EA044699B1 - THERAPEUTIC VACCINES AGAINST BETA-AMYLOID - Google Patents

Description

Field of technology

The present invention relates to therapeutic vaccines against beta-amyloid and their uses for the treatment and prevention of disease. Vaccines contain Άβ-derived peptide B-cell antigens and T-cell epitopes.

Disclosure of the present invention

Alzheimer's disease (AD) is a severe, progressive degenerative disorder characterized by loss of cognitive functions, including memory, and loss of the ability to perform normal daily activities. AD affects approximately 40 million patients worldwide, and this number is growing rapidly as the population ages. The main neuropathological change in the brain of patients with AD is the death of neurons, mainly in areas associated with memory and cognition (Soto, 1999). One of the most striking pathological features of AD is the abundant presence of beta-amyloid (Άβ) plaques in the brains of patients (Soto, 1999). Άβ plaques are formed by the 39 to 43 amino acid long Άβ peptide, which in its natural non-pathological form is in a random helix conformation. Upon transition to a pathological state, it transforms mainly into the secondary structure of the β-sheet, spontaneously aggregating into insoluble plaques.

The few currently available therapies for AD are considered primarily symptomatic in action. Despite significant efforts over the years to develop therapies, there are no approved AD-modifying therapies to date. Efforts have been made to develop an immunotherapy that neutralizes pathological Άβ in the affected brain over an extended period of time (Winblad, 2014). Vaccines have the advantage of stimulating the immune system to produce a pool of slightly different but very specific antibodies, while the response can be induced by repeated additional vaccinations if necessary.

However, the active immunization (vaccination) approach against Άβ has several major problems. Amyloid beta is a so-called autoantigen to which the human body is constantly exposed. Therefore, it is quite difficult to break immune tolerance and induce an immune response against it. In addition, it is quite difficult to induce a strong immune response to the vaccine in older and sick people, such as AD patients, due to their weakened immune system and reduced number of immune cells.

Despite these problems, in an initial study, a vaccine based on full-length Άβ1-42 (AN1792) induced an immune response and showed promising efficacy, with slower cognitive decline in patients who received the vaccine than in patients who received placebo (Gilman, 2005). However, 6% of treated patients developed meningoencephalitis, an inflammatory reaction believed to be a consequence of a T cell-mediated response to full-length Άβ1-42 (Orgogozo, 2003).

Another known Άβ vaccine, ACI-24, contains a 15 amino acid sequence completely identical to the human amyloid beta 1-15 sequence (WO 2007/068411). This peptide antigen is bound to a liposomal carrier to stimulate anti-Άβ antibodies while avoiding meningoencephalitis and hemorrhage (Muhs, 2007, Pihlgren, 2013). The choice of the Aβ1-15 peptide to serve as the antigen was based on the fact that this sequence contains the B cell epitope but lacks the strong T cell reaction site of full-length Άβ1-42 (Monsonego, 2003), which is considered to be a cause of adverse inflammatory reactions . ACI-24 has been shown to act through the simultaneous activation of Aβ1-15-specific B cell receptor and Toll-like receptor 4 (TLR4), activated by monophosphoryl lipid A (MPLA), an adjuvant present in the ACI-24 vaccine ( Pihlgren, 2013).

Activation of proliferation and immunoglobulin (Ig) production of B cells occurs through cross-linking of the Ig receptor on the surface of B cells. In order to enhance antibody production, a second signal may be provided by a helper T cell activated by a T cell epitope. T cell epitopes presented by major histocompatibility complex (MHC) molecules (in humans referred to as human leukocyte antigen (HLA)) on the surface of the antigen presenting cell (APC) promote the differentiation of cognate helper T cells capable of producing IFNy and IL-4. Cytokine release and co-stimulatory signals between activated T and B cells enhance immune responses and class switching. After primary vaccination, naïve T cells proliferate and differentiate into effector cells. A small proportion of these cells will form a pool of long-lived memory T cells capable of rapidly proliferating when re-encountered with the cognate peptide after re-vaccination (Sallusto, 2010). So-called universal T cell epitopes are specific to T cells that are present in the vast majority of the human population. They usually come from antigens that people are routinely exposed to during their lives (eg, tetanus, influenza virus, etc.). The ability of a T cell epitope to activate T cells is the result of at least two complementary properties: i) its HLA groove binding affinity, meaning the strength of binding, and ii) its ability to bind different HLA haplotypes in a non-selective manner, meaning the ability cover very different human populations with respect to differences in the expression of HLA molecules.

There is a need to develop an Av vaccine that is highly immunogenic while maintaining a good safety profile. This need was addressed by incorporating a universal T cell epitope into the ACI-24 liposomal vaccine. Since the ACI-24 vaccine is characterized by the presence of Ae1-15 on the surface of the liposome, the incorporation of universal T cell epitopes on the surface of the liposome was considered by the present inventors as a first choice route to improve the effectiveness of the vaccine. However, it was unexpectedly found that the inclusion of universal T-cell epitopes on the surface of the liposome did not lead to increased (or significantly increased) vaccine efficacy. Thus, as disclosed herein, an encapsulation approach was adopted which has been shown to provide increased efficiency. Incorporation of a universal T-cell epitope within a liposomal vaccine has been shown herein to increase (or substantially hemorrhage) vaccine efficacy while maintaining a good safety profile due to T-cell activation that is not directed to Av. However, several problems arose in developing this approach. First, the universal T cell epitopes developed according to the present invention tend to be hydrophobic, making liposome encapsulation difficult. Second, multiple universal T cell epitopes are often combined to enhance immunogenicity. However, the yield of peptide synthesis and the success rate decrease as the length of the peptides increases. Third, the charge of the selected universal T cell epitopes influences the efficiency of encapsulation and the experimental conditions required to ensure encapsulation due to the negatively charged liposomal membrane.

Accordingly, the present invention relates to a liposomal vaccine composition containing:

a) β-amyloid (Ab)-derived peptide B cell antigen presented on the surface of the liposome, and

b) a peptide containing a universal T-cell epitope encapsulated within a liposome.

A particularly preferred vaccine composition comprises an ACI-24 vaccine modified to include a peptide containing a universal T cell epitope encapsulated within a liposome. A liposome is an example of a carrier. Thus, the carrier is typically a liposome, but may be any carrier that is suitable for presenting the Ab-derived peptide antigen to a surface in the same manner as is achieved by a liposome (in which the Av-derived peptide antigen adopts a predominantly β-sheet conformation ), and also encapsulates a peptide containing a universal T-cell epitope. Examples include vesicles and corpuscular bodies.

The term universal T cell epitope means an epitope that is specific to T cells that are present in the majority of the human population. They usually originate from antigens that people are routinely exposed to during their lives. Examples include antigens included in commonly used vaccines. Specific examples are the T cell epitopes included in tetanus, influenza virus, and diphtheria, as well as keyhole limpet hemocyanin (KLH) and Epstein-Barr virus (EBV). The universal ability of a T cell epitope to activate T cells is the result of at least two complementary properties: i) its binding affinity to the HLA groove, meaning the strength of binding, and ii) its ability to bind different HLA haplotypes in a non-selective manner, meaning the ability to span very different differences in the expression of HLA molecules in human populations. Universal T cell epitopes can bind to most MHC class II alleles present in the human population. Thus, universal T cell epitopes included in the vaccine compositions of the present invention may be capable of stimulating a CD4 T cell response. Thus, universal T cell epitopes included in the vaccine compositions of the present invention may be capable of stimulating a helper T cell response that enhances the production of (Ab-specific) antibodies by B cells.

The universal T cell epitopes included in the vaccine compositions of the present invention are typically synthesized by solid phase synthesis. Thus, in some embodiments, universal T cell epitopes are synthesized by solid phase synthesis. This and other practical encapsulation problems mean that, in some non-limiting embodiments, the universal T cell epitope-containing peptide is no more than 85, 80, 75, or 70 amino acids in length. The minimum T cell epitope peptide length to provide sufficient immunogenicity is typically approximately 10 amino acids. Thus, the minimum peptide length is typically approximately 10 amino acids to ensure production of a T cell epitope with sufficient immunogenicity. In some embodiments, the peptide is at least 20 amino acids in length. In other embodiments, the peptide has a length of from 30 to 60 amino acids, which is based on the preferred minimum length of the universal T cell epitope and the preference for a peptide containing at least two, three, or four (linked) universal T cell epitopes.

It has also been found that generic T cell epitopes suitable for use in accordance with the present invention are typically hydrophobic. This poses additional challenges for their synthesis, purification and liposome encapsulation due to their interaction with lipids. Percent hydrophobicity is calculated by dividing the total number of hydrophobic amino acids (Phe, Ile, Leu, Met, Val, Tip, Ala, and Pro) by the total number of amino acids either in the peptide as a whole containing the universal T cell epitope (when considering the entire peptide) or in a particular T cell epitope (considering each universal T cell epitope separately) and multiplying by 100. Hydrophobic amino acids in the context of the present invention are defined as leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp), valine (Val), methionine (Met), proline (Pro) and alanine (Ala).

Thus, in general, a peptide containing a universal T cell epitope contains at least 30% hydrophobic amino acids. This means that at least 30% of the amino acids in the overall peptide containing the universal T cell epitope are hydrophobic amino acids. Most of the tested peptides containing universal T-cell epitopes contain up to 50% hydrophobic amino acids. In some cases, the peptide may contain at least 35, 40, 45 or 50% hydrophobic amino acids.

