Vaccine for treating anti a bata

Abstract

To provide a high immunogenicity anti Aβ vaccine while maintaining an excellent safety profile.SOLUTION: A liposome vaccine composition includes a peptide antigen derived from β-amyloid (Aβ) presented on a surface of a liposome. The vaccine composition further includes a peptide including a universal T-cell epitope encapsulated into the liposome. The vaccine composition also may form a part of the liposome, and includes an adjuvant that may be at least partially presented on the surface of the liposome. The vaccine compositions are used for treating or preventing a disease or a condition related to amyloid beta, or symptoms characterized in loss of cognitive memory ability of a subject or a condition related thereto, inducing protective immune response to the symptom, or alleviating the symptoms. The vaccine composition may be provided as a kit. A related method for producing a liposome vaccine composition is also provided.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to vaccines for anti-A beta therapy and their use in the treatment and prevention of diseases. The vaccines contain Aβ-derived peptide B cell antigens and T cell epitopes.

Background Art

[0002] Alzheimer's disease (AD) is a devastating and progressive neurodegenerative disease characterized by the loss of cognitive functions including memory and the loss of the ability to perform normal daily activities. AD affects approximately 400 million patients worldwide, and the number is rapidly increasing as the population ages. The main neuropathological change in the brains of AD patients is mainly neuronal death in the memory and cognition-related regions (Non-Patent Document 1: Soto, 1999). One of the most prominent pathological features of AD is the abundant presence of amyloid beta (Aβ) plaques in the brains of affected individuals (Non-Patent Document 1: Soto, 1999). Aβ plaques are formed by Aβ peptides that are 39-43 amino acids in length and have a random coil conformation in their original non-pathological form. During the transition to the diseased state, the peptide mainly changes to a β-sheet secondary structure and spontaneously aggregates into insoluble deposits.

[0003] Several currently available treatments for AD are thought to be mainly symptomatic in their action. Despite the great efforts that have been made over the years in developing treatments, disease-modifying treatments for AD have not been approved to date. Attempts have been made for a long time to develop immunotherapies that neutralize pathological Aβ in the diseased brain (Non-Patent Document 2: Winblad, 2014). Vaccines have the advantage of stimulating the immune system to generate a pool of slightly different but highly specific antibodies, and the response can be further evoked by additional vaccinations if necessary. However, there are several major problems with the prophylactic vaccination (vaccination) approach to Aβ. Amyloid beta is a so-called self-antigen, and the human body is constantly exposed to it. Therefore, it is very difficult to break immune tolerance and induce an antibody response against it. Furthermore, elderly and sick people such as AD patients have a weak immune system and a small number of immune cells, so it is very difficult to induce a strong immune response to the vaccine.

[0004] Despite these problems, in the first study, the full-length Aβ1-42 vaccine (AN1792) induced an antibody response and promising efficacy, and the rate of cognitive decline was slower in vaccinated patients than in those receiving placebo treatment (Non-Patent Document 3: Gilman, 2005). However, 6% of the treated patients developed meningoencephalitis, which was an inflammatory reaction thought to be caused by a T cell response to full-length Aβ1-42 (Non-Patent Document 4: Orgogozo, 2003).

[0005] Another known anti-Aβ vaccine, ACI-24, contains a 15-amino acid sequence that is identical to the human sequence 1-15 of Aβ (Patent Document 1: WO2007 / 068411). This peptide antigen is bound to a liposome carrier for the purpose of avoiding meningoencephalitis and hemorrhage and stimulating antibodies against Aβ (Non-Patent Document 5: Muhs, 2007; Non-Patent Document 6: Pihlgren, 2013). The selection of the Aβ1-15 peptide that functions as an antigen is based on the rationale that this sequence contains a B cell epitope but lacks a strong T cell response site of full-length Aβ1-42 (Non-Patent Document 7: Monsonego, 2003), and the latter is considered to be the cause of an undesirable inflammatory reaction. ACI-24 has been shown to act through the simultaneous activation of the B cell receptor specific for Aβ1-15 and Toll-like receptor 4 (TLR4) activated by monophosphoryl lipid A (MPLA), an adjuvant present in the ACI-24 vaccine (Non-Patent Document 6: Pihlgren, 2013). B cells are activated to proliferate and produce immunoglobulins (Ig) by cross-linking the Ig receptors on the B cell surface. To increase antibody production, a second signal can be provided by T helper cells activated by T cell epitopes. T cell epitopes presented by major histocompatibility complex (MHC) molecules (referred to as human leukocyte antigens (HLA) in humans) on the surface of antigen-presenting cells (APCs) promote the differentiation of naive T cells capable of producing IFNγ and IL-4 into activated T cells. Cytokine release and co-stimulatory signals between activated T cells and B cells increase the antibody response and class switch. After the primary vaccination, naive T cells proliferate and differentiate into effector cells. A small fraction of these cells form a pool of long-lived memory T cells and can rapidly proliferate upon re-encountering the homologous peptide after booster vaccination (Non-Patent Document 8: Sallusto, 2010). So-called "universal" T cell epitopes are specific for T cells present in the majority of the human population. They generally originate from antigens (such as tetanus, influenza) to which humans are normally exposed throughout their lives.The ability of a T cell epitope to activate T cells is the result of at least the following two complementary properties: i) the affinity for binding to the HLA groove, i.e., the strength of the binding, and ii) the ability to bind to different HLAs in an undiscriminating manner, i.e., the ability to cover a very diverse human population with respect to differences in the expression of HLA molecules.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Non-Patent Documents

[0007]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Summary of the Invention

Problems to be Solved by the Invention

[0008] It is necessary to develop an anti-Aβ vaccine with high immunogenicity while maintaining a good safety profile.

Means for Solving the Problems

[0009] This need was met by incorporating a universal T cell epitope within the liposomal ACI-24 vaccine. Since the ACI-24 vaccine presents Aβ1-15 on the surface of the liposome, including a universal T cell epitope on the surface of the liposome was considered by the inventors as the first choice route for improving the efficacy of the vaccine. However, surprisingly, including a universal T cell epitope on the surface of the liposome was unable to increase (or substantially increase) the potency of the vaccine. Therefore, as described herein, an encapsulation approach that has been shown to provide improved potency was adopted. Incorporation of the universal T cell epitope within the liposomal vaccine has been shown herein to increase (or substantially increase) the efficacy of the vaccine while maintaining a favorable safety profile through T cell activation not directed against Aβ. However, there were several challenges in the development of such an approach. First, the universal T cell epitopes developed herein tend to be hydrophobic, which makes encapsulation into liposomes difficult. Second, multiple universal T cell epitopes were often combined to improve immunogenicity. However, as the length of the peptide increases, the yield and success rate of peptide synthesis decrease. Third, the charge of the selected universal T cell epitope affects the efficiency of encapsulation and the experimental conditions required to ensure encapsulation. This is due to the negatively charged liposomal membrane.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0011] Accordingly, the present invention provides a liposomal vaccine composition comprising the following: a. a β-amyloid (Aβ)-derived peptide B cell antigen presented on the surface of the liposome; and b. a peptide containing a universal T cell epitope encapsulated within the liposome.

[0012] Particularly preferred vaccine compositions include the ACI-24 vaccine modified to include a peptide containing a universal T cell epitope encapsulated within liposomes. Liposomes are an example of a carrier. Thus, the carrier is generally a liposome, but can be any carrier suitable for presenting an Aβ-derived peptide antigen on its surface in the same manner achieved by liposomes (the Aβ-derived peptide antigen predominantly adopts a β-sheet conformation) and can encapsulate a peptide containing a universal T cell epitope. Examples include vesicles and particulate bodies.

[0013] "Universal T cell epitope" means an epitope specific for T cells present in a majority of the human population. They generally derive from antigens to which humans are normally exposed throughout their lives. Examples include antigens incorporated into routinely administered vaccines. Specific examples include T cell epitopes contained in tetanus, influenza, diphtheria, keyhole limpet hemocyanin (KLH), Epstein-Barr virus (EBV), etc. The "universal" ability of a T cell epitope to activate T cells is the result of at least two complementary properties: i) the affinity for binding to the HLA groove, i.e., the strength of the binding, and ii) its ability to bind to this HLA haplotype in an indiscriminate manner, i.e., the ability to cover a very diverse human population with respect to differences in the expression of HLA molecules. A universal T cell epitope may bind to a majority of the MHC class II alleles present in the human population. The universal T cell epitope contained in the vaccine composition of the present invention may stimulate a CD4 T cell response. The universal T cell epitope contained in the vaccine composition of the present invention may stimulate a helper T cell response that enhances (Aβ-specific) antibody production by B cells.

[0014] The universal T cell epitopes contained in the vaccine composition of the present invention are typically synthesized by solid-phase synthesis. Thus, in some embodiments, the universal T cell epitopes are synthesized by solid-phase synthesis. This or other encapsulation practices mean that, in some non-limiting embodiments, the peptide containing the universal T cell epitope is 85, 80, 75, or 70 amino acids or less in length. The minimum length of the T cell epitope peptide to ensure sufficient immunogenicity is typically about 10 amino acids. Thus, the minimum length of the peptide is typically about 10 amino acids to ensure that a sufficiently immunogenic T cell epitope is generated. In some embodiments, the peptide is at least 20 amino acids in length. In other embodiments, the peptide is 30 to 60 amino acids in length. This is based on the preferred minimum length per universal T cell epitope and the preference for peptides containing at least two, three, or four (linked) universal T cell epitopes.

