TECHNICAL FIELD

The present invention relates to compositions and methods for preventing and treating Alzheimer's disease, stimulating uptake of beta amyloid and/or preventing or reducing amyloid deposition.

BACKGROUND

Amyloid-beta peptide (Αβ) is believed to play a central role in the neuropathology of Alzheimer's disease. Immunization of young PDAPP mice with synthetic human Αβ1 -42 has been shown to reduce the extent and progression of AD-like neuropathologies (Schenk D. et al, 1999 Nature 400:173-7). One possible mechanism of action for the activity of this vaccine is that anti- Αβ antibodies facilitate clearance of amyloid-β, either before deposition or after plaque formation. These early preclinical observations were the basis for the development of a vaccine containing a pre-aggregated preparation of synthetic human Αβ42 peptides combined with QS21 (with or without polysorbate) which was tested in clinical trials on patients with Alzheimer's disease (clinical trial AN1792). The phase 2 of this trial was halted due to the occurrence of subacute meningoencephalitis in 18 out of 300 patients in the treatment group (Orgogozo J. M. et al. Neurology 2003; 61 : 46-54). It has been proposed that the cause of the meningo encephalitis was due to the helper T cell of type 1 (TH1 ), which are pro- inflammatory. That immune response has been postulated to be triggered by the long Αβ42 peptide that contains T cell epitopes within amino acids 15 to 42 (Monsonego, A. et al. J. Clin. Invest. 2003; 112: 415-422).

SUMMARY OF THE INVENTION

The present invention provides a composition comprising an amyloid beta mimotope and an adjuvant, wherein the composition is suitable for treatment or prevention of Alzheimer's disease, and/or for stimulating uptake of beta amyloid, and/or preventing or reducing amyloid deposition.

The invention further provides a method for the treatment or prevention of Alzheimer's disease, or for stimulating phagocytosis of beta amyloid, and/or for preventing or reducing amyloid deposition, the method comprising delivery of an effective amount of the composition of the invention.

The invention further provides a kit comprising an adjuvant, such as a TLR agonist, and an amyloid beta mimotope for separate, simultaneous or sequential delivery for stimulating an immune response to beta amyloid in an individual, the kit being for use, or suitable for use, in treatment or prevention of Alzheimer's disease. The invention further provides a method for stimulating an immune response to beta amyloid or for preventing or treating Alzheimer's disease, the method comprising delivering to an individual a composition comprising a TLR agonist and, separately, a composition comprising an amyloid beta mimotope.

The invention further provides a TLR agonist and an amyloid beta mimotope, for use in stimulating an immune response to beta amyloid or for preventing or treating Alzheimer's disease, wherein a composition comprising the TLR agonist and a composition comprising the mimotope are delivered separately

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Higher Αβ42 specific Immunogenicity promoted by TLR4 containing adjuvants such as

AS01 B compared to water-oil emulsion (AS03) when adjuvants are combined with the Αβ1 -6 CRM conjugate.

Figure 2: Dose-response study showing different level of Αβ42-3ρβοίίίο immunogenicity promoted by TLR containing adjuvants such as MPL or CRX601 (TLR4 ligands), AS15 (a TLR4 and TLR9 ligand) compared to appropriate controls (non-adjuvanted Αβ40/42 alone at ratio 4:1 ), non-adjuvanted Αβ1- 6CRM alone or with water-oil emulsion (AS03) or in combination with AS03-CRX601. Figure 3: Ex-vivo Αβ42 uptake capacity is promoted in the monocytes from mouse previously injected with MPL containing adjuvant (AS01 B). Moreover, the ex-vivo Αβ42 uptake is up-regulated by the addition of Αβ-specific antibody, e.g., monoclonal antibody 2E7 used herein.

Figure 4: Dose-escalation study using different doses of Αβ-specific polyclonal sera (anti Αβ1-

6CRM sera) to measure the ex-vivo Αβ42 uptake capacity promoted in the monocytes from mouse previously injected with or without MPL containing adjuvant (AS01 B).

Figure 5: In vivo Αβ uptake in the peripheral blood is promoted following the injection of a TLR- containing Αβ1-6 CRM conjugate compared to non-immunized control or Αβ42-3ρβοίίίο passive immunization only.

Figure 6: Comparison of different adjuvants to increase the number of peripheral blood monocytes 24 hrs following a single intramuscular injection.

Figure 7: Dose-escalations using 3 different doses of injected AS01 B on their respective capacity to increase the number of peripheral blood monocytes 24 hrs post injection. Figure 8: Αβ42 ex vivo uptake by peripheral blood monocytes from mice pre-injected with TLR4- containing adjuvants such as AS01 B (MPL 5 ug per mouse) or a synthetic TLR4 agonist (CRX601 , 2ug dose) in human microglial cell line CHME.