To enhance immunogenicity levels, it is preferred that the vaccine composition contains at least two different universal T cell epitopes encapsulated within a liposome. Due to liposomal capacity combined with peptide hydrophobicity and synthesis limitations, ideally each universal T cell epitope is typically no more than 30 amino acids in length, preferably no more than 20 amino acids in length, and even more preferably is a region approximately 10-20 amino acids in length . As explained later herein, the present inventors have discovered that longer universal T cell epitopes can be effectively shortened to 10-20 amino acids in length while maintaining immunogenicity. The truncated peptides were designed by selecting within each individual T cell epitope sequence the most immunogenic shorter subsequence, typically approximately 15 amino acids in length, based on in silico predicted T cell epitope hotspots. Various software are available to perform this assay, including the EpiVax immunogenicity screening platform (available at http://www.epivax.com). Other examples include SYFPEITHI (see Hans-Georg Rammensee, Jutta Bachmann, Niels Nikolaus Emmerich, Oskar Alexander Bachor, Stefan Stevanovic: SYFPEITHI: database for MHC ligands and peptide motifs. Immunogenetics (1999) 50: 213-219; available at http ://www.syfpeithi.com), SVMHC (https://www.ncbi.nlm.nih.gov/pubmed/16844990) and the IEBD database (Vita R, Overton JA, Greenbaum JA, Ponomarenko J, Clark JD, Cantrell JR, Wheeler DK, Gabbard JL, Hix D, Sette A, Peters B. The immune epitope database (IEDB) 3.0. Nucleic Acids Res. 2014 Oct 9. pii: gku938. [Epub ahead of print] PubMed PMID: 25300482; available at http://www.iedb.org/).

In some embodiments, each universal T cell epitope contains at least 30% hydrophobic amino acids. This means that at least 30% of the amino acids in a single universal T cell epitope are hydrophobic amino acids. For specific epitopes, this value can be up to 80% hydrophobic amino acids. In some cases, there may be at least 40, 45, 50, 55, 60, 65, 70, 75 or 80% hydrophobic amino acids. In some embodiments, a maximum of 80% hydrophobic amino acids may be present, which means that the maximum range may be 30 to 80% hydrophobic amino acids.

In order to balance improved immunogenicity with practical issues of encapsulation, the vaccine composition may contain two, three or four different universal T cell epitopes encapsulated in a carrier. If a larger number of different universal T cell epitopes are encapsulated (especially 3 or 4), they are preferably shortened to a length of about 10-20 amino acids, such as about 15 amino acids. Preferably, a plurality of different universal T cell epitopes are included in the same peptide that is encapsulated. Thus, synthetic peptide constructs containing a variety of different universal T cell epitopes are a preferred embodiment of the present invention. In some embodiments, the peptide contains at least two different universal T cell epitopes. In more specific embodiments, the peptide contains two, three, or four universal T cell epitopes. If at least two universal T cell epitopes are included in the synthetic peptide construct, they can be connected by a linker. A linker is used to physically link universal T cell epitopes to each other in a manner that does not reduce the immunogenicity of the linked epitopes. Suitable linkers for connecting amino acids to each other are well known in the art. Preferred linkers are themselves amino acid-based linkers, that is, peptide linkers. Thus, they can connect universal T cell epitopes to each other via peptide bonds. A linker is a linker that ensures correct processing of universal T cell epitopes. Antigen presentation by MHC class II molecules requires entry of antigens into the endosomal-lysosomal compartment. These antigens are then processed by proteolytic enzymes, of which the lysosomal cysteine proteases of the papain family constitute an important subgroup. The generated peptides bind to MHC class II molecules and are then presented on the surface of professional antigen-presenting cells (APCs), including macrophages, dendritic cells (DCs), and B cells (Lutzner and Kalbacher 2008). Thus, preferably the linker contains a substrate for a lysosomal cysteine protease of the papain family. The linker may contain a substrate for one or more of cathepsin S, cathepsin B, and cathepsin L. In some embodiments, the linker contains, consists essentially of, or consists of at least two or at least three amino acids. In some embodiments, the linker contains, consists essentially of, or consists of the amino acids VVR, TVGLR, KVSVR, PMGAP, or PMGLP.

Therefore, peptides containing two universal T cell epitopes may be linear peptides in the form:

[universal T-cell epitope 1]-[linker]-[universal T-cell epitope 2].

Therefore, peptides containing three universal T cell epitopes may be linear peptides in the form:

[universal T-cell epitope 1]-[linker]-[universal T-cell epitope 2]-[linker][universal T-cell epitope 3].

Therefore, peptides containing the four universal T cell epitopes may be linear peptides in the form:

[universal T-cell epitope 1]-[linker]-[universal T-cell epitope 2]-[linker][universal T-cell epitope 3]-[linker]-[universal T-cell epitope 4].

It should be noted that the linkers do not need to be the same between each pair of linked universal T cell epitopes. Thus, for example, the linker between universal T cell epitope 1 and universal T cell epitope 2 may be different from the linker between universal T cell epitope 2 and universal T cell epitope 3. In the case of four universal T cell epitopes, each of the three linkers may be different, or two linkers can be the same and the third linker can be different (in any order). In some embodiments, where multiple linkers are included in the peptide, they are all the same.

In developing suitable peptides for encapsulation, the present inventors have explored a number of sources of universal T cell epitopes. In some embodiments, the universal T cell epitopes are derived from diphtheria toxin, tetanus toxin, Epstein-Barr virus, influenza virus hemagglutinin, and/or keyhole limpet hemocyanin. Therefore, specific preferred combinations of universal T cell epitopes are selected from:

a) combinations of a universal T-cell epitope of diphtheria toxin and tetanus toxin;

b) combinations of the universal T-cell epitope of the Epstein-Barr virus and tetanus toxin;

c) combinations of the universal T-cell epitope of the Epstein-Barr virus, tetanus toxin and hemocyanin of the snail lymph; or

d) combinations of the universal T-cell epitope hemagglutinin of the influenza virus, diphtheria toxin, tetanus toxin and Epstein-Barr virus.

Such combinations are preferably presented in the form with a linker, as explained above. For the avoidance of doubt, although the combinations are preferably included in the order shown, they may be included in an alternative order.

For example, if there are three universal T cell epitopes A, B, and C, they may be included in any of the orders ABC, ACB, BAC, BCA, CAB, or CBA.

In another aspect, the present invention provides specific peptides containing a variety of different universal T cell epitopes. Such peptides are preferably included in the vaccine compositions of the present invention. Thus, the peptides used according to the present invention contain, consist essentially of, or consist of an amino acid sequence selected from SEQ ID NO: 1 (SAT42), SEQ ID NO: 2 (SAT43), SEQ ID NO: 3 (SAT44) , SEQ ID HET: 4 (SAT47). The composition of these peptides is explained in more detail below with reference to Table. 2.

In a further aspect, the present invention provides specific peptides containing a single universal T cell epitope. Such peptides are preferably included in the vaccine compositions of the present invention. Thus, the peptides used according to the present invention contain, consist essentially of, or consist of an amino acid

- 4 044699 sequence selected from SEQ ID NO: 5 (SAT6), SEQ ID NO: 6 (SAT13), SEQ ID NO: 7 (SAT15), SEQ ID HET: 8 (SAT17). The composition of these peptides is explained in more detail below with reference to Table. 1. Combinations of these peptides, optionally shortened to 10-20 amino acids in length, can also be included in the vaccine compositions of the present invention. The combination peptides are preferably connected by one or more linkers, as defined herein.

The Av-derived peptide antigen is presented on the surface of the liposome. Typically, this is accomplished by insertion into the outer surface of the liposome. Insertion into the outer surface of the liposome can be facilitated by attaching an Ab-derived peptide antigen to a moiety that is inserted into the outer surface of the liposome. A liposome can be any liposome that is suitable for presenting an Ab-derived peptide antigen on a surface and also encapsulates a peptide containing a universal T cell epitope. Typically, this moiety contains a hydrophobic moiety to facilitate insertion into the lipid bilayer of the liposome. The moiety may be any suitable moiety, but is preferably a fatty acid. The fatty acid may contain a palmitoyl residue. The preferred design, as in ACI-24, contains an Ab-derivative peptide antigen (Ab(1-15) in ACI-24) attached to two palmitoyl residues in the N- and C-terminal regions of the peptide. Thus, the peptide antigen is tetrapalmitoylated. This may be facilitated by the inclusion of two lysine residues in the N- and C-terminal regions of the A-derived peptide antigen. Lysine residues are palmitoylated.

In some embodiments, the liposome has a negative surface charge and the liposome is anionic. Preferably, the liposome contains phospholipids and, even more preferably, the phospholipids contain dimyrsitoylphosphatidylcholine (DMPC) and dimyrsitoylphosphatidylglycerol (DMPG). The liposome may additionally contain cholesterol. The molar ratios of these three components may be 9:1:7 in some embodiments.

Therefore, the most preferred design contains the Ab-derived peptide antigen reconstituted in a liposome. Accordingly, these compositions of the present invention may be generally referred to herein as the liposomal vaccine composition of the present invention.

