responses from immunized animals, as described above in Examples 5-12.

Colorimetric ELISA was used to determine the highest dilution of serum that had defined antigen-specific antibody, as represented by a positive signal. Serial dilution was obtained from serum samples and tested in the analysis. In some analyses as positive controls or standards used monoclonal antibodies specific to phospho-Tau-peptide. In cachestatistics control used sera from unvaccinated mice (BALB/c, TG4510+/+ or Tg4510-/-). On 96-well polystyrene tablets with high binding CoStar 9018) were applied in 100 μl of peptide diluted in 0.1 M sodium carbonate, pH of 8.2 (Sigma S7795) at 4°C for 18-21 hours. All peptides were at a concentration of 0.3 ág/ml, with the exception of S-1R and s-1, which were at a concentration of 3 μg/ml On the next day the solution for coating was removed and the plates were blocked with PBS solution (EMD OmniPure 6507)containing 0.05% Tween 20 (Sigma P2287) and 1% BSA (Sigma A9418), shaking using Heildolph Titramax 1000 at 600 rpm for 1 hour at room temperature. The blocking solution was removed before adding the samples in tablets.

Mouse serum and monoclonal antibodies used as standards were serially diluted using 0.5 or 1 log dilution in PBS containing 0.5% Tween 20 (PBS-T). Six or eight dilutions ranging from 1:500, 1:5000 or 1:15800, serum samples were tested for each sample. Monoclonal antibodies used as standards, and positive controls were: anti-Tau 396 (Zymed 35-5300) for A-1, at-180 (Thermo Pierce MN1040) for IN-1R; at-8 (Thermo Pierce MN1020) for D-1P and E-1R; ATA-100 (Thermo Pierce MN1060)-1P. Used concentrations of monoclonal antibodies for the standard curve represented 50, 15,8, 5, 1,58, 0,5, 0,158 and 0.05 ng per well.

Samples and standards were added to the tablets 100 μl per well in duplicate link is H. The plates were incubated for 1 hour at room temperature, shaking at 600 rpm Tablets then washed 3 times with PBS-T and added to the secondary antibody (HRPO-conjugated IgG against mouse, Caltag #M)diluted to 1:3000 in PBS-T at a concentration of 100 μl/well. Different secondary antibodies used for the detection of IgG1(Caltag #M32107 1:2000), IgG2a(Caltag #M32307 1:2000) and IgM (Caltag #31507 1:3000). The secondary antibody was left contact on the tablets for 1 hour at room temperature with shaking. Tablets are again washed 3 times with PBS-T, and tablets blotted dry after the final rinse. To develop in each well was added 100 μl of TMB substrate (3,3',5,5'-tetramethylbenzidine) Buffer EIA (enzyme-linked immunosorbent assay with peroxidase) (Bio-Rad #172-1067) for 11 minutes at room temperature. To terminate the reaction, each well was added 100 μl of 1 N. sulfuric acid. The absorption was read at 450 nm on a Molecular Devices Spectramax plus 384. The threshold value OD was calculated for each plate by taking the average of all wells treated with PBS-T, and adding 3 times the standard deviation of these holes. If the standard deviation cannot be calculated, then the value of the double-PBS-T OD was used as the threshold value. The titer of the sample was determined from the dilution of the first sample with the value of the absorption at 450 nm, greater than the calculated threshold value. For n is which analyses the standard curve, based on the relevant dilutions of positive control monoclonal antibodies were used to calculate the concentration of the caption relative to the standard curve. Is the smallest breeding or testing standard used for calculations when the signal was not determined, and the value of the highest dilution or test standard used when the largest breeding was positive. Average titers were calculated when N was greater than 2, whereas individual values were prodemonstrirovany when N was equal to 1 or 2. The selectivity ratio was determined by dividing the titer of the sample is phosphorylated peptide on the title nefosfaurilirovanna versions of the same peptide for each sample, and then averaging ratio for different samples. Values greater than 10 or less than 0.1 was considered selective. The first positive breeding to determine the selectivity was the most conservative way. The use of other methods, such as threshold OD 1 or premaxilla OD, will give, perhaps, values greater selectivity.

Example 14: ELISPOT analysis of IFN-γ

A set of IFN-γ ELISPOT (BD Biosciences; 551083) was used to measure T-cell responses after immunization with the use of peptide-Qbeta-the VLP. ELISPOT was performed on pooled spleens (N=3) from A-8P,-3P, TO-3P, IS-2P (in the presence of a low dose of alum or without alum)-immunized mice, and unimmunized mice. On 96-well ELISPOT tablets was applied at 5 μg/ml exciting (capture) antibody against mouse IFN-γ during the night at 4°C. antibody-Coated tablets were washed and blocked in complete medium RPMI 1640 (Invitrogen 11875-119)containing 10% fetal bovine serum (VWRA15-204).