Figure 9: Phagocytosis of Αβ 1 -42 peptide in human microglial cell line (CHME) is promoted by the stimulation with TLR2 containing adjuvants such as Protollin or Pam3CysLip peptide.

Figure 10: QS21 + liposome stimulates in vivo an increase in monocyte number (panel A) and monocyte activation state (Ly6C high) (panel B) after 24hrs in the C57BL/6 mouse peripheral blood following intra muscular injection. QS21 + liposome stimulates the ex vivo Αβ uptake by mouse peripheral blood monocytes after 24 hrs of the intra muscular injection in the C57BL/6 mouse (panel C). QS21 + liposome + Αβ1-6 CRM197 peptide-carrier triggers higher anti Αβ immunogenicity compared to QS21 + Aβ1-6CRM197 formulation (panel D). Graph plot in panel D includes individual mouse data and standard deviation (SD). Multiple comparison tests denote a significant (P value less than 1 %) improvement of Αβ1-6 CRM197 + QS21 + liposome over the non-liposome formulation (Αβ1-6 CRM197 + QS21 ). Figure 11 : 2 month regimen vaccination in amyloid transgenic animals using AS01 B combined with mimotope (e.g., p1252) or related native amyloid peptide (Αβ1-6) in presence of Αβ-specific antibodies triggers higher amyloid uptake capacity ex vivo.

Figure 12A: The Αβ level within the monocytes is decreased after a 9 month schedule of immunotherapy treatment in APP/PS1 transgenic animals using AS01 B either combined with Αβ1-6 CRM or Aβpyro3-6 CRM or Aβ3-8CRM + AS01 B or CRM APP/PS1 transgenic animals.

Figure 12B: The soluble Αβ1-40 level within the brain is decreased after a 9 month schedule of immunotherapy treatment in APP/PS1 transgenic animals using AS01 B either combined with Αβ1-6 CRM or in lower extent with CRM alone (CRM197 carrier).

Figure 12C: The soluble Αβ1-42 level within the brain is decreased after a 9 month schedule of immunotherapy treatment in APP/PS1 transgenic animals using AS01 B either combined with Αβ1-6 CRM or in lower extent with Aβ3-8CRM or CRM alone (CRM197 carrier).

Figure 13A and 13B: Αβ-specific immunogenicity from dose/range of mimotopes vs native Αβ1-6. All vaccines contain a constant dose of AS01 B.

Figure 13C: Comparison between mimotopes and Αβ native peptides for their ΑβΙ-42-specific immunogenicity using the optimal dose of 1 μg peptide for intra muscular injection. Figure 13D: Shows that all vaccines used in Figure 13C elicited a high immune response against the vaccine itself as demonstrated by the CRM-specific antibody response.

Figure 14: High Αβ42 specific immunogenicity promoted by TLR2 agonist containing adjuvants such as Pam3CysLip peptide fused with a model Αβ fragment, e.g., Αβ1-6.

Figure 15: A TLR2 agonist containing formulation could improve the working memory (right-left discrimination T-water maze test) in amyloid deposition model (TASTPM).

Figure 16: The usage of the TLR2 agonist, i.e. Pam3CysLip Αβ1 -6 peptide, improves the survival rate of TASTPM mouse model.

Figure 17A: The results from 4 consecutive immunizations show that the last immunization triggers higher number of peripheral blood monocytes when a TLR4-containing adjuvant is used in the final composition such as AS01 B or CRX601 combined with AS03 emulsion.

Figure 17B: The results from 4 consecutive immunizations show that the last immunization triggers higher number of amyloid beta phagocytic cells in the peripheral blood only when a TLR4-containing adjuvant is used in the final composition such as AS01 B or CRX601 combined with AS03 emulsion.

Figure 17C: The results from 4 consecutive immunizations using different adjuvants combined with Aβ1-6CRM shows that all vaccines elicited specific Αβ1-42 antibody titers in a range between 55000 to 85000.

Figure 18A: Advantage of AS01 B vs Alum adjuvant in term of immunogenicity in C57BL/6 mice using Aβ1-6CRM as an amyloid-containing vaccine model.

Figure 18B: Advantage of AS01 B vs Alum adjuvant in term of the capacity of the monocytes from the vaccinated mice on the amyloid uptake capacity. C57BL/6 mice using Aβ1 -6CRM as an amyloid- containing vaccine model was used herein.

Figure 19A: Higher in vivo Αβ uptake capacity observed in amyloid transgenic mice after Aβ1-6CRM or mimotope p1252-CRM when combined with AS01 B.