The Av-derived peptide antigen induces a B cell response in the subject. It is a B cell antigen. As already explained, Av plaques are formed by the Ab peptide, 39 to 43 amino acids long, which in its natural non-pathological form is in a random helix conformation. Upon transition to a pathological state, it transforms mainly into the secondary structure of the b-sheet, spontaneously aggregating into insoluble plaques. Thus, an Ab-derived peptide antigen is defined herein as a peptide antigen derived from (maximum) 43 amino acids of amyloid beta, but which is not full-length Ab. More specifically, the Av-derived peptide antigen includes the immunodominant B cell epitope from Av(1-42), but does not contain the T cell epitope found in Av(1-42). Therefore, in some embodiments, the Ab-derived peptide antigen contains, consists essentially of, or consists of 13-15 contiguous amino acids of the 17 N-terminal amino acids of Ab. It should be noted that the Av-derived peptide antigen can be provided in the context of a larger peptide molecule, the remainder of which is not derived from the amino acid sequence of Av. For example, the peptide may include additional residues, such as lysine residues, to facilitate palmitoylation. These residues are usually found at the N- and C-termini of the peptide. In this context, the term consists essentially of meaning that the Ab-derived peptide antigen comprises 13 to 15 contiguous amino acids of the 17 N-terminal amino acids of Ab, but may include a limited number of additional residues, such as four lysine residues, to facilitate palmitoylation. A preferred Ab-derived peptide antigen contains, consists essentially of, or consists of amino acids 1-15 of amyloid beta, which may be referred to as Ab(1-15) (WO 2007/068411, ACI-24).

The Av-derived peptide antigen included in the compositions of the present invention adopts a secondary structure that reproduces the pathological form of Av. Preferably, the A-derived peptide antigen adopts a secondary structure having a B-sheet conformation. Even more preferably, the A-derived peptide antigen adopts a predominantly B-sheet conformation when presented on the surface of a liposome.

The compositions of the present invention typically contain at least one adjuvant. According to some embodiments of the present invention, the compositions of the present invention contain two adjuvants. The purpose of the adjuvant(s) is to enhance or stimulate the immune response in the subject. Preferably, the at least one adjuvant is part of the vehicle (as opposed to being encapsulated in the vehicle). Thus, the at least one adjuvant may form part of the liposome, it may form part of the lipid bilayer. Therefore, the adjuvant may be a lipid-based adjuvant. The adjuvant may be at least partially

- 5 044699 is present on the surface of the liposome, which may be due to the fact that the adjuvant is part of the lipid bilayer. In another embodiment, one or more adjuvants forming part of the liposome may be combined with the encapsulated adjuvant. In other embodiments, one or more adjuvants forming part of the liposome may be mixed with an additional adjuvant (such as Alum or CpG) when forming the liposomes. The carrier (liposome) can function as an adjuvant when monophosphoryl lipid A (MPLA) is added to the liposome, this term encompassing MPLA derivatives such as monophosphoryl hexaacyl lipid A, 3-deacyl (synthetic) (3D-(6-acyl) PHAD®), PHAD® (phosphorylated hexaacyl disaccharide), MPL. Thus, in certain embodiments, the compositions further comprise MPLA. MPLA is typically added during liposome formation (as described below). Thus, preferred liposomes contain dimyrsitoyl-phosphatidylcholine (DMPC), dimyrsitoyl-phosphatidylglycerol (DMPG), cholesterol and MPLA. In some embodiments, the molar ratios of these four components may be 9:1:7:0.05.

Other adjuvants that can be used according to the present invention include, but are not limited to, aluminum hydroxide (Alum) and/or CpG.

The vaccine compositions of the present invention are administered to subjects to treat, prevent, induce a protective immune response against, or alleviate symptoms associated with a disease or condition associated with beta-amyloid, or a condition characterized by or associated with cognitive loss of memory. Thus, vaccine compositions can have both prophylactic and therapeutic uses. The subject is a mammal and usually a human.

The beta-amyloid-related disease or condition may be a neurological disorder such as Alzheimer's disease (AD). Other examples of diseases or conditions associated with amyloid beta according to the present invention include mild cognitive impairment (MCI), Down syndrome, cardiac amyloidosis, cerebral amyloid angiopathy (CAA), multiple sclerosis, Parkinson's disease, dementia with Lewy bodies, ALS (lateral amyotrophic sclerosis), adult-onset diabetes, inclusion body myositis (IBM), ocular amyloidosis, glaucoma, macular degeneration, lattice dystrophy and optic neuritis. Many of these conditions are characterized by or associated with loss of cognitive memory ability. Thus, conditions characterized by or associated with loss of cognitive memory ability in accordance with the present invention include AD, mild cognitive impairment (MCI), Down syndrome, cardiac amyloidosis, cerebral amyloid angiopathy (CAA), multiple sclerosis, Parkinson's disease, dementia with Lewy bodies, ALS (amyotrophic lateral sclerosis) and inclusion body myositis (IBM).

Accordingly, the present invention relates to a method of treating, preventing, inducing a protective immune response against, or alleviating symptoms associated with a beta-amyloid-related disease or condition, or a condition characterized by or associated with loss of cognitive memory, the method comprising administering to a subject vaccine composition according to the present invention.

Such methods can also be carried out in the form of medical administration of the vaccine compositions according to the present invention. Accordingly, the present invention also relates to a vaccine composition according to the present invention for use in the treatment, prevention, induction of a protective immune response against or alleviation of symptoms associated with a disease or condition associated with beta-amyloid, or a condition characterized by loss of cognitive ability, memory or associated with her, at the subject.

Similarly, the present invention relates to the use of vaccine compositions according to the present invention for the preparation of a medicament for use in the treatment, prevention, induction of a protective immune response against or alleviation of symptoms associated with a disease or condition associated with beta-amyloid, or a condition characterized by the loss of cognitive ability of memory, or related thereto, in a subject.

All embodiments herein relate to such methods or medical applications, regardless of how they are performed. Administration of the vaccine composition of the present invention to a subject results in the production of typically polyclonal IgG antibodies that bind to pathological forms of Av. As already explained, these pathological forms of Av contain β-sheet multimers. Therefore, the antibodies produced can be called Av-specific antibodies.

The ability of an antibody to bind a target antigen is mainly governed by two parameters, affinity and avidity. Antibody affinity is a measure of the strength of the monovalent interaction between an antibody and its antigen. Antibody avidity involves increased binding through more than one point of interaction between an antigen and an antibody. The binding capacity of polyclonal sera induced by vaccination depends on both of the above-mentioned parameters (Siegrist, 2013). In general, this is called polyclonal response avidity because it is very difficult

- 6 044699 evaluate affinity and avidity independently of each other. As explained in more detail herein, see Example 4 (clause 4.2), the present inventors have developed an ELISA assay in which the total binding of sera containing polyclonal antibodies to lower and higher concentrations of antigen is assessed in parallel (Martineau, 2010). The ratio between low and high coverage signal (the signal is the concentration of bound antibody) is expressed as an avidity index. A higher index value (closer to 1) indicates improved overall binding strength compared to a lower index value (closer to 0). An increase in the avidity index over time indicates an overall maturation of the avidity of vaccine-induced antibodies. It is shown herein (see Example 4 and FIG. 4) that immunization with vaccine compositions of the present invention that contain an encapsulated peptide containing a universal T cell epitope provides an improved maturation effect compared to immunization with ACI-24 (without encapsulated peptide containing a universal T-cell epitope).

The vaccine compositions of the present invention can be administered to a subject by any suitable route of administration. As is known to one skilled in the art, vaccine compositions can be administered by topical, oral, rectal, nasal or parenteral (eg, intravenous, intradermal, subcutaneous or intramuscular) routes. In addition, vaccine compositions can be included in sustained release matrices, such as biodegradable polymers, with the polymers implanted near or in close proximity to the site where delivery is desired. However, in preferred embodiments, the vaccine composition is administered intramuscularly or subcutaneously.

The vaccine compositions of the present invention can be administered to a subject once to produce a protective immune response. However, in some embodiments, the vaccine compositions of the present invention are administered multiple times to the same subject. Thus, in accordance with the present invention, so-called prime-boost modes can be used. Vaccine administration is usually separated by an intervening period of at least 1 week and often approximately 1-12 months. Without wishing to be bound by any particular hypothesis, it is believed that it is likely that the addition of a universal T cell epitope to ACI-24 enhances the immune response against Av by providing a second signal from activated T cells specific for the cognate T cell epitope. The vaccine compositions of the present invention provide a powerful new therapeutic approach for the prevention and treatment of beta-amyloid-related disease or condition such as AD. In some embodiments, the same vaccine composition is administered each time, resulting in a homologous vaccination regimen. Homologous vaccination refers to an immunization regimen using the same vaccine for both the primary (first immunization) and booster (second or any subsequent immunization).