The splenocytes are then planted in the tablets are coated with an antibody against IFN-γ, at a concentration of 500,000 splenocytes per well was stimulated by 10 μg/ml peptide or protein antigen for 20-24 hours at 37°C in an incubator with 5% CO2. An unrelated control peptide represented the HBV peptide-1 (SEQ ID NO:77), and bovine serum albumin (Sigma Aldrich; A) was used as an unrelated protein control for Qbeta-the VLP. Stimulation of phorbol-12-myristate-13-acetate (0.5 μg/ml PMA, Sigma Aldrich; P8139) and ionomycin (0.5 μg/ml, Sigma Aldrich; I0634) of spleen cells, seeded at a concentration 55555 and 18520 cells per well, were used as positive controls. After 20-24 hours of incubation ELISPOT tablets twice washed with distilled water followed by three additional washes with wash buffer (1×PBS (Invitrogen 10010072)containing 0.05% Tween-20 (Sigma P2287)). Determination of cytokine IFN-y was carried out by incubating 2 μg/ml biotinylated detection (detection) antibody against IFN-y, diluted in PBS containing 10% FBS for 2 hours at to the room temperature, followed by incubation with 1:100 streptavidin-HRP diluted in PBS with 10% FBS. After washing tablets 4 times with wash buffer and 2 times with PBS, IFN-γ points (spots) were visualized using AEC Chromogen substrate (11-minute incubation at room temperature).

IFN-γ-positive point scanned, recorded and counted using analyzer Cellular ELISpot Technology and software 5.0 Professional Immunospot and medium pulses per well. An unrelated peptide was a negative control for peptide antigens, whereas BSA was a negative control for the VLP unconjugated. To be considered positive, the average value of the spots should be significantly higher (p<0.05)than the relevant negative control using T-student test.

Example 15: Adjuvant medication and immunization

Adjuvants used in the specific examples described in this application (for example, Examples 5-14), was prepared as follows. CpG-24555 was prepared at 2 mg/ml concentrate in water. Used alum was a Alhydrogel "85" (Brenntag Biosector)containing 10 mg/ml of aluminum. Alhydrogel "85" was mixed in a 1:1 ratio with 100 μg of peptide or the VLP conjugated peptide. Usually up to 25 ál (for intramuscular vaccination) or 50 ál (for subcutaneous vaccination) was added to the solution with 100 µg of the VLP and immediately vstra ivali and placed on ice. TiterMax Gold (Alexis Biochemicals) was added at a ratio of 1:1 solutions of peptides. 50 μl TiterMax Gold was added to 50 μl of 2 mg/ml solution of the peptide to 100 μl subcutaneous dose and emulsiable for 10 minutes at 4°C With Mixermill (SPEX model Rger). 25 μl (12 µg) AblSCO-100 (Isconova) was added to 100 g of a solution of the VLP-peptide and 5 μl (10 μg) CpG-24555, were shaken and placed on ice.

Immunization and treatment of animals carried out in the specific examples described in this application (for example, Examples 5-14), was carried out according to conventional methods. For vaccinations, up to 100 μl of vaccine were injected with subcutaneously in the base of the tail or 50 µl were injected with one or both of the rear part of the anterior tibial muscles. The blood collection was performed through the submandibular tube or at the end through the puncture of the heart. The spleen was removed after exsanguination and cervical dislocation and were placed in cold sterile HBBS (Invitrogen, cat. No. 14170) with 5% PBS and Penn/Strep (penicillin/streptomycin) (Invitrogen, cat. No. 15140-122). The spleen was washed on a sieve of 70 μm (Falcon). Cells were washed in ice-cold HBBS and erythrocytes were literally using lyse buffer ASC (Invitrogen). Splenocytes were counted on the Guava PCA 96 (Guava Technologies Inc.).

Example 16: optimizing the density of the conjugation of RTU-peptide with Qbeta/the VLP for the desired immune response

The experiment was performed in order to determine whether the density of the conjugation of RTU-pepti the aqueous epitope with Qbeta/the VLP (number of peptides in Monomeric subunit Qbeta) on RTU-specific or antibody-based test answers. Different combinations obtained by varying the molar excess of SMPH and excess of RTU-peptide was used for the production of 8 conjugates pTay/the VLP with different densities epitopes (table 4). Groups of 5 female mice of BalbC (8 weeks) were immunized at 0 days and 14 days (p/C) with 100 μg of each of the conjugates with different density of 750 μg of alum (Al(OH)3). Serum was collected on the 26th day. Or antibody-based test responses from immunized animals was investigated using analysis determination of antigen-specific titers as described in Example 13.

