EA044254B1 - COMPOSITIONS OF PHOSPHORYLATED TAU PEPTIDES AND THEIR APPLICATIONS - Google Patents

Description

Field of technology to which the invention relates

The invention relates to the field of medicine. In particular, the present invention relates to liposomes or tau peptide conjugates and their use for the prevention or treatment of tauopathy such as Alzheimer's disease.

Prerequisites for creating the invention

Alzheimer's disease (AD) is a progressive, debilitating neurodegenerative disease that affects approximately 44 million people worldwide (Alzheimers.net). Treatments for AD that are currently available in the clinic aim to slow the progression of clinical symptoms but do not target the pathogenic processes underlying the disease. Unfortunately, these treatments are only minimally effective, and there is therefore an urgent need to develop and test additional preventive and therapeutic measures.

The hallmarks of Alzheimer's disease pathology are the accumulation of extracellular plaques containing aggregated amyloid-beta protein and intracellular tangles or aggregates of hyperphosphorylated tau protein. The molecular events that lead to the accumulation of these proteins are poorly characterized. In the case of amyloid, aberrant cleavage of the amyloid precursor protein is thought to result in the accumulation of an aggregation-prone fragment comprising amino acids 1-42. For tau, dysregulation of either kinases, phosphatases, or both is hypothesized to result in aberrant phosphorylation of tau. Once tau becomes hyperphosphorylated, it loses its ability to effectively bind and stabilize microtubules and instead accumulates in the cytoplasm of the affected neuron. Unbound and hyperphosphorylated tau appears to form first oligomers and then higher order aggregates, the presence of which is thought to negatively affect the functioning of the neuron in which they form, possibly by interrupting normal axonal transport.

In developed countries, individuals diagnosed with Alzheimer's disease or other dementia-causing tauopathies are typically treated with cholinesterase inhibitors (eg, Aricept®) or memantines (eg, Namenda™). These drugs, although quite well tolerated, have very low effectiveness. For example, Aricept® delays the increase in symptom intensity by 6-12 months in approximately 50% of treated individuals. The rest of the treatment is non-pharmacological and aims to make patients more able to perform everyday tasks as their cognitive abilities decline.

Several published studies (Asuni AA et al., J Neurosci. 2007 Aug 22; 27(34):911529., Theunis S et al., PLoS One. 2013; 8(8): e72301., Kontsekova E et al. , Alzheimers Res Ther. 2014 Aug 1;6(4):44) demonstrates that active vaccines containing tau peptides can induce anti-tau immune responses in mice or rats; reduce the accumulation of pathological tau aggregates in the brain of rodents; and reduce the rate of progression of cognitive decline in animal models of Alzheimer's disease. An active vaccine against pathological tau proteins has been shown to be immunogenic in human patients with Alzheimer's disease (Novak P et al., Lancet Neurology 2017, 16:123-134). WO 2010/115843 describes an antigenic phosphopeptide mimicking the major pathological phosphoepitope of the tau protein and related compositions for therapeutic and diagnostic use in the treatment of tauopathies, including Alzheimer's disease. However, currently there are still no approved effective vaccines on the market to prevent tau-mediated disease. There are also no effective medications on the market to stop or slow down the disease once it begins. Therefore, there is an urgent need to identify new preventive measures (eg, vaccines) that can prevent these diseases.

Summary of the Present Invention

In one general aspect, the present invention provides a liposome comprising:

a) tau peptide, preferably the tau peptide is a tau phosphopeptide; And

b) an epitope for helper T cells, the tau peptide being present on the surface of the liposome.

In one embodiment, the liposome further contains at least one adjuvant containing a toll-like receptor ligand. Preferably, the liposome further contains at least one of toll-like receptor ligand 4 and toll-like receptor ligand 9.

In a preferred embodiment, the present invention relates to a liposome containing:

a) tau peptide, preferably the tau peptide is a tau phosphopeptide;

b) epitope for T helper cells; And

c) at least one of

i) a toll-like receptor 9 ligand, preferably a lipidated CpG oligonucleotide; and ii) a toll-like receptor 4 ligand, preferably a toll-like receptor 4 agonist,

- 1 044254 than the tau peptide is presented on the surface of the liposome.

In a further preferred embodiment, the present invention relates to a liposome containing:

a) tau phosphopeptide;

b) epitope for T helper cells;

c) lipidated CpG oligonucleotide; And

d) an adjuvant containing a toll-like receptor 4 ligand; wherein the tau phosphopeptide is presented on the surface of the liposome.

In another general aspect, the present invention provides a conjugate comprising a tau peptide, preferably a tau phosphopeptide, and an immunogenic carrier conjugated thereto, wherein the tau peptide is conjugated to the carrier via a linker. The linker may include one or more of polyethylene glycol (PEG), succinimidyl-3-(bromoacetamido)propionate (SBAP), and m-maleimidobenzoyl-Nhydroxysuccinimide ester (MBS). Examples of an immunogenic carrier useful for the present invention include, but are not limited to, keyhole limpet hemocyanin (KLH), tetanus toxoid (TT), CRM197, and a mixture of outer membrane proteins from N. meningitidis (OMP) or a derivative thereof.

In one preferred embodiment, the present invention relates to a conjugate having the structure of formula (I):

or the structure of formula (II):

where x is an integer from 0 to 10, preferably from 2 to 6, most preferably 3; and n is an integer from 2 to 11, preferably from 3 to 11.

Aspects of the present invention relate to a pharmaceutical composition comprising a liposome or conjugate of the present invention and a pharmaceutically acceptable carrier, methods for preparing the pharmaceutical composition, and use of the pharmaceutical composition for inducing an immune response against tau or for treating or preventing a neurodegenerative disease or disorder in a subject in need thereof. .

In one embodiment, the present invention provides a method for inducing an immune response in a subject suffering from a neurodegenerative disorder, or for treating or preventing a neurodegenerative disease or disorder in a subject in need thereof. The method includes administering to a subject a pharmaceutical composition containing a liposome of the present invention and a pharmaceutically acceptable carrier, or a pharmaceutical composition containing a conjugate of the present invention and a pharmaceutically acceptable carrier. Preferably, the method includes administering to the subject a pharmaceutical composition of the present invention for primary immunization and a pharmaceutical composition of the present invention for repeated immunization (booster immunization).

Other aspects, features and advantages of the present invention will be better understood after reading the following detailed description of the invention and the claims.

Brief description of drawings

The foregoing summary, as well as the following detailed description of preferred embodiments of the present invention, will be better understood when read in conjunction with the accompanying drawings. However, it should be understood that the invention is not limited to the exact embodiments shown in the drawings.

In fig. 1 illustrates new vaccines in accordance with embodiments of the present invention: Tau-containing liposome in accordance with an embodiment of the present

- 2044254 invention (top) and a tau conjugate in accordance with an embodiment of the present invention (bottom).

Fig. 2 illustrates that a vaccine containing a liposome in accordance with an embodiment of the present invention (2nd generation liposome) that contains an encapsulated T helper epitope (eg, tetanus polypeptide (tet)) activates T helper cells.

Fig. 3 illustrates that a vaccine containing a conjugate in accordance with an embodiment of the present invention that contains a non-self or immunogenic carrier protein activates T helper cells.

In fig. 4 shows that tau vaccines in accordance with embodiments of the present invention induce a sustained high titer of antibodies against phosphorylated tau in rhesus monkeys: the geometric mean of the titration endpoints for each group, determined by enzyme-linked immunosorbent assay (ELISA) over time, higher in the case of a vaccine containing a liposome (Liposome Z) in accordance with an embodiment of the present invention, or in the case of a vaccine containing a conjugate (Conjugate A) in accordance with an embodiment of the present invention, compared to a control vaccine containing a liposome without the epitope for T-helper cells.

In fig. 5 shows that serum from rhesus macaques immunized with a liposome (Liposome Z) in accordance with an embodiment of the present invention binds to pathological tau structures in brain sections from a person with Alzheimer's disease (AD) (left panel) compared to healthy brain sections person (right panel).

In fig. 6 shows that serum from rhesus macaques immunized with a conjugate (Conjugate A) in accordance with an embodiment of the present invention contained in a composition containing soluble CpG and aluminum hydroxide binds to pathological tau structures in human AD brain sections (top row) compared to brain slices from a healthy person (bottom row).

In fig. 7 shows antibody titers against phosphorylated tau in rhesus monkeys induced by liposome-based vaccines in accordance with embodiments of the present invention, liposomes X, Y, and Z, each containing an encapsulated T-cell-recognized T50 epitope and one or more adjuvants; titers were measured by ELISA and reported as titration endpoints over time in individual monkeys. In particular:

in fig. 7A shows antibody titers against phosphorylated tau induced by liposome X with TLR4 ligand, only MPLA (3D-6-acyl)PHAD®) as an adjuvant;

in fig. 7B shows antibody titers against phosphorylated tau induced by liposome Y with only TLR9 ligand (lipidated CpG oligonucleotide) as adjuvant;

in fig. 7C shows antibody titers against phosphorylated tau induced by liposome Z with a combination of TLR4 ligand, MPLA (3D-(6-acyl)PHAD®) and TLR9 ligand (lipidated CpGooligonucleotide) as adjuvants, which also shows that the combination of the two adjuvants causes less variability antibody titers among individual monkeys;

in fig. 7D presents the geometric mean of the antibody titers of the above immunization groups and a control liposome-based vaccine with the TLR4 ligand, MPLA, but without the T-cell recognized epitope, and shows that vaccines in accordance with embodiments of the present invention result in higher antibody titers against phosphorylated tau versus liposome-based control vaccine: titers were measured by ELISA and presented as geometric mean ±95% confidence interval of titration endpoints for each group over time.

In fig. 8 shows that immunization with a liposome-based vaccine (e.g., Liposome X, Y, or Z) or a conjugate-based vaccine (Conjugate A) in accordance with an embodiment of the present invention induces IgG antibody titers specific for the enriched paired helical strands ( ePHF) isolated from postmortem brains of patients with Alzheimer's disease: antibody titers were measured using Meso Scale Discovery (MSD) technology and presented as individual monkey values at day 50 and geometric mean ±95% confidence interval (CI) after the first immunization.

In fig. 9 shows that immunization with a liposome-based vaccine according to an embodiment of the present invention (Liposome Z) containing encapsulated T50 and a combination of TLR4 ligand (3D-(6-acyl)PHAD®) and TLR9 ligand (lipidated CpG oligonucleotide) as adjuvants, induces antibodies that primarily bind to the N-terminus of phosphorylated tau peptide SEQ ID NO: 2 (Fig. 9A), whereas in monkeys immunized with a conjugate vaccine according to an embodiment of the present invention (Conjugate A), IgG antibodies are produced that bind mainly to the C-terminal part of the peptide, both in the case of the phosphorylated peptide (left) and in the case of the non-phosphorylated peptide (right) (Fig. 9B).

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In fig. 10A and B show that vaccination with a liposome-based vaccine in accordance with an embodiment of the present invention containing an encapsulated T50 cell-recognized epitope and a TLR4 ligand (3D-(6-acyl)PHAD®) as an adjuvant (S liposomes) induces significantly more higher antibody titers than the liposome-based control vaccine (with the TLR4 ligand, 3D-(6-acyl)PHAD®, but without the T50 T-cell-recognized epitope, liposome R), as well as the liposome-based vaccine according to an embodiment of the present invention containing T57 T cell surface epitope (dipalmitoylated T50) and TLR4 ligand (3D-(6-acyl)PHAD®, T liposomes) in mice: antibody titers measured on days 21 (Fig. 10A) and 35 (Fig. 10B ) after the first immunization were determined by ELISA and presented as individual values and geometric mean for the group ±95% CI; (**: p<0.01, ***: p<0.001).

In fig. Figure 11 shows that encapsulation of T-cell-recognized T48 or T52 peptides into a liposome (liposome M or N, respectively) induces a T-cell response in mice that is specific for the encapsulated peptide: The T-cell response was assessed using IFNγ-specific (Figure 11A ) and IL-4 (Fig. 11B) ELISP0T.

In fig. 12 shows that a liposome-based vaccine containing an encapsulated T-cell-recognized epitope (L liposomes) and a liposome-based vaccine containing an anchored T-cell-recognized epitope (O liposomes) each induced higher titers of tau-specific antibody phosphopeptide than a control liposome-based vaccine without a T-cell recognized epitope; each of liposome L, liposome O and control liposome further contains MPLA as an adjuvant.

In fig. 13 shows that the tau conjugate (KLH-TAUVAC-p7.1 or KLH-TAUVAC-p22.1) stimulates TfH cells and induces robust anti-tau antibody titers in wild-type mice, specifically:

in fig. 13A illustrates that groups of adult female Balb/C mice (n=14 in each group) were immunized a total of four times with 100 μg of KLH-tau conjugate vaccine (KLH-TAUVAC-p7.1 or KLH-TAUVAC-p22 .1) adjuvanted, active placebo vaccine (KLH plus alum or Ribi) or inactive placebo (PBS) according to the regimen presented: four animals from each immunization group were sacrificed seven days after primary immunization, and draining lymph nodes were collected injection site;

in fig. 13B shows the geometric mean of the percentage of TfH by immunization group (n=4 mice per group, analyzed individually) in the draining nodes: all groups receiving active vaccines or placebo had measurable TfH; Moreover, animals receiving the KLH-TAUVAC-p7.1 vaccine, KLH-TAUVAC-p22.1, or active KLH placebo plus alum had significantly more TfH than animals receiving inactive placebo (p=0.0044 for KLH-TAUVAC -p7.1; p=0.0482 for KLH-TAUVAC-p22.1; p=0.0063 for KLH using ANOVA followed by Dunnett's adjustment for multiple comparisons);

in fig. 13C-H shows the change in serum titers from baseline (day 0) at four time points after immunization (days 14, 28, 56 and 84) for group means (n=5-10) with 95% confidence intervals : Asterisks indicate time points at which the KLH-TAUVAC-induced humoral response is significantly greater than the active placebo-induced humoral response (p < 0.05, as determined using analysis of variance followed by Tukey's adjustment for multiple comparisons), more specifically:

in fig. 13C shows the titers of antibodies binding to phosphorylated p7.1 tau peptide;

in fig. 13D shows titers of antibodies binding to phosphorylated p22.1 tau peptide;

in fig. 13E shows the titers of antibodies binding to unphosphorylated tau peptide 7.1;

in fig. 13F shows the titers of antibodies binding to unphosphorylated tau peptide 22.1; and in each of FIG. 13G and H show the titers of antibodies binding to the KLH carrier protein.

In fig. 14 shows that sera from mice immunized with the tau conjugate also bound pathological tau structures in the case of other tauopathies: pooled sera (n=6) from each vaccination group 84 days after primary immunization were used to stain brain tissue in the case of frontotemporal dementia with MART mutation (MART P301S, frontal cortex), in the case of Pick's disease (frontal cortex), progressive supranuclear palsy (PSP, caudate nucleus) and primary age-related tauopathy (PART, hippocampus); sera from animals receiving active vaccines stained tau-associated structures characteristic of each tauopathy, whereas sera from animals immunized with active placebo (KLH-Alum or KLH-Ribi) or inactive placebo (PBS) did not stain any of these structures ; immunostaining with AT8 of the corresponding area is shown as a control; scale bar = 50 µm.

In fig. 15 shows that vaccine-induced antibodies reduce the amount of aggregated tau in a model of accelerated tauopathy, specifically:

fig. 15A: Three-month-old P301b transgenic mice (n=15 in each group) received a stereotactic injection of human ePHF preincubated with purified IgG from mice immunized with either KLH-TAUVAC-p7.1 plus RIBI or active KLH plus placebo RIBI; two months after injection, all mice were sacrificed, and the amount of aggregated tau in mice was determined in the total and sarkosyl-insoluble fractions;

fig. 15B and 15C: total fractions (B) and sarkosyl-insoluble fractions (C) collected from the injected hemisphere of each animal: graphs show the amount of tau determined by MSD in both the total fraction and the insoluble fraction in the brain mice that received ePHF preincubated with IgG from mice immunized with KLHTAUVAC-p7.1 had significantly less aggregated tau than the brains of mice that received ePHF preincubated with control antibodies (p < 0.0001, using ANOVA with subsequent Holm-Bonferroni adjustment for multiple comparisons).

Fig. 16 shows that a tau conjugate (Conjugate B) in accordance with an embodiment of the present invention induces high titers of antibodies against phosphorylated tau and ePHF in non-human primates: rhesus monkeys were immunized with KLH-TAUVAC.p7.1 with alum and CpG as adjuvants (n=6) or KLH (n=2) on days 1, 29, 85 and 169; blood was collected every 14 days, in particular:

fig. 16A: sera from animals immunized with KLH-TAUVAC-p7.1 were tested for reactivity with the p7.1 immunizing peptide using ELISA;

fig. 16B: sera collected from all animals 50 days after primary immunization had measurable levels of antibodies against human ePHF using MSD, with 3 of 6 animals showing high reactivity with this antigen;

fig. 16C: sera collected from animals 50 days after primary immunization were applied to brain sections from healthy individuals or from AD patients, postimmune sera from the KLH-TAUVAC-p7.1 immunization group stained pathological tau structures, namely neurofibrillary tangles, neuropil threads and senile plaques, in brain tissue of AD patients, while sera from mice immunized with KLH did not show any reactivity, and no staining was observed in control tissue;

fig. 16D: When tested in a tau immunodepletion assay, animals treated with KLH-TAUVACp7.1 had antibodies capable of binding and depleting the tau source (p=0.03 at day 50, using ANOVA followed by Dunnett's adjustment for multiple comparisons), in whereas KLH immunization did not trigger the production of such antibodies;

fig. 16E: pre- and post-immunization sera were also tested in a neutralization assay as serially diluted individual samples; change from baseline (CFB) was calculated as the difference between FRET (fluorescence resonance energy transfer) values for readings on day -14 pre-vaccination (baseline) and days 50, 106 and 190 post-vaccination, respectively. The response on a given day after vaccination (day _i ) was then calculated as follows: response = %FRET_day _i - %FRET_baseline; A general linear mixed model for the above responses, with a random effect per animal, was run with the variables vaccination group, day, and serum levels treated as categorical variables and all their interactions.