[0015] The useful universal T cell epitopes of the present invention have typically been found to be hydrophobic. This provides additional challenges for their synthesis, purification, and encapsulation within liposomes by interaction with lipids. The percentage of hydrophobicity is calculated by dividing the total number of hydrophobic amino acids (Phe, Ile, Leu, Met, Val, Trp, Ala, and Pro) by the total number of amino acids in the entire peptide containing the universal T cell epitope (when considering the entire peptide) or by the total number of amino acids in each individual T cell epitope (when considering each individual universal T cell epitope separately), and then multiplying by 100. Hydrophobic amino acids suitable for the purposes of the present invention are defined as leucine (Leu), isoleucine (Ile), phenylalanine (Phe), tryptophan (Trp), valine (Val), methionine (Met), proline (Pro), and alanine (Ala).

[0016] Thus, generally, peptides containing universal T cell epitopes contain at least 30% hydrophobic amino acids. This means that at least 30% of the amino acids of the entire peptide constituting 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 examples, the peptide may contain at least 35%, 40%, 45%, or 50% hydrophobic amino acids.

[0017] To improve the level of immunogenicity, the vaccine composition preferably comprises at least two different universal T cell epitopes encapsulated within liposomes. In combination with the hydrophobicity and synthetic constraints of the peptides, depending on the capacity of the liposomes, ideally each universal T cell epitope is typically 30 amino acids or less in length, preferably 20 amino acids or less in length, and even more preferably in the range of about 10 to 20 amino acids in length. As further described herein, the inventors have found that longer universal T cell epitopes can be effectively trimmed to a length of 10 to 20 amino acids while retaining immunogenicity. The trimmed peptides are designed by selecting the most immunogenic short sub-sequences, typically about 15 amino acids in length, based on the predicted T cell epitope hotspots in silico in the sequences of the individual T cell epitopes. Various software programs are available to assist in performing this analysis, such as the EpiVax immunogenicity screening platform (accessible from http: / / www.epivax.com).Further examples include SYFPEITHI (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; accessed via http: / / www.svfpeithi.com), SVMHC (https: / / www.ncbi.nlm.nih.gov / pubmed / 16844990), and the Immune Epitope Database (IEDB) (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; accessed via http: / / www.iedb.org / ).

[0018] In some embodiments, each universal T cell epitope comprises at least 30% hydrophobic amino acids. This means that at least 30% of the amino acids of an individual universal T cell epitope are hydrophobic amino acids. For a particular epitope, this value may be as high as 80% hydrophobic amino acids. In some instances, there may be at least 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80% hydrophobic amino acids. In some embodiments, the maximum value may be 80% hydrophobic amino acids, meaning that the broadest range may be from 30% to 80% hydrophobic amino acids.

[0019] To balance improved immunogenicity with the practical challenges of encapsulation, the vaccine composition may contain two, three, or four different universal T cell epitopes encapsulated within a carrier. When multiple different universal T cell epitopes are encapsulated (especially three or four), they are preferably trimmed to a length of about 10-20 amino acids, such as about 15 amino acids. Preferably, the multiple different universal T cell epitopes are included in the same peptide. Thus, synthetic peptide constructs containing multiple different universal T cell epitopes represent a preferred embodiment of the present invention. In certain 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. When at least two universal T cell epitopes are included in a synthetic peptide construct, they may be linked by a linker. A linker is used to physically connect the universal T cell epitopes to each other in a way that does not impair the immunogenicity of the linked epitopes. Suitable linkers for joining amino acids to each other are well known in the art. Preferred linkers are themselves amino acid-based linkers, i.e., peptide linkers. Thus, they can join universal T cell epitopes to each other via peptide bonds. The linker is a linker that allows for proper processing of the universal T cell epitopes. Antigen presentation by MHC class II molecules requires entry of the antigen into the endosome-lysosome compartment. These antigens are then processed by proteolytic enzymes, and within the proteolytic enzymes, the papain family of lysosomal cysteine proteases constitutes an important subset. The peptides generated bind to MHC class II molecules and are presented on the surface of professional antigen-presenting cells (APCs) such as macrophages, dendritic cells (DCs), and B cells (Lutzner and Kalbacher 2008). Thus, preferably, the linker contains a substrate for the papain family of lysosomal cysteine proteases.The linker may contain one or more substrates of cathepsin S, cathepsin B, and cathepsin L. In some embodiments, the linker comprises, consists essentially of, or consists of at least two or at least three amino acids. In some embodiments, the linker comprises, consists essentially of, or consists of the amino acids VVR, TVGLR, KVSVR, PMGAP, or PMGLP.

[0020] Thus, a peptide containing two universal T cell epitopes may be a linear peptide of the following form. [Universal T cell epitope 1]-[Linker]-[Universal T cell epitope 2] Thus, a peptide containing three universal T cell epitopes may be a linear peptide of the following form. [Universal T cell epitope 1]-[Linker]-[Universal T cell epitope 2]-[Linker]-[Universal T cell epitope 3] Thus, a peptide containing four universal T cell epitopes may be a linear peptide of the following form. [Universal T cell epitope 1]-[Linker]-[Universal T cell epitope 2]-[Linker]-[Universal T cell epitope 3]-[Linker]-[Universal T cell epitope 4]

[0021] It should be noted that the linker need not 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, two may be the same and the third different (in any order). In some embodiments, where multiple linkers are included in the peptide, in some embodiments they are all the same.

[0022] In devising peptides suitable for encapsulation, the inventors screened a series 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 hemagglutinin and / or keyhole limpet hemocyanin. Accordingly, certain preferred combinations of universal T cell epitopes are selected from the following. a. A combination of universal T cell epitopes of diphtheria toxin and tetanus toxin b. A combination of universal T cell epitopes of Epstein-Barr virus and tetanus toxin c. A combination of universal T cell epitopes of Epstein-Barr virus, tetanus toxin, keyhole limpet hemocyanin, or d. A combination of universal T cell epitopes of influenza hemagglutinin, diphtheria toxin, tetanus toxin, and Epstein-Barr virus

[0023] Such combinations are preferably provided in the linker format described above. To avoid doubt, the combinations are preferably included in the specified order, although they may be included in a different order. For example, if there are three universal T cell epitopes A, B, and C, they may be included in any order of ABC, ACB, BAC, BCA, CAB, or CBA.

[0024] Certain peptides containing multiple different universal T cell epitopes form a further aspect of the invention. Such peptides are preferably included in the vaccine compositions of the invention. Accordingly, peptides useful in the present invention comprise, 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 NO: 4 (SAT47). The composition of these peptides will be described in more detail with reference to Table 2 shown later.

[0025] Certain peptides containing a single universal T cell epitope also form a further aspect of the present invention. Such peptides are preferably included in the vaccine compositions of the present invention. Thus, peptides useful in the present invention comprise, consist essentially of, or consist 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). The composition of these peptides is described in more detail with reference to Table 1 shown later. Combinations of these peptides trimmed to a length of 10 to 20 amino acids may also be included in the vaccine compositions of the present invention, if desired. The combined peptides are preferably linked by one or more linkers as defined herein.

[0026] The Aβ-derived peptide antigen is presented on the surface of the liposome. Typically, this is by insertion into the outer surface of the liposome. Insertion into the outer surface of the liposome may be facilitated by attachment of the Aβ-derived peptide antigen to a portion inserted into the outer surface of the liposome. The liposome is suitable for presenting the Aβ-derived peptide antigen on its surface and may be any liposome encapsulating a peptide containing a universal T cell epitope. Typically, the portion contains a hydrophobic moiety to ensure insertion into the lipid bilayer of the liposome. The portion may be any suitable portion, but is preferably a fatty acid. The fatty acid may contain a palmitoyl residue. Preferred structures, such as in the case of ACI-24, include the Aβ-derived peptide antigen (Aβ(1-15) in ACI-24) linked to two palmitoyl residues in the N- and C-terminal regions of the peptide. Thus, the peptide antigen is tetra-palmitoylated. This may be facilitated by incorporating two lysine residues into the N- and C-terminal regions of the Aβ-derived peptide antigen. The lysine residues are palmitoylated.

[0027] In some embodiments, the liposomes have a negative surface charge. The liposomes are anionic. Preferably, the liposomes contain phospholipids, and more preferably, the phospholipids include dimyristoyl phosphatidylcholine (DMPC) and dimyristoyl phosphatidylglycerol (DMPG). The liposomes may further contain cholesterol. In some embodiments, the molar ratio of these three components may be 9:1:7.

[0028] Accordingly, the most preferred structure contains the Aβ-derived peptide antigen reconstituted in the liposomes. Accordingly, these compositions of the present invention may generally be referred to herein as "the liposomal vaccine compositions of the present invention."