Based on the results of the titles on the 26th day, shown in Table 4, the density of the conjugation of 2.3 for A-8P/QBeta gave immune response with a higher titer than the form of higher (3,6) density conjugation. For various conjugates IN-3P/Qbeta titers were similar and most forms of density conjugates of 2.2 and 3.6. For C-2P/Qbeta, density conjugation epitopes of 2.2 and 3.5 gave similar titles, which were slightly higher than the uniform density conjugation of 4.3. The results show that the density of conjugation epitopes may affect or antibody-based test answers antigen-specific manner and that in General terms the combination, resulting in a density of conjugating RTU-peptide epitopes 2-3 on the monomer Qbeta, are preferred.

In the following table, and as indicated earlier in this application, phosphorylated amino acids are indicated in bold and underlined.

Table 5:
Brief description perednia sequences
SEQ NO:DESCRIPTIONSEQUENCE
1pThr-231/pSer-235 phospho-Tau epitopeTPPKS
2Alternative pThr-231/pSer-235 phospho-Tau epitopePPKS
3pSer-202/pThr-205 phospho-Tau epitopeSPGT
4Peptide A-1R without linkerEIVYKSPWSGDTSPRHLS
5Peptide A-2P without linkerRENAKAKTDHGAEIVYKSPWSGDTSPRHLS
6Peptide A-3P without linkerEIVYKSPWS
7Peptide A-4P without the of incera GDTSPRH
8Peptide A-5P without linkerKSPWSGDTSP
9Peptide A-6R without linkerEIVYKSP
10Peptide A-7Đ without linkerIVYKSPV

SEQ NO:DESCRIPTIONSEQUENCE
11Peptide A-8P without linkerVYKSPW
12Peptide A-9R without linkerYKSPWS
13Peptide A-10P without linkerKSPWSG
14The peptide-1R without linkerKVAVVRTPPKSPSSAKS
15Peptide-2P without linkerVRTPPKSPS
16The peptide-3P without linker VVRTPPKSP
17Peptide-4P without linkerRTPPKSPSS
18Peptide-5P without linkerRTPPKSP
19The peptide-6R without linkerPPKSPSS
20Peptide WITH-1R without linkerSRSRTPSLPTPPT
21The peptide C-2P without linkerSRTPSLP
22The peptide C-3P without linkerRTPSLPT
23The peptide C-4P without linkerRSRTPSL
24Peptide-5P without linkerPGSRSRTPSLP
25Peptide D-1P without linkerGYSSPGSPGTPGSRS
26Peptide F-1R without linkerGYSSPGSPGTPGSRSRTPSLPTPPT
27CpG 79095' TCGTCGTTTTGTCGTTTTGTCGTT 3'
28CpG 101035' TCGTCGTTTTTCGGTCGTTTT 3'
29CpG 245555' TCGTCGTTTTTCGGIGCTTTT 3'
30Tau isoform 2 personGenbank no access NP005901
31Peptide A-1R linkerCGGEIVYKSPWSGDTSPRHLS
32Peptide A-2P linkerCGGRENAKAKTDHGAEIVYKSPVVSGDTSPRHLS
33Peptide A-3P linkerCGGEIVYKSPWS
34Peptide A-4P linkerCGGGDTSPRH
35Peptide A-5P linkerCGGKSPWSGDTSP
36Peptide A-6 linkerCGGEIVYKSP
37Peptide A-7Đ with the linker is m CGGIVYKSPV
38Peptide A-8P linkerCGGVYKSPVV
39Peptide A-9R linkerCGGYKSPVVS
40Peptide A-10P linkerCGGKSPVVSG

SEQ NO:DESCRIPTIONSEQUENCE
41Peptide A-R linkerEIVYKSPWSGDTSPRHLSGGC
42The peptide-1R linkerCGGKVAWRTPPKSPSSAKS
43Peptide-2P linkerCGGVRTPPKSPS
44The peptide-3P linkerCGGWRTPPKSP
45Peptide-4P linkerCGGRTPPKSPSS
46Peptide-5P linker/td> CGGRTPPKSP
47The peptide-6R linkerCGGPPKSPSS
48Peptide WITH-1R linkerCGGSRSRTPSLPTPPT
49The peptide C-2P linkerCGGSRTPSLP
50The peptide C-3P linkerCGGRTPSLPT
51The peptide C-4P linkerCGGRSRTPSL
52Peptide-5P linkerCGGPGSRSRTPSLP
53Peptide D-1P linkerCGGYSSPGSPGTPGSRS
54Peptide F-1R linkerCGGYSSPGSPGTPGSRSRTPSLPTPPT
55Peptide a-1 linker - CGGEIVYKSPWSGDTSPRHLS
56Peptide a-2 linker CGGRENAKAKTDHGAEIVYKSPVVSGDTSPRHLS
57Peptide a-3 linkerCGGEIVYKSPVVS
58Peptide a-4 linkerCGGGDTSPRH
59Peptide A-5 linker - CGGKSPVVSGDTSP
60Peptide A-6 linkerCGGEIVYKSP
61Peptide a-7 linkerCGGIVYKSPV
62Peptide a-8 linkerCGGVYKSPVV
63Peptide A-9 linkerCGGYKSPVVS
64Peptide A-10 linker - CGGKSPVVSG
65Peptide-1 linker - CGGKVAVVRTPPKSPSSAKS
66Peptide-2 linkerCGGVRTPPKSPS
67Peptide b-3 linkerCGGVVRTPPKSP
68Peptide b-4 linkerCGGRTPPKSPSS
68Peptide B-5 linker - CGGRTPPKSP
69Peptide-6 linkerCGGPPKSPSS