In fig. 17 shows that mice immunized with a conjugate vaccine (Conjugate A) according to an embodiment of the present invention and a combination of aluminum hydroxide (alum) with a CpG oligonucleotide (CpG) as adjuvants produce higher titers of antibodies against the vaccine peptide; adult female C57BL/6 mice (n=5-6 in each group) were immunized intramuscularly with either 2 μg or 0.2 μg of conjugate A vaccine, and the conjugate vaccine was administered either alone, with alum, with CpG, or with a combination of alum with CpG; all mice received a primary immunization on day 0 of the study, followed by a single boost on day 28; the dose of alum as adjuvant was 500 μg/mouse per injection, and the dose of CpG as adjuvant was 20 μg/mouse per injection; graphs show ELISA binding results using sera collected from mice immunized with the T3.5 vaccine peptide used as coating antigen, with the average T3.5-specific antibody titration endpoints plotted for each group, prior to immunization ( day 0) and at two time points after immunization (days 28 and 42), and with whiskers representing the mean squared error; the tables present statistical analysis of the results, in which antibody titers were compared using the nonparametric Kruskal-Wallis test, and pairwise group comparisons were assessed using the Wilcoxon signed-rank test based on the results obtained using the Kruskal-Wallis test; in particular:

fig. 17A: mice were immunized with 2 μg of conjugate A vaccine; and figs. 17B: Mice were immunized with 0.2 μg of conjugate A vaccine.

In fig. 18 shows that tau vaccines according to embodiments of the present invention with varying ratios of tau peptide to T-cell recognized epitope induce robust and high titers of antibodies against phosphorylated tau in rhesus monkeys.

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Detailed Description of the Present Invention

Various publications, articles and patents are cited or described in the Background section and throughout the specification; each of these references is incorporated herein by reference in its entirety. The discussion of documents, acts, materials, devices, articles or the like that have been included in the present description is intended to provide background to the present invention. Such discussion is not an admission that any or all of this material is part of the prior art with respect to any disclosed or claimed inventions.

Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention relates. Otherwise, certain terms used herein have the meanings set forth in the description.

It should be noted that, as used herein and in the accompanying claims, the singular forms a, an and the include the plural unless the context clearly dictates otherwise.

Unless otherwise indicated, any numerical values, such as concentration or concentration range, described herein should be understood to be referred to in all cases as approximately. Thus, the numerical value usually includes ±10% of the given value. For example, a concentration of 1 mg/ml includes 0.9 to 1.1 mg/ml. Likewise, the concentration range from 1 to 10% (w/v) includes 0.9% (w/v) to 11% (w/v). As used herein, a numeric range expressly includes all possible subranges, all individual numeric values within that range, including integers within such ranges, and fractions of values, unless the context clearly indicates otherwise.

Unless otherwise specified, the term at least preceding a series of elements should be understood to refer to each element in that series. Those skilled in the art will recognize, or be able to determine, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Such equivalents are intended to be covered by the present invention.

As used herein, the terms contains, contains, includes, includes, has, has, contains or contains, or any other variant thereof, will be understood to imply the inclusion of the specified integer or group of integers, but not the exclusion of any other integer or group of integers numbers and are assumed to be non-exclusive or open.

For example, a composition, mixture, process, method, article or device that contains a list of elements is not necessarily limited to those elements only, but may include other elements not expressly listed or not inherent in such composition, mixture, process, method, article or device. In addition, unless expressly stated otherwise, or refers to inclusive or non-exclusive or. For example, a condition A or B is satisfied by any of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A , and B are true (or present).

It should also be understood that the terms about, approximately, generally, substantially and similar terms used herein when referring to the size or characteristic of a component of a preferred invention indicate that the described size, characteristic is not a strict boundary or parameter and is not exclusive of minor deviations from it that are functionally the same or similar, as recognized by one of ordinary skill in the art. At a minimum, such references that include a numerical parameter will include variations that, using mathematical and industrial principles accepted in the art (e.g., rounding, measurement or other systematic errors, manufacturing tolerances, etc.), do not will change the least significant digit.

As used herein, the term tau or tau protein, also known as microtubule-associated protein tau, MART, neurofibrillary tangle protein, paired coiled-coil tau, PHFtau, MAPTL, MTBT1, refers to a protein present in abundance in the central and peripheral nervous system that has many isoform. In the human central nervous system (CNS), there are six major tau isoforms ranging in size from amino acids 352 to 441 due to alternative splicing (Hanger et al., Trends Mol Med. 15:112-9, 2009). Examples of tau include, but are not limited to, tau isoforms in the CNS, such as the longest tau isoform of 441 amino acids (4R2N), which has four repeats and two insertions, and the shortest (embryonic) isoform of 352 amino acids (3R0N), which has three repeats and no inserts. Examples of tau also include the large tau isoform expressed in peripheral nerves, which contains 300 additional residues (exon 4a). Friedhoff et al., Biochimica et Biophysica Acta 1502 (2000) 122-132. Examples of tau include human large tau, which is a 758 amino acid long protein encoded by a 6762 nucleotide long mRNA transcript (NM_016835.4), or isoforms thereof. The amino acid sequence of the exemplary human large tau is provided under GenBank accession number -NP_058519.3. As used herein, the term tau includes tau homologues from non-human species such as Macaca fascicularis (cynomolgus macaques) or Pan troglodytes (chimpanzees). As used herein, the term tau includes proteins containing mutations, such as point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type tau. The term tau also covers post-translational modifications of the amino acid sequence of tau. Post-translational modifications include, but are not limited to, phosphorylation.

As used herein, the term peptide or polypeptide refers to a polymer consisting of amino acid residues, related naturally occurring structural variants and their synthetic non-naturally occurring analogs linked by peptide bonds. The term refers to a peptide of any size, structure or function. Typically, the peptide is at least three amino acids in length. The peptide may be naturally occurring, recombinant or synthetic, or any combination thereof. Synthetic peptides can be synthesized, for example, using an automatic polypeptide synthesizer. Examples of tau peptides include any tau protein peptide of about 5 to about 30, preferably about 10 to about 25, more preferably about 16 to about 21 amino acids in length. In the present invention, peptides are listed from N-terminus to C-terminus using standard three- or one-letter amino acid abbreviations, with phosphorylated residues designated p. Examples of tau peptides useful in the present invention include, but are not limited to, tau peptides comprising the amino acid sequence of any of SEQ ID NOs: 1-12, or tau peptides having an amino acid sequence that is at least 75, 80, 85, 90 or 95% identical to the amino acid sequence of any of SEQ ID NO: 1-12.

As used herein, the term phosphopeptide or phosphoepitope refers to a peptide that is phosphorylated at one or more amino acid residues. Examples of tau phosphopeptides include any tau peptide comprising one or more phosphorylated amino acid residues. Examples of tau phosphopeptides useful in the present invention include, but are not limited to, tau phosphopeptides comprising the amino acid sequence of any of SEQ ID NO: 1-3 or 5-12, or tau phosphopeptides having an amino acid sequence that is is at least 75, 80, 85, 90 or 95% identical to the amino acid sequence of any of SEQ ID NO: 1-3 or 5-12.

The tau peptides of the present invention can be synthesized by solid-phase peptide synthesis or by recombinant expression systems. Automated peptide synthesizers are commercially available from numerous suppliers such as Applied Biosystems (Foster City, CA). Recombinant expression systems may include bacteria such as E. coli, yeast, insect cells or mammalian cells. Procedures for recombinant expression are described in Sambrook et al., Molecular Cloning: A Laboratory Manual (CSHP Press, NY 2d ed., 1989).

Tau is a human protein. This means that, in principle, all lymphocytes bearing the tau-specific receptor should have been eliminated during the development of central tolerance or rendered tolerant by the mechanism of development of peripheral tolerance. This problem has been shown to be a significant obstacle to the development of vaccines against self- or altered self-proteins (eg, antitumor antigens).

The production of high-quality antibodies against an antigen (self or infectious) requires the action of not only the B lymphocytes that produce the antibody, but also CD4+ helper T lymphocytes. CD4+ T cells provide critical survival and maturation signals to B cells, and CD4+ deficient animals are profoundly immunosuppressed. CD4+ T cells are also subject to tolerance mechanisms, and an additional obstacle to generating strong humoral responses to self-protein (eg, tau) is that tau-reactive CD4+ T cells may also be rare or non-existent in the human animal's repertoire.

Without wishing to be bound by any theory, it is believed, but without limiting in any way the scope of the present invention, that this problem is solved by the vaccine compositions of the present invention.

In one embodiment, a liposome is prepared containing tau peptide (one example is shown in FIG. 1; top), which also contains a T cell-recognized epitope that is capable of binding to most or all HLA-DR (leukocyte antigen D-related) molecules human antigen). The epitope recognized by T cells is then able to activate CD4+ T cells and provides the necessary maturation and survival signals for tau-specific B cells (Fig. 2). In another embodiment, a tau peptide-carrier protein conjugate is prepared (one example is shown in FIG. 1; bottom) that induces a potent T helper response (FIG. 3). This embodiment utilizes uncoupled recognition in which host-specific T cells provide survival and maturation signals to self-reactive B cells. Accordingly, tau-specific B cells receive critical signals to initiate affinity maturation, immunoglobulin class switching, and the creation of a pool of long-term memory cells. Tau liposomes and tau conjugates can be used to generate high quality anti-tau antibodies in homologous or heterologous immunization regimens, with the liposomes or conjugates being used in primary and/or booster immunizations.

Liposomes.

In one general aspect, the present invention provides a liposome comprising:

a) the tau peptide, preferably the tau peptide is a tau phosphopeptide; And

b) an epitope for helper T cells, the tau peptide being present on the surface of the liposome.

Liposomes in accordance with embodiments of the present invention are also referred to herein as improved liposomes, improved liposome-containing vaccines or liposome-based vaccines in accordance with embodiments of the present invention, or tau liposomes or optimized liposome-containing 2nd generation liposome-based vaccines.

As used herein, the term liposome generally refers to a lipid vesicle that is made from materials high in lipids, eg phospholipids, cholesterol. The lipids of these vesicles are typically organized into the form of lipid bilayers. Lipid bilayers typically encapsulate a volume that is either distributed among multiple onion-shaped shells of lipid bilayers, forming multilamellar lipid vesicles (MLVs), or contained in an amorphous central cavity. Lipid vesicles having an amorphous central cavity are unilamellar lipid vesicles, i.e. lipid vesicles with one peripheral bilayer surrounding the cavity. Large unilamellar vesicles (LUVs) typically have a diameter ranging from 100 nm to several micrometers, such as 100-200 nm or more, while small unilamellar lipid vesicles (SUVs) typically have a diameter of less than 100 nm. for example 20-100 nm, usually 15-30 mm.

In accordance with specific embodiments, the liposome contains one or more taupeptides. In accordance with specific embodiments, the tau peptides in the liposome may be the same or different.

Any suitable tau peptide known to those skilled in the art may be used in the present invention in light of the present invention. In certain embodiments, the one or more tau peptides comprise the amino acid sequence of one of SEQ ID NOs: 1-12. In other embodiments, one or more tau peptides comprise an amino acid sequence that is at least 75, 80, 85, 90, or 95% identical to the amino acid sequence of one of SEQ ID NOs: 1-12, wherein none of the amino acid residues is phosphorylated, or one or more amino acid residues are phosphorylated.

In certain embodiments, the one or more tau peptides is a tau phosphopeptide. In certain embodiments, the one or more tau phosphopeptides comprise an amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, or an amino acid sequence that is at least 75, 80, 85, 90, or 95% identical to the amino acid sequence one of SEQ ID NOs: 1-3 or 5-12, wherein one or more of said amino acid residues are phosphorylated. Preferably, the tauphosphopeptide comprises the amino acid sequence of one of SEQ ID NOs: 1-3. The tau peptide may have an amidated C-terminus.

In accordance with embodiments of the present invention, the tau peptide is presented on the surface of the liposome. Tau peptide, preferably tau phosphopeptide, can be presented on the surface of the liposome using methods known in the art in light of the present invention. See, for example, the corresponding disclosure in US Pat. Nos. 8,647,631 and 9,687,447, the contents of which are incorporated herein by reference. In accordance with specific embodiments, one or more tau peptides, including phosphopeptides, further comprise one or more modifications, such as palmitoylation or dodecyl modification, to allow the tau peptides to be presented on the surface of the liposome. Additional amino acid residues such as Lys, Cys or sometimes Ser or Thr can be added to the taupeptide to facilitate modification. The position of lipid anchors has been reported to give rise to different conformations of the peptide sequence (Hickman et al., J. Biol. Chem. Vol. 286, NO. 16, pp. 13966-13976, April 22, 2011). Without wishing to be limited by theory, it is believed that the addition of hydrophobic moieties at both ends may increase the pathological conformation of the tau peptide beta sheet. Thus, one or more tau peptides further include hydrophobic moieties at both ends. The modified tau peptide may have an amidated C-terminus. Preferably, the tau peptide present on the surface of the liposome consists of the amino acid sequence of one of SEQ ID NO: 27 to SEQ ID NO: 38.

As used herein, the term helper T cell epitope refers to a polypeptide containing

- 8 044254 epitope that can be recognized by a helper T cell. Examples of epitopes for T helper cells include, but are not limited to, tetanus toxoid (e.g., P2 and P30 epitopes, also called T2 and T30, respectively), hepatitis B virus surface antigen, cholera toxin B, toxoid, diphtheria toxoid, viral F protein measles, major outer membrane protein

Chlamydia trachomatis, Circumsporosity protein T of Plasmodium falciparum, CS P antigen.

falciparum, triosephosphate isomerase from Schistosoma mansoni, Bordetella pertussis, Clostridium tetani, Pertusaria trachythallina, TraT Escherichia coll and influenza virus hemagglutinin (HA).

Any suitable epitope for T helper cells known to those skilled in the art may be used in the present invention in light of the present invention. In certain embodiments, the T helper cell epitope includes at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23 - SEQ ID NO: 26. Preferably, the T helper cell epitope includes two or more amino acids sequences from SEQ ID NO: 23 to SEQ ID NO: 26 fused together through a linker, such as a peptide linker comprising one or more amino acids, for example Val (V), Ala (A), Arg (R), Gly (G) , Ser (S), Lys (K). The length of the linker may vary, preferably 1-5 amino acids. Preferably, the T helper epitope comprises three or more amino acid sequences from SEQ ID NO: 23 to SEQ ID NO: 26 fused together through one or more linkers selected from the group consisting of VVR, GS, RR, RK. The epitope for T helper cells may have an amidated C-terminus.

In accordance with embodiments of the present invention, epitopes for T helper cells can be included on the surface of the liposome, for example, anchored by a covalently linked hydrophobic moiety, wherein said hydrophobic moiety is an alkyl group, a fatty acid, a triglyceride, a diglyceride, a steroid, a sphingolipid, a glycolipid or a phospholipid, in particular an alkyl group or a fatty acid, in particular with a carbon skeleton of at least 3 carbon atoms, in particular at least 4 carbon atoms, in particular at least 6 carbon atoms, in particular according to at least 8 carbon atoms, in particular at least 12 carbon atoms, in particular at least 16 carbon atoms. In one embodiment of the present invention, the hydrophobic moiety is palmitic acid. Alternatively, epitopes for T helper cells can be encapsulated in liposomes. In certain embodiments, the T helper cell epitope is encapsulated in a liposome.

The T helper epitope can be modified to position it as desired in liposomes using methods known in the art, subject to the present invention. In certain embodiments, a T helper epitope useful for the present invention comprises an amino acid sequence of one of SEQ ID NO: 39 to SEQ ID NO: 44. Preferably, the T helper epitope consists of an amino acid sequence selected from the group , consisting of SEQ ID NO: 13-SEQ ID NO: 17.

In certain embodiments, the liposome contains tau peptide and T helper epitope in a weight ratio of 1:1, 2:1, 3:1, 4:1, 5:1, or 6:1.

In one embodiment, the liposome further contains at least one adjuvant containing a toll-like receptor ligand. Thus, in another general aspect, the present invention provides a liposome containing:

a) tau peptide, preferably tau phosphopeptide;

b) epitope for T helper cells; And

c) at least one of

i) toll-like receptor ligand 9 and ii) toll-like receptor ligand 4.

As used herein, the term toll-like receptor or TLR refers to a class of pattern recognition receptor (PRR) proteins that play a key role in the innate immune response. TLRs recognize pathogen-associated molecular patterns (PAMs) of microbial pathogens such as bacteria, fungi, parasites and viruses, which can be distinguished from host molecules. TLRs are membrane-spanning proteins that typically function as dimers and are expressed by cells involved in the innate immune response, including antigen-presenting dendritic cells and phagocytic macrophages. There are at least ten members of the human TLR family, TLR1-TLR10, and at least twelve members of the mouse TLR family, TLR1-TLR9 and TLR11-TLR13, and they differ in the types of antigens they recognize. For example, TLR4 recognizes lipopolysaccharides (LPS), a component present in many Gram-negative bacteria, as well as viral proteins, polysaccharides, and endogenous proteins such as low-density lipoprotein, beta-defensins, and heat shock protein; a TLR9 is a nucleotide-sensitive TLR that is activated by unmethylated single-stranded and double-stranded cytosine-phosphate-guanine (CpG) dinucleotides that are abundant in prokaryotic genomes but rare in vertebrate genomes. TLR activation results in a series of signaling events leading to the production of type I interferons (IFNs), inflammatory cytokines and chemokines, and the induction of immune responses. Ultimately, this inflammation also activates the adaptive immune system, which then leads to the clearance of colonizing pathogens and infected cells.

As used herein, the term ligand refers to a molecule that forms a complex with a biomolecule (eg, a receptor) for a biological purpose. In accordance with specific embodiments, the toll-like receptor ligand is a toll-like receptor agonist.

As used herein, the term agonist refers to a molecule that binds to one or more TLRs and induces a receptor-mediated response. For example, an agonist may induce, stimulate, increase, activate, facilitate, enhance, or upregulate receptor activity. Such actions are called agonistic actions. For example, a TLR4 or TLR9 agonist can activate or enhance cell signaling through the associated receptor. Agonists include, but are not limited to, nucleic acids, small molecules, proteins, carbohydrates, lipids, or any other molecules that bind or interact with receptors. Agonists can mimic the activity of a naturally occurring receptor ligand. Agonists may be homologous to these naturally occurring receptor ligands in terms of sequence, conformation, charge, or other characteristics such that they can be recognized by the receptors. This recognition can lead to physiological and/or biochemical changes within the cell such that the cell responds to the presence of the agonist in the same way as if a naturally occurring receptor ligand were present. In certain embodiments, the toll-like receptor agonist is at least one of a toll-like receptor 4 agonist and a toll-like receptor agonist 9.