Figure 19B: Αβ-specific antibody level within the plasma from animals described in Figure 19A.

Figure 20: A 2 months treatment window in amyloid mouse model (TASTPM) could impact the monocytes number (Figure 20A), the exogenous amyloid uptake by peripheral blood monocytes in vivo (Figure 20B). Figure 20C shows the Αβ-specific antibody response elicited by the different mimotopes- CRM and Αβ1 -6 CRM + AS01 B vaccines. Figure 21 : Biochemical analysis (western blot) of the low molecular weight Αβ oligomers from the brain soluble homogenates of amyloid mouse model (TASTPM) after a 2 months treatment using the 3 mimotopes vaccines: p4381-CRM + AS01 B; p4390-CRM + AS01 B; p4715-CRM + AS01 B.

Figure 22: Shows examples for in vivo characterisations of the immune response elicited by mimotope/MIMOTOPE vaccination (injected peptide/Αβ peptide) using Alum or oil in water emulsions as adjuvant in BALB/c mice.

Figure 23: Shows examples for in vivo characterisations of the immune response elicited by mimotope vaccination (injected peptide/ Αβ peptide) using oil in water emulsions in hAPP over- expressing animals (strain: Tg2576). Figure 24: Shows examples for in vivo characterisations of the immune response elicited by mimotope vaccination (injected peptide/ Αβ peptide); using Alum or an adjuvant containing a TLR4 agonist in BALB/c mice.

Figure 25: Shows examples for in vivo characterisations of the immune response elicited by mimotope vaccination (injected peptide/ Αβ peptide); using an adjuvant containing a TLR4 agonist in C57/BI6 mice.

Figure 26: Shows examples for in vivo characterisations of the immune response elicited by mimotope vaccination (injected peptide/Αβ peptide); using an adjuvant containing a TLR4 agonist in hAPP over-expressing animals (strain Tg2576).

DETAILED DESCRIPTION Sequences

Αβ1-6 : DAEFRH (SEQ ID NO: 1 ) Αβ2-7 : AEFRHD (SEQ ID NO: 2) Αβ3-8 : EFRHDS (SEQ ID NO: 3) Αβ3-8 pyr: pyrEFRHDS (SEQ ID NO: 4) Αβ11 -16: EVHHQK (SEQ ID NO: 5) SQEPAAPAAEAT PAAEAP (SEQ ID NO: 6) SQEPAAPAAEATPAAEAPDAEFRH (SEQ ID NO: 7) Αβ1-42: DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA (SEQ ID NO: 8)

TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO:9) Amyloid beta mimotopes

The present invention concerns mimotopes of the amyloid beta peptide Αβ1 -42 (SEQ ID NO: 8). These are peptides that have been selected to mimic the amyloid beta peptide or epitopes or fragments thereof. In one embodiment, the mimotope is capable of inducing an antibody response that is reactive with Αβ1- 42 but not with the native amyloid precursor protein (APP). The mimotope may be identified by screening a peptide library with an antibody, such as a monoclonal antibody. A monoclonal antibody may be used that does not recognize APP but detects only different Αβ species with an amino-terminal aspartic acid. Such antibodies are commercially available, as are suitable peptide libraries. In contrast to Αβ vaccines that comprise the native amyloid beta sequence, no autoimmune reactions are expected to occur during treatment with a vaccine containing a mimotope because of differences in amino acid sequence and lack of T cell epitopes.

The mimotope sequence may be from 5 to 15 amino acids in length, such as from 6 to 12 or 9 to 11 amino acids in length. The mimotope may be coupled (covalently or non-covalently) to a carrier, optionally via a linker molecule. The linker and/or carrier may consist of or contain T-cell helper epitopes. Suitable carriers include protein carriers, for example tetanus toxoid (TT), diphtheria toxid (DT), detoxified diphtheria toxoid such as Cross-Reacting Material 197 (CRM197), keyhole limpet hemocyanin (KLH), cholera toxin, virus-like particles, exoprotein A, albumin binding protein or bovine serum albumin. In one embodiment, the mimotope includes an additional cysteine residue at the C-terminus to enable conjugation to a carrier. Any suitable conjugation methodology may be used and such techniques are well known in the art. Where more than one mimotope is used, each mimotope may be conjugated to different carrier molecules or to the same carrier molecule. For example, each mimotope may be conjugated to separate lots of the same carrier molecule and then mixed. WO 2004/062556, WO 2006/005707, WO 2009/149486 and WO 2009/149485 disclose suitable amyloid beta mimotopes for use in the present invention.