SEQ NO:DESCRIPTIONSEQUENCE
70Peptide-1 linker - CGGSRSRTPSLPTPPT
71The peptide C-2 linkerCGGSRTPSLP
72The peptide C-3 linkerCGGRTPSLPT
73The peptide C-4 linkerCGGRSRTPSL
74The peptide C-5 linker - CGGPGSRSRTPSLP
75Peptide D-1 linker - CGGYSSPGSPGTPGSRS
76Peptide E-1 linker CGGYSSPGSPGTPGSRSRTPSLPTPPT
77The HBV peptide-1IPQSLDSWWTSL
78C' -sequence Qbeta containing Xhol site5'-GTATTAATGACTCGAG-3'
79LinkerGGGGGC
80LinkerGGGGC
81LinkerGGGC
82LinkerGGGGGK
83LinkerGGGGK
84LinkerGGGK
85LinkerGGGGSC
86LinkerGGGSC
87LinkerGGSC
88LinkerCSGGGG
89LinkerCSGGG
90LinkerCSGG
91LinkerCGGGG
92LinkerCGGG
93LinkerCGGGGG
94LinkerCGDKTHTSPP
95LinkerDKTHTSPPCG
96LinkerCGGPKPSTPPGSSGGAP
97LinkerPKPSTPPGSSGGAPGGCG
98LinkerGCGGGG
99LinkerGGGGCG

SEQ NO:DESCRIPTIONSEQUENCE
100 LinkerCGKKGG
101LinkerCGDEGG
102LinkerGGKKGC
103LinkerGGEDGC
104LinkerGGCG
105Peptide F-1P without linkerAGTYGLG
106Peptide F-1P linkerCGGAGTYGLG
107Peptide F-1 linker - CGGAGTYGLG
108Peptide F-2P without linkerDHAGTYG
109Peptide F-3P without linkerHAGTYGL
110Peptide F-4P without linkerGTYGLGD
111Peptide F-5P without linkerTYGLGDR
Peptide F-6P without linkerDHAGTYGLGDR
113Peptide F-2P linkerCGGDHAGTYG
114Peptide F-3P linkerCGGHAGTYGL
115Peptide F-4P linkerCGGGTYGLGD
116Peptide F-5P linkerCGGTYGLGDR
117Peptide F-6P linkerCGGDHAGTYGLGDR
118Peptide F-2 linkerCGGDHAGTYG
119Peptide F-3 linkerCGGHAGTYGL
120Peptide F-4 linkerCGGGTYGLGD
121Peptide F-5 linker - CGGTYGLGDR
122Peptide F-6 linkerCGGDHAGTYGLGDR

1. Immunogen having the ability to induce an immune response against autoantigens Tau and containing antigenic Tau peptide associated with immunogenic carrier, where the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID NO:6, 8-19, 21-26, 105, 108-112, and where specified antigenic Tau peptide covalently associated with the specified immunogenic carrier via a linker represented by the formula (G)nC, where specified, the linker is either on the C-end (peptide-(G)nC)or N-(C(G)n-peptide) of the peptide, and where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

2. The immunogen according to claim 1, where the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID NO:6 and 8-13.

3. The immunogen according to claim 2, where the specified antigenic Tau peptide consists of the amino acid sequence represented in SEQ ID NO:11.

4. The immunogen according to claim 1, where the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID NO:14-19.

5. The immunogen according to claim 4, where the specified antigenic Tau peptide consists of the amino acid sequence represented in SEQ ID NO:16.

6. The immunogen according to claim 1, where the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID nos:21-24.

7. The immunogen according to claim 6, where the specified antigenic Tau-p is ptid consists of the amino acid sequence, presented in SEQ ID NO:21.

8. The immunogen according to claim 1, where the specified antigenic Tau peptide consists of an amino acid sequence selected from SEQ ID NO:105 and 108 to 112.

9. The immunogen of claim 8, where the specified antigenic Tau peptide consists of the amino acid sequence represented in SEQ ID NO:105.

10. The immunogen according to any one of claims 1 to 9, where specified immunogenic carrier is a virus-like particle selected from the group consisting of HBcAg the VLP, the VLP HBsAg and Qbeta the VLP.