As used herein, the term toll-like receptor 4 agonist refers to any compound that acts as a TLR4 agonist. Any suitable toll-like receptor 4 agonist known to those skilled in the art in view of the present invention can be used in the present invention. Examples of a toll-like receptor 4 ligand useful for the present invention include a TLR4 agonist, including, but not limited to, monophosphoryl lipid A (MPLA). As used herein, the term monophosphoryl lipid A or MPLA refers to a modified form of lipid A that is the biologically active portion of the endotoxin lipopolysaccharide (LPS) of Gram-negative bacteria. MPLA is less toxic than LPS while maintaining immunostimulatory activity. As a vaccine adjuvant, MPLA stimulates both cellular and humoral responses to the vaccine antigen. Examples of MPLA include, but are not limited to, 3-O-deacyl-4'monophosphoryl lipid A, monophosphoryl hexaacyl lipid A, 3-deacylated, monophosphoryl-3-deacyl lipid A, and structurally related variants thereof. MPLA useful for the present invention can be prepared using methods known in the art or from a commercial source, for example 3D-(6-acyl)PHAD®, PHAD®, PHAD®-504, 3D-PHAD® from Avanti Polar Lipids (Alabaster, Alabama, USA) or MPL™ from various commercial sources. In certain embodiments, the toll-like receptor 4 agonist is MPLA. As used herein, the term toll-like receptor 9 agonist refers to any compound that acts as a TLR9 agonist. Any suitable toll-like receptor 9 agonist known to those skilled in the art in light of the present invention may be used in the present invention. Examples of a toll-like receptor 9 ligand useful for the present invention include a TLR9 agonist, including, but not limited to, CpG oligonucleotides.

As used herein, the term CpG oligonucleotide, CpG oligodeoxynucleotide, or CpGODN refers to an oligonucleotide comprising at least one CpG motif. As used herein, the term oligonucleotide, oligodeoxynucleotide or ODN refers to a polynucleotide formed from a plurality of linked nucleotide units. Such oligonucleotides can be obtained from existing sources of nucleic acids or can be obtained by synthetic methods. As used herein, the term CpG motif refers to a nucleotide sequence that contains unmethylated cytosine-phosphate-guanine (CpG) dinucleotides (i.e., cytosine (C) followed by guanine (G)) linked via a phosphate bond or phosphodiester backbone or other internucleotide bonds.

In certain embodiments, the CpG oligonucleotide is lipidated, i.e. conjugated (covalently bound) to the lipid component.

As used herein, the term lipid moiety refers to a moiety containing a lipophilic structure. Lipid moieties such as an alkyl group, fatty acid, triglyceride, diglyceride, steroid, sphingolipid, glycolipid or phospholipid, in particular a sterol such as cholesterol, or fatty acids, when attached to highly hydrophilic molecules such as nucleic acids, can significantly increase binding to plasma proteins and, therefore, half-life in the bloodstream of hydrophilic molecules. In addition, binding to certain plasma proteins, such as lipoproteins, has been shown to increase absorption in specific tissues,

- 10 044254 expressing the corresponding lipoprotein receptors (for example, HDL receptor, receptor

HDL or phagocytic receptor SR-B1). In particular, a lipid moiety conjugated to phosphopeptides and/or a CpG oligonucleotide allows said peptides and/or oligonucleotides to be anchored in the liposome membrane through the hydrophobic moiety.

In certain embodiments of the present invention, the CpG oligonucleotide may include any suitable internucleotide linkages.

As used herein, the term internucleotide bond refers to a chemical bond connecting two nucleotides through their sugars, consisting of a phosphorus atom and a charged or neutral group between adjacent nucleosides. Examples of internucleotide linkages include phosphodiester (po), phosphorothioate (ps), phosphorodithioate (ps2), methylphosphonate (mp), and methylphosphorothioate (rp) linkages. Phosphorothioate, phosphorodithioate, methylphosphonate and methylphosphorothioate bonds are stabilizing internucleotide bonds, while the phosphodiester bond is a naturally occurring internucleotide bond. Phosphorothioate oligonucleotides are typically synthesized as a random racemic mixture of Rp and Sp phosphorothioate bonds.

Any suitable CpG oligonucleotide known to those skilled in the art can be used in the present invention in light of the present invention. Examples of such CpG oligonucleotides include, but are not limited to, CpG2006 (also known as CpG7909), CpG1018, CpG2395, CpG2216, or CpG2336.

The CpG oligonucleotide can be lipidated using methods known in the art in light of the present invention. In some embodiments, the 3' end of the CpG oligonucleotide is covalently linked to a cholesterol molecule via a phosphate bond, optionally through a linker including PEG. Another lipophilic moiety may also be covalently linked to the 3' end of the CpG oligonucleotide. For example, a CpG oligonucleotide can be covalently linked to a lipid anchor of the same length as the phospholipids from the liposome: one palmitic acid chain (using Pal-OH or an analogue activated for binding) or two palmitic acids (for example, using 1,2- dipalmitoyl-sn-glycero-3-phosphoethanolamine-N(succinyl) or a binding-activated analogue), optionally via a linker including PEG. See, for example, the related disclosure in US Pat. No. 7,741,297, the contents of which are incorporated herein by reference. The length of the PEG can vary, for example from 1 to 5 PEG units.

Other linkers can also be used to covalently link a CpG oligonucleotide to a lipophilic moiety (such as a cholesterol molecule), examples of which include, but are not limited to, an alkyl spacer containing from 3 to 12 carbon atoms. A short linker compatible with oligonucleotide chemistry is required in the form of an aminodiol. In some embodiments, the linker is not used for covalent linking. See, for example, Ries et al., Convenient synthesis and application of versatile nucleic acid lipid membrane anchors in the assembly and fusion of liposomes, Org. Biomol. Chem., 2015, 13, 9673, the relevant disclosure of which is incorporated herein by reference.

In certain embodiments, a lipidated CpG oligonucleotide useful for the present invention includes a nucleotide sequence selected from the group consisting of SEQ ID NO: 18-SEQ ID NO: 22, wherein the nucleotide sequence contains one or more phosphorothioate internucleotide linkages, and the nucleotide sequence is covalently linked to at least one cholesterol through a linker. Any suitable linkers can be used to covalently link the CpG oligonucleotide to the cholesterol molecule. Preferably, the linker includes polyethylene glycol (PEG).

In accordance with specific embodiments, the liposome contains:

a) tau phosphopeptide,

b) epitope for T helper cells,

c) lipidated CpG oligonucleotide and

d) toll-like receptor 4 ligand;

wherein the tau phosphopeptide is presented on the surface of the liposome, and the epitope for T-helper cells is encapsulated in the liposome.

In accordance with specific embodiments, the liposome contains:

a) a tau peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 27-SEQ ID NO: 38;

b) a T helper epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 39-SEQ ID NO: 44, preferably a T helper epitope consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13-SEQ ID NO: 17;

c) a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to

- 11 044254 with at least one cholesterol via a linker; And

d) monophosphoryl lipid A (MPLA).

In certain embodiments, the liposome further comprises one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl -3'-rac-glycerol (DMPG) and cholesterol.

In accordance with specific embodiments, the liposome further comprises a buffer. Any suitable buffer known to those skilled in the art in light of the present invention may be used in the present invention. In one embodiment, the liposome contains phosphate buffered saline. In certain embodiments, the buffer contains histidine and sucrose.

In certain embodiments, the liposome contains DMPC, DMPG, cholesterol, tau phosphopeptide, and T helper cell epitope in a molar ratio of 9:1:7:0.07:0.04.

The liposomes of the present invention can be prepared using methods known in the art in light of the present invention.

An exemplary liposome of the present invention is illustrated in FIG. 1. More specifically, tetrapalmitoylated tau phosphopeptide (pTau peptide T3, SEQ ID NO: 28) is presented on the surface of the liposome by two palmitic acids at each end of the taupeptide. A lipidated CpG-containing TLR-9 ligand (CpG7909-Chol adjuvant) is introduced into the liposome membrane via covalently bound cholesterol. TLR-4 ligand (3D-(6-acyl)PHAD® adjuvant) is also included in the membrane. The epitope for T helper cells (pan-DR-binding T50) is encapsulated.

Conjugates.

In one general aspect, the present invention provides a conjugate comprising a tau peptide and an immunogenic carrier conjugated thereto.

In accordance with specific aspects, the conjugate has the following structure:

or the structure of formula (II):

where x is an integer from 0 to 10, and n is an integer from 2 to 15, preferably 3-11.

In certain embodiments, x is an integer from 1 to 10, 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, 2 to 4, or 2 to 3 In accordance with specific embodiments, x is equal to 3.

In certain embodiments, n is 2 to 15, 3 to 11, 3 to 9, 3 to 8, or 3 to 7.

In accordance with specific embodiments, the conjugate includes one or more taupeptides. In certain embodiments, the tau peptides of the conjugate may be the same or different.

In accordance with specific embodiments, in view of the present invention, any suitable tau peptides can be used in the present invention. In certain embodiments, the one or more tau peptides comprise an amino acid sequence of one of SEQ ID NOs: 1-12, or an amino acid sequence that is at least 75, 80, 85, 90, or 95% identical to the amino acid sequence of one of SEQ ID NOs: NO: 1-12, wherein none of the amino acid residues is phosphorylated, one or more amino acid residues are phosphorylated.

In certain embodiments, the one or more tau peptides is a tau phosphopeptide. In certain embodiments, the one or more tau phosphopeptides comprise an amino acid sequence of one of SEQ ID NOs: 1-3 or 5-12, or an amino acid sequence that is at least 75, 80, 85, 90, or 95% identical to the amino acid sequence one of SEQ ID NOs: 1-3 or 5-12, wherein one or more of said amino acid residues are phosphorylated.

In certain embodiments, the tau phosphopeptide consists of the amino acid sequence of one of SEQ ID NOs: 1-3.

As used herein, the term immunogenic carrier refers to an immunogenic substance that may be associated with tau peptide. The immunogenic moiety bound to tau peptide can trigger an immune response and cause the production of antibodies that can specifically bind tau peptide. Immunogenic moieties are functional moieties that include proteins, polypeptides, glycoproteins, complex polysaccharides, particles, nucleic acids, polynucleotides, and the like, which are recognized as foreign and thereby induce an immunological response in the host. Any suitable immunogenic carrier known to those skilled in the art in light of the present invention may be used in the present invention. In certain embodiments, the immunogenic carrier is keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197 (a non-toxic form of diphtheria toxin), a mixture of N. meningitidis outer membrane proteins (OMP), or a derivative thereof. In certain embodiments, the immunogenic carrier is KLH or CRM197.

In certain embodiments, the tau peptide is conjugated to a carrier via a linker. As used herein, the term linker refers to the chemical moiety that attaches the immunogenic carrier to the tau peptide. Any suitable linker known to those skilled in the art in light of the present invention may be used in the present invention. The linkers may be, for example, a single covalent bond, a substituted or unsubstituted alkyl moiety, a substituted or unsubstituted heteroalkyl moiety, a polyethylene glycol (PEG) linker, a peptide linker, a sugar-based linker, or a cleavable linker such as a disulfide bond or a protease cleavage site, or amino acid, or a combination thereof. Examples of the linker may include one or more of polyethylene glycol (PEG), succinimidyl-3(bromoacetamido)propionate (SBAP), m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or one or more amino acids such as Cys, Lys or sometimes Ser or Thr, or a combination thereof.

In certain embodiments _{, the linker comprises (C2H4O ) x} -cysteine acetamidopropionamide or m-maleimidobenzoyl-N-hydroxysuccinimide ester-cysteine-( _C2H4O ) _x , where x is an integer from ₀ to 10, for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

In certain embodiments, the carrier is covalently linked to the N-terminus of the tau peptide through a linker.

In other specific embodiments, the carrier is covalently linked to the C-terminus of the taupeptide via a linker.

In accordance with specific embodiments, the conjugate has the structure:

where n is an integer from 2 to 15, preferably 3-11, more preferably 3-7.

The conjugates of the present invention can be prepared by methods known in the art in light of the present invention. For example, the above conjugate can be prepared by reacting succinimidyl-3-(bromoacetamido)propionate (SBAP):

with the amino group of CRM197 to form an amide bond. This CRM197 precursor can subsequently react with a tau peptide (e.g., phosphorylated tau peptide of SEQ ID NO: 2) conjugated at its N-terminus or at its C-terminus to a PEG-cysteine linker to a free nucleophilic thiol group to form a tau phosphopeptide conjugate.

An exemplary conjugate according to an embodiment of the present

- 13 044254 invention is illustrated in Fig. 1. More specifically, a variety of tau phosphopeptides (pTau peptide

T3.76) are covalently linked to the carrier protein CRM197.

Pharmaceutical compositions.

In one general aspect, the present invention relates to pharmaceutical compositions containing a therapeutically effective amount of a liposome or conjugate of the present invention together with a pharmaceutically acceptable excipient and/or carrier. Pharmaceutically acceptable excipients and/or carriers are well known in the art (see Remington's Pharmaceutical Science (15th ed.), Mack Publishing Company, Easton, PA, 1980). The preferred composition of the pharmaceutical composition depends on the intended route of administration and therapeutic use. The compositions may include pharmaceutically acceptable, non-toxic carriers or diluents, which are defined as those typically used to formulate pharmaceutical compositions for administration to animals or humans. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, phosphate buffered saline, Ringer's solutions, dextrose solution and Hank's solution. In addition, the pharmaceutical composition or preparation may also include other carriers, adjuvants or non-toxic, non-therapeutic, non-immunogenic stabilizers and the like. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application.

The pharmaceutical composition may contain a mixture of the same immunogenic taupeptide. Alternatively, the pharmaceutical composition may contain a mixture of various immunogenic tau peptides of the present invention.

Another concern with neuronal disease vaccines is that exceptionally high antibody titers may be required to be effective. This is because the target antigen for the vaccine is located in the brain. The brain is separated from the bloodstream by a specialized cellular structure called the blood-brain barrier (BBB). The BBB limits the passage of substances from the bloodstream into the brain. This prevents the entry of toxins, germs, etc. into the central nervous system. The BBB also has the potentially less desirable effect of preventing the effective entry of immune mediators (such as antibodies) into the interstitial and cerebrospinal fluid that surrounds the brain.

Approximately 0.1% of antibodies present in the systemic circulation cross the BBB and enter the brain. This means that the systemic titers induced by a vaccine targeting a CNS antigen must be at least 1000 times the minimum effective titer to be effective in the brain.

In accordance with specific embodiments, the pharmaceutical compositions of the present invention therefore further contain one or more suitable adjuvants. Thus, the tau peptides of the present invention, present in a liposome or conjugate, can be administered in combination with a suitable adjuvant to achieve the desired immune response in a subject. Suitable adjuvants may be administered before, after, or simultaneously with the administration of the liposome or conjugate of the present invention. Preferred adjuvants enhance the intrinsic response to an immunogen without causing conformational changes in the immunogen that affect the characteristic shape of the response. Examples of adjuvants are aluminum salts (alum), such as aluminum hydroxide, aluminum phosphate and aluminum sulfate. Such adjuvants can be used with or without other specific immunostimulating agents, such as the MPLA class (3-de-Oacylated monophosphoryl lipid A (MPL™), monophosphoryl hexaacyl lipid A 3-deacylated synthetic (3D-(6-acyl)PHAE))® ), PHAD™, PHAD®-504, 3D-PHAD®), lipid A), polymeric or monomeric amino acids such as polyglutamic acid or polylysine. Such adjuvants may be used with or without other specific immunostimulating agents, such as muramyl peptides (e.g., N-acetylmuramyl-L-threonyl-L-isoglutamine (thr-MDP), N-acetylnormuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)ethylamine (MTP-PE), N-acetylglucosaminyl-Nacetylmuramyl-L-Al- D-isoglu-L-Ala-dipalmitoxypropylamide (DTP-DPP) Theramide™) or other bacterial cell wall components. Oil-in-water emulsions include adjuvant MF59 (see WO 90/14837) containing 5% squalene, 0.5% Tween 80 and 0.5% Span 85 (optionally containing varying amounts of MTP-PE) formulated into submicron particles using a microfluidizer; SAF containing 10% squalene, 0.4% Tween 80, 5% Pluronic-blocked polymer L121 and thr-MDP, either microfluidized into a submicron emulsion or vortexed to form a larger particle size emulsion; and Ribi™ Adjuvant System (RAS) (Ribi ImmunoChem, Hamilton, Mont.) 0.2% Tween 80 and one or more bacterial cell wall components selected from the group consisting of monophosphoryl lipid A (MPL™), trehalose dimycolate (TDM) and cell wall skeleton (CWS), preferably MPL™+CWS (Detox™). Other adjuvants include Freund's complete adjuvant (CFA) and cytokines such as interleukins (IL-1, IL-2

- 14 044254 and IL-12), macrophage colony stimulating factor (M-CSF) and tumor necrosis factor (TNF).

As used herein, the term combination in connection with administering two or more therapies to a subject refers to the use of more than one therapy. Use of the term in combination does not limit the order in which the therapies are administered to a subject. For example, the first therapy (e.g., the composition described herein) can be administered before (e.g., 5 minutes, 15, 30, 45 minutes, 1 hour, 2,4,6, 12, 16, 24,48,72,96 hours, 1 week, 2, 3, 4, 5, 6, 8 or 12 weeks, simultaneously or after (for example, after 5, 15, 30, 45 minutes, 1 hour, 2, 4, 6, 12, 16, 24, 48, 72.96 hours, 1 week, 2, 3, 4, 5, 6, 8, or 12 weeks) administration of the second therapy to the subject.

The pharmaceutical compositions of the present invention can be formulated in accordance with methods well known in the art. The optimal ratios of each component in the compositions can be determined using methods well known to those skilled in the art in light of the present invention.

Methods of application.

Another general aspect of the present invention relates to methods of inducing an immune response against tau protein in a subject suffering from a neurodegenerative disease, disorder or condition, comprising administering to the subject a pharmaceutical composition in accordance with an embodiment of the present invention. In certain aspects, an immune response is induced against phosphorylated tau protein, preferably ePHF.

Another general aspect of the present invention relates to methods of treating or preventing a neurodegenerative disease, disorder or condition, comprising administering to a subject a pharmaceutical composition in accordance with an embodiment of the present invention.