In one aspect of the invention, the mimotope is selected to mimic the beta amyloid N-terminal epitope DAEFRH (Αβ1-6). Such mimotopes may consist of or comprise one of the following sequences: DKELRI, DWELRI, YAEFRG, EAEFRG, DYEFRG, DRELRI, GREFRN, EYEFRG, DWEFRDA, SWEFRT, DKELR, DAEFRWP, DNEFRSP, GSEFRDY, GAEFRFT, SAEFRTQ, SAEFRAT, SWEFRNP, SWEFRLY,

SWELRQA, SVEFRYH, SYEFRHH, SQEFRTP, SSEFRVS, DWEFRD, DAELRY, DWELRQ, SLEFRF, GPEFRW, GKEFRT, AYEFRH, DKE(Nle)R, DKE(Nva)R and DKE(Cha)R. In another aspect the mimotope is selected to mimic the N-terminal truncated epitope EFRHDS (Αβ3-8). Such mimotopes may consist of or comprise one of the following sequences: SEFKHG, TLHEFRH, ILFRHG, TSVFRH, SQFRYH, LMFRHN, SPNQFRH, ELFKHHL, THTDRFH, DEHPFRH, QSEFKHW, ADHDFRH, YEFRHAQ and TEFRHKA. In a further aspect the mimotope is selected to mimic the N-terminal truncated epitope pEFRHDS (Αβρ(Ε)3-8). Such mimotopes may consist of or comprise one of the following sequences: IRWDTP, VRWDVYP, IRYDAPL, I R YD MAG, IRWDTSL, IRWDQP, IRWDG and IRWDGG.

In a yet further aspect the mimotope is selected to mimic the N-terminal truncated epitope EVHHQK (Αβ11-16). Such mimotopes may consist of or comprise one of the following sequences: EVWHRHQ, ERWHEKH, EVWHRLQ, ELWHRYP, ELWHRAF, ELWHRA, EVWHRG, EVWHRH and ERWHEK.

In a yet further aspect the mimotope is selected to mimic the N-terminal truncated epitope HQKLVFFAED (Αβ14-23). Such mimotopes may consist of or comprise one of the following sequences: SHTRLYF, SGEYVFH, SGQLKFP, SGQIWFR, SGEIHFN, GQIWFIS, NDAKIVF, GQIIFQS, GQIRFDH, HMRLFFN, GEMWFAL, GELQFPP, GELWFP, SHQRLWF, HQKMIFA, GEMQFFI, GELYFRA, GEIRFAL, GMIVFPH, GEIWFEG, GEIYFER, AIPLFVM, GDLKFPL, GQILFPV, GELFFPK, GQIMFPR, HMRMYFE, GSLFFWP, GEILFGM, GQLKFPF, KLPLFVM, GTIFFRD, THQRLWF, GQIKFAQ, GTLIFHH, GEIRFGS, GQIQFPL, GEIKFDH, GEIQFGA, QLPLFVL, HQKMIF, GELFFEK, GEIRFEL, SGEIYFER and AGEIYFER

The mimotope sequence may include an additional cysteine residue at the C-terminus, which may be used for conjugation to a carrier. In one embodiment of the invention, the mimotope is selected from the following sequences: DKELRI, SWEFRT, GAEFRFT, DWEFRD, SLEFRF, GREFRN, SEFKHG, ILFRHG, TLHEFRH, IRWDTP and HQKMIFA. In a further embodiment of the invention, the composition comprises combinations of more than one mimotope. The combination may consist of or comprise two different N-terminal mimotopes; an N-terminal mimotope and an N-terminal truncated mimotope; or two different mimotopes of an N-terminal truncated epitope. Specifically contemplated combinations include: DKELRI and SEFKHG; DKELRI and ILFRHG; DKELRI and TLHEFRH; DKELRI and IRWDTP; DKELRI and HQKMIFA; SWEFRT and SEFKHG; SWEFRT and ILFRHG; SWEFRT and TLHEFRH; SWEFRT and IRWDTP; SWEFRT and HQKMIFA; GAEFRFT and SEFKHG; GAEFRFT and ILFRHG; GAEFRFT and TLHEFRH; GAEFRFTand IRWDTP; and GAEFRFT and HQKMIFA. In one aspect the mimotope is able to trigger at least 0.1 ug per ml of specific antibody in the blood or serum when delivered in a suitable form to an individual in need of treatment. In another aspect the antibody raised to the mimotope is of the lgG1 , lgG2, lgG3, and/or lgG4 isotypes. Combinations of mimotopes and adjuvants