[0029] An Aβ-derived peptide antigen induces a B cell response in a subject. It is a "B cell antigen". As already explained, Aβ plaques are formed by Aβ peptides that are 39-43 amino acids in length, and the Aβ peptides are in a random coil conformation as their native non-pathological form. During the transition to a pathological state, it mainly changes to a secondary structure of a β-sheet and spontaneously aggregates into insoluble deposits. Thus, an Aβ-derived peptide antigen is defined herein as a peptide antigen derived from (up to) 43 amino acids of Aβ, but not the full-length Aβ. More specifically, an Aβ-derived peptide antigen contains the immunodominant B cell epitope of Aβ(1-42) but lacks the T cell epitope found in Aβ(1-42). Thus, in some embodiments, an Aβ-derived peptide antigen comprises, consists essentially of, or consists of 13 to 15 contiguous amino acids derived from the first 17 amino acids at the N-terminus of Aβ. It should be noted that an Aβ-derived peptide antigen may be provided in connection with a larger peptide molecule, the remainder of which is not derived from the Aβ amino acid sequence. For example, the peptide can contain additional residues such as lysine residues to facilitate palmitoylation. Typically, these residues are found at the N-terminus and C-terminus of the peptide. In this context, the term "consists essentially of" means that an Aβ-derived peptide antigen contains 13 to 15 contiguous amino acids derived from the first 17 amino acids at the N-terminus of Aβ but can contain a limited number of additional residues such as four lysine residues to facilitate palmitoylation. A preferred Aβ-derived peptide antigen comprises, consists essentially of, or consists of amino acids 1-15 of Aβ, which may be referred to as "Aβ(1-15)" (WO2007 / 068411, ACI-24).

[0030] The Aβ-derived peptide antigen contained in the composition of the present invention adopts a secondary structure that replicates pathological Aβ. Preferably, the Aβ-derived peptide antigen adopts a secondary structure that includes a β-sheet conformation. Even more preferably, the Aβ-derived peptide antigen preferentially adopts a β-sheet conformation when displayed on the surface of a liposome.

[0031] The composition of the present invention typically includes at least one adjuvant. In some embodiments of the present invention, the composition of the present invention includes two adjuvants. The purpose of the adjuvant is to increase or stimulate the immune response of the subject. Preferably, at least one adjuvant is part of the carrier (as opposed to being encapsulated within the carrier). Thus, at least one adjuvant may form part of the liposome. It may form part of the lipid bilayer. Thus, the adjuvant may be a lipid-based adjuvant. The adjuvant may be presented, at least in part, on the surface of the liposome. This may be the result of the adjuvant-forming portion of the lipid bilayer. In some embodiments, one or more adjuvants that form part of the liposome may be combined with encapsulated adjuvants. In other embodiments, one or more adjuvants that form part of the liposome may be mixed with additional adjuvants (such as alum or CpG) when forming the liposome. The carrier (liposome) may function as an adjuvant with added monophosphoryl lipid A (MPLA), and the term includes MPLA-derivatives such as monophosphoryl hexaacyl lipid A, 3-deacyl (synthetic) (3D-(6-acyl)PHAD (registered trademark)), PHAD (registered trademark) (phosphorylated hexaacyl disaccharide), MPL. Thus, according to certain embodiments, the composition further includes MPLA. MPLA is typically added during liposome formation (as further described herein). Thus, preferred liposomes include dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), cholesterol, and MPLA. The molar ratio of these four components may be 9:1:7:0.05 in some embodiments.

[0032] Other adjuvants that can be used according to the present invention include, among others, aluminum hydroxide (alum) and / or CpG.

[0033] The vaccine composition of the present invention is administered to a subject to treat, prevent, or induce a protective immune response against or improve symptoms associated with an amyloid-beta related disease or condition, or a condition characterized by or associated with loss of cognitive memory ability. Thus, the vaccine composition can have both prophylactic and therapeutic uses. The subject is a mammal, typically a human.

[0034] The amyloid-beta related disease or condition may be a neuropathy such as Alzheimer's disease (AD). Other examples of amyloid-beta related diseases or conditions 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 (amyotrophic lateral 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, the conditions characterized by or associated with loss of cognitive memory ability according to 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).

[0035] Accordingly, the present invention provides a method of treating, preventing, or inducing a protective immune response against or improving symptoms associated with an amyloid-beta related disease or condition, or a condition characterized by or associated with loss of cognitive memory ability in a subject, the method comprising administering the vaccine composition of the present invention to the subject.

[0036] Such a method can also be expressed in the form of a medical use of the vaccine composition of the present invention. Accordingly, the present invention also provides a vaccine composition of the present invention for use in treating, preventing, or inducing a protective immune response against or ameliorating symptoms associated with an amyloid beta-related disease or condition, or a condition characterized by or associated with loss of cognitive memory ability in a subject.

[0037] Similarly, the present invention provides the use of the vaccine composition of the present invention in the manufacture of a medicament for use in treating, preventing, or inducing a protective immune response against or ameliorating symptoms associated with an amyloid beta-related disease or condition, or a condition characterized by or associated with loss of cognitive memory ability in a subject.

[0038] All embodiments herein apply to such methods or medical uses, as expressed. When the vaccine composition of the present invention is administered to a subject, typically polyclonal IgG antibodies that bind to pathological forms of Aβ are produced. As already explained, those pathological forms of Aβ contain β-sheet multimers. Accordingly, the antibodies produced may be referred to as "Aβ-specific" antibodies.

[0039] The ability of an antibody to bind to its target antigen is mainly controlled by two parameters: affinity and avidity. The affinity of an antibody measures the strength of the monovalent interaction between the antibody and its antigen. The avidity of an antibody includes the strengthening of binding through multiple interaction points between the antigen and the antibody. The binding ability of polyclonal sera induced by vaccination depends on both of the above parameters (Siegrist, 2013). Since it is very difficult to evaluate affinity and avidity independently, it is generally referred to as the avidity of the polyclonal response. As described in more detail herein, see Example 4 (Section 4.2), the inventors developed an ELISA assay in which the overall binding of sera containing polyclonal antibodies to lower and higher concentrations of antigen is evaluated in parallel (Martineau, 2010). The ratio of the low coating signal to the high coating signal (the signal represents the concentration of the bound antibody) is expressed as the avidity index. A higher index score (close to 1) indicates an improved overall binding strength compared to a lower index score (close to 0). The increase in the avidity index over time provides an indicator of the overall avidity maturation of vaccine-induced antibodies. Vaccination with the vaccine composition of the present invention containing encapsulated peptides comprising universal T cell epitopes results in an improved maturation effect compared to vaccination with ACI-24 (without encapsulated peptides comprising universal T cell epitopes), as shown herein (see Example 4 and Figure 4).

[0040] The vaccine composition of the present invention may be administered to a subject by any suitable route of administration. As known to those skilled in the art, the vaccine composition may be administered by local, oral, rectal, nasal or parenteral (such as intravenous, intradermal, subcutaneous, or intramuscular) routes. Further, the vaccine composition may be incorporated into a sustained release matrix such as a biodegradable polymer, and the polymer is implanted near or at the location where delivery is desired. However, in a preferred embodiment, the vaccine composition is administered intramuscularly or subcutaneously.

[0041] The vaccine composition of the present invention can be administered to a subject in a single dose to elicit a protective immune response. However, in some embodiments, the vaccine composition of the present invention is administered to the same subject multiple times. Thus, a so-called prime-boost regimen can be used according to the present invention. The administrations of the vaccine are typically separated by an intervening period of at least one week and often about 1 to 12 months. Without wishing to be bound by a particular hypothesis, the addition of a universal T cell epitope to ACI-24 is likely to enhance the anti-Aβ antibody response by providing a second signal from activated T cells specific for the cognate T cell epitope. The vaccine composition of the present invention represents a powerful new therapeutic option for the prevention and treatment of amyloid-beta related diseases or conditions such as AD. In some embodiments, the same vaccine composition is administered each time - an isologous vaccination regimen. Isologous vaccination refers to a vaccination regimen in which the same vaccine is used for both the prime (first vaccination) and the boost (subsequent vaccinations).

[0042] On the one hand, in heterologous prime-boost vaccination, it is necessary to use different vaccines for the primary vaccination and at least some of the follow-up vaccinations. In some embodiments, the vaccine composition of the present invention is administered multiple times to the same subject in a heterologous prime-boost combination with another "anti-Aβ" vaccine carrying a peptide antigen derived from any part of the Aβ protein, which may include a peptide antigen derived outside the Aβ(1-15) region. In some embodiments, the vaccine composition of the present invention is administered multiple times to the same subject in a heterologous prime-boost combination with another "anti-Aβ" vaccine carrying the same peptide antigen as that contained in the liposomal vaccine composition of the present invention, which may include the Aβ(1-15) peptide antigen. In some embodiments, the vaccine composition of the present invention, preferably containing the Aβ(1-15) peptide antigen, is administered multiple times to the same subject in a heterologous prime-boost combination with another "anti-Aβ" vaccine carrying the corresponding Aβ-derived peptide antigen, preferably the Aβ(1-15) peptide antigen. Examples of "anti-Aβ" vaccines that can be administered in heterologous prime-boost vaccination together with the vaccine composition of the present invention containing an Aβ-derived antigen include, but are not limited to, Aβ(1-15)-PADRE vaccine (Agadjanyan et al., 2005; Ghochikyan et al., 2006), Aβ(1-15)-diphtheria toxoid (DT) or CRM vaccine (WO2010016912), tandem repeat of lysine-linked Aβ(1-15) (Maier et al., 2006), dendritic Aβ(1-15) vaccine (Seabrook et al., 2006), Aβ(1-15)DT conjugate (Liu et al., 2013), Aβ(1-6) conjugated to bacteriophage Qβ coating protein (Windblad et al., 2012), Aβ(1-7)-CRM (Arai et al., 2015), NtermAβ-KLH (Schneeberger et al, 2010).