11. The composition having the ability to induce an immune response against autoantigens Tau and containing at least two of the immunogen, each of which contains an antigenic Tau peptide associated with immunogenic carrier, where the specified antigenic Tau peptide covalently associated with the specified immunogenic carrier via a linker represented by the formula (G)nC, where specified, the linker is either C-end (peptide-(G)nC)or N-(C(G)n-peptide) of the peptide, and where n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10,
where this song contains one of the following combinations:
(1) (a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:6 and 8-13; and
b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID NO:14-19; or
(2) (a)antigenic Tau peptide of the first immunogen consists of an amino acid sequence, selected from SEQ ID NO:6 and 8-13; and
b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID nos:21-24; or
(3) (a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:14-19; and
b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID nos:21-24; or
(4) (a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:6 and 8-13; and
b) antigenic Tau peptide of the second immunogen selected from SEQ ID NO:105 and 108 to 112; or
(5) (a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO: 14-19; and
b) antigenic Tau peptide of the second immunogen selected from SEQ ID NO:105 and 108 to 112; or
(6) (a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID nos:21-24; and
b) antigenic Tau peptide of the second immunogen selected from SEQ ID NO:105 and 108 to 112; or
(7) (a) antigenic Tau peptide of the first immunogen consists of an amino acid sequence selected from SEQ ID NO:6 and 8-13;
b) antigenic Tau peptide of the second immunogen consists of an amino acid sequence selected from SEQ ID NO:14-19; and
C) antigenic Tau peptide of the third immunogen consists of an amino acid sequence which, selected from SEQ ID nos:21-24;
d) antigenic Tau peptide of the fourth immunogen consists of an amino acid sequence selected from SEQ ID NO:105 and 108 to 112.

12. Pharmaceutical composition for treating Tau-associated neurological disorders, containing the immunogen according to any one of claims 1 to 10 or a composition according to claim 11 and a pharmaceutically acceptable excipient.



 

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7 cl, 9 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to muteins of human tear lipocalin, and can be used in medicine. Mutein of human tear lipocalin (hTLc) has identifiable affinity of binding with human receptor Met (c-Met) receptor tyrosine kinase, or its domain, or fragment of human c-Met. Mutein contains from 6 to 18 amino acid substitutions relative to amino acid sequence of mature lipocalin of human tear liquid (SWISSPROT DATABANK ENTRY P31025; SEQ ID NO:36), selected from group, consisting of Arg 26→Thr, Val, Pro, Ser, Gly; Glu 27→Gln, Gly, Val, Ser; Phe 28→Met, Asp; Pro 29→Leu, Ile, Ala, Trp; Glu 30→Leu, Gly, Arg, Phe; Met 31→Ser; Asn 32→Leu, Arg, Val, Gln; Leu 33→Tyr, Val, Ile, Thr, Phe; Glu 34→Val, Arg, Ala; Leu 56→Asn; Ile 57→Gln; Ser 58→Ile, Val; Asp 80→Tyr; Lys 83→Ala; Glu 104→Asp; Leu 105→Thr; His 106→Trp and Lys 108→Gly. Mutein can also additionally contain the following substitutions: Cys 61→Ser; Cys 101→Ser; Cys 153→Ser; Arg 111→Pro; Lys 114→Trp; Thr 37→Ser; Met 39→Ile, Leu; Asn 48→Ser; Lys 52→Thr, Met; Met 55→Leu; Lys 65→Arg, Leu; Ala 79→Leu, Ser; Ala 86→Thr; Ile 89→Ser, Gln, Thr, His; Thr 40→Cys; Glu 73→Cys; Arg 90→Cys; Asp 95→Cys; Lys 121→Cys; Asn 123→Cys and Glu 131→Cys.

EFFECT: invention makes it possible to efficiently treat pathological disorders, which involve pathway HGF/c-Met, as well as to perform identification of human c-Met in sample.

40 cl, 16 dwg, 9 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is disclosed is a vaccine representing four RNAs coding a prostate-specific antigen (PSA), a prostate-specific membrane antigen (PSMA), a prostate stem cell antigen (PSCA) and a six-transmembrane epithelial antigen of the prostate (STEAP). The vaccine is applicable for treating prostate carcinoma, preferentially neo-adjuvant and/or hormone resistant prostate carcinoma, as well as related diseases or disorders. Using the vaccine and a kit are also disclosed. The invention can be used in medicine.

EFFECT: preparing the vaccine for treating prostate carcinoma.

16 cl, 23 dwg, 8 ex

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

7 cl, 13 dwg, 7 ex

FIELD: biotechnologies.

SUBSTANCE: invention refers to conjugate antibody-medical preparations containing charged linkers, stitching reagents, pharmaceutical compositions including conjugates antibody-medical preparations.

EFFECT: improvement of reagent properties.

51 cl, 72 dwg, 3 tbl, 8 ex

FIELD: biotechnologies.