According to the present invention, the amyloid beta mimotopes are combined with an adjuvant. In one aspect, the adjuvant is capable of inducing a systemic activation of the innate immune system. The adjuvant may also contribute to the immunogenicity of the antigen, for example by increasing the titre or affinitiy of antibodies raised against the antigen. This dual role of the adjuvant is expected to improve the treatment efficacy of the mimotope. Therefore in one aspect the invention provides an immunogenic composition that can induce a specific antibody immune response in a subject whilst at the same time inducing a system activation of the innate immune response. Accordingly, the invention also relates to the use of such compositions for improved uptake, such as improved phagocytosis, of amyloid beta. Amyloid beta (Αβ) in the brain is thought to be removed across the blood-brain-barrier by low-density lipoprotein receptor related protein-1 (LRP). LRP binds Αβ in the brain and then transports it into the blood in a process called transcytosis. This process is in equilibrium with partitioning back into the brain. The peripheral sink hypothesis suggests that removal of the Αβ from the blood shifts the equilibrium to bias it towards removal of Αβ from the brain. (Nature Medicine 13, 1029 - 1031 (2007)). The present invention shows that the use of a composition of the invention is able to increase the phagocytosis of amyloid, and therefore reduce the amount of Αβ present within the blood and hence within the central nervous system. A similar increase in the phagocytic activity of the resident microglial cells or freshly recruited resulting phagocytic cells in the brain may improve the clearance of the pathogenic Αβ.

Thus in one aspect the invention relates to a method of preventing and/or reducing amyloid deposition in a subject comprising stimulating the innate immune system of an individual using the methods and compositions of the invention, under conditions effective to prevent or reduce amyloid deposits. This prevention or reduction may be achieved by enhancing uptake, and optionally intracellular degradation, of the amyloid in cells of the immune system.

The stimulated cells may be monocytes, the circulating precursors of macrophages, microglial cells or their precursors and dendritic cells, or any phagocytic cell as disclosed herein. Reference to phagocytosis and stimulation of phagocytosis herein, may be read more generally to include uptake of a material (e.g. an antigen) into a cell of the immune system. Likewise reference to cells capable of phagocytosis includes reference to immune cells capable of antigen uptake, for example uptake of antigen bound to an antibody. As examples of uptake mechanisms, phagocytosis and macropinocytosis are specific examples, and the invention specifically contemplates both.

In one aspect of the invention, the adjuvant is a TLR agonist. Without wishing to be bound by theory, the use of a TLR agonist (such as an aminoalkyl glucosaminide phosphate, 3D-MPL or MPL or AS01 B) is thought to stimulate the innate immune system. Stimulation of the immune system may result in improved phagocytosis of antibodies which are bound to an antigen. In one aspect the TLR agonist is a TLR1 , TLR2, TLR 3, TLR 4, TLR 5, TLR 6, TLR 7, TLR 8 or TLR 9, agonist, such as a TLR2, TLR4 or TLR9 agonist. In one embodiment the TLR2 agonist is Pam3Cys - SQ EPAAPAAEAT PAAEAP (SEQ ID NO: 6; see WO 2010/028246). The TLR agonist may be used alone, suitably formulated, or comprised within a composition with other components, such as other pharmaceutically active agents. In one aspect the TLR agonist is a TLR4 agonist. In one aspect the TLR agonist is not a TLR9 agonist. In one aspect the TLR agonist is not coupled to an antigen. In one aspect the TLR agonist is not a TLR9 agonist coupled to an antigen.

Suitable TLR4 agonists include MPL and 3D-MPL, which are less toxic than Lipid A. U.S. Patent No. 4,436,727 discloses monophosphoryl lipid A [MPL] and its manufacture. U.S. Patent No. 4,912,094 and reexamination certificate B1 4,912,094 discloses 3-O-deacylated monophosphoryl lipid A [3D MPL] and a method for its manufacture.

In one aspect the TLR agonist or adjuvant comprises 3D-MPL in combination with a saponin, such as QS21 , and liposomes. In one aspect, the TLR agonist or adjuvant comprises QS21 and liposomes. In one aspect the TLR agonist or adjuvant consists or consists essentially of AS01 B which contains 50 μg MPL and 50 μg QS21 per human dose in a liposome formulation (see for example EP822831 ).

In one aspect the composition for use in the invention comprises a combination of a TLR4 agonist such as monophosphoryl lipid A, and a saponin derivative, particularly the combination of QS21 and 3D-MPL as disclosed in WO 94/00153, or a less reactogenic composition where the QS21 is quenched with cholesterol as disclosed in W096/33739. An adjuvant formulation involving QS21 3D-MPL and tocopherol in an oil in water emulsion is described in W095/17210 and is a suitable formulation.