[0043] The present invention further provides a kit comprising the vaccine composition according to the present invention. Accordingly, a kit for inducing a protective immune response for treating, preventing, or counteracting or ameliorating symptoms associated with an amyloid beta-related disease or condition, or a condition characterized by or associated with loss of cognitive memory ability in a subject, comprising the (liposomal) vaccine composition of the present invention described herein is provided. Such a kit may be accompanied by appropriate instructions for use. The instructions for use may describe the administration schedule of the composition. Accordingly, the kit may comprise a plurality of (separate) doses of the vaccine composition of the present invention. The instructions for use may further describe the storage conditions of the composition, particularly during the period between administrations of the doses of the vaccine composition. These kits may be applied to all relevant methods of the present invention disclosed herein.

[0044] The present invention further provides a method for manufacturing the liposomal vaccine composition of the present invention. Such a method may include the following steps: a. Obtaining a lipid membrane b. Rehydrating the lipid membrane in a buffer containing a peptide comprising a universal T cell epitope c. Obtaining liposomes encapsulating a peptide comprising a universal T cell epitope from the rehydrated lipid film to obtain a solution comprising liposomes encapsulating the universal T cell epitope d. Adding a β-amyloid (Aβ)-derived peptide antigen to the solution and maintaining the solution under conditions that result in insertion of the β-amyloid (Aβ)-derived peptide antigen into the lipid bilayer of the liposomes.

[0045] Such methods are exemplified herein and the details may be applicable to these aspects of the invention. Generally speaking, the method may include the formation of a thin lipid film followed by homogenization and extrusion. Thus, in some embodiments, the lipid film is produced by dissolving the lipid in ethanol and then evaporating the ethanol under vacuum. Preferred lipid components are described in connection with the liposomal vaccine compositions of the invention and include DMPC, DMPG, cholesterol and MPLA (as an adjuvant). The molar ratio of these components may be 9:1:7:0.05. Such a molar ratio is also applicable to the liposomal vaccine compositions of the invention. The lipid component may need to be solubilized at an elevated temperature. The elevated temperature may be between 40°C and 80°C, for example about 60°C.

[0046] In step b, the buffer used for rehydration may depend on which peptide containing the universal T cell epitope is used. Generally, any suitable buffer may be used. In some embodiments, the buffer includes sodium acetate or PBS. When encapsulating SAT42, the buffer may be sodium acetate. When encapsulating any one or more of SAT43, SAT44, or SAT47, the buffer may be PBS. In all cases, DMSO such as 5% DMSO may be added to the buffer. Rehydration may be performed while stirring the sample.

[0047] In step c, the liposomes may be obtained by vortexing in the presence of beads. Any suitable beads may be used. The beads may be, for example, glass beads. This step may result in a multilamellar vesicle, which is subsequently converted into a liposome containing a lipid bilayer. This conversion may depend on several, 5 - 15, preferably 10 freeze - thaw cycles. Homogenization may be performed after the freeze - thaw cycles. Subsequently, size - based extrusion may be performed. In some embodiments, the liposomes are extruded through pores having a diameter (or maximum dimension) of about 0.08 - 0.1 μm. This may be done through a membrane such as a polycarbonate membrane. The extruded liposomes may be concentrated using a form of filtration such as ultrafiltration, for example.

[0048] Step d results in the insertion of a β - amyloid (Aβ) - derived peptide antigen into the lipid bilayer of the liposomes. The required conditions may include stirring at a temperature of 25 - 35 °C, for example about 30 °C, for 10 - 60 minutes, for example about 30 minutes. A preferred β - amyloid (Aβ) - derived peptide antigen is a tetrapalmitoylated peptide containing Aβ1 - 15. This peptide contains 2 lysine residues at either end to obtain the tetrapalmitoylated peptide. The peptide may be pre - dissolved in disodium hydrogen phosphate before injection into the liposome solution.

[0049] As a final step, the method may further include filtering the vaccine composition. This may be done aseptically. The filtration may be performed through a membrane with a pore size of 0.2 μm. Suitable membranes include polyethersulfone (PES) membranes, which may be provided in the form of a syringe filter. The resulting vaccine composition may then be stored under appropriate conditions, for example refrigerated (e.g., at about 5 °C) until use.

[0050] Alternative methods for manufacturing the liposomal vaccine compositions of the present invention may rely on cross-flow injection, as exemplified herein. Accordingly, the present invention further provides a method for manufacturing the liposomal vaccine compositions of the present invention by cross-flow injection. These methods are particularly applicable to compositions encapsulating SAT44 or SAT47. Such methods may include the following steps: a. Dissolve the lipids (and adjuvants in the case of lipid-based) forming the liposomes in a solution b. Dissolve a peptide containing a universal T cell epitope in the solution c. Using a cross-flow injection module, mix the solutions obtained in steps a and b to obtain intermediate liposomes encapsulating a peptide containing a universal T cell epitope d. Extrude the intermediate liposomes through a membrane to reduce their size and polydispersity e. Using a cross-flow injection module, mix a solution containing a β-amyloid (Aβ)-derived peptide antigen with the solution obtained in step d to cause insertion of the β-amyloid (Aβ)-derived peptide antigen into the lipid bilayer of the liposomes.

[0051] Such methods are exemplified herein and the details thereof may be applicable to these aspects of the present invention. Generally, this method uses cross-flow injection to encapsulate a peptide containing a universal T cell epitope and insert a peptide antigen derived from β-amyloid (Aβ) into the lipid bilayer of the liposomes.

[0052] In step a, a lipid (which may include an adjuvant such as MPLA adjuvant as described herein) is typically dissolved in ethanol. The ethanol may be 90 - 100% ethanol, for example 96% ethanol. Dissolution may be facilitated by heating at a temperature between 40°C and 80°C, for example about 60°C. Preferred lipid components are described in relation to the liposomal vaccine composition of the present invention and include DMPC, DMPG, cholesterol and MPLA (as an adjuvant). The molar ratio of these components may be 9:1:7:0.05. Such a molar ratio is also applicable to the liposomal vaccine composition of the present invention.

[0053] In step b, a peptide containing a universal T cell epitope is dissolved. In some embodiments, the peptide may be dissolved in a suitable buffer (such as His - sucrose buffer) with the aid of agitation such as sonication.

[0054] In step c, the solutions obtained in steps a and b are mixed using a cross - flow injection module to obtain intermediate liposomes encapsulating the peptide containing the universal T cell epitope. Prior to this step, the solutions obtained in steps a and b may be filtered. The appropriate pore size of the filter is about 0.2 μm. The solutions may be used at any appropriate concentration. After filtration, the solution may be heated to a temperature between 30°C and 60°C, for example 40°C. Liposomes are formed by injecting the two solutions (obtained in steps a and b) through a cross - flow module (where the two solutions meet). This is generally carried out at a specific flow rate and temperature, as will be readily understood by those skilled in the art (the appropriate temperature is described above). In some embodiments, following liposome formation, typically a buffer may be added to reduce the ethanol concentration.

[0055] In step d, the intermediate liposomes are extruded through a membrane to reduce their size and polydispersity. Liposomes formed in solution encapsulate peptides containing universal T cell epitopes. Any suitable membrane can be used. A suitable pore size may be about 100 nm. A suitable membrane type is a polycarbonate membrane. This step may be carried out at any suitable temperature, preferably at room temperature (e.g., about 25 °C). Following this step, a filtration step such as ultrafiltration / diafiltration may be performed to remove ethanol. Any suitable membrane such as a hollow fiber membrane with a cut-off molecular weight of about 500 kD may be used in this step. A buffer exchange step may be carried out to serve as the dispersion buffer. A preferred dispersion buffer is PBS. PBS may be at a suitable pH, for example, between 6 and 8, particularly about 6.9. This may require an exchange of 5 to 15 times, for example, about 10 times the volume. Prior to step e, the liposomes may be diluted to the desired concentration with the dispersion buffer. The desired concentration may be in the range of 0.1 to 10 mg / ml, for example, about 1 mg / ml. Prior to step e, the liposome-containing solution may be heated to a suitable temperature, for example, between 30 °C and 60 °C, preferably about 35 °C.