SUBSTANCE: invention represents a method for obtaining recombinant DNAse I of a human or its mutein, as well as their conjugates with polyethylene glycol, using a bacterium belonging to Escherichia class, transformed with expression plasmid, containing a promoter functioning in a bacterial cell, DNA fragment coding a hexahistidine cluster, a fragment coding enterokinase recognition sequence amalgamated in frame with human DNAse I or its functionally active mutein containing replacements of asparagine with cysteine, transcription termination section, vector pET28a(+) fragment containing initiation section of replication of bacteriophage fl, sequence coding aminoglycoside-3'-phosphotransferase, area of beginning of plasmid pBR322 replication, gene RNA-organising protein Rop, sequence coding lactose operon repressor.

EFFECT: invention allows obtaining recombinant human DNAse I or its mutein with high yield.

18 cl, 7 dwg, 1 tbl, 12 ex

Vns-met-histones // 2498997

FIELD: biotechnologies.

SUBSTANCE: nucleic acid molecule codes a polypeptide consisting of two residues of methionine as the first and the second N-end amino-acid residues connected through a peptide link to a mature eucariotic histone. Polypeptide is obtained by cultivation of a host cell transformed by an expression vector including the above molecule of nucleic acid. Polypeptide is used as part of pharmaceutical composition for therapy of cancer, bacterial, virus or fusarium infections. Besides, polypeptide is used as part of composition for diagnostics of a patient in relation to response to pharmaceutical composition containing the above polypeptide, or in relation to curability using it.

EFFECT: invention allows improving efficiency of recombinant expression and simplifying determination of the above polypeptide in presence of endogenic histones at preservation of biologic activity of mature eucariotic histone.

17 cl, 3 dwg, 6 tbl, 7 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology, and represents a method of producing a recombinant mutein [C112S] of human enterokinase light chain, using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a mutein [C112S] inactive precursor of human enterokinase light chain, comprising a sequence encoding a cleavable N-terminal peptide comprising a hexahistidine cluster and the enterokinase recognition sequence, and a sequence fused with it in frame encoding a precursor of mutein [C112S] of human enterokinase light chain with non-cleavable C-terminal hexahistidine cluster under control of a promoter operating in the bacterial cell.

EFFECT: invention enables to produce the recombinant mutein of human enterokinase light chain with a high yield.

11 cl, 5 dwg, 1 tbl, 6 ex

FIELD: biotechnology.

SUBSTANCE: invention represents a method of production of precursor of a recombinant fragment of human tissue plasminogen activator (tPA) (reteplase) using a bacterium belonging to the genus Escherichia, transformed with an expression plasmid containing a DNA fragment encoding a precursor of the recombinant fragment of human tissue plasminogen activator (tPA) (reteplase), comprising a sequence encoding a cleavable N-terminal peptide comprising decahistidine cluster and sequence of enterokinase recognition fused in frame, or a fragment of DNA encoding the [-1] methionyl of fragment of human tPA (reteplase), under the control of a promoter operating in the said bacterial cell.

EFFECT: invention enables to obtain a recombinant fragment of human tissue plasminogen activator with a high yield.

10 cl, 6 dwg, 1 tbl, 8 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to field of biochemistry. Claimed is method of separation and purification of target protein by chromatography, in which chromatography removes or reduces content of prions (PrPSc). Realised is interaction of potentially PrPsc-contaminated sample, which contains target protein, with combined chromatographic material, which contains ligand. Ligand is selected from: positively charged N-benzyl-N-methylethanolamine ligand; negatively charged ligand of 2-(benzoylamino)butamoic acid; phenylpropyl ligand; N-hexyl ligand; 4-mercaptoethylpyridine ligand; ligand of 3-[{3-methyl-5-((tetra hydrofuran-2-ylmethyl)amino)-phenyl}amino]benzoic acid. Than buffer conditions in chromatographic conditions are created, such as, target protein binds with combined chromatographic material, and PrPsc does not bind with combined chromatographic material. Then target protein is eluted. Also claimed is fraction of pharmaceutically applicable target protein with reduced content of prion protein.

EFFECT: invention makes it possible to obtain fraction of target protein with reduced content of PrPsc from >1 to 4 lg(10) by application of single chromatographic resin.

9 cl, 3 tbl, 2 ex

FIELD: chemistry.

SUBSTANCE: described are novel 2-pyridyl-substituted imidazoles of general formula (I) , where Ra is C1-6alkyl; m = 1; A1 = N; A2 = NR1, where R1 is hydrogen; X is bond, -NR2-, -O- or -S-, where R2 is hydrogen or C1-3alkyl; Rb independently is H, halogen, C1-6alkyl, C2-6alkenyl, C2-6alkinyl, -(CH2)q-OR3, where R3 - C1-6alkyl or C1-6halogene alkyl, and q=0,1, -(CH2)q-NR3R4, where R3 and R4 independently are C1-6alkyl or together with nitrogen atom - pyrrolidinyl or morpholinyl, and q=0-2; -SR3, where R3 - C1-6alkyl, -(CH2)q-CN, where q=0 or 1, -COR3 or -CO2R3, where R3 is C1-6alkyl, -CONR3R4, where R3 and R4 is hydrogen, -NHCOR3 or -NHSO2R3, where R3 stands for C1-6alkyl; n equals 0, 1, 2, 3, 4 or 5; or their pharmaceutically acceptable salts, or hydrates and pharmaceutically acceptable compositions for treatment or abatement of metastasis of tumour cells, carcinomas, fibrosis by inhibiting pathways of transmission of signal of TGF-β or activin, or both.