As used herein, the terms induce and stimulate and variations thereof refer to any measurable increase in cellular activity. Induction of an immune response may include, for example, activation, proliferation, or maturation of a population of immune cells, increased production of cytokines, and/or other indication of increased immune function. In some embodiments, induction of an immune response may include increased B cell proliferation, production of antigen-specific antibodies, increased proliferation of antigen-specific T cells, improved antigen presentation by dendritic cells, and/or increased expression of certain cytokines, chemokines, and co-stimulatory markers.

The ability to induce or stimulate an immune response against tau when administered to an animal or human can be assessed either in vitro or in vivo using various assays that are standard in the art. For a general description of the methods available to assess the initiation and activation of the immune response, see, for example, Coligan et al. (1992 and 1994, Current Protocols in Immunology; ed. J Wiley & Sons Inc, National Institute of Health). Determination of cellular immunity can be performed by methods fully known in the art, for example, by measuring the profiles of cytokines secreted by activated effector cells, including those derived from CD4+ and CD8+ T cells (for example, quantitative analysis of IL-4 or IFN-γ-producing cells ELISPOT method), by determining the activation state of immune effector cells (for example, T cell proliferation assay using classical [3H]thymidine uptake), by analyzing antigen-specific T lymphocytes in a sensitized subject (for example, peptide-specific lysis in a cytotoxicity assay, etc. .).

The ability to stimulate a cellular and/or humoral response can be determined by testing a biological sample (e.g., blood, plasma, serum, PBMC, urine, saliva, feces, cerebrospinal fluid, or lymph fluid) from the subject for the presence of antibodies directed to the immunogenic tau peptide (s) included in a pharmaceutical composition (see, for example, Harlow, 1989, Antibodies, Cold Spring Harbor Press). For example, titers of antibodies produced in response to administration of a composition providing an immunogen can be measured using an enzyme-linked immunosorbent assay (ELISA), dot blots, SDS-PAGE, ELISPOT, or an antibody-dependent cellular phagocytosis (ADCP) assay.

The term subject as used here refers to an animal. In certain embodiments, the subject is a mammal, including a non-primate (e.g., camel, donkey, zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea pig, or mouse) or a primate ( e.g. monkey, chimpanzee or human). In accordance with specific embodiments, the subject is a human.

As used herein, the term therapeutically effective amount refers to the amount of an active ingredient or component that produces a desired biological or drug response in a subject. The therapeutically effective amount can be determined empirically and routinely depending on the intended purpose. For example, in vitro assays may not necessarily be used to help determine optimal dosage ranges. The selection of a specific effective dose can be determined (eg, through clinical trials) by those skilled in the art based on consideration of several factors, including the disease being treated or prevented, the symptoms involved, the patient's body weight, the patient's immune status, and

- 15 044254 other factors known to a person skilled in the art. The exact dose to be used in the drug will also depend on the route of administration and the severity of the disease, and should be chosen according to the judgment of the practitioner and the circumstances of each patient. Effective doses can be extrapolated from dose response curves obtained from in vitro test systems or animal models.

As used herein, the terms treat, treatment, and treatment are intended to all refer to the improvement or reversal of at least one measurable physical parameter associated with a neurodegenerative disease, disorder, or condition that is not necessarily apparent in the subject, but may be discernible in the subject . The terms treat, treat, and treat may also refer to causing regression, preventing progression, or at least slowing the progression of a disease, disorder, or condition. In a specific embodiment, the term treat, treatment, and treatment refers to alleviating, preventing the development or occurrence of, or reducing the duration of one or more symptoms associated with a neurodegenerative disease, disorder, or condition. In a specific embodiment, treat, treat, and cure refer to preventing the recurrence of a disease, disorder, or condition. In a specific embodiment, treat, treat, and cure refer to increasing the survival of a subject having a disease, disorder, or condition. In a specific embodiment, treat, treat, and cure refer to eliminating a disease, disorder, or condition in a subject.

In certain embodiments, a therapeutically effective amount refers to an amount of therapy that is sufficient to achieve one, two, three, four or more of the following effects: (i) attenuating or reducing the severity of the disease, disorder or condition being treated or associated with him symptom; (ii) reducing the duration of the disease, disorder or condition being treated, or a symptom associated therewith; (iii) preventing the progression of the disease, disorder or condition being treated, or a related symptom; (iv) causing regression of the disease, disorder or condition being treated, or a related symptom; (v) preventing the development or occurrence of the disease, disorder or condition being treated, or a symptom related thereto; (vi) preventing recurrence of the disease, disorder or condition being treated, or a related symptom; (vii) reducing the incidence of hospitalization of a subject having the disease, disorder or condition being treated, or a related symptom; (viii) reducing the length of hospitalization of a subject having the disease, disorder or condition being treated, or a symptom associated therewith; (ix) increasing the survival of a subject with the disease, disorder or condition being treated, or a symptom associated therewith; (x) suppressing or ameliorating the disease, disorder or condition being treated, or a related symptom in the subject; and/or (xi) enhancing or improving the prophylactic or therapeutic effect(s) of another therapy.

As used herein, the term neurodegenerative disease, disorder or condition includes any neurodegenerative disease, disorder or condition known to those skilled in the art in light of the present invention. Examples of neurodegenerative diseases, disorders or conditions include neurodegenerative diseases or disorders caused by or associated with the formation of neurofibrillary lesions, such as tau-associated diseases, disorders or conditions called tauopathies. In certain embodiments, the neurodegenerative disease, disorder, or condition includes any of the diseases or disorders that are accompanied by the coexistence of tau and amyloid pathologies, including, but not limited to, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Boxer's dementia, Down's disease, Gerstmann-Straussler-Scheckner disease, myositis with inclusion bodies, prion protein, cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, parkinsonism-dementia-Guam complex, non-Guam motor neuron disease with neurofibrillary tangles, dementia characterized by the appearance of argyrophilic grains, corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, diffuse neurofibrillary tangles with calcification, frontotemporal dementia, preferably frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar dementia, Hallerwarden disease- Spatz, multiple system atrophy, Niemann-Pick disease type C, Pick disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, dementia with predominant neurofibrillary tangles, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PART) or Lewy body dementia (LBD). In certain embodiments, the neurodegenerative disease, disorder, or condition is Alzheimer's disease or other tauopathy.

The present invention also provides a method for accelerating the clearance of tau aggregates from the brain of a subject, the method comprising administering to the subject a pharmaceutical composition in accordance with an embodiment of the present invention under conditions effective to accelerate the clearance of tau aggregates from the brain of the subject. In certain embodiments, the tau aggregates are neurofibrillary tangles or pathological tau precursors thereof.

The present invention also provides a method of slowing the progression of a behavioral phenotype associated with taupathology in a subject, the method comprising administering to the subject a pharmaceutical composition in accordance with an embodiment of the present invention under conditions effective to slow the progression of a behavioral phenotype associated with taupathology in the subject.

In a preferred embodiment of the present invention, administration of tau peptide through administration of a pharmaceutical composition in accordance with an embodiment of the present invention induces an active immune response in a subject to tau peptide and the pathological form of tau, thereby promoting the clearance of associated tau aggregates, slowing the progression of behavior associated with taupathology, and/or treating the underlying tauopathy. In accordance with this aspect of the present invention, the immune response includes the development of a beneficial humoral (antibody-mediated) response directed against the tau peptide and a cellular (mediated by antigen-specific T cells or their secretion products) response directed against the T cell-recognized epitope or immunogenic vehicle .

As used herein, tau pathology-associated behavioral phenotype includes, but is not limited to, cognitive impairment, early personality change and disinhibition, apathy, abulia, mutism, apraxia, perseveration, stereotyped movements/behavior, hyperoralism, disorganization, inability to plan or organize sequential tasks , selfishness/callousness, antisocial traits, lack of empathy, stopping, ungrammatical speech with frequent paraphasic errors but relatively preserved comprehension, impaired comprehension and difficulty finding words, slowly progressive unsteadiness in gait, retropulsion, freezing, frequent falls, not responsive to levodopa axial muscle rigidity, supranuclear gaze paresis, square wave saccades, slow vertical saccades, pseudobulbar palsy, limb apraxia, dystonia, loss of cortical sensation, and tremor.

When performing the methods of the present invention, it is preferable to select a subject having or at risk of developing Alzheimer's disease or other tauopathy, a subject having tau aggregates in the brain, or a subject exhibiting a tangle-related behavioral phenotype prior to administration of the immunogenic peptides or antibodies of the present invention . Treatable subjects include individuals at risk of developing the disease but not exhibiting symptoms, as well as patients currently exhibiting symptoms. In the case of Alzheimer's disease, almost anyone is at risk of suffering from Alzheimer's disease. Therefore, the present methods can be applied prophylactically to the general population without the need for any risk assessment in the patient in question. The methods of the present invention are particularly useful for individuals with a known genetic risk of developing Alzheimer's disease. Such individuals include individuals who have relatives who have had the disease and individuals whose risk is determined by analysis of genetic or biochemical markers.

For asymptomatic patients, treatment can begin at any age (eg, 10, 20, 30 years). Usually, however, there is no need to start treatment until the patient reaches the age of 40, 50, 60 or 70 years. Treatment usually involves multiple doses over a period of time. Treatment can be monitored by testing antibodies or activated T cell or B cell responses to the therapeutic agent over time. If the response decreases, a booster dose is indicated.

For prophylactic applications, pharmaceutical compositions containing tau peptides are administered to a patient susceptible or otherwise at risk of developing Alzheimer's disease or other tauopathy in an amount sufficient to eliminate or reduce the risk of, reduce the severity of, or delay the onset of the disease, including biochemical, histological, and /or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes recorded during the development of the disease. In the case of therapeutic applications, pharmaceutical compositions containing tau peptide are administered to a patient suspected of or already suffering from such a disease in an amount sufficient to cure or at least partially suppress the symptoms of the disease (biochemical, histological and/or behavioral), including its complications and intermediate pathological phenotypes during the development of the disease.

Effective doses of the pharmaceutical composition of the present invention for the prevention and/or treatment of a neurodegenerative disease, disorder or condition will vary depending on many different factors, including the route of administration, the destination, the physiological condition of the patient, other drugs administered and whether the treatment is prophylactic or

- 17 044254 therapeutic. The amount of peptides depends on whether an adjuvant is administered, with higher doses required in the absence of an adjuvant. The timing of injections can vary significantly from once a day to once a year or once a decade. A typical regimen consists of immunization followed by booster injections at intervals such as 6-week intervals. Another regimen consists of immunization followed by booster injections at 1, 2, 6, 9, and 12 months. Another regimen involves an injection every two months for the rest of your life. Alternatively, repeat injections may be irregular, as indicated by monitoring the immune response.

Those skilled in the art will appreciate that the schedule for initial and repeat administrations can be adjusted based on the determination of immune responses following administration. For example, booster compositions are typically administered weeks or months after administration of the primary composition, for example, 2-3, or 4, or 8, or 16, or 20, or 24, or 26, or 28, or 30, or 32 , or 36 weeks, or one to two years after the introduction of the primary composition.

Peptides can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterial, intracranial, intraperitoneal, intradermal, intranasal, or intramuscular for prophylactic and/or therapeutic treatment. The most typical route of administration of an immunogenic agent is subcutaneous or intramuscular injection. This last type of injection is most often performed in the muscles of the arms or legs.

According to specific aspects, one or more booster immunizations may be administered. The antigens in the respective primary and booster compositions, however multiple booster compositions are used, need not be identical, but must have common antigenic determinants or be substantially similar to each other.

The composition may, if desired, be presented in a kit, package or dispenser, which may contain one or more unit dosage forms containing the active ingredient. The kit may, for example, contain metal foil or polymer film, such as a blister pack. The kit, package or dispenser may be accompanied by instructions for administration.

In accordance with specific embodiments, the kit contains at least one of a pharmaceutical composition comprising a liposome in accordance with an embodiment of the present invention and a pharmaceutical composition containing a conjugate in accordance with an embodiment of the present invention.

Embodiments of the Invention

The present invention also provides the following non-limiting embodiments.

Embodiment 1 is a liposome containing:

a) tau peptide; And

b) epitope for T helper cells;

wherein the tau peptide is present on the surface of the liposome.

Embodiment 2 is the liposome of Embodiment 1, in which the tau peptide is a tau phosphopeptide.

Embodiment 3 is the liposome of Embodiment 1 or 2, further comprising a toll-like receptor ligand.

Embodiment 4 is the liposome of Embodiment 3, wherein the toll-like receptor ligand includes at least one of toll-like receptor ligand 4 and toll-like receptor ligand 9.

Embodiment 5 is the liposome of Embodiment 3 or 4, wherein the toll-like receptor ligand is toll-like receptor ligand 4.

Embodiment 6 is the liposome of Embodiment 5, wherein the toll-like receptor 4 ligand includes monophosphoryl lipid A (MPLA).

Embodiment 7 is the liposome of Embodiment 3 or 4, wherein the toll-like receptor ligand is toll-like receptor ligand 9.

Embodiment 8 is the liposome of Embodiment 7, wherein the toll-like receptor 9 ligand includes a lipidated CpG oligonucleotide.

Embodiment 9 is the liposome of Embodiment 1 containing:

a) tau peptide;

b) epitope for T helper cells; And

c) at least one of

i) toll-like receptor ligand 9 and ii) toll-like receptor ligand 4.

Embodiment 10 is the liposome of Embodiment 9, wherein the tau peptide is a tau phosphopeptide.

Embodiment 11 is the liposome of Embodiment 9 or 10, in

- 18 044254 in which the toll-like receptor 9 ligand is a lipidated CpG oligonucleotide.

Embodiment 12 is a liposome as in any one of embodiments 911 that contains toll-like receptor 4 ligand and toll-like receptor ligand 9.

Embodiment 13 is the liposome of Embodiment 12, wherein the toll-like receptor 4 ligand includes monophosphoryl lipid A (MPLA).

Embodiment 14 is a liposome containing:

a) tau phosphopeptide;

b) epitope for T helper cells;

c) lipidated CpG oligonucleotide; And

d) an adjuvant containing a toll-like receptor 4 ligand; wherein the tau phosphopeptide is presented on the surface of the liposome.

Embodiment 15 is the liposome of Embodiment 14, wherein the toll-like receptor 4 ligand includes monophosphoryl lipid A (MPLA).

Embodiment 16 is a liposome as in any one of embodiments 115, wherein the helper T cell epitope is encapsulated in the liposome.

Embodiment 16a is a liposome as in any one of embodiments 1-15, wherein the helper T cell epitope is included in the liposome membrane.

Embodiment 16b is a liposome as in any one of embodiments 1-15, wherein the epitope for T helper cells is presented on the surface of the liposome.

Embodiment 17 is a liposomal composition comprising:

a) tau phosphopeptide;

b) epitope for T helper cells;

c) lipidated CpG oligonucleotide; And

d) monophosphoryl lipid A (MPLA);

wherein the tau phosphopeptide is presented on the surface of the liposome, and the epitope for T-helper cells is encapsulated in the liposome.

Embodiment 17a is the liposome of Embodiment 17, wherein the MPLA is 3-O-deacyl-4'-monophosphoryl lipid A, preferably MPL™.

Embodiment 17b is the liposome of Embodiment 17, wherein the MPLA is monophosphoryl hexaacyl lipid A, 3-deacylylated, preferably 3D-(6-acyl)PHAD®.

Embodiment 17c is the liposome of Embodiment 17, wherein the MPLA is monophosphoryl-3-deacyllipid A, preferably 3D-PHAD®.

Embodiment 18 is a liposome as in any one of Embodiments 117c, further comprising one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero -3-phosphoryl-3'-racglycerol (DMPG) and cholesterol.

Embodiment 19 is a liposome as in any one of embodiments 118, wherein the tau peptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 12 or at least 85, 90 or 95% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 12.

Embodiment 19-1 is the liposome of Embodiment 19, wherein the tau peptide is a phosphopeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-3 and 5-12.

Embodiment 19-2 is the liposome of Embodiment 19-1, wherein the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 1.

Embodiment 19-3 is the liposome of Embodiment 19-1, wherein the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 2.

Embodiment 19-4 is the liposome of Embodiment 19-1, wherein the tau phosphopeptide comprises the amino acid sequence of SEQ ID NO: 3.

Embodiment 19a is a liposome as in any one of Embodiments 19, 19-1, 19-2, 19-3, and 19-4, wherein the amino acid sequence further contains one or more modifications to allow the tau peptide to be presented on the surface of the liposome .

Embodiment 19b is the liposome of Embodiment 19a, wherein the one or more modifications include at least one of palmitoylation and dodecyl modification.

Embodiment 19c is the liposome of Embodiment 19a or 19b in which the tau peptide is modified at its N-terminus by one or more modifications.

Embodiment 19d is a liposome as in any one of embodiments 19a to 19c, wherein the tau peptide is modified at its C-terminus by one or more modifications.

- 19 044254

Embodiment 19e is the liposome of Embodiment 19d in which the tau peptide is palmitoylated at both its N-terminus and its C-terminus.

Embodiment 19f is a liposome according to any one of embodiments

19a-19e, wherein the tau peptide further contains one or more additional amino acids to facilitate one or more modifications.

Embodiment 19g is the liposome of Embodiment 19f, wherein one or more additional amino acids are selected from the group consisting of Lys, Cys, Ser and Thr.

Embodiment 19h is a liposome as in any of embodiments 19-19g, wherein the tau peptide is amidated at its C-terminus.

Embodiment 19i is a liposome as in any one of embodiments 19-19h, wherein the tau peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 27 through SEQ ID NO: 38.

Embodiment 19j is the liposome of any one of embodiments 19-19i, wherein the tau peptide consists of the amino acid sequence SEQ ID NO: 27.

Embodiment 19k is a liposome as in any one of embodiments 19-19i, wherein the tau peptide consists of the amino acid sequence of SEQ ID NO: 28.

Embodiment 19i is a liposome as in any one of embodiments 19-19i, wherein the tau peptide consists of the amino acid sequence of SEQ ID NO: 29.

Embodiment 20 is a liposome as in any one of Embodiments 119l, wherein the T helper cell epitope comprises at least one amino acid sequence selected from the group consisting of: SEQ ID NO: 23-SEQ ID NO: 26.