[0056] Step e involves mixing a solution containing a β-amyloid (Aβ)-derived peptide antigen with the solution obtained in step d using a crossflow injection module. As discussed herein, the β-amyloid (Aβ)-derived peptide antigen is preferably lipidated (e.g., tetra-palmitoylated), and this discussion applies with the necessary modifications. Prior to mixing, typically, the β-amyloid (Aβ)-derived peptide antigen is in 10 mM Na 2 HPO 4Dissolve it in a suitable buffer such as a 10% w / v solution of Beta-OG in pH 11.4 buffer. Typically, heat the solution to a suitable temperature, for example, between 30 °C and 80 °C, for example, about 60 °C. The solution may be further diluted if necessary to ensure an appropriate concentration of the β-amyloid (Aβ)-derived peptide antigen. The appropriate concentration may be in the range of 0.1 to 10 mg / ml, for example, about 1 mg / ml. Typically, the pH is maintained in the range of 11 to 12, for example, about 11, preferably 11.4. Using a cross-flow injection module, mix the solution containing the β-amyloid (Aβ)-derived peptide antigen with the solution obtained in step d, whereby the β-amyloid (Aβ)-derived peptide antigen is inserted into the outer lipid bilayer of the liposome. To facilitate the insertion of the β-amyloid (Aβ)-derived peptide antigen into the lipid bilayer of the liposome, the mixture may be incubated at a suitable temperature for a certain period of time. The appropriate time may be in the range of 20 to 120 minutes, for example, about 30 minutes. The appropriate temperature is between 30 °C and 60 °C, for example, about 30 minutes. Incubation may be carried out with agitation such as stirring.

[0057] Following step e, the product can be recovered and included in the composition of the present invention. Thus, the product may be formulated in the liposomal vaccine composition of the present invention. This may include an ultrafiltration / diafiltration step to remove Beta-OG from the buffer. Any suitable membrane such as a hollow fiber membrane with a cut-off molecular weight of about 500 kD may be used in this step. Ultrafiltration / diafiltration may include a buffer exchange step to the final buffer. A preferred final buffer is His-sucrose buffer, which may be 10 mM histidine, 250 mM sucrose. This may require an exchange of 5 - 15 times, for example about 10 times the volume. A concentration step may be performed to achieve the preferred final volume. A final (sterile) filtration step may be performed. A cartridge filter may be used for this. The filtration step may be performed through a filter with any suitable pore size such as about 0.2 μm. The filtration may be performed aseptically. The resulting vaccine composition may then be stored under suitable conditions such as refrigeration (e.g., at about 5°C) until use.

[0058]

Table 1

[0059] The present invention will be further understood with reference to the following non-limiting examples.

Examples

[0060] Example 1. Design of a novel T cell epitope The ability of a T cell epitope to activate T cells (immunogenicity score) is the result of two complementary properties: i) affinity for HLA and ii) the ability to bind indiscriminately to different HLA haplotypes. For the purpose of selecting peptides with the highest immunogenicity scores, an in silico evaluation (Epivax) of several T cell epitopes of different origins was performed. In a preliminary step, 10 different peptides from different origins (keyhole limpet hemocyanin - KLH, diphtheria toxin, influenza virus, Epstein - Barr virus and herpes virus) were evaluated. Based on the predicted HLA affinity and HLA haplotype coverage, peptides with the highest immunogenicity scores (above 10) were selected as they are likely to be highly immunogenic in humans (the selected peptide sequences are shown in Table 1).

Table 2

[0061] Following the screening results of individual peptides, we designed a combination of promiscuous peptides composed of two or three immunogenic T cell epitopes (named SAT42, SAT43, and SAT44) from different origins and promiscuous peptides composed of trimmed peptides (such as SAT47 and SAT43) (Table 2). Based on the hotspots of T cell epitopes predicted in silico, trimmed peptides were designed by selecting the most immunogenic 15-mer peptide sequences in the sequences of individual T cell epitopes. The goal was to increase the immunogenicity score without increasing the size of the final promiscuous peptides due to the constraints of peptide synthesis and vaccine encapsulation processes. Briefly, the yield and success rate of peptide synthesis decrease with the length of the peptide, especially when the length exceeds 30 amino acids. Moreover, like the T cell epitope peptides disclosed herein, they are mainly composed of hydrophobic residues. Furthermore, the encapsulation rate of the peptide decreases as the length of the peptide increases. This is because as the length of the peptide increases, the opportunity to accommodate the peptide in the lumen of the liposome decreases. The in silico immunogenicity scores of these four promiscuous T cell epitopes were very high, and importantly, they were higher than the scores of the individual component peptides, confirming that combining peptides from different origins can improve HLA affinity and HLA haplotype coverage (the promiscuous T cell epitope sequences are shown in Table 2).

Table 3

Example

[0062] Example 2. Synthesis and Formulation of Vaccine General Methods for the Synthesis and Purification of Universal T Cell Epitope Peptides T cell peptides were produced by linear solid-phase peptide synthesis (SPPS) on 2-chlorotrityl resin using standard Fmoc chemistry. Standard coupling procedures were carried out at room temperature for 1 h in the presence of 3.0 equivalents of base in DMF using 3.0 equivalents of amino acid and coupling reagent. For difficult coupling sequences, the reaction time was extended to implement double coupling. After completion of amino acid coupling, an acetylation capping step was introduced using 5.0 equivalents of Ac 2 O in pyridine to terminate unwanted peptide chain elongation. The resin was washed with DMF and the Fmoc group was removed using 20% piperidine in DMF for 5 min. After completion of SPPS, overall deprotection and peptide cleavage from the resin were carried out at room temperature for 2 h using a standard cleavage cocktail (TFA / TIS / water). The resin was filtered off and washed with TFA. Subsequently, the crude product was precipitated with 10-fold excess of cold isopropyl ether / hexane, the solid was filtered off using a glass frit and dried under vacuum. The crude peptide was purified on a reverse-phase C18 column using a gradient of solvent A (water, 0.1% TFA) and solvent B (acetonitrile, 0.1% TFA) on a preparative HPLC system. HPLC fractions containing the desired peptide with purity exceeding 90% were pooled together, diluted with water and subjected to ion exchange. The desired ion exchange fractions were lyophilized. The identity and purity of the final peptide were characterized and confirmed by HPLC-MS analysis.

[0063] Preparation of ACI-24.043 / ACI-24.044 / ACI-24.045 / ACI-24.046 / Vaccine (Thin Lipid Film) Vaccines containing encapsulated T cell epitope peptides were produced by the thin-film method followed by homogenization and extrusion. First, DMPC, DMPG (Lipoid, Germany), cholesterol and monophosphoryl hexaacyl lipid A, 3-deacyl synthetic or 3D-(6-acyl)PHAD in a molar ratio of 9:1:7:0.05 respectively TM were solubilized in ethanol at 60 °C (Avanti Polar Lipids, USA). Ethanol was evaporated under a vacuum rotary evaporator to obtain a thin lipid film. The lipid film was rehydrated in one of these buffers (varying by the T cell epitope peptide encapsulated). · 5% DMSO (Sigma Aldrich) in MilliQ water containing 20 mM sodium acetate pH 4 (Fluka) and 0.8 mg / mL T cell epitope peptide SAT42, or · 5% DMSO (all Sigma - Aldrich) in MilliQ water containing 0.1x PBS pH 7.4 and 0.3 - 0.4 mg / mL T cell epitope peptides SAT43, SAT44, or SAT47. The solution was gently stirred for 15 minutes. The sample was further vortexed vigorously in the presence of glass beads. The resulting multilamellar vesicles were subjected to 10 freeze - thaw cycles (liquid nitrogen and 37 °C water bath), homogenized, and then successively extruded through a polycarbonate membrane with pore sizes of 0.1 / 0.08 μm (Whatman, UK). Both the homogenization and extrusion steps were performed using an EmulsiFlex - C5 (Avestin, Canada). The extruded liposomes were concentrated by ultrafiltration and the buffer was exchanged to PBS pH 7.4 by diafiltration (10 exchanges). The resulting liposomes were diluted with PBS pH 7.4, heated to 30 °C, and then Pal1 - 15 was added. Tetra - palmitoylated human peptide Pal1 - 15 (Bachem AG, Switzerland) was dissolved in 10 mM Na 2 HPO 4 with pH 11.4 in MilliQ water containing 1% β - OG (Sigma - Aldrich, USA), injected into the liposome solution at 30 °C, stirred for 30 minutes, and then subjected to a concentration step by ultrafiltration and dilution with PBS pH 7.4 by diafiltration. Next, the resulting liposomes were sterile - filtered through a 0.2 μm polyethersulfone (PES) membrane syringe filter and stored at 5 °C.

[0064] Preparation of ACI-24.043 Vaccine (Cross-Flow Infusion) Lipids (DMPG, DMPC, cholesterol, 3D - (6 - acyl)PHAD TM(Avanti Polar Lipids, USA) was dissolved in 96% EtOH in a heating cabinet at 60 °C. After the lipid was completely dissolved, the solution was filtered through a 0.2 μm pore size filter and placed into an injection system heated to 60 °C. Specifically, an appropriate amount of ACI-24.043 (SAT47) was dispersed in EtOH at room temperature using sonication (the EtOH concentration was typically 2% v / v of the final SAT47 solution). After the peptide was completely dispersed, His-sucrose buffer (10 mM histidine, 250 mM sucrose) was added to achieve a drug ratio of 1 / 50 by mass to the lipid. The SAT47 solution was filtered through a 0.2 μm pore size filter (Sartoscale filter) and placed into an injection buffer bottle and heated to 40 °C. Liposomes were formed at the injection site when the lipid / ethanol solution and the injection buffer were mixed. Immediately after liposome formation, an online dilution step with 10 mM histidine, 250 mM sucrose was performed to lower the EtOH concentration. The intermediate liposomes were extruded through a 100 nm pore size polycarbonate membrane (1 pass) at room temperature. Ultrafiltration / diafiltration (UDF) using a hollow fiber membrane (MWCO: 500 kD) was performed to remove EtOH and exchange the buffer to PBS pH 6.9 (10-fold volume exchange). Next, the SAT47 liposomes were diluted to a total lipid concentration of 1 mg / mL using dispersion buffer (PBS pH 6.9) and warmed to 35 °C. Pal1-15 was 10 mM Na 2 HPO 4A 10% w / v solution of beta OG in pH 11.4 buffer was dissolved at 60 °C and further diluted with the same buffer to a final concentration of 1 mg / mL. The pH was adjusted to 11.4. After mixing these two solutions using a cross-flow injection module, the liposome suspension was further incubated with stirring at 35 °C for 30 minutes to fully insert Pal1-15. A second UDF step was performed using a hollow fiber membrane (MWCO: 500 kD) to remove beta OG and exchange the buffer to 10 mM histidine, 250 mM sucrose (10-fold volume exchange). The product was concentrated to the final volume and filtered through a 0.2 μm Acrodisc mPES syringe filter.