On the other hand, heterologous prime-boost immunization requires that different vaccines be used in the primary and at least some subsequent immunizations. In some embodiments, vaccine compositions of the present invention are administered multiple times to the same subject in a heterologous prime-boost combination with other anti-Ab vaccines carrying peptide antigens derived from any portion of the Ab protein, which may include peptide antigens derived from outside Av(1-15) regions. In some embodiments, vaccine compositions of the present invention are administered multiple times to the same subject in a heterologous prime-boost combination with other anti-Av vaccines carrying the same peptide antigens included in the liposomal vaccine compositions of the present invention, which may contain Av (1-15) peptide antigens. In some embodiments, the vaccine compositions of the present invention, preferably containing Av(1-15) peptide antigens, are administered multiple times to the same subject in a heterologous prime-boost combination with other anti-Av vaccines carrying the corresponding Av-derived peptide antigens, preferably Ab(1-15) peptide antigens. Examples of anti-Ab vaccines that can be administered in heterologous prime-boost vaccination with vaccine compositions of the present invention containing Av-derived antigens include, but are not limited to, the Aβ(1-15)-PADRE vaccine (Agadjanyan et al., 2005; Ghochikyan et al. ., 2006), Av(1-15)-diphtheria toxoid (DT) vaccine or CRM vaccine (WO 2010016912), lysine-linked tandem repeat Av(1-15) vaccine (Maier et al., 2006), vaccine based on dendrimeric Av(1-15) (Seabrook et al., 2006), vaccine based on Aβ(1-15)-DT conjugate (Liu et al. 2013), vaccine based on Av(1-6) associated bacteriophage QP coated protein (Windblad et al. 2012), Aβ(1-7)-CRM vaccine (Arai et al. 2015), Nterm Aβ-KLH vaccine (Schneeberger et al. 2010).

In addition, the present invention relates to kits containing vaccine compositions according to the present invention. Accordingly, the present invention relates to a kit for treating, preventing, inducing a protective immune response against or alleviating symptoms associated

- 7 044699 with a disease or condition associated with beta-amyloid, or a condition characterized by or associated with loss of cognitive ability, containing a (liposomal) vaccine composition according to the present invention, as described herein. Such kits may be provided with suitable instructions for use. The instructions for use may explain the schedule for administration of the compositions. Therefore, kits may include multiple (individual) doses of the vaccine compositions of the present invention. The instructions for use may further explain the storage conditions of the compositions, especially during the period of time between administration of doses of the vaccine compositions. These kits can be used for all relevant methods according to the present invention, as disclosed herein.

In addition, the present invention relates to a method for preparing liposomal vaccine compositions according to the present invention. Such methods may involve the following stages:

a) obtaining a lipid film;

b) rehydrating the lipid film in a buffer containing a peptide containing a universal T cell epitope;

c) preparing liposomes from a rehydrated lipid film that encapsulates a peptide containing a universal T cell epitope to form a solution containing liposomes that contain an encapsulated universal T cell epitope;

d) adding the β-amyloid (Ab)-derived peptide antigen to the solution and maintaining the solution under conditions resulting in insertion of the β-amyloid (Av)-derived peptide antigen into the lipid bilayer of the liposomes.

Provided herein are examples of such methods, features of which may be applied to these aspects of the present invention. In general, the methods may involve forming a thin lipid film followed by homogenization and extrusion. Thus, in some embodiments, the lipid film is prepared by dissolving the lipid in ethanol and then evaporating the ethanol in vacuo. Preferred lipid components are explained in relation to the liposomal vaccine compositions of the present invention and include DMPC, DMPG, cholesterol and MPLA (as an adjuvant). The molar ratios of these components can be 9:1:7:0.05. Such molar ratios are also applicable to the liposomal vaccine compositions of the present invention. It may be necessary to solubilize lipid components at elevated temperatures. The elevated temperature may be from 40 to 80°C, such as approximately 60°C.

In step b, the buffer used for rehydration may depend on the universal T cell epitope containing peptide used. In general, any suitable buffer can be used. In some embodiments, the buffer includes sodium acetate or PBS. If SAT42 is to be encapsulated, the buffer may be sodium acetate. If it is necessary to encapsulate one or more of SAT43, SAT44 or SAT47, the buffer may be PBS. In all cases, DMSO can be added to the buffer, such as 5% DMSO. Rehydration can be carried out by stirring the sample.

In step c, liposomes can be prepared by shaking in the presence of beads. You can use any suitable balls. For example, the balls can be glass. At this stage, multilayer vesicles can be formed, which subsequently turn into liposomes containing a lipid bilayer. This transformation can occur over several, such as 5-15, preferably 10 freeze-thaw cycles. Freeze-thaw cycles may be followed by homogenization. This can be followed by extrusion to size. In some embodiments, the liposomes are extruded through pores with a diameter (or maximum size) of approximately 0.08-0.1 microns. This may be accomplished by means of a membrane such as a polycarbonate membrane. Extruded liposomes can be concentrated, for example, using a form of filtration such as ultrafiltration.

Step d results in the insertion of β-amyloid (Ab)-derived peptide antigen into the lipid bilayer of the liposomes. Required conditions may include stirring for 10-60 minutes, such as about 30 minutes, at a temperature of 25-35°C, such as about 30°C. A preferred β-amyloid (Ab)-derived peptide antigen is a tetrapalmitoylated peptide containing Ae1-15. This peptide includes two lysine residues at each end to form a tetrapalmitoylated peptide. The peptide can be pre-dissolved in disodium hydrogen phosphate before injection into the liposomal solution.

The method may further comprise filtering the vaccine composition as a final step. It can be carried out under sterile conditions. Filtration can be carried out through a membrane with a pore size of 0.2 microns. Suitable membranes include polyethersulfone (PES) membranes, which may be in the form of a syringe filter. The resulting vaccine composition can then be stored under suitable conditions until use, e.g.

- 8 044699 in a refrigerator (for example, at a temperature of about 5°C).

Alternative methods for preparing the liposomal vaccine compositions of the present invention may rely on cross-flow injection, as illustrated herein. Accordingly, the present invention also relates to methods for preparing liposomal vaccine compositions according to the present invention by cross-flow injection. These methods may be particularly applicable to compositions encapsulating SAT44 or SAT47. Such methods may involve the following stages:

a) dissolving the lipids (and the adjuvant if lipid based) forming the liposomes in solution;

b) dissolving the peptide containing the universal T cell epitope in a solution;

c) mixing the solutions from steps a and b, using a cross-flow injection module, to form intermediate liposomes that encapsulate a peptide containing a universal T cell epitope;

d) extruding intermediate liposomes through a membrane to reduce their size and polydispersity;

e) mixing the solution containing the β-amyloid (Av)-derived peptide antigen with the solution obtained in step d using a cross-flow injection module, resulting in the insertion of the β-amyloid (Av)-derived peptide antigen into the lipid bilayer of the liposomes .

Provided herein are examples of such methods, features of which may be applied to these aspects of the present invention. In general, the methods use lateral flow injection to encapsulate a peptide containing a universal T cell epitope and to insert the β-amyloid (Ab)-derived peptide antigen into the lipid bilayer of liposomes.

In step a, the lipids (which may contain an adjuvant such as MPLA adjuvant as described herein) are typically dissolved in ethanol. Ethanol can be 90-100% ethanol, for example 96% ethanol. Dissolution can be accelerated by heating, for example to a temperature of from 40 to 80°C, such as about 60°C. Preferred lipid components are explained in relation to the liposomal vaccine compositions of the present invention and include DMPC, DMPG, cholesterol and MPLA (as an adjuvant). The molar ratios of these components can be 9:1:7:0.05. Such molar ratios are also applicable to the liposomal vaccine compositions of the present invention.

In step b, the peptide containing the universal T cell epitope is dissolved. The peptide can be dissolved in a suitable buffer (such as His-sucrose buffer) using agitation, such as sonication, according to some embodiments.

In step c, the solutions from steps a and b are mixed using a cross-flow injection module to form intermediate liposomes that encapsulate the peptide containing the universal T cell epitope. Before this step, the solutions from steps a and b may be subjected to filtration. A suitable pore size for the filter may be around 0.2 µm. The solutions can be used in any suitable concentration. After filtration, the solutions can be heated to a temperature of 30 to 60°C, such as approximately 40°C. Liposomes are formed by injecting two solutions (from step a and b) through the cross-flow module (where the 2 solutions meet). This is usually carried out at a certain flow rate and temperature, as is easily understood by one skilled in the art (suitable temperatures are indicated above). In some embodiments, a buffer may be added after liposome formation, typically to reduce the ethanol concentration. Any suitable buffer, such as His-sucrose buffer, can be used.

In step d, the intermediate liposomes are extruded through the membrane to reduce their size and polydispersity. The resulting liposomes in solution encapsulate the peptide that constitutes the universal T-cell epitope. Any suitable membrane can be used. A suitable pore size may be around 100 nm. A suitable type of membrane is a polycarbonate membrane. This step can be carried out at any suitable temperature, preferably at room temperature (eg approximately 25°C). After this step, a filtration step, such as ultra/diafiltration, can be carried out to remove ethanol. Any suitable membrane can be used for this step, such as a hollow fiber membrane with a molecular weight cut-off of approximately 500 kDa. The buffer exchange step can be performed in a dispersed buffer. The preferred dispersion buffer is PBS. PBS may have a suitable pH value such as 6 to 8, in particular about 6.9. This may require from 5 to 15, for example approximately 10 volume exchanges. Before step e, the liposomes can be diluted in a dispersion buffer to the desired concentration. The desired concentration may be in the range of 0.1-10 mg/ml, for example approximately 1 mg/ml. Before step e, the liposome-containing solution can be heated to a suitable temperature, for example 30 to 60°C, preferably about 35°C.