Embodiment 20a is the liposome of Embodiment 20, wherein the T helper cell epitope comprises at least two amino acid sequences selected from the group consisting of: SEQ ID NO: 23-SEQ ID NO: 26.

Embodiment 20b is the liposome of Embodiment 20, wherein the T helper cell epitope comprises at least three amino acid sequences selected from the group consisting of: SEQ ID NO: 23 to SEQ ID NO: 26.

Embodiment 20c is the liposome of Embodiment 20, wherein the T helper cell epitope comprises the four amino acid sequences: SEQ ID NO: 23-SEQ ID NO: 26.

Embodiment 20d is a liposome as in any one of embodiments 20a to 20c, wherein two or more amino acid sequences selected from the group consisting of SEQ ID NO: 23 to SEQ ID NO: 26 are covalently linked by a linker.

Embodiment 20e is the liposome of Embodiment 20d, wherein the linker includes one or more amino acids selected from the group consisting of Val (V), Ala (A), Arg (R), Gly (G), Ser (S) , Lys (K).

Embodiment 20f is the liposome of Embodiment 20e, wherein the linker includes an amino acid sequence selected from the group consisting of VVR, GS, RR and RK.

Embodiment 20g is a liposome as in any one of embodiments 20-20f, wherein the helper T cell epitope is amidated at its C-terminus.

Embodiment 20h is a liposome as in any one of embodiments 20-20g, wherein the T helper epitope is modified to be inserted into the membrane of the liposome, presented on the surface of the liposome, or encapsulated within the liposome, depending on the intended location of the T helper epitope.

Embodiment 20i is a liposome as in any one of embodiments 20-20h, wherein the T helper cell epitope consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 13-SEQ ID NO: 17.

Embodiment. 20j is a liposome as in any one of embodiments 1-20i that contains tau peptide and T helper cell epitope in a 6:1 weight ratio.

Embodiment 20k is a liposome as in any one of embodiments 1-20i that contains tau peptide and T helper epitope in a 5:1 weight ratio.

Embodiment 20l is a liposome as in any one of embodiments 1-20i, which contains tau peptide and T helper cell epitope in a 4:1 weight ratio.

Embodiment 20m is a liposome as in any one of embodiments 1-20i, which contains tau peptide and T helper cell epitope in a 3:1 weight ratio.

Embodiment 20n is a liposome as in any one of embodiments 1-20i, which contains tau peptide and T helper epitope in a 2:1 weight ratio.

Embodiment 20o is a liposome as in any one of embodiments 1-20i, which contains tau peptide and T helper cell epitope in a 1:1 weight ratio.

Embodiment 21 is a liposome as in any one of Embodiments 120o, wherein the lipidated CpG oligonucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NO: 18-SEQ ID NO: 22.

Embodiment 21a is the liposome of Embodiment 21 in which

A CpG oligonucleotide has one or more phosphorothioate internucleotide linkages.

Embodiment 21b is the liposome of Embodiment 21a in which the CpG oligonucleotide has all phosphorothioate internucleotide linkages.

Embodiment 21c is a liposome as in any one of embodiments 21-21b, wherein the lipidated CpG oligonucleotide includes a CpG oligonucleotide covalently linked to at least one lipophilic group through a linker.

Embodiment 21d is the liposome of Embodiment 21c, wherein the linker comprises (C ₂ H ₄ O) _n , where n is an integer from 0 to 10.

Embodiment 21e is the liposome of Embodiment 21c, wherein the linker contains an alkyl spacer having from 3 to 12 carbon atoms.

Embodiment 21f is a liposome as in any one of embodiments 21-21e, wherein at least one lipophilic group is cholesterol.

Embodiment 21g is a liposome as in any one of Embodiments 21-21f, wherein the lipidated CpG oligonucleotide comprises the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19 covalently linked to a cholesterol molecule through a linker containing (C ₂ H ₄ O) _n , where n is an integer between 3 and 5.

Embodiment 22 is a liposome containing:

a) a tau peptide having an amino acid sequence selected from the group consisting of SEQ ID NO:27-SEQ ID NO:38;

b) a T helper epitope having an amino acid sequence selected from the group consisting of SEQ ID NO: 39-SEQ ID NO: 44, preferably a T helper epitope consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13-SEQ ID NO: 17;

c) a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide contains one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to at least at least one cholesterol via a linker; And

d) monophosphoryl lipid A (MPLA).

Embodiment 22a is the liposome of Embodiment 22 containing:

a) a tau phosphopeptide consisting of the amino acid sequence of SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29;

b) an epitope for T helper cells consisting of the amino acid sequence SEQ ID NO: 13

c) a lipidated CpG oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 18 or SEQ ID NO: 19, covalently linked to cholesterol through a linker containing (C ₂ H ₄ O) _n , where n is an integer from 3 to 7 ; And

d) monophosphoryl lipid A (MPLA).

Embodiment 22b is the liposome of Embodiment 22 or 22a, wherein the MPLA is 3-O-deacyl-4'-monophosphoryl lipid A, preferably MPL™.

Embodiment 22c is the liposome of Embodiment 22 or 22a, wherein the MPLA is preferably 3D-(6-acyl)PHAD®.

Embodiment 22d is a liposome according to embodiment 22 or 22a, wherein the MPLA is preferably 3D-PHAD®.

Embodiment 23 is a liposome as in any one of embodiments 22-22d, wherein the helper T cell epitope is encapsulated in the liposome.

Embodiment 24 is a pharmaceutical composition comprising the liposome of any one of embodiments 1-23 and a pharmaceutically acceptable carrier.

Embodiment 25 is a conjugate comprising a tau phosphopeptide and an immunogenic carrier conjugated thereto through a linker having the following structure:

where x is an integer from 0 to 10; And

- 21 044254 n represents an integer from 2 to 15.

Embodiment 25a is a conjugate comprising a tau phosphopeptide and an immunogenic carrier conjugated thereto through a linker having the structure of formula (II):

where x is an integer from 0 to 10; and n is an integer from 2 to 15.

Embodiment 26 is a conjugate of Embodiment 25 or 25a, wherein x is an integer from 2 to 6.

Embodiment 27 is a conjugate of Embodiment 25 or 25a in which x is 3.

Embodiment 28 is a conjugate of any of embodiments 25-25a, wherein n is from 3 to 7.

Embodiment 29 is the conjugate of any one of embodiments 25-28, wherein the carrier is an immunogenic carrier selected from the group consisting of keyhole limpet hemocyanin (KLH), tetanus toxoid, CRM197, and a mixture of outer membrane proteins from N. meningitides ( OMR) or their derivative.

Embodiment 30 is the conjugate of any one of embodiments 25-29, wherein the tau phosphopeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 1-SEQ ID NO: 12.

Embodiment 30a is a conjugate of Embodiment 30, wherein the tau phosphopeptide consists of the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3.

Embodiment 31 is a conjugate of any of embodiments 25-30 wherein the carrier is CRM197.

Embodiment 32 is a conjugate of Embodiment 25 having the structure:

where n is 3-7.

Embodiment 32a is the conjugate of _Embodiment 25, which is KLH-[m-maleimidobenzoyl-N-hydroxysuccinimide ester-cysteine-( _C2H4O ) _x -taupeptide]n

wherein the tau peptide consists of SEQ ID NO: 1 or SEQ ID NO: 3;

x is an integer from 0 to 10; and n is an integer from 2 to 15.

Embodiment 33 is a pharmaceutical composition comprising a conjugate of any of embodiments 25-32a and a pharmaceutically acceptable carrier.

Embodiment 33a is the pharmaceutical composition of claim 33, further comprising an adjuvant.

Embodiment 33b is a pharmaceutical composition according to claim 33, wherein

- 22 044254 the adjuvant includes at least one of a TLR-4 ligand and a TLR-9 ligand.

Embodiment 34 is a method of inducing an immune response in a subject suffering from a neurodegenerative disorder, comprising administering to the subject at least one of the pharmaceutical compositions of any one of embodiments 24 and 33-33b.

Embodiment 35 is the method of Embodiment 34, comprising administering to a subject at least one of the pharmaceutical compositions of Embodiments 24 and 33-33b for primary immunization and administering to the subject at least one of the pharmaceutical compositions of Embodiments 24 and 33-33b for booster immunization.

Embodiment 36 is a method of treating or preventing a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject at least one of the pharmaceutical compositions of Embodiment 24 or 33.

Embodiment 37 is the method of Embodiment 36, comprising administering to a subject at least one of the pharmaceutical compositions of Embodiments 24 and 33-33b for primary immunization and administering to the subject at least one of the pharmaceutical compositions of Embodiments 24 and 33-33b for booster immunization.

Embodiment 38 is the method of any one of embodiments 3437, wherein the neurodegenerative disease or disorder is caused by or is associated with the formation of neurofibrillary lesions.

Embodiment 39 is the method of any one of embodiments 3438, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Boxer's dementia, Down syndrome, Gerstmann-Straussler-Scheickner disease, inclusion body myositis, prion protein, cerebral amyloid angiopathy, traumatic brain injury, amyotrophic lateral sclerosis, parkinsonism dementia - Guam complex, non-Guam motor neuron disease with neurofibrillary tangles, dementia characterized by the appearance of argyrophilic grains, corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, diffuse neurofibrils lary balls with calcification, frontotemporal dementia, preferably frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17), frontotemporal lobar dementia, Hallerwarden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, disease Pica, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, dementia with predominant neurofibrillary tangles, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PART) or dementia with Lewy bodies (LBD).

Embodiment 40 is the method of any one of embodiments 3439, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down syndrome, progressive supranuclear palsy (PSP), frontotemporal dementia, and parkinsonism associated with chromosome 17 (FTDP-17 ), Pick's disease, corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, myotonic dysphasia, chronic traumatic encephalopathy (CTE), cerebral angiopathy, primary age-related tauopathy (PART) or dementia with Lewy bodies (LBD).

Embodiment 40b is the method of any one of embodiments 3439, wherein the neurodegenerative disease or disorder is Alzheimer's disease, progressive supranuclear palsy (PSP), frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), or disease Pica and PART (primary age-related tauopathy).

Embodiment 40c is the method of any one of embodiments 3439, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down syndrome, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, Lewy dementia, amyotrophic lateral sclerosis, myotonic dysphasia, chronic traumatic encephalopathy (CTE), cerebral angiopathy, primary age-related tauopathy (PART) or dementia with Lewy bodies (LBD).

Embodiment 41 is a kit containing at least one of the pharmaceutical composition of embodiment 24 and the pharmaceutical composition of embodiment 33, 33a, or 33b.

Embodiment 42 is an epitope for T helper cells consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13-SEQ ID NO: 17.

Embodiment 43 is a pharmaceutical composition containing the T helper epitope of Embodiment 42.

Embodiment 44 is a method of enhancing an immune response to an antigen in a subject in need thereof, comprising administering the antigen to the subject along with the pharmaceutical composition of Embodiment 43.

Examples

The following examples of the present invention serve to further illustrate the essence

- 23 044254 of the present invention. It should be understood that the following examples do not limit the present invention, and that the scope of the invention is to be determined by the appended claims.

The experimental methods used in the following examples, unless otherwise noted, are all conventional methods. The reagents used in the following embodiments, unless otherwise noted, are all purchased from common reagent suppliers.

Example 1. Preparation of liposome-based vaccines.

Preparation of a control vaccine based on liposomes (method of introduction into ethanol).

A control liposome-based vaccine was prepared by infusion of ethanol (EtOH) followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, the Netherlands) and MPLA (Avanti Polar Lipids, AL, USA) were solubilized in a molar ratio of 9:1:7:0. 05 in a mixture of EtOH and tert-butanol (t-BuOH) in a volume ratio of 20:1 at 60°C. A solution of the lipid in ethanol was diluted in phosphate-buffered saline (PBS) pH 7.4 at 60°C with a 10% EtOH concentration and this resulted in the formation of multilamellar liposome vesicles (MLVs). The MLV was then extruded 5 times through three 0.08 μm polycarbonate filters (Whatman) in sequence using Emulsiflex-C5 (Avestin, Canada). The resulting liposomes were diluted in PBS pH 7.4 and heated to 60°C to obtain a liposome solution before adding tau peptide.

Tetrapalmitoylated phosphorylated tau peptide acetate SEQ ID NO: 2 (Bachem AG, Switzerland), herein referred to as the active pharmaceutical ingredient (API), was dissolved in PBS at pH 11.4 with 2.0% octyl-sO-glucopyranoside (Sigma-Aldrich, USA) to a concentration of 1 mg/ml, and the peptide solution was introduced into the liposome solution at 60°C, followed by stirring for 30 min at 60°C. Concentration was accomplished by ultrafiltration to the target final volume, and buffer exchange was performed 10 times using PBS pH 7.4 during diafiltration. The resulting liposomes with API present on the surface of the liposomes were then filter sterilized by passing through two 0.2 μm polycarbonate syringe filters sequentially, and the final product was stored at 5°C.

Preparation of vaccines based on liposomes X, Y, Z u Z ⁺ .

Liposome X and Y based vaccines were prepared using lipid thin film technology followed by homogenization and extrusion.

Liposome Z ⁺ vaccines with a final API concentration of 1200 μg/ml and a final T50 concentration of 1200 μg/ml were prepared by ethanol injection followed by extrusion, and Liposome Z vaccines with a final API concentration of 400 µg/ml, and a final T50 concentration of 100 µg/ml was obtained using lipid thin film technology followed by homogenization and extrusion.

Z++ liposome-based vaccines with a final API concentration of 400 μg/ml and a final T50 concentration of 400 μg/ml were prepared using lipid thin film technology followed by homogenization and extrusion.

Z ⁺⁺⁺ liposome vaccines with a final API concentration of 1200 μg/ml and a final T50 concentration of 300 μg/ml were prepared by ethanol injection followed by extrusion.

Preparation of vaccines based on liposomes X, Y, Z and Z ⁺⁺ using the thin lipid film method.

Vaccines based on liposomes X, Y, Z and Z ⁺⁺ were prepared using lipid thin film technology followed by homogenization and extrusion. First DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, the Netherlands) and monophosphorylhexaacyl lipid A 3-deacylated synthetic (3D-(6-acyl)PHAD)® (Avanti Polar Lipids, AL, USA) were solubilized in a molar ratio of 9:1:7:0.05 in EtOH at 60°C, with the exception of liposome Y, which did not contain 3D-(6-acyl) PHAD®. Ethanol was evaporated under vacuum in a rotary evaporator to obtain a thin lipid film.

The lipid film was rehydrated using PBS pH 7.4, 5% DMSO (all Sigma-Aldrich) containing 0.15 mg/ml T50 peptide (Peptides & Elephants, Germany). The sample was mixed gently for 15 min and further shaken vigorously to dissolve the thin lipid film. The resulting multilamellar vesicles were subjected to 10 freeze-thaw cycles (liquid _N2 and 37°C water bath) and homogenized followed by sequential extrusion through 0.08-μm polycarbonate membranes (Whatman, UK). Both homogenization and extrusion steps were performed in EmulsiFlex-C5 (Avestin, Canada). Extruded liposomes with encapsulated T50 peptide were concentrated by ultrafiltration, and buffer exchanged to PBS pH 7.4 by diafiltration. The resulting liposomes were diluted in PBS pH 7.4 and heated to 60°C to obtain a liposome solution before adding tau peptide and adjuvant.

CpG2006-cholesterol (CpG2006-Chol) (Microsynth, Switzerland) is a DNA oligonucleotide with all internucleotide bonds in the form of thiophosphate, which is modified at the 5' end with a cholesterol molecule through a phosphate bond through a spacer including PEG.

- 24 044254

CpG2006-cholesterol (CpG2006-Chol) (Microsynth, Switzerland) was dissolved in PBS pH 7.4 to 1 mg/ml and added to liposome solutions (except for liposome X, which does not contain CpG2006-Chol) followed by incubation for 15 min before inserting the API.

API (Bachem AG, Switzerland) was dissolved in PBS pH 11.4 with 2% octyl-β-D-glucopyranoside (Sigma-Aldrich, USA) to a concentration of 1 mg/ml, and the peptide solution was poured into the liposome solution at 60°C with followed by stirring for 30 minutes at 60°C. Concentration was carried out by ultrafiltration to obtain the target value (400 μg/ml API and 100 μg/ml T50 for liposome X, Y, Z; and 400 μg/ml API and 400 μg/ml T50 for liposome Z ⁺⁺ ), and buffer exchange carried out 10 times using PBS pH 7.4 during diafiltration. The resulting liposomes with API present on the surface of the liposomes were then filter sterilized through 0.2 μm polycarbonate syringe filters and the final product was stored at 5°C.

Preparation of liposome O by the method of introduction into ethanol.

The vaccine based on liposome O was prepared by injection into ethanol (EtOH) followed by extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, the Netherlands) and MPLA (Avanti Polar Lipids, AL, USA) were solubilized in a molar ratio of 9:1:7:0. 05 in a mixture of EtOH and tert-butanol (t-BuOH) in a volume ratio of 20:1 at 60°C. A solution of the lipid in ethanol was diluted in phosphate-buffered saline (PBS) pH 7.4 at 60°C with a 10% EtOH concentration and this resulted in the formation of multilamellar liposome vesicles (MLVs). The MLV was then extruded 5 times through three 0.08 μm polycarbonate filters (Whatman) in sequence using Emulsiflex-C5 (Avestin, Canada). The resulting liposomes were diluted in PBS pH 7.4 and heated to 60°C to obtain a liposome solution before adding tau peptide.

T46 peptide (Pepscan, Netherlands) was dissolved in PBS pH 7.4 to 1 mg/ml and poured into liposome solutions, followed by incubation for 15 min before adding API.

API (Bachem, Switzerland) was dissolved in PBS pH 11.4 with 2% octyl-sO-glucopyranoside (SigmaAldrich, USA) to obtain a concentration of 1 mg/ml, and the peptide solution was poured into the liposome solution at 60°C, followed by stirring for 30 min at 60°C. Concentration was carried out by ultrafiltration to obtain the target value (400 μg/ml API and 100 μg/ml T46), and buffer exchange was carried out 10 times using PBS pH 7.4 during diafiltration. The resulting liposomes with API present on the surface of the liposomes were then filter sterilized through 0.2 μm polycarbonate syringe filters and the final product was stored at 5°C.

Preparation of vaccines based on liposome Z ⁺ and liposome Z + by introducing into ethanol.