[0065] Preparation of ACI-24.046 Vaccine (Cross-Flow Infusion) Lipids (DMPG, DMPC, cholesterol, 3D-(6-acyl)PHAD TM (Avanti Polar Lipids, USA)) were dissolved in 96% EtOH in a heating cabinet at 60 °C. After the lipids were completely dissolved, the solution was filtered through a filter with a pore size of 0.2 μm and placed into an injection system heated to 60 °C. In parallel, ACI-24.046 (SAT44) was dissolved in an injection buffer (10 mM histidine, 250 mM sucrose) at 40 °C. After SAT44 was completely dissolved, the solution was filtered through a filter with a pore size of 0.2 μm (Sartoscale) and placed into an injection buffer bottle heated to 40 °C. Liposomes are formed at the injection site when the lipid / ethanol solution and the injection buffer are mixed. Immediately after liposome formation, an online dilution step with 10 mM histidine, 250 mM sucrose was performed to lower the EtOH concentration. The intermediate liposomes were extruded through a polycarbonate membrane with a pore size of 100 nm at room temperature (1 pass). Ultrafiltration / diafiltration (UDF) using a hollow fiber membrane (MWCO: 500 kD) was performed to remove EtOH and exchange the buffer to PBS pH 6.9 (10-fold volume exchange). Next, the SAT44 liposomes were diluted to a total lipid concentration of 1 mg / mL using a dispersion buffer (PBS pH 6.9) and warmed to 35 °C. Pal1-15 was dissolved in 10 mM Na 2HPO 4 It was dissolved at 60 °C in a 10% w / v solution of beta OG in pH 11.4 buffer and further diluted with the same buffer to a final concentration of 1 mg / mL. The pH was checked and carefully adjusted back to 11.4. After mixing these two solutions using an injection module, the liposome suspension was incubated at 35 °C for 30 minutes with further stirring to complete the insertion of Pal1-15. A second UDF step using a hollow fiber membrane (MWCO: 500 kD) was performed to remove beta OG and exchange the buffer to 10 mM histidine, 250 mM sucrose (10-fold volume exchange). The product was concentrated to the final volume and finally filtered through a 0.2 μm Acrodiscm PES syringe filter.

Example

[0066] Example 3. Proof of Concept (PoC) Study of the In Vivo Immunogenicity of Vaccines with Encapsulated T-Cell Epitopes After successful encapsulation of different T-cell epitopes, the immunogenicity of vaccines containing encapsulated T-cell epitopes with high immunogenicity scores SAT42, SAT44, and SAT47 compared to the ACI-24 vaccine (ACI-24.044, ACI-24.046, and ACI-24.043 vaccines, respectively) was tested in vivo. Wild-type C57BL / 6 mice were subcutaneously (sc) inoculated three times at days 0, 14, and 28 with ACI-24, ACI-24.044 (using encapsulated SAT42), ACI-24.046 (using encapsulated SAT44), and ACI-24.043 (using encapsulated SAT47). Blood samples were collected at days -21 (ACI-24.046) or -7 (ACI-24, ACI-24.043, ACI-24.044) (prebleed), 7, 21, and 35, and Aβ1-42-specific IgG titers were measured by ELISA.

[0067] Plates were coated overnight at 4°C with 10 μg / ml of human Aβ1-42 peptide film (Bachem, Switzerland). 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 phosphatase (AP)-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, PA, USA) at 37°C for 2 hours. After final washing, the plates were incubated with AP substrate (pNPP) for 2.5 hours and read at 405 nm using an ELISA plate reader. Results were shown with reference to serial dilutions of a commercially available antibody (6E10, Biolegend, UK, catalog number 803002). Figure 1A shows the Aβ1-42-specific IgG titers induced by the ACI-24 vaccine over time, regardless of the presence or absence of the encapsulated T cell epitope. The ACI-24 vaccine showed the highest Aβ1-42-specific IgG titer on day 7, but when the T cell epitope was encapsulated in the ACI-24 vaccine, an increase in antibody titer was observed after the first, second, and third vaccinations. The results in Figure 1b show that vaccination with the ACI-24 vaccine containing the encapsulated T cell epitope induced an increase in Aβ-specific antibody titer compared to ACI-24, and was statistically significant for the group vaccinated with ACI-24.043 (with encapsulated SAT47).

[0068] The encapsulated SAT42, SAT43, SAT44, or SAT47 vaccines were tested in cynomolgus monkey studies. Four monkeys per group were given a 3-month sc vaccination with ACI-24.044 (a total of 8 monkeys in the encapsulated SAT42-2 group), ACI-24.046 vaccine (a total of 8 monkeys in the encapsulated SAT44-2 group) by inoculation (on days 1, 29, and 57), ACI-24.045 vaccine (4 monkeys in the encapsulated SAT43) or ACI-24.043 vaccine (4 monkeys in the encapsulated SAT47). Blood was collected before the first inoculation (day 1) and 1 week and 3 weeks after each inoculation (days 8, 22, 36, 50, 64, 78), and the Aβ1-42 specific IgG titer was measured by ELISA.

[0069] Plates were coated overnight at 4°C with a 10 μg / ml human Aβ1-42 peptide film (Bachem, Switzerland). After washing with 0.05% Tween 20 / PBS and blocking with 1% BSA / 0.05% Tween 20 / PBS, two-fold serial dilutions (8-fold) of the serum were added to the plates and incubated at 37°C for 2 hours. After washing, the plates were incubated with horseradish peroxidase (HRP)-labeled anti-monkey IgG antibody (KPL, catalog number 074 11021) at 37°C for 2 hours. After washing, the plates were incubated with 50 μl of ABTS / H 2 O 2 (2,2’-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (HRP substrate) and read at 405 nm after 1 hour using an ELISA plate reader. The results are expressed with reference to the serial dilutions of a positive monkey sample used as a standard.

[0070] The immunogenicity of vaccines with different T cell epitopes was compared with the ACI-24 vaccine. Table 3 shows the fold increase in Aβ-specific antibody titers compared with the ACI-24 vaccine 1 week after the third vaccination. All the vaccines tested, ACI-24.046 (SAT44), ACI-24.043 (SAT47), ACI-24.045 (SAT43), and ACI-24.044 (SAT42), induced at least a 7-fold increase in antibody titers compared with the titers induced by the ACI-24 vaccine (ACI-24.044 with encapsulated SAT42). The ACI-24.043 vaccine (with encapsulated SAT47) and ACI-24.046 (with encapsulated SAT44) induced significantly higher Aβ-specific antibody titers compared with ACI-24 1 week after the third vaccination (Table 3). The ACI-24.043 vaccine (with encapsulated SAT47) and ACI-24.046 (with encapsulated SAT44) have high Epivax scores (142.89 and 57.2, respectively).

Table 4

[0071] After in vivo results (Figure 1) were obtained for ACI-24.046 (encapsulated SAT44) and ACI-24.043 (encapsulated SAT47) obtained by the thin lipid film method, the in vivo immunogenicity of the same vaccines obtained by the cross-flow injection method was tested. Wild-type C57BL / 6 mice were subcutaneously (sc) inoculated a total of 3 times at days 0, 14, and 28 with ACI-24, ACI-24.046 (with encapsulated SAT44), or ACI-24.043 (with encapsulated SAT47). Blood samples were collected at days -7, 7, 21, and 35, and Aβ1-42-specific IgG titers were measured by ELISA. The results in Figure 6 show that inoculation with the ACI-24 vaccine containing the encapsulated T cell epitope induced a significant increase in Aβ-specific antibody titers compared with ACI-24.

Example

[0072] Example 4. Quality of the Induced Aβ-Specific Antibodies 4.1 In Vitro Inhibition of Human Aβ1-42 Self-Association The quality of the induced Aβ-specific antibodies was tested in vitro by measuring the inhibition of Aβ1-42 self-association / aggregation. This assay is based on the ability of pre- and post-immunization mouse plasma to impair the natural self-association propensity of human Aβ1-42.