Step e involves mixing the solution containing the β-amyloid (Ab)-derived peptide antigen with the solution from step d using a cross-flow injection module. How

- 9 044699 disclosed herein, the β-amyloid (Ab)-derived peptide antigen is preferably lipidated (eg, tetrapalmitoylated), which applies mutatis mutandis. Before mixing, the β-amyloid (Av)-derived peptide antigen is typically dissolved in a suitable buffer solution, such as 10% v/w. Beta-OG solution in 10 mM Na ₂ HPO ₄ buffer, pH 11.4. The solution is usually heated to a suitable temperature, for example, to a temperature of from 30 to 80°C, such as approximately 60°C. If necessary, the solution can be further diluted to provide the appropriate concentration of β-amyloid (Av)-derived peptide antigen. A suitable concentration may be in the range of 0.1-10 mg/ml, for example about 1 mg/ml. The pH value is usually maintained in the range of 11-12, for example about 11, preferably 11.4. Mixing the solution containing the β-amyloid (Aβ)-derived peptide antigen with the solution from step d using a cross-flow injection module results in the insertion of the β-amyloid (Aβ)-derived peptide antigen into the outer lipid bilayer of the liposomes. The mixture can be incubated for a fixed period of time at a suitable temperature to facilitate the insertion of the amyloid β (Aβ)-derived peptide antigen into the lipid bilayer of the liposomes. A suitable period of time may be in the range of 20-120 minutes, for example approximately 30 minutes. A suitable temperature may be from 30 to 60°C, preferably about 35°C. Incubation can be carried out with agitation, for example, by stirring.

After step e, the product can be recovered for inclusion in the compositions of the present invention. Thus, the product can be formulated as a liposomal vaccine composition according to the present invention. This may include an ultra/diafiltration step to remove beta-OG from the buffer solution. Any suitable membrane can be used for this step, such as a hollow fiber membrane with a molecular weight cut-off of about 500 kDa. Ultra/diafiltration may include a buffer exchange step with a final buffer. The preferred final buffer is His-sucrose buffer, which may contain 10 mM histidine, 250 mM sucrose. This may require from 5 to 15, for example approximately 10 volume exchanges. A concentration step may be carried out to achieve the preferred final volume. A final (sterile) filtration step can also be performed. At this stage, you can use a cartridge filter. The filtration step can be carried out through a filter of any suitable pore size, for example approximately 0.2 microns. Filtration can be done under sterile conditions. The resulting vaccine composition can then be stored under suitable conditions until use, for example in a refrigerator (eg at about 5°C).

Brief description of drawings

Figure 1. (A) Analysis of Aβ 1-42-specific IgG antibodies by ELISA in plasma of C57BL/6 mice on day 21 (ACI-24.046) or day 7 (ACI-24, ACI-24.043, aCi-24.044) before (collection blood before immunization) and on days 7, 21 and 35 after the ^1st immunization with the indicated vaccines (arrows indicate the time points of immunization). Results are expressed as geometric mean with +/- 95% confidence interval (CI) in ng/ml for n=5 mice per group. The X-axis shows the days of treatments/blood collections, and the Y-axis shows antibody titers expressed in ng/ml. (B) Analysis of Aβ1-42-specific IgG antibodies by ELISA in plasma of C57BL/6 mice on day 21 (ACI-24.046) or day 7 (ACI-24, ACI24.043, ACI-24.044) before (blood collection before immunization) and day 21 after ^{the 1st} immunization with the indicated vaccines. Results are expressed as geometric mean with confidence interval +/- 95% CI in ng/ml for n=5 mice per group. Statistical test among different groups at 21 days: Kruskal-Wallis test with Dunn's multiple comparisons. * p<0.05; ** p<0.01. The X-axis shows individual plasma from groups immunized with the indicated vaccines, while the Y-axis shows antibody titers expressed in ng/ml.

Figure 2. (A) Analysis of inhibition of Aβ1-42 self-association by ELISA analysis of IgG antibodies in the plasma of C57BL/6 mice on day 21 or day 7 before (dashed lines) and day 21 after ^1st immunization (thick lines) with the indicated vaccines. Results are expressed as the mean +/- standard deviation of 5 mice per group for percentage inhibition of Aβ1-42 self-association. The X axis shows serial dilutions of plasma, while the Y axis shows the percentage inhibition of Aβ1-42 self-association. (B) Inhibition of Aβ1-42 self-association, shown as percentage (%) inhibition on day 21 minus % inhibition on day -21 or day -7 (baseline - pre-immunization blood draw) at a 1/25 plasma dilution. The X axis shows the groups treated with the indicated vaccines, while the Y axis shows the percentage inhibition of Aβ1-42 self-association after subtracting the baseline value.

Figure 3. Analysis of Aβ1-42 oligomer-specific IgG antibodies by ELISA in plasma of C57BL/6 mice on day 21 (ACI-24.046) or day 7 (ACI-24, ACI-24.043, ACI-24.044) before and day 21 after 1 ^oh immunization with the specified vaccines. Results are expressed as geometric mean with confidence interval +/- 95% CI in ng/ml for 5 mice per group. Statistical test among different groups on day 21: Kruskal-Wallis test with Dunn's multiple comparisons. * p<0.05; ** p<0.01. The X axis shows the groups immunized with the indicated vaccines, while the X axis

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Y shows antibody titers expressed in ng/ml.

Figure 4. Analysis of Αβ1-42 - avidity of IgG antibodies by ELISA in the plasma of C57BL/6 mice on days 7 and 21 after the ^1st immunization with the indicated vaccines. Results are expressed as geometric mean with confidence interval +/- 95% CI for avidity index for 5 mice per group. Statistical test among different groups on day 21: Kruskal-Wallis test with Dunn's multiple comparisons. * p<0.05; ** p<0.01. The X-axis shows the groups immunized with the indicated vaccines, while the Y-axis shows the avidity index.

Figure 5. Analysis of Ab oligomer-specific antibodies by MSD in cynomolgus serum before the first immunization (day 1) and 1 week after the third immunization (day 64) in macaques immunized with ACI-24.046 (SAT44, n=8), ACI- 24.045 (SAT43, n=4) or ACI-24.043 (SAT47, n=4). Results are expressed as geometric mean with confidence interval +/- 95% CI in AU/ml. The X-axis shows individual plasma from groups immunized with the indicated vaccines, while the Y-axis shows antibody titers expressed in AU/ml.

Figure 6. Analysis of Ae 1-42-specific IgG antibodies by ELISA in the plasma of C57BL/6 mice 7 days after the ^3rd immunization (day 36) with ACI-24 and ACI-24.046 (SAT44) vaccines (A) or ACI-24 vaccines and ACI-24.043 (SAT47) (B). Results are expressed as geometric mean with confidence interval +/- 95% CI in ng/ml for n=10 mice per group. The X-axis shows the vaccines used to immunize each specific group, and the Y-axis shows antibody titers expressed in ng/ml. statistical test: Mann-Whitney test between ACI-24 and the indicated vaccine. *p<0.05;**p<0.01,***p<0.001.

Table of abbreviations_________________________________________________________________

ABTS 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) Αβ amyloid beta (abeta) Ac2O acetic anhydride AD Alzheimer's disease AR alkaline phosphatase ARS antigen presenting cells B.S.A. bovine serum albumin AU/ml arbitrary units/ml 01 confidence interval DMF dimethylformamide DMPC 1,2-dimir isto yl-sn - gl and cero-Z -phospho ol in DMPG 1,2-dimyristoyl-sn-glycero-3-phosphoryl gl and cerine

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DMSO dimethyl sulfoxide EL1SA enzyme immunosorbent assay HLA human leukocyte antigen HPLC high performance liquid chromatography NKR horseradish peroxidase 1g immunoglobulin KLH snail lymph hemocyanin MPLA monophosphoryl lipid A ms mass spectrometry MSD Meso Scale Discovery Ra! 1-15 tetralal mitoylated A β 1 -15 PBS phosphate buffered saline solution PES polyethersulfonate pNPP p-nitrophen and lphosph at S.C. subcutaneously TMV hetram ethylbenzidine T.F.A. trifluoroacetic acid T1S 'gr and isopropyl yl an TLR4 Toll-like receptor 4 Beta-OG n-octyl-p-P-glucopyranoside

The present invention will become better understood through the following non-limiting examples.

Example 1. Construction of new T-cell epitopes.

The ability of a T cell epitope to activate T cells (immunogenicity index) is the result of two complementary properties: i) affinity for HLA, and ii) its ability to bind different HLA haplotypes in a non-selective manner. In silico evaluation (Epivax) of several T-cell epitopes of different origins was performed to select peptides with the highest immunogenicity index. At the preliminary stage, 10 different peptides of different origins (snail limpet hemocyanin-KLH, diphtheria toxin, influenza virus, Epstein-Barr virus and herpes virus) were evaluated. Peptides with the best immunogenicity index (above 10) were selected based on their likely high immunogenicity in humans based on their predicted HLA affinity and HLA haplotype coverage (selected peptide sequences are shown in Table 1).