Z ⁺ liposome and Z+++ liposome vaccines were prepared using an ethanol injection-based process. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, The Netherlands) and 3D-(6-acyl)PHAD® (Avanti Polar Lipids, AL, USA) were solubilized in a molar ratio approximately 9:1:7:0.04 in EtOH at 60°C. Peptide T50 (Bachem AG, Switzerland) was dissolved in 10 mM His/270 mM sucrose (pH 5.8-6.0). The ethanol solution of the lipid was then poured into the solution containing the T50 peptide and stirred gently for 15 min, resulting in the formation of multilamellar vesicles (MLVs). MLV was homogenized (6 times in the case of Z ⁺ liposome and without homogenization in the case of Z+++ liposome) followed by sequential extrusion through polycarbonate membranes (Whatman, UK) with a pore size of 0.08 μm (5 passes in the case of Z ⁺ liposome, 3-5 times in the case of liposomes Z^ ⁺ ). Both homogenization and extrusion steps were performed in EmulsiFlex-C5 (Avestin, Canada) for Z ⁺ liposome. Extrusion of the Z+++ liposome was carried out using a LIPEX filter extruder. Extruded liposomes were concentrated by ultrafiltration, and the buffer was exchanged to 20 mM His/145 mM NaCl pH 7.4 by diafiltration. The resulting T50 peptide-encapsulated liposomes were diluted in 20 mM His/145 mM NaCl pH 7.4 and heated to 60°C to obtain a liposome solution before adding API and adjuvant.

CpG2006-Chol (Microsynth, Switzerland for Z ⁺ liposome; Avecia, USA for Z+++ liposome) was dissolved in 20 mM His/145 mM NaCl pH 7.4 to 1 mg/ml and poured into the liposome solution, followed by incubation for 15 min before introduction of API.

API (Bachem AG, Switzerland) was dissolved in carbonate buffer pH 10.2 with 1% octyl-β-D-glucopyranoside (Sigma-Aldrich, USA) to obtain a concentration of 1 mg/ml, and the peptide solution was poured into the Z ⁺ liposome solution at 60° C, followed by stirring for 30 minutes at 60°C. The peptide solution was mixed with the Z ⁺ liposome solution using T-Line Mixing at 60°C, followed by stirring for 30 min at 60°C. Concentration was carried out by ultrafiltration to obtain the target value (1200 μg/ml API and 1200 μg/ml T50 for Z ⁺ liposome; and 1200 μg/ml API and 300 μg/ml T50 for Z+++ liposome), and buffer exchange was carried out 10 times using 10 mM His/270 mM sucrose pH 6.5 during diafiltration. The resulting Z ⁺ liposomes with API present on the liposome surface and the resulting Z+++ liposomes with API present on the liposome surface were then filter sterilized through 0.2 μm polycarbonate syringe filters and the final product was stored at 5°C. WITH.

Preparation of vaccines based on liposomes L, M and N.

Vaccines based on L, M and N liposomes were prepared using thin lipid film technology followed by homogenization and extrusion. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, the Netherlands) and MPLA (Avanti Polar Lipids, AL, USA) were solubilized in a molar ratio of 9:1:7:0. 05 in EtOH at 60°C. Ethanol was evaporated under vacuum in a rotary evaporator to obtain a thin lipid film.

The lipid film was rehydrated using PBS pH 7.4, 5% DMSO (all Sigma-Aldrich) containing either:

0.15 mg/ml T48 peptide (Peptides & Elephants, Germany) - for liposome M; or

0.13 mg/ml peptide T50 (Peptides & Elephants, Germany) - for liposome L; or

0.15 mg/ml T52 peptide (Peptides & Elephants, Germany) - for liposome N.

The sample was mixed gently for 15 min and further shaken vigorously to dissolve the thin lipid film. The resulting multilamellar vesicles were subjected to 10 freeze-thaw cycles (liquid _N2 and 37°C water bath) and homogenized followed by sequential extrusion through 0.08-μm polycarbonate membranes (Whatman, UK). Both homogenization and extrusion steps were performed in EmulsiFlex-C5 (Avestin, Canada). Extruded liposomes were concentrated by ultrafiltration and buffer exchanged to PBS pH 7.4 by diafiltration. The resulting liposomes encapsulated with T48, T50 or T52 peptide were diluted in PBS pH 7.4 and heated to 60°C to obtain a liposome solution before adding tau peptide.

API (Bachem AG, Switzerland) was dissolved in PBS pH 11.4 with 2% octyl-β-D-glucopyranoside (Sigma-Aldrich, USA) to obtain a concentration of 1 mg/ml, and the peptide solution was poured into the liposome solution at 60°C followed by stirring for 30 minutes at 60°C. Concentration was carried out by ultrafiltration to obtain the target value (400 μg/ml API and 100 μg/ml T48, T50 or T52), and 10 buffer exchanges were performed with PBS pH 7.4 during diafiltration. The resulting liposomes with API present on the surface of the liposomes were then filter sterilized through 0.2 μm polycarbonate syringe filters and the final product was stored at 5°C.

Preparation of vaccines based on liposomes R, S and T.

R, S and T liposome vaccines were prepared using an ethanol injection followed by extrusion process. First, DMPC (Lipoid GmbH, Ludwigshafen, Germany), DMPG (Lipoid GmbH, Ludwigshafen, Germany), cholesterol (Dishman, the Netherlands) and 3D-(6-acyl)PHAD)® (Avanti Polar Lipids, AL, USA) were solubilized at molar ratio 9:1:7:0.04 in EtOH at 60°C. For liposomes R and T, the above lipid solution in ethanol was mixed with 10 mM histidine pH 5.8 supplemented with 270 mM sucrose to achieve a 10% solvent concentration (EtOH) and then incubated for 30 min at 60°C. For liposome S, T50 peptide (Bachem AG, Switzerland) was dissolved in 10 mM His/270 mM sucrose (pH 5.8–6.0). Buffer-lipid mixtures belonging to liposomes R, S and T were mixed gently for 15 min, resulting in the formation of multilamellar vesicles (MLVs). The resulting multilayer vesicles were extruded through polycarbonate membranes (Whatman, UK) with a pore size of 0.08 μm (5X) in an EmulsiFlex-C5 high-pressure system (Avestin, Canada).

Extruded liposomes were concentrated by ultrafiltration, and the buffer was exchanged to 20 mM His/145 mM NaCl pH 7.4 by diafiltration. The resulting liposomes with encapsulated T50 in the case of liposome S and the resulting liposomes R and T were further diluted in 20 mM His/145 mM NaCl pH 7.4 and heated to 60°C to obtain a liposome solution before adding API and T57 in the case of liposome T.

In the case of liposome T, T57 was dissolved to 1 mg/ml in 1% octyl-in-O-glucopyranoside (Sigma-Aldrich, USA) in deionized distilled water and introduced into the liposome, followed by incubation for 15 min at 60°C before adding API .

API (Bachem AG, Switzerland) was dissolved in carbonate buffer pH 10.2 with 1% octyl-β-D-glucopyranoside (Sigma-Aldrich, USA) to obtain a concentration of 1 mg/ml, and the peptide solution was mixed into the liposome solution at 60°C with followed by stirring for 30 minutes at 60°C. Concentration was carried out by ultrafiltration to obtain the following target value:

1200 µg/ml API for liposome R;

1200 µg/ml API and 300 µg/ml T50 for liposome S; And

1200 µg/ml API and 300 µg/ml T57 for T liposome.

Buffer exchange was performed 10 times using 10 mM His/270 mM sucrose pH 6.5 during diafiltration. The resulting liposomes with the API present on the surface of the liposomes were then filter sterilized by passing through 0.2 μm polycarbonate syringe filters, and

- 26 044254 the final product was stored at 5°C.

Example 2. Preparation of a vaccine based on a conjugate.

Peptides and adjuvants.

The sequences of two polyphosphorylated peptide epitopes (TAUVAC-p7.1 and TAUVAC-p22.1, which have three and two phosphorylated amino acids, respectively) were improved by optimizing the length so that they could better bind to B cell surface immunoglobulin, and so that the sequences did not contain epitopes predicted to bind with high affinity to human HLA class IA, B, and C molecules. The latter criterion was important to avoid the induction of a cytotoxic CD8+ T cell response to tau, which could potentially cause significant neuronal damage. Using the T-cell epitope prediction tool from the Immune Epitope Database and analysis resources, the TAUVAC-p7.1 peptide did not identify predicted epitopes capable of binding to human HLA class IA, B, C and HLA class II molecules with high affinity, while the TAUVAC-p22.1 peptide was predicted to contain epitopes that bind to HLA class II DQ and DR molecules with medium/high affinity (data not shown).

Phosphorylated tau peptides (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) used in this study were prepared synthetically (Pepscan, NL) by adding phosphoric acid residues during synthesis. A conjugate comprising a phosphorylated tau peptide having the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 3 covalently linked to a KLH carrier through a linker is herein referred to as conjugate B or conjugate C, respectively. A conjugate comprising a phosphorylated tau peptide having the amino acid sequence of SEQ ID NO: 2 covalently linked to a CRM carrier via a linker is herein referred to as conjugate A.

To obtain conjugates B and C, vaccine peptides were conjugated to the KLH carrier protein through an m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) linker and an additional cysteine at the N-terminus of the peptide. Unbound peptide was removed using a Sephadex G25 column before concentrating the conjugate. The conjugates were mixed before injection with either a strong multicomponent adjuvant (Sigma Adjuvant System, Sigma-Aldrich) or a single-component depotadjuvant (aluminum hydroxide, Alhydrogel®, Invivogen) according to the manufacturer's instructions.

The vaccine peptides were conjugated to the CRM197 carrier protein via a polyethylene glycol (PEG)-cysteine-acetamidopropionamide linker.

Phosphorylated tau peptide having the amino acid sequence SEQ ID NO: 2 was produced synthetically (Polypeptide Laboratories SAS) by adding phosphoric acid residues and a PEG3 spacer during synthesis. Conjugate A was prepared by conjugating the CRM197 carrier protein via a succinimidyl-3-(bromoacetamide) propionate (SBAP) linker to a cysteine at the N-terminus of the peptide. SBAP was ligated to the primary amines of the CRM197 protein (-NH2) via reaction chemistry with an activated NHS ester cross-linker. Excess linker as SBAP was removed using ultrafiltration and diafiltration (UF/DF). The CRM197-SBAP intermediate was conjugated to phosphorylated tau peptide, and after completion of the reaction, the conjugation reaction was stopped by adding an excess amount of L-cystine to quench the reaction. The crude CRM197-peptide conjugate product was purified using a Capto Q ImpRes chromatography column (GE Healthcare) and eluted using a salt isocratic method. The purified CRM197 peptide product was then spiked into 20 mM Tris, 250 mM sucrose, pH 8.1 to a concentration of 0.5 mg/mL using ultrafiltration/diafiltration. Drug substance (DS) CRM197-tau peptide was prepared by adding 10% PS80 basal buffer to achieve a final concentration=0.01% PS80. The solution was thoroughly mixed before filtering.

Example 3 Vaccine-induced IgG antibodies specific for tau peptide.

All animal experiments were authorized and conducted in accordance with local animal experimentation laws. Rhesus monkeys (Masaca mulatta) were obtained from Kunming Biomed International Ltd, China, Yunnan Yinmore Bio-Tech Co. LTD, China and Yunnan Laboratory Primates Inc., China. Animals were between two and five years old at the start of immunization and had a minimum weight of 2.5 kg. Detailed clinical examination was performed before treatment and weekly thereafter. In addition, the macaques were observed twice daily and clinical signs were recorded.

Adult rhesus monkeys (n=3 males and 3 females per group) were immunized subcutaneously with 1800 μg tetrapalmitoylated phosphorylated tau peptide acetate SEQ ID NO: 2 per dose of liposome control vaccine (tetrapalmitoylated phosphorylated tau peptide liposomes SEQ ID NO: 2 and MPLA) or a liposome-based vaccine according to an embodiment of the present invention, for example, Liposome Z (tetrapalmitoylated phosphorylated tau peptide liposome SEQ ID NO: 2, 3D-(6-acyl)PHAD® lipidated CpG oligonucleotide CpG 2006 and T-cell recognized peptide T50) or 15 μg per dose of a vaccine based on a conjugate in accordance with an embodiment of the present invention (for example, conjugate A, phosphorylated tau peptide SEQ ID NO: 2 linked to CRM197) , administered together with alum and CpG oligonucleotide CpG 2006 on days 1, 29 and 85. Blood draws were performed before immunization and on days 8, 22, 36, 50, 64, 78, 92, 106, 120, 134 and 148, and serums were isolated.

Specific IgG antibody titers were determined by ELISA using phosphorylated tau peptide SEQ ID NO: 2 as the coating antigen. Serum from an individual immunized monkey was serially diluted in assay buffer (PBS, 0.05% Tween 20, 1% BSA) and added to 96-well plates that had been coated with the appropriate peptide. After two hours of incubation, samples were removed and plates were washed in PBST (PBS, 0.05% Tween 20). Antibodies were detected using HRP-conjugated anti-simian IgG (KPL) followed by ABTS substrate (Roche). All samples were tested in eight 2-fold dilutions, with positive and negative controls included in each plate. Data were presented as the geometric mean of titration endpoints (last serum dilution showing a positive reaction) for each group.

As shown in FIG. 4, both the liposome Z vaccine and conjugate A induced higher titers of phosphopeptide-specific IgG antibodies compared to the control liposome vaccine.

Example 4: Vaccine-induced antibodies specific for pathological tau structures in the human brain.

All brain tissues were obtained from the Netherlands Brain Bank (NBB) and collected from donors after signing informed consent for brain autopsy and the use of samples and clinical information about them for research purposes. Paraffin-embedded brain sections from non-demented (healthy) controls, donors with Alzheimer's disease (AD), frontotemporal dementia with tau pathology (FTD-tau), Pick's disease, primary age-related tauopathy (PART) and progressive supranuclear palsy (PSP) were used. Brain regions included the parietal cortex, middle frontal gyrus, hippocampus, or caudate nucleus.

Specifically, formalin-fixed paraffin-embedded sections of parietal cortex from a human control subject (healthy) and a human Alzheimer's disease subject (AD Braak V/VI) were stained with macaque postimmune serum diluted 1:100 in standard antibody diluent ( immunological). Sections were then washed and stained with HRP-conjugated goat anti-monkey antibody (Abcam). Finally, staining was visualized using 3,3′-diaminobenzidine (DAB; Dako), which leaves a brown specific stain in the presence of horseradish peroxidase (HRP). Slides were counterstained with hematoxylin, dehydrated, and mounted in Quick D histology medium (Klinipath). Pictures were taken using a Leica DC500 microscope.

The results in Fig. 5 show that postimmune sera from rhesus macaques immunized with liposome Z (liposome with tetrapalmitoylated phosphorylated tau peptide SEQ ID NO: 2, 3D-(6-acyl)PHAD®, lipidated CpG oligonucleotide CpG 2006 and T50 T cell recognition peptide) were stained pathological tau structures in human brain slices. Serum was collected from rhesus monkeys on day 106 after primary immunization with improved liposomes. This macaque was immunized three times at 0, 1, and 3 months before serum collection. The left panel (AD Braak V/VI) shows staining in the parietal cortex from a Braak stage V donor. Arrows indicate tau tangle staining. The right (healthy donor) panel shows staining of the parietal cortex from a Braak stage 0 donor. Serum was applied to sections at a 1:100 dilution followed by the addition of goat anti-simian antibody at a 1:100 dilution, and the staining was visualized using DAB developer.

The results in Fig. 6 show that sera from rhesus macaques immunized with Conjugate A, which contains phosphorylated tau peptide SEQ ID NO: 2 plus soluble CpG and aluminum hydroxide, binds to pathological tau structures in human Alzheimer's disease (AD) brain slices. Serum was collected on day 106 after primary immunization with the CRM conjugate vaccine. These macaques were immunized three times at 0, 1, and 3 months before serum collection. Upper (AD) panels show staining of the parietal cortex, including tau tangles, from a Braak stage V donor. Lower (CTRL) panels show staining of the parietal cortex from a Braak stage 0 donor. Serum was applied to sections at a 1:100 dilution followed by the addition of goat anti-simian. antibodies were diluted 1:100, and staining was visualized using DAB developer.

Example 5: Liposome-based vaccines with one or two adjuvants.

The addition of two adjuvants to an improved liposome-based vaccine increases the level of tau phosphopeptide-specific IgG antibody titers as well as the consistency of the humoral response between individuals.

Adult rhesus monkeys (n=3 males and 3 females per group) were immunized subcutaneously on days 1, 29, 85, and 169 with 1800 μg tetrapalmitoylated phosphorylated taupeptide acetate SEQ ID NO: 2 per dose of liposome-based control vaccine or improved vaccine based on a liposome with an encapsulated T50 epitope recognized by T cells, containing or

- 28 044254 3D-(6-acyl)PHAD® adjuvant only (X liposomes, Fig. 7A), CpG 2006 lipidated oligonucleotide adjuvant only (Y liposomes, Fig. 7B), or as 3D-(6-acyl)PHAD®, and lipidated oligonucleotide CpG 2006 as adjuvants (liposomes Z, Fig. 7C). Blood draws were performed before immunization and on days 8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, and 190, and sera were isolated. IgG specific antibody titers in the sera were determined by ELISA using phosphorylated tau peptide SEQ ID NO: 2 as the coating antigen and a second anti-simian IgG antibody. The resulting antibody levels are presented as titration endpoints (the last serum dilution showing a positive reaction) for each individual monkey over time. Each immunization group is presented in one panel (Fig. 7A-C). The geometric mean of the titration endpoints for each group ±95% confidence interval is presented in Fig. 7D. Thus, in FIG. 7A-D show that the inclusion of two adjuvants in a liposome-based vaccine containing encapsulated T50 improved the level and consistency of the humoral response to tau phosphopeptide, resulting in less variability in the humoral response in individual monkeys. More specifically, as shown in FIG. 7D, improved liposome-based vaccines with phosphorylated tau peptide SEQ ID NO: 2, T50 epitope recognized by T cells (liposomes X, Y and Z) and 1 or 2 adjuvants induced higher anti-tau phosphopeptide antibody titers than the control vaccine based on a liposome without an epitope recognized by T cells. All monkeys responded to each of the improved liposome-based vaccines, whereas 4 of 6 animals responded to the control liposome-based vaccine.