[0073] Standard ELISA plates were coated overnight at 4 °C with 1 μg / mL of Aβ1-42. The plates were washed four 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, two-fold serial dilutions of four sets of plasma were added to the plates with stirring for 20 minutes at room temperature. Biotinylated Aβ1-42 was added to each well to a final concentration of 0.1 μg / mL and incubated for 2 hours at room temperature with stirring. Biotinylated Aβ1-42 without plasma was used as a positive control for Aβ1-42 self-association (considered 100% self-association and 0% inhibition). After the washing step, the plates were incubated with horseradish peroxidase (HRP) conjugated to streptavidin (R&D Systems, Canada, Ref. 890803). After washing, the plates were incubated with Sure Blue Reserve TMB substrate (Seracare, catalog number 5120-0081) for 10 minutes. The reaction was stopped using a betyl stop solution (Bethyl Laboratories, Inc., catalog number E115), and the plates were read at 450 nm using an ELISA plate reader. The inhibition rate of self-association was calculated using biotinylated Aβ1-42 without plasma as a reference positive control (0% inhibition).

[0074] The results showed that the Aβ-specific antibodies generated after two vaccinations with all vaccines containing T cell epitopes impaired the self-association of Aβ1-42 more efficiently than the antibodies induced by ACI-24 (Figure 2A). Since pre-bleeding plasma induced background inhibition of self-association, the percentage on day 21 was normalized by subtracting the background of pre-bleeding plasma. The Aβ1-42-specific antibodies generated by vaccination with all ACI-24 vaccines containing T cell epitopes showed higher inhibition against Aβ1-42 self-association compared to ACI-24. This inhibition was statistically significant in the group vaccinated with ACI-24.046 (with encapsulated SAT44) (Figure 2B).

[0075] 4.2 Generation of antibodies recognizing Aβ oligomers To evaluate the specificity of antibodies induced in C57BL / 6 mice binding to pathological Aβ, the Aβ1-42-oligomer-specific IgG response was examined by ELISA. Plates were coated with 10 μg / ml oligomers prepared overnight at 4°C as described above (Adolfsson, 2012). 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 an alkaline phosphatase (AP)-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, catalog number 115-055-164, PA, USA) at 37°C for 2 hours. After the final wash, the plates were incubated with AP substrate (pNPP) for 2.5 hours and read at 405 nm using an ELISA plate reader. The results were expressed with reference to serial dilutions of a commercially available antibody (6E10, Biolegend, UK, catalog number 803002).

[0076] Each sample was tested in eight or four two-fold serial dilutions starting at 1 / 100, 1 / 400, 1 / 800, or 1 / 1600 dilution based on the Aβ1-42 antibody titer. The results in Figure 3 show that vaccination with all ACI-24 vaccines containing T cell epitopes induced an increase in the Aβ1-42 oligomer-specific antibody titer compared to ACI-24, and reached statistical significance for the group vaccinated with the ACI-24.043 vaccine (with encapsulated SAT47). The avidity index of the antibodies induced in C57BL / 6 mice 7 and 21 days after vaccination was examined by ELISA assay. Half of the standard ELISA plates were coated with a 10 μg / mL Aβ1-42 peptide film, and the other half was coated with a 1 μg / mL Aβ1-42 peptide film at 4 °C overnight. After washing with 0.05% Tween 20 / PBS and blocking with 1% BSA / 0.05% Tween 20 / PBS, eight-fold serial dilutions of plasma were added to both coating conditions and incubated at 37 °C for 2 hours. After the washing step, the plates were incubated with an alkaline phosphatase (AP)-labeled anti-mouse IgG antibody (Jackson ImmunoResearch, catalog number 115-055-164, PA, USA) at 37 °C for 2 hours. After the final wash, the plates were incubated with an AP substrate (pNPP) for 2.5 hours and read at 405 nm using an ELISA plate reader. The results are shown with reference to serial dilutions of a commercially available antibody (6E10, Biolegend, UK, catalog number 803002).

[0077] To determine the avidity index, the AU / mL was calculated for each sample of both coatings using a standard curve obtained with 10 μg / mL Aβ1-42 peptide. O.D. values between 0.6 and 2.8 were used for back-calculating the concentration. The avidity index was calculated as the ratio of the antibody concentrations at the low coating concentration (1 μg / mL Aβ1-42 peptide) and the saturated coating (10 μg / mL Aβ1-42 peptide).

[0078] The results in Figure 4 showed that vaccination with all ACI-24 vaccines containing T cell epitopes induced Aβ1-42-specific antibody avidity maturation between the first and second vaccinations (days 7 and 21, respectively), and they achieved statistical significance in the groups vaccinated with ACI-24.044 (using encapsulated SAT42) and ACI-24.043 (using encapsulated SAT47).

[0079] To evaluate the specificity of antibodies induced in cynomolgus monkeys to bind pathological Aβ, Aβ1-42 oligomer-specific IgG titers in the sera of cynomolgus monkeys inoculated with ACI-24.046 (a total of 8 monkeys in 2 groups containing encapsulated SAT44), ACI-24.045 vaccine (encapsulated SAT43 - 4 monkeys), or ACI-24.043 vaccine (encapsulated SAT47 - 4 monkeys) were measured by the Mesoscale Discovery (MSD) method on day 64 (1 week after the third inoculation). MSD streptavidin plates were saturated overnight at 4°C with 5% Blocking Agent A (MSD, Ref. R93BA-4). The next day, the plates were washed 4 times with 0.05% Tween 20 / PBS and coated with 25 μl of the capture antibody biotinylated 6E10 (Biolegend, Ref. 803008) in PBS at 0.5 μg / ml for 1 hour at 37°C on a shaker. After washing, the plates were incubated with 25 μl of Aβ1-42 oligomers (Adolfsson, 2012) in PBS at 10 μg / ml for 1 hour at 37°C on a shaker. The plates were washed and incubated with 8 two-fold dilutions of monkey sera (starting with a 1 / 50 dilution in 1% skim milk / 0.05% Tween / PBS). Samples were incubated for 2 hours at 37°C on a shaker. The plates were washed 4 times, and an anti-human IgG detection antibody labeled with SULFO-TAG (Jackson, Ref. 109-005-098) was added and diluted at 37°C for 1 hour in 1% skim milk / 0.05% Tween20 / PBS on a shaker. After washing 4 times, MSD Read Buffer T 2X (MSD, Ref. R92TC-2) was added and the plates were read within 5 minutes. Results are presented with reference to a serial dilution of a monkey sample used as a standard.

[0080] Results showed that all the vaccines tested, ACI-24.046 (encapsulated SAT44), ACI-24.043 (encapsulated SAT47), and ACI-24.045 (encapsulated SAT43), induced an increase in antibodies capable of recognizing Aβ oligomers on day 64 (1 week after the third inoculation) compared to day 1 (before the first inoculation). See Figure 5.