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Table 1

Name Subsequence Origin of the peptide SAT6 STLEYFI.YDPIFFLHHSNTDRLWAIWQAI. QKYRGKPYNTANCAIVRHDTY (SEQ ID NO 5) KLH SAT13 VEmNTEEIVAQSIALSSLMV (SEQ GO NO: 6) Diphtheria toxin SAT15 roGVKLESMGVYQrLAIYSWASSL (SEQ GO NO: 7) Hemagglutinin influenza virus SAT17 VYGGSKTSLYNLRRGTALAI (SEQ GO NO: 8) Epstein-Barr virus

According to the results of screening of individual peptides, combined mixed peptides consisting of 2 or 3 immunogenic T-cell epitopes of different origin (named SAT42, SAT43 and SAT44) and mixed peptides consisting of truncated peptides (for example, SAT47 and SAT43) were designed (Table 2 ). Truncated peptides were designed by selecting within each individual T cell epitope sequence the most immunogenic 15-mer peptide sequence based on in silico predicted T cell epitope hotspots. The challenge was to increase the immunogenicity index without increasing the size of the final mixed peptide due to limitations of peptide synthesis and the vaccine encapsulation method. Briefly, the yield of peptide synthesis and the likelihood of success decrease as the length of peptides increases, especially to lengths greater than 30 amino acids, and, in addition, peptides consist primarily of hydrophobic residues, as disclosed herein for T cell epitope peptides. In addition, the encapsulation ratio of a peptide decreases with increasing peptide length because the chances of placing it in the liposome lumen decrease as the peptide length increases. The in silico immunogenicity index of these 4 mixed T-cell epitopes was very high and, importantly, higher than that of the single-component peptides, confirming that combining peptides of different origins can improve HLA affinity and HLA haplotype coverage (mixed T-cell epitope sequences) are given in Table 2).

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table 2

Name Subsequence Peptide model Origin peptide SAT42 VHHNTEEIVAQSIALSSLMVPMG APQYIKANSKF1GITEL (SEQIDNO: 1) SAT13+PMGAP+ Tetanus toxin Diphtheria and n+tetanus TOXIN SAT43 VYGGSKTSLYNLRRGTALAIVV RQYIKANSKFIGITELVVRPIFFLH HSNTDRLWAI (SEQ ID NO.2) SAT17+VVR+ Tetanus TOXIN+ VVR+ SAT6 EpsteinBarr virus+ Column toxin-!- KLH SAT44 VYGGSKTSLYNLRRGTALAIVV RQYIKANSKF1G1TEL (SEQIDNO: 3) SAT17+VVR+ Tetanus toxin Epstein-Barr virus + Tetanus toxin SAT47 SMGVYQ1LAIYSTVVRIVAQSIAL SSWRYTKANSKFIGVVRLYNLR RGTAL (SEQIDNO: 4) SAT15+VVR+SA T13+WR*Ctoa6 NURSE TOXIN-!· WR+ SAT 17 Hemagglutinia influenza virus Diphtheria current and n-!-Column- Ny toxin*- Epstein virus- Barr

Example 2. Synthesis and formulation of the vaccine.

General method for the synthesis and purification of a universal T cell epitope peptide.

T cell peptides were prepared by linear solid-phase peptide synthesis (SPPS) on 2-chlorotrityl resin using standard Fmoc chemistry. A standard coupling procedure was performed using 3.0 equivalents of amino acid and coupling reagent in the presence of 3.0 equivalents of base in DMF for 1 hour at room temperature. For difficult-to-bind sequences, double binding with extended reaction times was used. After completion of amino acid coupling, a capping acetylation step was performed using 5.0 equivalents of Ac ₂ O in pyridine to stop unwanted peptide chain extension. The resin was washed with DMF and the Fmoc group was removed using 20% piperidine in DMF for 5 min. After completion of SPPS, general deprotection was performed and the peptide was cleaved from the resin using a standard deprotection solution (TFA/TIS/water) for 2 h at room temperature. The resin was filtered and washed with TFA. The crude product was then precipitated with 10 times excess volume of cold isopropyl ether/hexane and the solid was filtered using a glass frit and dried in vacuo. The crude peptide was purified on a C18 reverse phase column using a gradient of solvent A (water, 0.1% TFA) and solvent B (acetonitrile, 0.1% TFA) in a preparative HPLC system. HPLC fractions containing the desired peptide at greater than 90% purity were pooled, diluted with water, and ion-exchanged. The desired ion exchange fractions were lyophilized. The identity and purity of the final peptide was determined and confirmed by HPLC-MS analysis.

Preparation of vaccines ACI-24.043/ACI-24.044/ACI-24.045/ACI-24.046/ (thin lipid film).

Vaccines containing encapsulated peptide T-cell epitopes were prepared by the lipid thin film method followed by homogenization and extrusion. Primarily through the solubilization of DMPC, DMPG (Lipoid, Germany), cholesterol and monophosphorylhexaacyl-lipid A, 3deacyl synthetic or 3D-(6-acyl) PHAD™ (Avanti Polar Lipids, USA) at molar ratios of 9:1:7:0.05 in ethanol at 60°C, respectively. Ethanol was evaporated on a vacuum rotary evaporator to obtain a thin lipid film.

The lipid film was rehydrated with one of the following buffers (depending on the T-14 044699 peptide of the cellular epitope that needs to be encapsulated):

mM sodium acetate, pH 4 (Fluka), 5% DMSO (Sigma Aldrich) in MilliQ water containing 0.8 mg/ml SAT42 T cell epitope peptide, or

0.1 x PBS, pH 7.4, 5% DMSO (all Sigma-Aldrich) in MilliQ water containing 0.3-0.4 mg/ml SAT43, SAT44, or SAT47 T cell epitope peptide.

The solution was stirred gently for 15 minutes. The sample was then shaken vigorously in the presence of glass beads. The resulting multilayer vesicles were subjected to 10 freeze-thaw cycles (liquid N2 and water bath at 37°C) and homogenized followed by sequential extrusion through polycarbonate membranes (Whatman, UK) with a pore size of 0.1/0.08 μm. Both homogenization and extrusion steps were performed using EmulsiFlex-C5 (Avestin, Canada). Extruded liposomes were concentrated by ultrafiltration, and the buffer was exchanged to PBS, pH 7.4, by diafiltration (10-fold exchange). The resulting liposomes were diluted in PBS, pH 7.4, and heated to 30°C before adding Pal1-15.

Tetrapalmitoylated human peptide Pal1-15 (Bachem AG, USA) was dissolved in 10 mM Na ₂ HPO ₄ , pH 11.4, in MilliQ water with 1% β-OG (Sigma-Aldrich, USA), injected into a liposomal solution at 30° C and stirred for 30 min followed by steps of concentration by ultrafiltration and dilution in PBS, pH 7.4, by diafiltration. The resulting liposomes were then sterile filtered through 0.2 μm polyethersulfone (PES) syringe filters and stored at 5°C.

Preparation of vaccine ACI-24.043 (cross-flow injection).

Lipids (DMPG, DMPC, cholesterol and 3D-(6-acyl) PHAD™ (Avanti Polar Lipids, USA)) were dissolved in 96% EtOH in a heating oven at 60°C. After complete dissolution of the lipids, the solution was filtered through a 0.2 μm filter into the injection system, which was heated to 60°C. In detail, an appropriate amount of ACI-24.043 (SAT47) was dispersed in EtOH at room temperature by sonication (EtOH concentration is typically 2% v/v of the final SAT47 solution). Once peptide dispersion was complete, His-sucrose buffer (10 mM histidine, 250 mM sucrose) was added to achieve a drug to lipid weight ratio of 1/50. The SAT47 solution was filtered through a 0.2 μm filter (Sartoscale filter) into a buffer injection vessel, which was then heated to 40°C. Liposomes were formed at the injection site when the lipid/EtOH solution and the injection buffer were mixed. Immediately after liposome formation, an operational dilution step was performed using 10 mM histidine, 250 mM sucrose to reduce the EtOH concentration. Intermediate liposomes were extruded through polycarbonate membranes with a pore size of 100 nm (1 pass) at room temperature. Ultra/diafiltration (UDF) using a hollow fiber membrane (MWCO: 500 kDa) was performed to remove EtOH, and the buffer was exchanged with PBS, pH 6.9 (10 volume exchanges). SAT47 liposomes were then diluted using dispersion buffer (PBS, pH 6.9) to a total lipid concentration of 1 mg/mL and heated to 35°C. Pal1-15 was dissolved in 10% w/v. beta-OG solution in 10 mM Na ₂ HPO ₄ buffer, pH 11.4, at 60°C, and then dissolved with the same buffer to a final concentration of 1 mg/ml. The pH value was adjusted to 11.4. After mixing the two solutions using a cross-flow injection module, the liposomal suspension was then incubated at 35°C for 30 min with mixing to ensure complete insertion of Pal1-15. A second UDF step using a hollow fiber membrane (MWCO: 500 kDa) was performed to remove beta-OG and buffer exchange with 10 mM histidine, 250 mM sucrose (10 volume exchanges). The product was concentrated to its final volume and filtered through 0.2 μm Acrodisc mPES syringe filters.

Preparation of vaccine ACI-24.046 (cross-flow injection).