Example 6: Vaccine-induced antibodies specific for enriched paired helical filament (ePHF).

Groups of rhesus macaques (n=3 males and 3 females per group) were immunized subcutaneously by vaccination on day 1 and day 29 with (i) an improved liposome-based vaccine containing the T-cell recognized T50 epitope and only 3D-( 6-acyl)PHAD® (X liposomes), (ii) an improved liposome-based vaccine containing a T cell-recognized T50 epitope and only a lipidated CpG 2006 adjuvant (Y liposomes), (iii) an improved liposome-based vaccine containing a T-recognized epitope -cells T50 epitope and two adjuvants (3D-(6-acyl)PHAD® and lipidated CpG 2006, Z liposomes) or (iv) a conjugate vaccine (phosphorylated tau peptide SEQ ID NO: 2 associated with CRM197) administered together with alum and CpG oligonucleotide CpG 2006 (conjugate A).

Enriched paired helical filament (ePHF) preparations were prepared from postmortem brain tissue of patients with histologically confirmed AD by sarcosyl extraction of insoluble tau using a modified method of Greenberg and Davies (Greenberg and Davies, 1991, Proc Natl Acad Sci USA, 87 (15): 5827-31). Enriched paired helical strand (ePHF)-specific antibody titers were assessed using the Mesoscale Discovery (MSD) platform. Streptavidin MSD plates were coated with a biotinylated tau capture antibody (HT7-biotin, ThermoScientific) prior to incubation with ePHF isolated from Alzheimer's disease patients, while ePHF-specific IgG antibodies were further detected using SulfoTag-labeled antibody against Human IgG, which cross-reacts with monkey IgG antibodies. More specifically, ePHF was added to 96-well plates (MSD) with streptavidin in small sections of MSD Gold, preblocked with 1% BSA and coated with biotinylated HT-7 (Thermo Scientific). After a one-hour incubation, the plates were washed with PBST, serial dilutions of sera were added, and incubated for two hours. Bound antibodies were detected using SulfoTag-labeled anti-human IgG followed by a fixation step in 1% PFA before adding reading buffer T. The plates were analyzed using a Sector Imager (MSD). Results were presented in arbitrary units per milliliter (AU/ml) for each individual monkey along with the geometric mean for the group. The titers of ePHF-specific antibodies at day 50 after the first immunization are presented.

Fig. 8 shows that all vaccines induced high titers of ePHF-specific IgG antibodies.

Similar results with high titers of ePHF-specific IgG antibodies were also obtained with other liposomes, such as Z+ liposome, administered to rhesus monkeys by intramuscular injection.

Example 7 Degree of specificity of tau phosphopeptide-specific antibody induced by liposome-based vaccine and conjugate-based vaccine in rhesus monkeys.

Groups of rhesus macaques (n=3 males and 3 females in each group) were immunized subcutaneously on day 1 and day 29 with (i) an improved liposome-based vaccine containing encapsulated T50 and two adjuvants: TLR4 ligand (3D-(6- acyl)PHAD®) and lipidated oligonucleotide CpG 2006 (Z liposomes), and (ii) a conjugate vaccine (phosphorylated tau peptide) SEQ ID NO: 2 associated with CRM) (conjugate A) administered together with alum and CpG -oligonucleotide CpG 2006. The profile of epitope recognition by antibodies was determined using epitope mapping using

- 29 044254 by ELISA three weeks after the second immunization (on day 50), using a library of N-terminally biotinylated 8-mer peptides shifted by one amino acid and covering the phosphorylated tau peptide sequence SEQ ID NO: 2 as well as the sequence SEQ ID NO: 4 (VYKSPVVSGDTSPRHL, a non-phosphorylated tau peptide having the same amino acid sequence as SEQ ID NO: 2), and the corresponding biotinylated full-length peptides.

Fig. 9 shows that monkeys immunized with liposome Z produced IgG antibodies that bind primarily to the N-terminal portion of the phosphorylated peptide SEQ ID NO: 2 (Fig. 9A), whereas monkeys immunized with a conjugate vaccine (phosphorylated tau- peptide SEQ ID NO: 2 associated with CRM) produced IgG antibodies that bind primarily to the C-terminal portion of the tau peptide SEQ ID NO: 2 (Fig. 9B).

Example 8. Increased titers of IgG antibodies specific for tau phosphopeptide induced by a liposome-based vaccine with an encapsulated epitope recognized by T cells.

Three groups of C57BL/6J mice (n=10 in each group) were immunized subcutaneously on days 0 and 14 (i) with a liposome-based vaccine containing the TLR4 agonist (3D-(6-acyl)PHAD®), (R liposomes), ( ii) a liposome-based vaccine containing an encapsulated T-cell-recognized T50 epitope and a TLR4 ligand (3D-(6-acyl)PHAD®) as an adjuvant (S liposomes) or (iii) a liposome-based vaccine containing an anchored, a T57 epitope recognized by T cells on the surface of the liposome (i.e. dipalmitoylated T50) and a TLR4 ligand (3D-(6-acyl)PHAD®) as an adjuvant (T liposomes). The level of IgG antibodies specific for phosphorylated tau peptide SEQ ID NO: 2 was measured 21 and 35 days after the first injection in mouse plasma using ELISA; results are presented as values for individual mice along with group geometric mean ±95% CI presented in arbitrary units (AU) per ml. As shown in FIG. 10A, vaccination with a liposome-based vaccine containing encapsulated T50 (S liposomes) induced significantly higher antibody titers than a control liposome-based vaccine (R liposomes) and a liposome-based vaccine containing an anchored T cell-recognized epitope (Liposomes T) 21 days after the first immunization (Kruskal-Wallis test: p=0.0089 and p=0002, respectively), as well as higher antibody titers than vaccination with the control liposome-based vaccine, and significantly higher antibody titers, than when vaccinated with a liposome-based vaccine containing an anchored T-cell recognized epitope, 35 days after the first immunization (Kruskal-Wallis test: p=0.7591 and p=0053, respectively) (Fig. 10B).

Example 9 Liposome Vaccine-Induced T Cell Response Specific for the T Cell Epitope Incorporated.

Three groups of C57BL/6J mice (n=5 in each group) were immunized subcutaneously on days 0, 14 and 28 (i) with an improved liposome-based vaccine encapsulated with T-cell-recognized T48 peptide (containing the T-cell-recognized epitopes PADRE, T2, T30 and T17 separated by a GS linker) and a TLR4 agonist adjuvant (MPLA), (M liposomes), (ii) an improved liposome-based vaccine with encapsulated T52 (containing the T cell recognized epitopes PADRE, T2 and T30, separated by an RK linker ) and TLR4 agonist (MPLA) as an adjuvant (N liposomes) or (iii) PBS. Mice spleens were collected 42 days after the first immunization for analysis of T cell responses using IL-4- and IFN-γ-specific ELISPOT. Single cell suspensions were incubated with medium, T48 or T52 peptide at a concentration of 10 μg/ml for 48 hours. The plates were incubated with biotinylated monoclonal antibodies against mouse IL-4 or IFN-γ and streptavidin alkaline phosphatase (AP). Halos were developed by adding AR substrate. Fig. 11 shows that restimulation of mouse splenocytes with the same liposome-encapsulated peptide stimulated IL-4 (FIG. 11B) and IFN-γ (FIG. 11A) halo-forming cells, whereas splenocytes from PBS-injected mice did not. were stimulated. This confirmed that adding a T-cell-recognized epitope to the vaccine caused activation of specific T cells, allowing them to further assist tau-specific B cells in producing antibodies.

Example 10 Liposome-based vaccines containing an encapsulated T-cell-recognized epitope and an anchored T-cell-recognized epitope.

Groups of rhesus macaques (n=6 in each group) were immunized subcutaneously on days 1, 29, 85, and 169 (i) with a liposome-based vaccine containing an encapsulated T-cell-recognized T50 epitope and TLR4 ligand (MPLA) as an adjuvant, (L liposomes), (ii) a liposome-based vaccine containing a T-cell-anchored T46 epitope and TLR4 ligand (MPLA) as an adjuvant (O liposomes), and (iii) a control liposome-based vaccine containing TLR4 ligand (MPLA ) as an adjuvant and does not contain a T cell epitope. Blood draws were performed prior to immunization (on day −14) and on days 8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, and 190, and sera were isolated. Specific IgG antibody titers were determined by ELISA using phosphorylated tau peptide SEQ ID NO: 2 as the coating antigen and a secondary anti-simian IgG antibody. The resulting antibody levels were calculated as titration endpoints (last

- 30 044254 dilution of serum showing a positive reaction), and the data were presented as the geometric mean for each group. As shown in FIG. 12, a liposome-based vaccine containing an encapsulated T-cell-recognized epitope (L liposomes) and a liposome-based vaccine containing an anchored T-cell-recognized epitope (O liposomes) each induced higher titers of tau-specific phosphopeptide antibodies than the control liposome-based vaccine without the T-cell epitope.

Example 11. Humoral response in mice induced by a conjugate vaccine.

Female BALB/c mice (14 mice in each group) were immunized with Conjugate B or Conjugate C (containing SEQ ID NO: 1 or SEQ ID NO: 3 covalently linked to KLH) according to the schedule shown in FIG. 13A, and using vaccine candidates that are supplemented with either a strong multi-component adjuvant (Sigma Adjuvant System®, Sigma-Aldrich, henceforth called Ribi) or a single-component depot adjuvant (Alhydrogel®, 2% adjuvant or aluminum hydroxide gel, InvivoGen, henceforth called alum). The amino acid sequence of SEQ ID NO: 1 contains only one amino acid difference compared to the mouse protein, while the sequence of SEQ ID NO: 3 is 100% conserved between humans and mice. Thus, the selected epitopes can reasonably be considered self-proteins in the case of mice, and mice should be an appropriate model to study the limitations that immune tolerance may impose on immunogenicity.

As a first measure of vaccine immunogenicity, flow cytometry was used to measure the induction of follicular T helper (TfH) cells in the cervical lymph nodes draining the site of vaccine administration (four mice in each group). TfH are a specialized population of CD4+ T cells characterized by the expression of CXCR5, PD-1, and ICOS among other molecules. The amount of TfH increases following exposure to vaccine or other immune stimuli, and TfH support B cell affinity maturation in the germinal center (Crotty, 2011, Annual Reviews of Immunology. Vol 29:p621-663). The amount of stimulated TfH is positively correlated with the protective efficacy of vaccines in humans (Bentebibel et al., 2013, Sci Transl Med., 5(176): 176ra32; Spensieri et al., 2013, Proc Natl Acad Sci U S A., 110(35) : 14330-5) and small animals. As shown in FIG. 13B, both vaccines, as well as a control immunization with KLH plus adjuvant, resulted in measurable TfH in vaccinated mice. In addition, all animals given active vaccine (Conjugate B and Conjugate C immunization groups) or active placebo (KLH) plus alum had significantly more TfH than animals given inactive placebo (PBS immunization group) when draining cervical lymph nodes were collected seven days after the first immunization (P=0.0044 for KLH-TAUVAC-p7.1; P=0.0482 for KLH-TAUVAC-p22.1; P=0.0063 for KLH, using dispersive analysis (ANOVA) followed by Dunnett's adjustment for multiple comparisons).

ELISA was performed to determine the serum titer of antibodies binding to tauphosphopeptides and to KLH on day 0 and at four additional time points after immunization (days 14, 28, 56 and 84, see Fig. 13C, D, G and H). As shown in FIG. 13C, immunization with conjugate B induced binding antibodies that reacted with the corresponding vaccine peptide. For animals immunized with Conjugate B plus Ribi adjuvant, binding antibody titers against the vaccine peptide were significantly higher than binding antibody titers induced by active placebo (compare Conjugate B plus Ribi with KLH plus Ribi) at all time points determined (P<0.0001 using analysis of variance followed by Tukey's correction for multiple comparisons). For the group receiving alum as an adjuvant, the difference was significant only on days 56 and 84 (p=0.001 and 0.012, respectively).

Tau-specific antibody production in response to conjugate C (FIG. 13D) was generally lower in magnitude than that in response to conjugate B, although differences in assays (different coating peptide) precluded direct statistical comparisons between the two vaccines. However, anti-Conjugate C antibody titers were significantly higher in mice vaccinated with Conjugate C plus Ribi than in mice receiving active KLH plus Ribi placebo at days 28 and 84 (P=0.001 and 0.008, respectively) postimmunization; titers in the group receiving alum as an adjuvant were not significantly different from those in the active placebo group.

Although the carrier protein protects the phosphopeptide from degradation to some extent in vivo, it is likely that phosphatase cleavage of peptide antigens in vivo could result in exposure of some unphosphorylated peptide to the immune system. To determine whether this exposure resulted in the production of antibodies capable of binding the non-phosphorylated peptide in Conjugate B and Conjugate C, an ELISA was performed using the non-phosphorylated peptides as the coating antigen. As shown in FIG. 13E-F, the response to non-phosphorylated tau peptides was weak, comparable to the active placebo response to the same non-phosphorylated peptide. In addition, in animals immunized with conjugate B and Ribi, titers of binding antibodies against phosphorylated peptide were significantly higher than titers of binding antibodies against non-phosphorylated peptide at all time points (P=0.009 on day 14; P<0.009). 0001 on day 28, and 84 using ANOVA). For the group receiving alum as an adjuvant, the difference was significant only on days 56 and 84 (P=0.0002 and 0.001, respectively). For animals immunized with conjugate C, responses to phosphorylated peptide were higher only with Ribi adjuvant (P<0.0001 on day 28; P=0.001 on days 56 and 84).

Example 12 Antibodies induced by conjugate vaccines bind to physiologically relevant forms of altered tau.

To further determine whether vaccine-induced antibodies could bind to physiologically relevant forms of altered tau, we used postimmune sera from vaccinated mice to stain brain sections collected postmortem from either Alzheimer's disease patients (5 cases of AD) or , affected by other tauopathies (3 cases of PART, FTD, Pick's disease and PSP), or from age-matched healthy animals as controls (5 control cases, CTRL). As expected, sera from control animals (PBS and active placebo immunization groups) did not bind to brain slices, while AT8, a monoclonal antibody that binds to pTau [pSer202, pThr 205], derived from a mouse clone, exhibited strong immunoreactivity in case of tau pathology in the adjacent tissue section of the corresponding area (Fig. 14). Sera from animals immunized with active vaccines, conjugate B and conjugate C, bound pathological tau structures not only in sections from AD patients (data not shown) but also in other tauopathies (Fig. 14). Antibodies induced by conjugate B reacted with (pre)tangles, neuropil filaments and senile plaques in AD cases. These postimmune sera were also able to immunoreact with neurofibrillary tangles and neuropil threads in the brain tissue of patients with PART, inclusion bodies in neurons and neuropil threads in tissue of a patient with FTD-tau (MART P301S), inclusion bodies and astrocytes in some cases of Pick's disease and , finally, with inclusion bodies in neurons, neuropil filaments and astrocytes characteristic of PSP. Conjugate C-induced polyclonal serum antibodies also reacted with pathological tau structures characteristic of each tauopathy. In AD cases, staining was mainly concentrated in neurofibrillary tangles and to a lesser extent in senile plaques and neuropil threads. Lower magnification of the corresponding areas showed similar results (data not known).

Example 13 Vaccine-induced antibodies are functional in mice.

The protective efficacy of the Conjugate B vaccine was tested in an injection model of tauopathy (Peeraer et al., 2015, Neurobiol Dis., 73:83-95). In this model, mice affected by tauopathy through a genetic mutation (P301L) receive an intracerebral injection of enriched PHF isolated from the brain of an AD patient according to the time frame indicated in FIG. 15A. An injection that is given before the onset of transgene-induced tauopathy accelerates the development of tauopathy in these animals. Conversely, when the ePHF source is premixed with an antibody capable of inhibiting the activity of the tau source, such as AT8, the induction of tauopathy is reduced (unpublished data, not shown).

Following the diagram in Fig. 15A, the present inventors assessed the development of tauopathy following stereotactic injection of enriched human PHFs premixed with IgG purified from the serum of animals immunized with Conjugate B, Ribi, or the active control KLH plus Ribi. Two months after injection, the brains of these mice were harvested, and the amount of aggregated tau in the total and sarkosyl-insoluble fractions was determined using a standard biochemical assay. The findings showed that when mice were injected with ePHF that had been premixed with IgG from mice vaccinated with Conjugate B, there was significantly less aggregated phospho-tau in both total (Figure 15B) and sarkosyl-insoluble (Figure 15C) ) fractions compared to control-injected animals (p < 0.0001 KLH Ribi versus KLH-TAUVAC-p7.1 plus Ribi, using ANOVA followed by Holm-Bonferroni adjustment for multiple comparisons). It is generally accepted that sarkosyl-insoluble tau correlates with pathological features of tauopathy, so this result demonstrates that antibodies induced by KLH-TAUVAC-p7.1 vaccination are protective in vivo.

Example 14 Vaccine-induced antibodies are functional in non-human primates.

Rhesus monkeys were immunized with alum conjugate B and CpG oligonucleotide adjuvants (n=6) or KLH (n=2) on days 1, 29, 85 and 169. Blood was collected every 14 days and sera from animals immunized with conjugate B , were tested for reactivity with the immunizing peptide using ELISA (Fig. 16A) and human ePHF using MSD (Fig. 16B). Immunization with conjugate B resulted in a long-lasting and durable humoral response to the vaccine phosphopeptide. Moreover, all animals had measurable levels of antibodies against human ePHF, with 3 of 6 animals showing high reactivity with this antigen. Sera collected from animals 50 days after primary immunization were applied to brain sections from healthy individuals or

- 32,044,254 patients with AD (Fig. 16C). Postimmune sera from the conjugate B immunization group stained pathological tau structures, namely neurofibrillary tangles, neuropil filaments, and senile plaques, in the brain tissue of AD patients, while sera from KLH-immunized mice did not show any reactivity. No staining was observed in control tissue. When tested in the tau immunodepletion assay, conjugate B-treated animals had antibodies capable of binding and depleting sources of tau (p=0.03 on day 50, using ANOVA followed by Dunnett's adjustment for multiple comparisons), although KLH immunization did not trigger production of such antibodies (Fig. 16D). Pre- and post-immunization sera were also tested in a neutralization assay as serially diluted individual samples (Fig. 16E). Change from baseline (CFB) was calculated as the difference between FRET (fluorescence resonance energy transfer) values for readings on day -14 pre-vaccination (baseline) and days 50, 106 and 190 post-vaccination, respectively. The response on a given day after vaccination (day _i ) was then calculated as follows:

response = %FRET_day _i - %FRET_baseline.