[0081] Literature Adolfsson O., Pihlgren M., Toni N., Varisco Y., Buccarello A.L., Antoniello K., Lohmann S., Piorkowska K., Gafner V., Atwal J.K., Maloney J., Chen M., Gogineni A., Weimer R.M., Mortensen D.L., Friesenhahn M., Ho C., Paul R., Pfeifer A., Muhs A., Watts R.J., An effector-reduced anti-β-amyloid (Aβ) antibody with unique aβ binding properties promotes neuroprotection and glial engulfment of Aβ. J Neurosci. Jul 11;32(28):9677-89 (2012). Agadjanyan M.G., Ghochikyan A., Petrushina I, Vasilevko V., Movsesyan N., Mkrtichyan M., Saing T. and Cribbs D.H., Prototype Alzheimer’s Disease Vaccine Using the Immunodominant B Cell Epitope from β-Amyloid and Promiscuous T Cell Epitope Pan HLA DR-Binding Peptide. J Immunol 174 (3) 1580-1586 (2005). Arai H, Suzuki H, Yoshiyama T. Vanutide cridificar and the QS-21 adjuvant in Japanese subjects with mild to moderate Alzheimer's disease: results from two phase 2 studies. Curr Alzheimer Res. 12(3):242-54 (2015). Ghochikyan A., Mkrtichyan M., Petrushina I., Movsesyan N., Karapetyan A., Cribbs D.H., Agadjanyan M.G., Prototype Alzheimer's disease epitope vaccine induced strong Th2-type anti-Abeta antibody response with Alum to Quil A adjuvant switch. Vaccine. 20;24(13):2275-82 (2006). Gilman S., Koller M., Black R.S., Jenkins L., Griffith S.G., Fox N.C., Eisner L., Kirby L., Boada Rovira M., Forette F., Orgogozo J.M., Clinical effect of Aβ immunization (AN1792) in patients with AD in an interrupted trial. Neurology 64, 1553-1562 (2005). Liu B., Frost J.L., Sun J., Fu H., Grimes S., Blackburn P., Lemere C.A., MER5101, a novel Aβ1-15:DT conjugate vaccine, generates a robust anti-Aβ antibody response and attenuates Aβ pathology and cognitive deficits in APPswe / PS1ΔE9 transgenic mice. J Neurosci. 33(16):7027-37 (2013). Lutzner N., Kalbacher H., Quantifying Cathepsin S Activity in Antigen Presenting Cells Using a Novel Specific Substrate. J. Biol. Chem. Vol. 283 No. 52 p. 36185 (2008). Maier M., Seabrook T.J., Lazo N.D., Jiang L., Das P., Janus C, Lemere C.A., Short amyloid-beta (Abeta) immunogens reduce cerebral Abeta load and learning deficits in an Alzheimer's disease mouse model in the absence of an Abeta-specific cellular immune response. J Neurosci. 3;26(18):4717-28 (2006). Martineau P, chapter 41: Affinity Measurements by Competition ELISA, Pages 657-665, from book: Antibody engineering, Vol.1; R. Kontermann and S. Dubel (2010) Monsonego A., Weiner H.L., Immunotherapeutic approaches to Alzheimer's disease. Science. 31;302(5646):834-8 (2003). Muhs A., Hickman D.T., Pihlgren M., Chuard N., Giriens V., Meerschman C., van der Auwera I., van Leuven F., Sugawara M., Weingertner M.-C., Bechinger B., Greferath R., Kolonko N., Nagel-Steger L., Riesner D., Brady R.O., Pfeifer A., Nicolau C., Liposomal vaccines with conformation-specific amyloid peptide antigens define immune response and efficacy in APP transgenic mice. PNAS, 104 23:9810-9815 (2007). Orgogozo J.M., Gilman S., Dartigues J.F., Laurent B., Puel M., Kirby L.C., Jouanny P., Dubois B., Eisner L., Flitman S., Michel B.F., Boada M., Frank A., Hock C., Subacute meningoencephalitis in a subset of patients with AD after Abet42 immunization. Neurology 61: 46-54 (2003). Pihlgren M., Silva A.B., Madani R., Giriens V., Waeckerle-Men Y., Fettelschoss A., Hickman D.T., Lopez-Deber M.P., Ndao D.M., Vukicevic M., Buccarello A.L., Gafner V., Chuard N., Reis P., Piorkowska K., Pfeifer A., Kundig T.M., Muhs A., Johansen P., TLR4- and TRIF-dependent stimulation of B lymphocytes by peptide liposomes enables T cell-independent isotype switch in mice. Blood. Jan 3;121(1):85-94 (2013). Sallusto F., Lanzavecchia A., Araki K., Ahmed R., From vaccines to memory and back. Immunity. Oct 29;33(4):451-63 (2010). Schneeberger A., Mandler M., Mattner F., Schmidt W., AFFITOME(registered trademark) technology in neurodegenerative diseases: the doubling advantage. Hum Vaccin. 11:948-52 (2010) Seabrook T.J., Thomas K., Jiang L., Bloom J., Spooner E., Maier M., Bitan G., Lemere C.A., Dendrimeric Abeta1-15 is an effective immunogen in wildtype and APP-tg mice. Neurobiol Aging. 28(6):813-23 (2006). Siegrist CA, Chapter 2: Vaccine Immunology, Pages 14 - 32 from book: Vaccine (6th Edition, 2013).n, Walter A. Orenstein and Paul) Soto C., Plaque busters: strategies to inhibit amyloid formation in Alzheimer’s disease. Molecular Medicine Today (vol 5), August 1999. Winblad B., Graf A., Riviere M.E., Andreasen N., Ryan J.M., Active immunotherapy options for Alzheimer's disease. Alzheimers Res Ther. 2014 Jan 30;6(1):7. Winblad B., Andreasen N., Minthon L., Floesser A., Imbert G., Dumortier T., Maguire R.P., Blennow K., Lundmark J., Staufenbiel M., Orgogozo J.M., Graf A., Safety, tolerability, and antibody response of active Aβ immunotherapy with CAD106 in patients with Alzheimer's disease: randomised, double - blind, placebo - controlled, first - in - human study. Lancet Neurol. 11(7):597 - 604 (2012).

[0082] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are hereby incorporated by reference in their entirety for all purposes related to this invention.

[0083] The present invention should not be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those of ordinary skill in the art from the foregoing description and the accompanying drawings. Such modifications are intended to be included within the scope of the appended claims. Furthermore, all aspects and embodiments of the invention described herein are widely applicable and are considered to be combinable with any other consistent embodiments, including those taken from other aspects of the invention as necessary.

[0084]

Claims

1. A liposomal vaccine composition comprising: a. A peptide antigen derived from β-amyloid (Aβ) presented on the surface of liposomes b. A peptide comprising at least two different universal T cell epitopes, wherein the universal T cell epitopes stimulate a helper T cell response that enhances antibody production by B cells, said peptide being encapsulated in a liposome, said peptide being 20 to 60 amino acids in length, and said peptide being i. a universal T-cell epitope of diphtheria toxin consisting of 10-20 amino acids of SEQ ID NO:6; and a universal T-cell epitope of tetanus toxin consisting of 10-15 amino acids of the amino acid sequence QYIKANSKFIGITEL; ii. a universal T-cell epitope of Epstein-Barr virus consisting of 10 to 20 amino acids of SEQ ID NO: 8; A universal T-cell epitope of tetanus toxin consisting of 10 to 15 amino acids of the amino acid sequence QYIKANSKFIGITEL; and A universal T cell epitope of keyhole limpet hemocyanin consisting of 10 to 20 amino acids of SEQ ID NO:5 iii. A universal T-cell epitope of Epstein-Barr virus consisting of 10 to 20 amino acids of SEQ ID NO: 8; and a universal T-cell epitope of tetanus toxin consisting of 10 to 15 amino acids of the amino acid sequence QYIKANSKFIGITEL; or iv. a universal T-cell epitope of influenza hemagglutinin consisting of 10 to 20 amino acids of SEQ ID NO: 7; A universal T-cell epitope of diphtheria toxin consisting of 10 to 20 amino acids of SEQ ID NO: 6; A universal T-cell epitope of tetanus toxin consisting of the amino acid sequence YIKANSKFIG; and A peptide comprising a universal T-cell epitope of Epstein-Barr virus consisting of 10 to 20 amino acids of SEQ ID NO: 8; and C. Adjuvants.

2. The liposomal vaccine composition described in claim 1, wherein the peptides containing at least two different universal T cell epitopes contain at least 30% hydrophobic amino acids.

3. A liposomal vaccine composition as described in claim 1 or 2, comprising two, three, or four different universal T cell epitopes encapsulated within the liposome.

4. A liposomal vaccine composition described in any one of claims 1 to 3, wherein the peptides comprising at least two different universal T cell epitopes comprise two, three, or four universal T cell epitopes.

5. A liposomal vaccine composition described in any one of claims 1 to 4, wherein at least two different universal T cell epitopes are linked by a linker.

6. The liposomal vaccine composition of claim 5, wherein the linker comprises at least two amino acids.

7. A liposomal vaccine composition described in claim 5 or 6, wherein the linker comprises the amino acid VVR or PMGAP (sequence number 11).

8. A liposomal vaccine composition described in any one of claims 1 to 7, wherein the peptide comprising at least two different universal T cell epitopes consists of an amino acid sequence selected from SEQ ID NO: 1 (SAT42), SEQ ID NO: 2 (SAT43), SEQ ID NO: 3 (SAT44), and SEQ ID NO: 4 (SAT47).

9. A liposomal vaccine composition comprising: a. A tetrapalmitrylated β-amyloid (Aβ)-derived peptide antigen containing amino acids 1-15 of Aβ, displayed on the surface of liposomes. b. A peptide comprising at least two universal T-cell epitopes encapsulated within a liposome, A universal T-cell epitope of Epstein-Barr virus consisting of 10 to 20 amino acids of SEQ ID NO:8; and A peptide comprising a universal T-cell epitope of tetanus toxin consisting of 10 to 15 amino acids of the amino acid sequence QYIKANSKFIGITEL; and C. Adjuvants.

10. A liposomal vaccine composition comprising: a. A tetrapalmitrylated β-amyloid (Aβ)-derived peptide antigen containing amino acids 1-15 of Aβ, displayed on the surface of liposomes. b. A peptide comprising at least two universal T-cell epitopes encapsulated within a liposome, A universal T cell epitope of influenza hemagglutinin consisting of 10 to 20 amino acids of SEQ ID NO: 7; A universal T-cell epitope of diphtheria toxin consisting of 10 to 20 amino acids of SEQ ID NO: 6; A universal T-cell epitope of tetanus toxin consisting of the amino acid sequence YIKANSKFIG; and A peptide comprising a universal T-cell epitope of Epstein-Barr virus consisting of 10 to 20 amino acids of SEQ ID NO: 8; and C. Adjuvants.

11. A liposomal vaccine composition according to any one of claims 1 to 10, wherein the adjuvant comprises monophosphoryl lipid A (MPLA), CpG, or both MPLA and CpG.

12. A liposomal vaccine composition according to any one of claims 1 to 11, wherein the adjuvant forms part of the liposome.

13. A liposomal vaccine composition according to any one of claims 1 to 12, wherein the adjuvant is at least partially presented on the surface of the liposome.

14. A liposomal vaccine composition described in any one of claims 1 to 13, wherein the peptides comprising at least two different universal T cell epitopes are 30 to 60 amino acids in length.

15. A vaccine composition according to any one of claims 1 to 14 for use in treating, preventing, inducing a protective immune response against, or alleviating symptoms associated with an amyloid beta-related disease or condition in a subject.

16. A kit for treating, preventing, inducing a protective immune response against, or alleviating symptoms associated with an amyloid beta-related disease or condition in a subject, the kit comprising a liposomal vaccine composition described in any one of claims 1 to 14.

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