EFFECT: improvement of composition properties.

6 cl, 6 tbl, 7 ex, 33 dwg

FIELD: chemistry.

SUBSTANCE: invention relates to a compound of general formula I , where R1 is a hydrogen atom, a lower alkyl, CD3, -(CH2)n-CHO, -(CH2)n-O-lower alkyl, -(CH2)n-OH, -(CH2)n-cycloalkyl or is a heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with O); R2 is a hydrogen atom, a halogen atom, hydroxy, lower alkyl, di-lower alkyl, -OCH2-O-lower alkyl or lower alkoxy; or the piperidine ring along with R2 forms a spiro-ring selected from 4-aza-spiro[2,5]oct-6-yl; Ar is an aryl or heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N), optionally having one, two or three substitutes selected from a halogen atom, lower alkyl, lower alkyl having as substitutes, a halogen atom, a lower alkoxy having as substitutes, a halogen atom, cycloalkyl, lower alkoxy, S-lower alkyl, heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with N), or optionally having as substitutes, phenyl, optionally having R' as substitutes, and R' is a halogen atom, CF3, lower alkyl, lower alkoxy or a lower alkoxy having as substitutes, a halogen atom, or is a heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N and S); R is a lower alkyl, heterocycloalkyl (where the heterocycloalkyl is a partially unsaturated ring containing up to 6 carbon atoms, at least one of which is substituted with O), aryl or heteroaryl (where the heteroaryl is a cyclic aromatic hydrocarbon radical consisting of one ring and containing 6 ring atoms, and which contains at least one heteroatom selected from N), Where the aryl and heteroaryl optionally have as substitutes, one or two R'; n equals 0, 1, 2 or 3; or to a pharmaceutically acceptable acid addition salt, a racemic mixture or a corresponding enantiomer and/or optical isomer of said compound. The invention also relates to pharmaceutical compositions based on a glycine reuptake inhibitor of a compound of formula I.

EFFECT: obtaining novel compounds and a pharmaceutical composition based thereon, which can be used in medicine to treat neurological and psychoneurological disorders.

22 cl, 1 tbl, 128 ex

FIELD: medicine.

SUBSTANCE: what is presented is using hypaconitine (a heterocyclic nitrogen organic compound recovered from plants of the genus Aconitum of the family Ranunculaceae with the known analgesic activity) as a cerebroprotective agent. The mode of action of the agent is activation of cerebral neural stem cells.

EFFECT: prevented animal death following suffered hypoxia.

3 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to pharmaceutical industry and represents medication, which has a nootropic action on an organism based on cyclic dipeptides, which contains a propyl-alanine sequence.

EFFECT: invention ensures extension of arsenal of medication with nootropic activity.

2 cl, 2 ex, 2 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to novel compounds of formula (I) and pharmaceutically acceptable salts thereof, having the capacity to bind amyloid peptides and/or amyloids. In formula (I), EDG is an electron donor group, πCE is a pi-coupling element and WSG is a water-soluble group. EDG is -NR4R5, where R4 and R5 independently denote hydrogen, R12-substituted or unsubstituted C1-C10alkyl, where R4 and R5 are optionally linked to form a R12-substituted or unsubstituted 6-member heterocycloalkyl, where said 6-member heterocycloalkyl optionally contains a heteroatom selected from O or N, R12 is an unsubstituted C1-C10alkyl or unsubstituted 6-member heterocycloalkyl, where said 6-member heterocycloalkyl contains a heteroatom selected from O or N. The pi-coupling element has the formula -L1-(A1)q-L2-(A2)r-L3-, in which q and r are independently equal to 0 or 1 and at least one of q and r is equal to 1, A1 and A2 denote an unsubstituted group , L1, L2 and L3 independently denote a bond or a binding group of formula , where x is an integer from 1 to 50. The water-soluble group is a R25-substituted C1-C5alkyl or group, where R25 is -OR26 or a R29-substituted 5-member heterocycloalkyl containing two O atoms, R26 is hydrogen, R29 is an unsubstituted C1-C10alkyl and y is an integer from 1 to 50.

EFFECT: invention relates to specific compounds of formula (I), pharmaceutical compositions and a method of detecting an amyloid peptide.