In one aspect the TLR4 agonist may be a synthetic TLR4 agonist such as a synthetic disaccharide molecule, similar in structure to MPL and 3D-MPL or may be synthetic monosaccharide molecules, such as the aminoalkyl glucosaminide phosphate (AGP) compounds disclosed in, for example, WO9850399, WO0134617, WO0212258, W03065806, WO04062599, WO06016997, WO0612425, WO03066065, and WO0190129 the disclosure of each of which is herein incorporated by reference. Such molecules have also been described in the scientific and patent literature as lipid A mimetics. Lipid A mimetics suitably share some functional and/or structural activity with lipid A, and in one aspect are recognised by TLR4 receptors. AGPs as described herein are sometimes referred to as lipid A mimetics in the art. Lipid A mimetics in one aspect are less toxic than lipid A. In one aspect the aminoalkyl glucosaminide phosphate (AGP) is one in which an aminoalkyl (aglycon) group is glycosidically linked to a 2-deoxy-2-amino-a-D- glucopyranose (glucosaminide) to form the basic structure of the claimed molecules. The compounds are phosphorylated at the 4 or 6 carbon on the glucosaminide ring. Further, the compounds possess three 3-alkanoyloxyalkanoyl residues comprising a primary and secondary fatty acyl chain, each carbon chain consisting of from 2-24 carbon atoms, and preferably from 7-16 carbon atoms. In one preferred aspect, each primary chain contains 14 carbon atoms and each secondary chain has between 10 and 14 carbon atoms.

In one aspect the AGP compounds are described by the general formula:

Such compounds comprise a 2-deoxy-2-amino-a-D-glucopyranose (glucosamine) in glycosidic linkage with an aminoalkyl (aglycon) group. Compounds are phosphorylated at the 4 or 6 carbon on the glucosamine ring and have three alkanoyloxyalkanoyi residues. The compounds are described generally by Formula I, wherein X represents an oxygen or sulfur atom, Y represents an oxygen atom or NH group, "n", "m", "p" and "q" are integers from 0 to 6, R1 , R2, and R3 represent normal fatty acyl residues having 7 to 16 carbon atoms, R4 and R5 are hydrogen or methyl, R6 and R7 are hydrogen, hydroxy, alkoxy, phosphono, phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl or carboxy and esters and amides thereof; R8 and R9 are phosphono or hydrogen. The configuration of the 3' stereogenic centers to which the normal fatty acyl residues are attached is R or S, but preferably R. The

stereochemistry of the carbon atoms to which R4 or R5 are attached can be R or S. All stereoisomers, both enantiomers and diastereomers, and mixtures thereof, are considered to fall within the scope of the subject invention. The heteroatom X of such compounds of the subject invention can be oxygen or sulfur. In a preferred embodiment, X is oxygen. Although the stability of the molecules could be affected by a substitution at X, the immunomodulating activity of molecules with these substitutions is not expected to change.

The number of carbon atoms between heteroatom X and the aglycon nitrogen atom is determined by variables "n" and "m". Variables "n" and "m" can be integers from 0 to 6. In a preferred embodiment, the total number of carbon atoms between heteroatom X and the aglycon nitrogen atom is from about 2 to about 6 and most preferably from about 2 to about 4.

Such compounds are aminoalkyi glucosamine compounds which are phosphorylated. Compounds can be phosphorylated at position 4 or 6 (R8 or R9) on the glucosamine ring and are most effective if phosphorylated on at least one of these positions. In a preferred embodiment, R8 is phosphono and R9 is hydrogen.

Such compounds are hexaacylated, that is they contain a total of six fatty acid residues. The aminoalkyi glucosamine moiety is acylated at the 2- amino and 3-hydroxyl groups of the glucosamine unit and at the amino group of the aglycon unit with 3-hydroxyalkanoyl residues. In Formula I, these three positions are acylated with 3-hydroxytetradecanoyl moieties. The 3-hydroxytetradecanoyl residues are, in turn, substituted with normal fatty acids (R1-R3), providing three 3-n- alkanoyloxytetradecanoyl residues or six fatty acid groups in total.

The chain length of normal fatty acids R1-R3 can be from about 7 to about 16 carbons. Preferably, R1-R3 are from about 9 to about 14 carbons. The chain lengths of these normal fatty acids can be the same or different. Although, only normal fatty acids are described, it is expected that unsaturated fatty acids (i.e. fatty acid moieties having double or triple bonds) substituted at R1 ,-R3 on the compounds would produce biologically active molecules. Further, slight modifications in the chain length of the 3-hydroxyalkanoyl residues are not expected to dramatically effect biological activity.

Specific examples of AGP's include: CRX- 527 which is disclosed in Stover et al., JBC 2004 279, No 6, page 4440 - 4449 (available at www.jbc.org/content/279/6/4440.full.pdf). WO0212258 and W03065806 disclose additional embodiments of AGPs having a cyclic aminoalkyi (aglycon) linked to a 2-deoxy-2- amino-a-D- glucopyranose (glucosaminide), commonly referred to as "cyclic AGP's."