Lipids (DMPG, DMPC, cholesterol and 3D-(6-acyl) PHAD™ (Avanti Polar Lipids, USA)) were dissolved in 96% EtOH in a heating oven at 60°C. After complete dissolution of the lipids, the solution was filtered through a 0.2 μm filter into the injection system, which was heated to 60°C. Simultaneously, ACI-24.046 (SAT44) was dissolved in injection buffer (10 mM histidine, 250 mM sucrose) at 40°C. Once dissolution of SAT44 was complete, the solution was filtered through a 0.2 μm pore size filter (Sartoscale) into a buffer injection vessel that was heated to 40°C. Liposomes were formed at the injection site when the lipid/EtOH solution and the injection buffer were mixed. Immediately after liposome formation, an operational dilution step was performed using 10 mM histidine, 250 mM sucrose to reduce the EtOH concentration. Intermediate liposomes were extruded through polycarbonate membranes with a pore size of 100 nm (1 pass) at room temperature. Ultra/diafiltration (UDF) using a hollow fiber membrane (MWCO: 500 kDa) was performed to remove EtOH, and the buffer was exchanged with PBS, pH 6.9 (10 volume exchanges). SAT44 liposomes were then diluted using dispersion buffer (PBS, pH 6.9) to a total lipid concentration of 1 mg/mL and heated to 35°C. Pal1-15 was dissolved in 10% w/v. a solution of beta-OG in 10 mM Na ₂ HPO ₄ buffer, pH 11.4, at 60°C, and then dissolved with the same buffer to a final concentration of 1 mg/ml. The pH value was checked and carefully adjusted to 11.4. After mixing the two solutions using the injection module, the liposomal suspension was then incubated at 35°C for 30 minutes with mixing to ensure complete insertion of Pal1-15. A second UDF step using a hollow fiber membrane (MWCO: 500 kDa) was performed to remove beta-OG and buffer exchange with 10 mM histidine, 250 mM sucrose (10 volume exchanges). The product was concentrated to its final volume and filtered through 0.2 μm Acrodisc mPES syringe filters.

Example 3: Conceptual (PoC) in vivo studies of the immunogenicity of vaccines with encapsulated T-cell epitopes.

Following successful encapsulation of various T cell epitopes, the immunogenicity of vaccines containing the high immunogenicity index T cell epitopes encapsulated SAT42, SAT44 and SAT47 (vaccines ACI-24.044, ACI-24.046 and ACI-24.043, respectively) was examined in vivo in comparison with the ACI vaccine -24. Wild-type C57BL/6 mice received a total of three subcutaneous (s.c.) immunizations on days 0, 14, and 28 with ACI-24, ACI-24.044 (with SAT42 encapsulated), ACI-24.046 (with SAT44 encapsulated), and ACI-24.043 ( with encapsulated SAT47). Blood samples were collected on day -21 (ACI-24.046) or day -7 (ACI-24, ACI-24.043, ACI-24.044) (pre-immunization blood draw), and days 7, 21 and 35 to measure Ae titers 1-42 -specific IgG by ELISA.

The plates were coated with 10 μg/ml human Ab 1-42 peptide film (Bachem, Switzerland) overnight at 4°C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween/PBS, serial dilutions of plasma were added to the plates and incubated at 37°C for 2 hours. After washing, the plates were incubated with alkaline-conjugated phosphatase (AP) with anti-mouse IgG antibodies (Jackson ImmunoResearch, PA, USA) for 2 h at 37°C. After the final wash, the plates were incubated for 2.5 h with AP substrate (pNPP) and read at 405 nm using an ELISA plate reader. Results are expressed with reference to serial dilutions of a commercially available antibody (6E10, Biolegend, UK, cat. no. 803002). In fig. 1A shows the Ab 1-42-specific IgG titers induced by the ACI-24 vaccine with or without an encapsulated T-cell epitope as a function of time. Although the ACI-24 vaccine showed the highest titers of Ae 1-42-specific IgG at day 7 after the ^1st immunization, increases in antibody titers were observed after the 2nd and 3rd immunizations when the T cell epitope was encapsulated in ACI-24 vaccine.

The results shown in Fig. 1B show that immunization with ACI-24 vaccines containing encapsulated T cell epitopes caused an increase in Av-specific antibody titers compared to ACI-24, which reached statistical significance for the group immunized with ACI-24.043 vaccine (with encapsulated SAT47).

Vaccines with encapsulated SAT42, SAT43, SAT44 or SAT47 were tested in a study in cynomolgus monkeys. Four macaques per group received subcutaneous immunization three times a month (days 1, 29 and 57) with ACI-24.044 (encapsulated SAT 42 - two groups with 8 macaques in total), ACI-24.046 vaccine (encapsulated SAT 44-2 two groups with 8 macaques in general), vaccine ACI-24.045 (encapsulated SAT 43-4 macaques) or vaccine ACI-24.043 (encapsulated SAT 47-4 macaques). Blood was drawn before the first immunization (day 1) and 1 and 3 weeks after each immunization (days 8, 22, 36, 50, 64, and 78) to measure Ae 1-42-specific IgG titers by ELISA.

The plates were coated with 10 μg/ml human Ab1-42 peptide film (Bachem, Switzerland) overnight at 4°C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween 20/PBS, 8 2-fold serial dilutions of serum were added to the plates and incubated at 37°C for 2 hours. After washing, the plates were incubated with horseradish peroxidase (HRP)-conjugated anti-monkey IgG antibody (KPL, cat. no. 07411021) for 2 h at 37°C. After washing, the plates were incubated with 50 µl ABTS/H2O2 (2,2'-azino-bis(3-ethylbenzothiazoline-6sulfonic acid) (HRP substrate) and read at 405 nm after one hour using an ELISA plate reader. Results are given with reference to serial dilutions of a positive pool of macaques used as a standard.

The immunogenicity of vaccines with different T cell epitopes was compared with the ACI-24 vaccine. In table Figure 3 shows a fold increase in the titer of Av-specific antibodies compared to the ACI-24 vaccine 1 week after the third immunization. All tested vaccines: ACI-24.046 (SAT44), ACI24.043 (SAT47), ACI-24.045 (SAT43) and ACI-24.044 (SAT42) caused an increase in antibody titers of at least 7 times (ACI-24.044 with encapsulated SAT42), according to compared with titers induced by the ACI-24 vaccine. Vaccine ACI-24.043 (with encapsulated SAT47) and ACI-24.046 (with encapsulated SAT44) induced significantly higher titers of Av-specific antibodies compared to ACI-24 1 week after the third immunization (Table 3). ACI-24.043 (with encapsulated SAT47) and ACI-24.046 (with encapsulated SAT44) each had high Epivax scores (142.89 and 57.2, respectively).

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Table 3

Fold increase in Aβ-specific antibody titer compared to ACI-24 (1 week after third immunization, day 64)

Vaccine ACI-24.046 (encapsulated SAT44) ACI-24.043 (encapsulated SAT47) ACI-24.045 (encapsulated SAT43) ACI-24.044 (encapsulated SAT42) multiple increase is not titre Aβ- specific koyu IgG compared to ACI-24 40 p-0.0027 (**) 144 p-0.0003 (***) 17 p-0.1408 (ns) 7 p-0.6003 (ns)

Statistical test: Kruskal-Wallis test with Dunn's multiple comparisons. *p<0.05; **p<0.01; ***p<0.001; ns: statistically insignificant.

Based on the in vivo results (Fig. 1) with vaccines ACI-24.046 (encapsulated SAT44) and ACI-24.043 (encapsulated SAT47) prepared using the lipid thin film technique, the in vivo immunogenicity of the same vaccines prepared using the lateral flow injection method was tested. . Wild-type C57BL/6 mice received a total of three subcutaneous (s.c.) immunizations on days 0, 14, and 28 with ACI-24, ACI-24.046 (with SAT44 encapsulated), or ACI-24.043 (with SAT47 encapsulated). Blood samples were collected on days -7, 7, 21 and 35 to measure Αβ1-42 specific IgG levels by ELISA.

The results in Fig. 6 show that immunization with ACI-24 vaccines containing encapsulated T-cell epitopes caused a significant increase in Αβ-specific antibody titers compared to ACI-24.

Example 4. Quality of induced Aβ-specific antibodies.

4.1. In vitro inhibition of human Αβ1-42 self-association.

The quality of the Αβ-specific antibodies induced was tested in vitro by measuring the inhibition of Αβ1-42 self-association/aggregation. This assay is based on the ability of mouse plasma before and after immunization to disrupt the natural propensity of human Aβ1-42 to self-associate.

Standard ELISA plates were coated with 1 μg/ml Αβ1-42 overnight at 4°C. The plates were washed 4 times with 300 μl of 0.05% Tween 20/PBS. Saturation was achieved by adding 0.5% BSA/PBS and incubating for 1 hour at 37°C. After washing, four 2-fold serial dilutions of plasma were added to the plates for 20 min at room temperature with shaking. Biotinylated Αβ1-42 was added to each well to a final concentration of 0.1 μg/ml and incubated at room temperature for 2 h with agitation. Biotinylated Αβ1-42 without plasma was used as a positive control for Αβ1-42 self-association (considered as 100% self-association, 0% inhibition). After the washing step, the plates were incubated with streptavidin-conjugated horseradish peroxidase (HRP) (R&D Systems, Canada, Ref. 890803) at a 1/200 dilution in 0.5% BSA/0.05% Tween 20/PBS for 1 h. at room temperature with shaking. After washing, the plates were incubated with Sure Blue Reserve TMB substrate (Seracare, cat. no. 5120-0081) for 10 min. The reaction was stopped with Bethyl Stop Solution (Bethyl Laboratories, Inc, Cat. No. El 15) and the plates were read at 450 nm using an ELISA plate reader. The percentage inhibition of self-association was calculated using biotinylated Αβ1-42 without plasma as a positive control (0% inhibition) as a reference.