A general linear mixed model for the above responses, with a random effect per animal, was run with the variables vaccination group, day, and serum levels treated as categorical variables and all their interactions. Given the exploratory nature of the study, multiple testing adjustments were not considered. Hypothesis testing was performed at a 5% significance level.

Example 15 Mice immunized with a conjugate vaccine in combination with alum and a CpG oligonucleotide as adjuvants produced higher titers of antibodies against the vaccine peptide.

Adult female C57BL/6 mice (n=5-6 in each group) were immunized intramuscularly with either 2 μg (FIG. 17A) or 0.2 μg (FIG. 17B) of conjugate A vaccine. The conjugate vaccine was administered either alone (without adjuvant), with aluminum hydroxide, with a CpG oligonucleotide, or with a combination of alum with a CpG oligonucleotide. All mice received a primary immunization on day 0 of the study, followed by a single boost on day 28. The alum adjuvant dose was 500 μg/mouse per injection, and the CpG oligonucleotide adjuvant dose was 20 μg/mouse per injection. Graphs in Fig. 17 shows binding ELISA results using sera collected from mice before immunization (day 0) and at two time points after immunization (days 28 and 42) with the T3.5 vaccine peptide used as coating antigen. The average T3.5-specific antibody titration endpoints were plotted with whiskers representing the root mean square error. The tables present statistical analysis of the results, in which antibody titers were compared using the nonparametric Kruskal-Wallis test, and pairwise group comparisons were assessed using the Wilcoxon signed-rank test for results obtained using the Kruskal-Wallis test.

Shown in FIGS. 17 results show that at both doses, the vaccine without adjuvant did not induce a strong immune response. The use of alum or CpG oligonucleotide or their combination improved the magnitude of the humoral response (p < 0.0152). Moreover, in animals immunized with 2 μg of vaccine, the combination of adjuvants produced significantly higher antibody titers than the individual adjuvants at day 28 (p=0.0028). The combination of alum and CpG oligonucleotide also performed better than CpG oligonucleotide alone in animals immunized with 0.2 μg of vaccine on day 42 (p=0.0497). These data support the use of a combination of alum and CpG oligonucleotide adjuvants.

Example 16. Liposome-based vaccines with varying ratios of tau peptide and T-cell recognized epitopes induce high and sustained titers of tauphosphopeptide-specific IgG antibodies.

Adult rhesus monkeys (n=6 in each group) were immunized subcutaneously on days 1, 29, 85, and 169 with 1,800 μg of tetrapalmitoylated phosphorylated tau peptide acetate SEQ ID NO: 2 per dose in an improved liposome-based vaccine with encapsulated, recognizable Cells epitope T50 containing both 3D-(6-acyl)PHAD® and lipidated oligonucleotide CpG 2006 as adjuvants, with: i) 400 μg/ml phosphorylated tau peptide SEQ ID NO: 2 and 100 μg/ml T50 ( liposome Z), ii) 1200 µg/ml phosphorylated tau peptide SEQ ID NO: 2 and 1200 µg/ml T50 (liposome Z+), iii) 400 µg/ml phosphorylated tau peptide SEQ ID NO: 2 and 400 µg/ml T50 (Z++ liposome), iv) 1200 μg/ml phosphorylated tau peptide SEQ ID NO: 2 and 300 μg/ml T50 (Z++ liposome). Blood drains were performed before immunization and on days 8, 22, 36, 50, 64, 78, 92, 106, 120, 134, 148, 162, 176, and 190, and sera were isolated. IgG specific antibody titers in the sera were determined by ELISA using phosphorylated tau peptide SEQ ID NO: 2 as the coating antigen and a secondary anti-simian IgG antibody. The resulting antibody levels were calculated as titration endpoints (the last serum dilution showing a positive reaction) for each individual monkey over time. The geometric mean of the titration endpoints for each group ±95% confidence interval is presented in Fig. 18, showing that all four liposome-based vaccines tested induced high and sustained antibody titers against tau phosphopeptide.

List of sequences

SEQ ID NO:1 - phospho-tau peptide (7.1) GDRSGYS[pS]PG[pS]PG[pT]PGSRSRT SEQ ID NO :2 - phospho-tau peptide (T3.5) VYK[pS]PVVSGDT[pS ]PRHL

SEQ ID NO:3 - phospho-tau peptide (22.1)

SSTGSIDMVD[pS]PQLA[pT]LA

SEQ ID NO:4 - tau peptide

VYKSPVVSGDTSPRHL

SEQ ID NO:5 - phospho-tau peptide

RENAKAKTDHGAEIVYK[pS]PVVSGDT[pS]PRHL

SEQ ID NO:6 - phospho-tau peptide

RQEFEVMEDHAGT[pY]GL

SEQ ID NO:7 - phospho-tau peptide

PGSRSR[pT]P[pS]LPTPPTR

SEQ ID NO:8 - phospho-tau peptide GYSSPG[pS]PG[pT]PGSRSR

SEQ ID NO:9 - phospho-tau peptide GDT[pS]PRHL[pS]NVSSTGSID SEQ ID NO: 10 - phospho-tau peptide PG[pS]PG[pT]PGSRSR[pT]P[pS]LP

SEQ ID NO: 11 - phospho-tau peptide HL[pS]NVSSTGSID

SEQ ID NO: 12 - phospho-tau peptide

VSGDT[pS]PRHL

SEQ ID NO: 13 - T50 epitope recognized by T cells

AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHL

E-NH ₂

SEQ ID NO: 14 - T46 T cell recognized epitope

AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLEK (Pal)K(Pal)-NH ₂

SEQ ID NO: 15 - T48 epitope recognized by T helper cells

AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVS ASHLEGS LINSTKIYSYFPSVISKVNQ-NH ₂

SEQ ID NO: 16 - T51 epitope recognized by T helper cells

- 34 044254

AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLEnh ₂

SEQ ID NO: 17 - T52 epitope recognized by T helper cells

AKFVAAWTLKAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLEnh ₂

SEQ ID NO: 18 - CpG 2006 (also known as CpG 7909) ' -tcgtcgttttgtcgttttgtcgtt-3 ' where the lowercase letter indicates phosphorothioate (ps) internucleotide linkages

SEQIDNO: 19 - CpG 1018 ’ -tgactgtgaacgttcgagatga-3 ’ where the lowercase letter means phosphorothioate internucleotide bonds

SEQ ID NO:20 - CpG2395 ’ -tcgtcgttttcggcgcgcgccg-3 ’ where the lowercase letter indicates phosphorothioate internucleotide bonds

SEQ ID NO:21 - CpG2216

5’-ggGGGACGATCGTCgggggg-3’ where lowercase letters indicate phosphorothioate internucleotide linkages and capital letters indicate phosphodiester (rho) linkages

SEQ ID NO:22 - CpG2336

5'- gggGACGACGTCGTGgggggg -3', where lowercase letters indicate phosphorothioate internucleotide bonds and capital letters indicate phosphodiester bonds.

SEQ ID NO:23 - Pan-DR-binding epitope (PADRE) peptide

AKFVAAWTLKAAA

SEQ ID NO:24 - P2

QYIKANSKFIGITEL

SEQ ID NO:25 - RZO

FNNFTVSFWLRVPKVSASHLE

SEQ ID NO:26 - TT ₅₈₆ -605

LINSTKIYSYFPSVISKVNQ

SEQ ID NO:27 - palmitoylated phospho-tau peptide (palmitoylated 7.1)

K(pal)K(pal)GDRSGYS[pS]PG[pS]PG[pT]PGSRSRTK(pal)K(pal)

SEQ ID NO:28 - palmitoylated phospho-tau peptide (T3, palmitoylated T3.5)

K(pal)K(pal)VYK[pS]PVVSGDT[pS]PRHLK(pal)K(pal)

SEQ ID NO:29 - palmitoylated phospho-tau peptide (palmitoylated 22.1)

K(pal)K(pal)SSTGSroMVD[pS]PQLA[pT]LAK(pal)K(pal)

SEQ ID NO:30 - palmitoylated tau peptide

- 35 044254

K(pal)K(pal)VYKSPVVSGDTSPRHLK(pal)K(pal)

SEQ ID NO:31 - palmitoylated phospho-tau peptide

K(pal)K(pal)RENAKAKTDHGAEIVYK[pS]PWSGDT[pS]PRHLK(pal)K(pal)

SEQ ID NO:32 - palmitoylated phospho-tau peptide K(pal)K(pal)RQEFEVMEDHAGT[pY]GLK(pal)K(pal)

SEQ ID NO:33 - palmitoylated phospho-tau peptide K(pal)K(pal)PGSRSR[pT]P[pS]LPTPPTRK(pal)K(pal)

SEQ ID NO:34 - palmitoylated phospho-tau peptide K(pal)K(pal)GYSSPG[pS]PG[pT]PGSRSRK(pal)K(pal)

SEQ ID NO:35 - palmitoylated phospho-tau peptide K(pal)K(pal)GDT[pS]PRHL[pS]NVSSTGSIDK(pal)K(pal) SEQ ID NO:36 - palmitoylated phospho-tau peptide K( pal)K(pal)PG[pS]PG[pT]PGSRSR[pT]P[pS]LPK(pal)K(pal)

SEQ ID NO:37 - palmitoylated phospho-tau peptide K(pal)K(pal)HL[pS]NVSSTGSIDK(pal)K(pal)

SEQ ID NO:38 - palmitoylated phospho-tau peptide K(pal)K(pal)VSGDT[pS]PRHLK(pal)K(pal)

SEQ ID NO:39 - T50 without C-terminal amide

AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHLE SEQ ID NO:40 - T46 without -Lys(Pal)-Lys(Pal)-NH ₂ at the C-terminus

AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVSASHLE SEQ ID NO:41 - T48 without C-terminal amide

AKFVAAWTLKAAAGSQYIKANSKFIGITELGSFNNFTVSFWLRVPKVS ASHLEGS LINSTKIYSYFPSVISKVNQ

SEQ ID NO:42 - T51 without C-terminal amide

AKFVAAWTLKAAARRQYIKANSKFIGITELRRFNNFTVSFWLRVPKVSASHLE SEQ ID NO:43 - T52 without C-terminal amide

AKFVAAWTLKAAARKQYIKANSKFIGITELRKFNNFTVSFWLRVPKVSASHLE SEQ ID NO:44 - T57

AKFVAAWTLKAAAVVRQYIKANSKFIGITELVVRFNNFTVSFWLRVPKVSASHL EK(Pal)K(Pal)-NH ₂

Bibliography

Asuni AA et al. J Neurosci. 2007 Aug 22;27(34):9115-29

Bentebibel et al., 2013, Sci Transl Med., 5(176): 176ra32

Crotty, 2011, Annual Reviews of Immunology. Vol 29:p621-663

Friedhoff et al., Biochimica et Biophysica Acta 1502 (2000) 122-132

Greenberg and Davies, 1991, Proc Natl Acad Sci USA, 87(15):5827-31

Hanger et al., Trends Mol Med. 15:112-9, 2009

Hickman et al., J. Biol. Chern, vol. 286, NO. 16, pp. 13966-13976, April 22, 2011

KontsekovaE et al., Alzheimers Res Ther. 2014 Aug 1;6(4):44

Novak P et al., Lancet Neurology 2017, 16:123-134

Peeraer et al., 2015, Neurobiol Dis., 73:83-95

Ries et al., 2015, Org. Biomol. Chern., 13:9673

Spensieri et al., 2013, Proc Natl Acad Sci U S A., 110(35):14330-5

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WO90/14837

WO2010/115843

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Claims (22)

CLAIM 1. Liposome containing: a) tau phosphopeptide, b) epitope for T helper cells; c) lipidated CpG oligonucleotide; And d) an adjuvant containing a toll-like receptor 4 ligand; wherein the tau phosphopeptide is presented on the surface of the liposome, has an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - SEQ ID NO: 3 and SEQ ID NO: 5 - SEQ ID NO: 12, and the amino acid sequence further comprises one or more modifications to allow the tau peptide to be presented on the surface of the liposome; an epitope for T helper cells includes at least one amino acid sequence selected from the group consisting of SEQ ID NO: 23 - SEQ ID NO: 26; the lipidated CpG oligonucleotide has a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide has one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to at least one lipophilic group; and the toll-like receptor 4 ligand is monophosphoryl lipid A (MPLA). 2. The liposome according to claim 1, which further contains one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl -3'-rac-glycerol (DMPG) and cholesterol. 3. Liposome containing: a) a tau peptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 27 - SEQ ID NO: 38; b) an epitope for T helper cells having an amino acid sequence selected from the group consisting of SEQ ID NO: 39 - SEQ ID NO: 44; c) a lipidated CpG oligonucleotide having a nucleotide sequence selected from the group consisting of SEQ ID NO: 18 to SEQ ID NO: 22, wherein the CpG oligonucleotide includes one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to at least at least one cholesterol via a linker; And d) monophosphoryl lipid A (MPLA). 4. Liposome containing: a) a tau peptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 2, 5, 9 and 12; b) an epitope for T helper cells comprising at least one amino acid sequence selected from the group consisting of SEQ ID NOs: 23, 24, 25 and 26; c) a lipidated CpG oligonucleotide having the nucleotide sequence of SEQ ID NO: 18, wherein the CpG oligonucleotide includes one or more phosphorothioate internucleotide linkages, and the CpG oligonucleotide is covalently linked to at least one cholesterol through a linker; And d) monophosphoryl lipid A (MPLA), wherein the tau peptide is present on the surface of the liposome. 5. Liposome according to claim 4, in which a) the tau peptide has an amino acid sequence selected from the group consisting of SEQ ID NOs: 28, 31, 35 and 38; b) the lipidated CpG oligonucleotide has the nucleotide sequence SEQ ID NO: 18; and c) an epitope for T helper cells comprising the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25, wherein the amino acid sequences are covalently linked together, optionally through one or more linkers. 6. The liposome of claim 5, wherein the T helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42 and 43. 7. The liposome of claim 5, wherein the T helper epitope comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, 17 and 44. 8. The liposome according to claim 4, which further contains one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl -3'-rac-glycerol (DMPG) and cholesterol. 9. Liposome containing: a) tau peptide comprising the amino acid sequence SEQ ID NO: 2; b) an epitope for T helper cells having an amino acid sequence selected from the group consisting of SEQ ID NOs: 39, 40, 41, 42 and 43; c) a lipidated CpG oligonucleotide having the nucleotide sequence SEQ ID NO: 18, wherein the CpG oligonucleotide is covalently linked to at least one cholesterol through - 37 044254 linker; And d) monophosphoryl lipid A (MPLA); wherein the tau peptide is present on the surface of the liposome. 10. The liposome according to claim 9, which further contains one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl -3'-rac-glycerol (DMPG) and cholesterol, and: a) tau peptide comprises the amino acid sequence of SEQ ID NO: 28; b) the epitope for T helper cells has the amino acid sequence of SEQ ID NO: 39; And c) the lipidated CpG oligonucleotide has the nucleotide sequence SEQ ID NO: 18. 11. The liposome according to claim 10, wherein the T helper epitope has the amino acid sequence of SEQ ID NO: 13. 12. The liposome according to claim 9, which further contains one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl -3'-rac-glycerol (DMPG) and cholesterol, and: a) tau peptide comprises the amino acid sequence of SEQ ID NO: 28; b) the epitope for T helper cells has the amino acid sequence SEQ ID NO: 40; And c) the lipidated CpG oligonucleotide has the nucleotide sequence SEQ ID NO: 18. 13. The liposome according to claim 12, wherein the T helper epitope has the amino acid sequence of SEQ ID NO: 14. 14. The liposome according to claim 9, which further contains one or more lipids selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dimyristoyl-sn-glycero-3-phosphoryl -3'-rac-glycerol (DMPG) and cholesterol, and: a) tau peptide comprises the amino acid sequence of SEQ ID NO: 28; b) the epitope for T helper cells has the amino acid sequence SEQ ID NO: 41; And c) the lipidated CpG oligonucleotide has the nucleotide sequence SEQ ID NO: 18. 15. The liposome according to claim 14, wherein the T helper epitope has the amino acid sequence of SEQ ID NO: 15. 16. A pharmaceutical composition containing a liposome according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier. 17. A method of inducing an immune response in a subject suffering from a neurodegenerative disorder, comprising administering to the subject a pharmaceutical composition according to claim 16. 18. A method of treating or preventing a neurodegenerative disease or disorder in a subject in need thereof, comprising administering to the subject a pharmaceutical composition according to claim 16. 19. The method of claim 17 or 18, wherein the neurodegenerative disease or disorder is caused by or is associated with the formation of neurofibrillary lesions. 20. The method of claim 19, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease, Boxer's dementia, Down syndrome, Gerstmann-Straussler-Scheckner disease, inclusion body myositis, prion protein-containing cerebral amyloid Angiopathy, traumatic brain injury, lateral amyotrophic sclerosis, Parkinsonism-Demence-Hoama complex, Neguaman disease of motor neuron with neurofibrillar clubs, dementia characterized by argyrophilic grains, corticobasal degeneration, Levy dementia, side amyotrophic sclerosis, diplomatic science Rofibrillar balls with calcification, Lobno -temporal dementia with parkinsonism associated with chromosome 17 (FTDP-17), Hallervorden-Spatz disease, multiple system atrophy, Niemann-Pick disease type C, Pick disease, progressive subcortical gliosis, progressive supranuclear palsy, subacute sclerosing panencephalitis, dementia with predominance neurofibrillary tangles, postencephalitic parkinsonism, myotonic dystrophy, chronic traumatic encephalopathy (CTE), primary age-related tauopathy (PART) or Lewy body dementia (LBD). 21. The method according to any one of claims 17 to 20, wherein the neurodegenerative disease or disorder is Alzheimer's disease, Parkinson's disease, Down syndrome, frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), corticobasal degeneration, dementia Lewy disease, amyotrophic lateral sclerosis, myotonic dysphagia, chronic traumatic encephalopathy (CTE), cerebral angiopathy, primary age-related tauopathy (PART) or dementia with Lewy bodies (LBD). 22. A kit comprising the pharmaceutical composition of claim 16 for treating or preventing a neurodegenerative disease or disorder in a subject in need thereof. -