Reference generally to AGPs herein includes both cyclic and non cyclic AGPs. Cyclic AGPs possess three 3-alkanoyloxyalkanoyl residues comprising a primary and secondary fatty acyl chain, each carbon chain consisting of from 2-24 carbon atoms, and preferably from 7-16 carbon atoms. In one preferred aspect each primary chain contains 14 carbon atoms and each secondary carbon chain has between 10 and 14 carbon atoms per chain. The cyclic AGPs are described by the general formula II:

These compounds comprise a 2-deoxy-2-amino-p-D-glucopyranose (glucosamine) glycosidically linked to an cyclic aminoalkyl (aglycon) group. The compounds are phosphorylated at the 4 or 6-position of the glucosamine ring and acylated with alkanoyloxytetradecanoyi residues on the aglycon nitrogen and the 2 and 3-positions of the glucosamine ring. The compounds are described generally by formula (II): and pharmaceutically acceptable salts thereof, wherein X is-O-or NH-and Y is-O-or -S-; R1 , R2, and R3 are each independently a (C2-C24) acyl group, including saturated, unsaturated and branched acyl groups; R4 is -H or -P0 ₃ R7R8, wherein R7 and R8 are each independently H or (C1-C4) alkyl ; R5 is -H, -CH ₃ or - PO ₃ R9R10, wherein R9 and RIO are each independently selected from-H and (CI-C4) alkyl ; R6 is independently selected from H, OH, (CI-C4) alkoxy, -P0 ₃ R11 R12, -OP0 ₃ R11 R12, -S0 ₃ R11 , -OS0 ₃ R1 1 , - NR1 1 R12, -SR1 1 , -CN, -N0 ₂ , -CHO, -CC½R11 , and -CONR11 R12, wherein R11 and R12 are each independently selected from H and (CI-C4) alkyl; with the proviso that when R4 is- Po ₃ R7R8, R5 is other than-PO R9R10, wherein" ^* 1 -3"and" ^** "represent chiral centers; wherein the subscripts n, m, p and q are each independently an integer from 0 to 6, with the proviso that the sum of p and m is from 0 to 6.

In some embodiments, these compounds contain an- -O-at X and Y, R4 is P0 ₃ R7R8, R5 and R6 are H, and the subscripts n, m, p, and q are integers from 0 to 3. In a more preferred embodiment, R7 and R8 are -H. In an even more preferred embodiment, subscript n is 1 , subscript m is 2, and subscripts p and q are 0. In yet an even more preferred embodiment, R1 , R2, and R3 are tetradecanoyl residues. In a still more preferred embodiment, ^* 1 -3 are in the R configuration, Y is in the equatorial position, and ^** is in the S configuration (N-[(R)-3-tetradecanoyloxytetradecanoyl]-(S)-2- pyrrolidinomethyl 2-deoxy-4-0- phosphono-2-[(R)-3- tetradecanoyloxytetradecanoylamino]-3-0-[(R)-3-tetradecanoyl oxytetradecanoyl]-p- D- glucopyranoside and pharmaceutically acceptable salts thereof

Preferred cyclic structures include:

[III]

[IV]

Formula V is CRX 590. In another aspect the TLR4 receptor ligand is an AGP having one or more ether linked rather than ester linked primary and/or secondary lipid groups. In this embodiment, R1-R3 represent straight chain alkyl groups and not acyl groups, making the groups R10-, R20-, and R30- alkoxy rather than alkanoyloxy groups and the attachment to the primary acyl chain an ether rather than an ester linkage. In the case of an ether-linked primary lipid group, the 3-alkanoyloxyalkanoyl residue attached to the 3-hydroxy group of the glucosamine unit is replaced with either a 3-alkanoyloxyalkyl moiety or a 3-alkoxyalkyl moiety, making the attachment of the primary lipid group to the glucosamine 3-position an ether rather than an ester linkage.

A general formula for ethers is that of formula IV of WO2006 016997. An example of a preferred compound is CRX601.

In another aspect, the AGP molecule may have different number of carbons in the molecule's primary chains and/or secondary chains. Such compounds are disclosed in WO04062599 and WO06016997. As with other AGPs, each carbon chain may consist of from 2-24 carbon atoms, and preferably from 7-16 carbon atoms. In one preferred aspect each primary chain contains 14 carbon atoms and each secondary carbon chain has between 10 and 14 carbon atoms per chain.