14 cl, 11 dwg, 5 tbl, 11 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to paediatrics and neonatology, and may be used for prevention of a developing perinatal CNS damage of the hypoxic-ischemic genesis in infants of a risk group. That is ensured by evaluating birth-time umbilical blood neuron-specific enolase in an infant of a high perinatal risk group of the developing perinatal CNS damage of the hypoxic-ischemic genesis; if the derived value is more than 10 mcg/l, L-carnitine 2 drops 2 times a day and glycine 1/4 of a tablet 2 times a day are administered orally in the early neonatal period for the first month of life.

EFFECT: method enables reducing the delayed manifestations of the CNS damage of the hypoxic-ischemic genesis in the infants of the risk group by conducting a preventive treatment in the early neonatal period.

4 ex

FIELD: teaching.

SUBSTANCE: learners are affected by electromagnetic radiation of extremely high frequency with the modulations of frequency range from 43 GHz to 44 GHz. Simultaneously with the electromagnetic radiation 0.2 g of caffeine is administered. This combined effect is carried out for 20 minutes in the morning hours daily as a course of 5 procedures.

EFFECT: effective recovery of mental capacity, long-lasting maintenance of the results achieved, easy in performing.

1 tbl

FIELD: medicine, pharmaceutics.

SUBSTANCE: there are described new compounds of general formula [I]: or their pharmacologically acceptable salts, wherein R1 is C1-6 alkyl; R2 is C1-6 alkoxy; m and n mean 1; W means N; the ring A represents fragments of formula , or that can be substituted; X1 is a single bond, C1-6 alkylene group or -C(O)NR3-, wherein R3 is hydrogen, C1-6 alkyl or phenyl; and the ring B represents fragments of formula [5]-[11]: that can be substituted, and a pharmaceutical composition containing them.

EFFECT: new compounds possess activity inhibiting the amyloid beta production, and are effective as a therapeutic agent for treating an Aβ-caused disease, such as Alzheimer disease or Down syndrome.

10 cl, 48 tbl, 399 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to compounds possessing a high effectiveness in modulation of NMDA receptor activity.

EFFECT: compounds are applicable in treating the diseases and disorders, such as disturbed learning, cognitive activities, as well as for relieving and/or reducing neuropathic pain.

26 cl, 21 dwg, 2 tbl, 9 ex

FIELD: chemistry.

SUBSTANCE: invention relates to 2-amino-5,5-difluoro-5,6-dihydro-4H-[1,3]oxazin-4-yl)-phenyl]-amide derivatives of formula I, having BACE1 and/or BACE2 inhibiting activity, pharmaceutical compositions based thereon and use thereof as therapeutically active substances. In general formula

,

R1 is selected from a group consisting of i) a 5-6-member heteroaryl containing 1-2 heteroatoms selected from nitrogen and oxygen, and ii) 5-6-member heteroaryl containing 1-2 heteroatoms selected from nitrogen and oxygen, substituted with 1-4 substitutes, individually selected from amide, cyano, cycloalkyl, cycloalkyl-lower alkynyl, cycloalkyl-lower alkyl, cycloalkyl-lower alkoxy, halogen atom, lower haloalkoxy, lower haloalkyl, lower alkyl and nitro; iii) lower alkyl; iv) lower alkyl substituted with 1-5 substitutes individually selected from a halogena tom and hydroxy; v) lower alkenyl substituted with furanyl; vi) cycloalkyl substituted with 1-4 substitutes individually selected from a halogen atom, lower haloalkyl; R2 is selected from a group consisting of i) hydrogen atom, ii) halogen atom, R3 is a lower alkyl; R4 is selected from a group consisting of i) a hydrogen atom, and ii) lower alkyl; R5 is selected from a group consisting of i) a hydrogen atom, and ii) lower alkyl; n equals 0, 1 or 2.

EFFECT: active compounds in the present invention are useful in therapeutic and/or preventive treatment, for example, of Alzheimer's disease and type 2 diabetes.

29 cl, 10 tbl, 83 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention discloses an immunogenic composition having an immunogenic activity on serogroup B and C Neisseria meningitidis containing (a) N. meningitidis serogroup C (NmC) oligosaccharide, (b) proteoliposome vesicles of the outer membrane of N.meningitidis (NmB) serogroup B and (c) NmB protein containing and an amino acid sequence presented in the description, or an immunogenic fragment of the above sequence, or a sequence min. 80% identical to the above sequence. The ingredient (a) may be conjugated with a carrier, e.g. with a protein, CRM197, a diphtherial anatoxin or a tetanus anatoxin. The immunogenic composition can contain aluminium hydroxide or MF59 as an adjuvant. What is described is a N.meningitidis serogroup B and C vaccine containing the described immunogenic composition.

EFFECT: using the invention enables preparing the combined vaccine eliciting an immune response on both serogroups of the agent.

6 cl, 4 dwg, 5 tbl, 6 ex

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