The results showed that Αβ-specific antibodies obtained after 2 immunizations with all vaccines containing the T-cell epitope disrupted Αβ1-42 self-association more effectively than antibodies induced by ACI-24 (Fig. 2A). Because preimmunization plasma causes basal inhibition of self-association, the percentage at day 21 was normalized by subtracting the baseline value for preimmunization plasma. Specific antibodies to Αβ1-42 obtained by immunization with all ACI-24 vaccines containing the T cell epitope showed higher inhibition of Αβ1-42 self-association compared to ACI-24, this inhibition reached statistical significance in the group immunized with ACI-24.046 (with encapsulated SAT44) (Fig. 2B).

4.2 Formation of antibodies that recognize Αβ-oligomers.

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To assess the specificity of the antibodies induced in C57BL/6 mice to pathological Άβ binding, IgG responses specific for Aβ 1-42 oligomers were determined by ELISA. The plates were coated with 10 μg/ml oligomers prepared as previously described ( Adolfsson, 2012 ) overnight at 4°C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween 20/PBS, serial dilutions of plasma were added to the plates and incubated at 37°C for 2 hours. After washing, the plates were incubated with conjugated alkaline phosphatase (ΆΡ) anti-mouse IgG antibody (Jackson ImmunoResearch, Cat: 115-055-164, PA, USA) for 2 hours at 37°C. After the final wash, the plates were incubated for 2.5 h with ΆΡ substrate (pNPP) and read at 405 nm using an ELISA plate reader. Results are expressed relative to serial dilutions of commercially available antibody.

Each sample was tested in eight or four 2-fold serial dilutions, starting at a dilution of 1/100, 1/400, 1/800, or 1/1600, based on anti-Ae1-42 antibody titers. The results in Fig. 3 show that immunization with all ACI-24 vaccines containing the T-cell epitope caused an increase in titers of antibodies specific to the Άβ1-42 oligomer compared to ACI-24, which reached statistical significance for the group immunized with the ACI-24.043 vaccine (with encapsulated SAT47).

The avidity index of induced antibodies in C57BL/6 mice 7 and 21 days after immunization was determined by ELISA assay. One half of a standard ELISA plate was coated with 10 μg/ml Άβ1-42 peptide film and the other half was coated with 1 μg/ml Άβ142 peptide film overnight at 4°C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween 20/PBS, eight 2-fold serial dilutions of plasma were added to both coatings and incubated at 37°C for two hours. After the washing step, the plates were incubated with alkaline phosphatase (ΆΡ)-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, Cat: 115-055-164, PA, USA) for 2 h at 37°C. After the final wash, the plates were incubated for 2.5 h with AP substrate (pNPP) and read at 405 nm using an ELISA plate reader. Results are expressed relative to serial dilutions of a commercially available antibody (6E10, Biolegend, UK, Cat. 803002).

To determine the avidity index, AU/mL was calculated for each sample on both coatings using a standard curve obtained for 10 μg/mL of Ae1-42 peptide. Absorbance values between 0.6 and 2.8 were used to back-calculate the concentration. The avidity index was calculated as the ratio between the antibody concentration of the lower coating concentration (1 μg/ml Άβ1-42 peptide) and the saturated coating concentration (10 μg/ml Άβ1-42 peptide).

The results shown in Fig. 4 show that immunization with all ACI-24 T-cell epitope-containing vaccines induced maturation of Άβ 1-42-specific antibody avidity between the ^1st and ^2nd immunizations (day 7 and day 21, respectively), which reached statistical significance at groups immunized with ACI-24.044 (with encapsulated SAT42) and ACI-24.043 (with encapsulated SAT47).

To assess the specificity of induced antibodies in cynomolgus monkeys for binding pathological Άβ, Άβ1-42 oligomer-specific IgG titers were measured using Meso Scale Discovery (MSD) technology on day 64 (1 week after the third immunization) in the serum of cynomolgus monkeys immunized with the vaccine ACI-24.046 (encapsulated SAT44 - 2 groups, total 8 macaques), ACI-24.045 vaccine (encapsulated SAT 43-4 macaques) or vaccine ACI-24.043 (encapsulated SAT 47-4 macaques). MSD streptavidin plates were saturated overnight with 5% blocker A (MSD, Ref. R93BA-4) at 4°C. The next day, plates were washed 4 times with 0.05% Tween 20/PBS and coated with 25 μl of biotinylated 6E10 capture antibody (Biolegend, Ref. 803008) in PBS at a concentration of 0.5 μg/ml for 1 hour at 37°C on a shaker. . After washing, the plates were incubated with 25 μl of Άβ1-42 oligomers (Adolfsson, 2012) at 10 μg/ml in PBS for 1 h at 37°C on a shaker. The plates were washed and incubated with eight 2-fold dilutions of macaque serum (initial dilution 1/50 in 1% skim milk/0.05% Tween/PBS). Samples were incubated for 2 hours at 37°C on a shaker. Plates were washed 4 times and SULFO-TAG-labeled anti-human IgG antibody (Jackson, Ref. 109-005-098) diluted in 1% skim milk/0.05% Tween 20/PBS was added for 1 h at 37°C. on a shaker. After 4 washes, MSD T 2X Reading Buffer (MSD, Ref. R92TC-2) was added and the plates were read within 5 min. Results are expressed relative to serial dilutions of the macaque pool used as a standard.

Results showed that all tested vaccines ACI-24.046 (encapsulated SAT44), ACI-24.043 (encapsulated SAT47) and ACI-24.045 (encapsulated SAT43) induced an increase in antibodies capable of recognizing Άβ oligomers at day 64 (1 week after immunization) compared from day 1 (before the first immunization), see Fig. 5.

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Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art of the present invention. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes related to the invention.

The scope of the present invention is not limited to the specific embodiments described herein. Indeed, various modifications of the present invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be within the scope of the appended claims. Moreover, all aspects and embodiments of the invention described herein are intended to be broadly applicable and compatible with any and all other consistent embodiments, including those taken from other aspects of the invention (including individually), as appropriate.

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Claims (16)

CLAIM 1. Liposomal vaccine composition containing: a) β-amyloid (Ab)-derived peptide antigen presented on the surface of the liposome; b) a peptide containing a universal T cell epitope encapsulated within a liposome, which is capable of stimulating a helper T cell response that enhances the production of antibodies by the B cells; c) adjuvant. 2. The liposomal vaccine composition according to claim 1, wherein the peptide containing the universal T-cell epitope contains at least 30% hydrophobic amino acids. 3. The liposomal vaccine composition according to claim 1 or 2, wherein the vaccine composition contains at least two different universal T-cell epitopes that are encapsulated within the liposome. 4. Liposomal vaccine composition according to any one of claims 1-3, where each universal T-cell epitope has a length of no more than 30 amino acids, no more than 20 amino acids, or no more than 10-20 amino acids. 5. The liposomal vaccine composition according to any one of claims 1 to 4, wherein the vaccine composition contains two, three or four different universal T-cell epitopes that are encapsulated within the liposome. 6. The liposomal vaccine composition according to any one of claims 1 to 5, wherein the universal T-cell epitope-containing peptide contains at least two different universal T-cell epitopes. 7. The liposomal vaccine composition according to any one of claims 1 to 6, wherein the universal T-cell epitope-containing peptide contains two, three or four universal T-cell epitopes. 8. Liposomal vaccine composition according to any one of claims 3 to 7, wherein at least two universal T-cell epitopes are connected by a linker. 9. The liposomal vaccine composition of claim 8, wherein the linker comprises at least two amino acids, wherein the linker optionally contains, consists essentially of, or consists of the amino acids VVR or PMGAP. 10. Liposomal vaccine composition according to any one of claims 1 to 9, where the universal T-cell epitopes are selected from: a) combinations of a universal T-cell epitope of diphtheria toxin and tetanus toxin; b) combinations of the universal T-cell epitope of the Epstein-Barr virus and tetanus toxin; c) combinations of the universal T-cell epitope of the Epstein-Barr virus, tetanus toxin and hemocyanin of the snail lymph; or d) combinations of the universal T-cell epitope hemagglutinin of the influenza virus, diphtheria toxin, tetanus toxin and Epstein-Barr virus. 11. The liposomal vaccine composition according to any one of claims 1 to 10, wherein the universal T cell epitope containing peptide contains, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NO: 1 (SAT42), SEQ ID NO : 2 (SAT43), SEQ ID NO: 3 (SAT44), SEQ ID NO: 4 (SAT47). 12. The liposomal vaccine composition according to any one of claims 1 to 10, wherein the universal T cell epitope contains, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NO: 5 (SAT6), SEQ ID NO: 6 (SAT13 ), SEQ ID NO: 7 (SAT15), SEQ ID NO: 8 (SAT17). 13. Liposomal vaccine composition containing: a) a tetrapalmitoylated amyloid beta (Ab)-derived peptide antigen presented on the surface of a liposome that contains, consists essentially of, or consists of amino acids 1 to 15 of amyloid beta; b) a peptide containing a universal T cell epitope encapsulated within a liposome, wherein the peptide containing a universal T cell epitope contains, consists essentially of, or consists of an amino acid sequence selected from SEQ ID NO: 3 (SAT44) and SEQ ID NO: 4 (SAT47). c) adjuvant. 14. Liposomal vaccine composition according to any one of claims 1 to 13, wherein the adjuvant forms part of the liposome. 15. Liposomal vaccine composition according to any one of claims 1 to 14, wherein the adjuvant is present at least partially on the surface of the liposome. 16. Liposomal vaccine composition according to any one of claims 1 to 15, wherein the adjuvant contains monophosphoryl lipid A (MPLA). -