Such compounds are represented by the following structures:

[VI]

wherein X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH; n, m, p and q are integers from 0 to 6; R1 , R2 and R3 are the same or different and are fatty acyl residues having from 1 to about 20 carbon atoms and where one of R1 , R2 or R3 is optionally hydrogen; R4 and R5 are the same or different and are selected from the group consisting of H and methyl; R6 and R7 are the same or different and are selected from the group consisting of H, hydroxy, alkoxy, phosphono, phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl and carboxy, and esters and amides thereof; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; R10, R1 1 and R12 are independently selected from straight chain unsubstituted saturated aliphatic groups having from 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof.

[VII]

wherein X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH ; n and m are 0; R1 , R2 and R3 are the same or different and are fatty acyl residues having from 1 to about 20 carbon atoms and where one of R1 , R2 or R3 is optionally hydrogen; R4 is selected from the group consisting of H and methyl; p is 1 and R6 is COOH or p is 2 and R6 is OP0 ₃ H ₂ ; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; and R10, R1 1 and R12 are independently selected from straight chain unsubstituted saturated aliphatic groups having from 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof.

[VIII]

wherein X is selected from the group consisting of O and S at the axial or equatorial position; Y is selected from the group consisting of O and NH; n, m, p and q are integers from 0 to 6; R1 , R2 and R3 are the same or different and are straight chain saturated aliphatic groups (i.e., straight chain alkyl groups) having from 1 to about 20 carbon atoms and where one of R1 , R2 or R3 is optionally hydrogen; R4 and R5 are the same or different and are selected from the group consisting of H and methyl; R6 and R7 are the same or different and are selected from the group consisting of H, hydroxy, alkoxy, phosphono, phosphonooxy, sulfo, sulfooxy, amino, mercapto, cyano, nitro, formyl and carboxy, and esters and amides thereof; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; R10, R11 and R12 are independently selected from straight chain unsubstituted saturated aliphatic groups having from 1 to 1 1 carbon atoms; or a pharmaceutically acceptable salt thereof. The general formula may also comprise an R5 group, at the same position as shown in formula VI above, wherein R5 is selected from the group consisting of H and methyl.

[IX]

Yet another type of compound of this invention has the formula (IV): wherein Y is now fixed as oxygen; X is selected from the group consisting of O and S at the axial or equatorial position; n and m are 0; R1 , R2 and R3 are the same or different and are fatty acyl residues having from 1 to about 20 carbon atoms and where one of R1 , R2 or R3 is optionally hydrogen; R4 is selected from the group consisting of H and methyl; p is 0 or1 and R6 is COOH, or p is 1 or 2 and R6 is OP03H2 ; R8 and R9 are the same or different and are selected from the group consisting of phosphono and H, and at least one of R8 and R9 is phosphono; and R10, R11 and R12 are independently selected from straight chain unsubstituted saturated aliphatic groups having from 1 to 10 carbon atoms; or a pharmaceutically acceptable salt thereof. These compounds thus have two acylated chains and one non-acylated ether chain.

Processes for making AGPs are also disclosed in WO0612425. Other AGP structures such as CRX 524 are disclosed in INFECTION AND IMMUNITY, May 2005, p. 3044-3052 Vol. 73, No. 5.

In one embodiment the TLR agonist for use in combination with the mimotope is combined with another adjuvant, wherein the adjuvant may be an oil in water emulsion, such as AS03 or MF59, or a saponin such as QS21 , or an aluminum salt or glucopyranosyl lipid A adjuvants (Immune Design). A combination of TLR agonists may be used. The present invention further shows that the use of a detoxified form of QS21 by the addition of liposome is able to increase the phagocytosis of amyloid, and therefore reduce the amount of Αβ present within the blood and hence within the central nervous system. A similar increase in the phagocytic activity of the resident microglial cells or freshly recruited resulting phagocytic cells in the brain may improve the clearance of the pathogenic Αβ.

Thus in one aspect the invention relates to a method of preventing and/or reducing amyloid deposition in a subject comprising stimulating the innate immune system of an individual using the methods and compositions of the invention, including but not limited to those comprising detoxified QS21 , under conditions effective to prevent or reduce amyloid deposits. This prevention or reduction may be achieved by enhancing uptake, and optionally intracellular degradation, of the amyloid in cells of the immune system. In a particular aspect the invention relates to use of compositions as disclosed herein to increase uptake of amyloid by monocytes in the peripheral blood. The mode of action of the immunotherapy may rely both on an increased number of monocytes and their degree of activation, as determined by the presence of CD11 b and/or Ly6C markers. Compositions of the invention may comprise the saponin QS21 (WO8809336A1 ; US5057540A). QS21 is well known in the art as a natural saponin derived from the bark of Quillaja saponaria Molina, which induces CD8+ cytotoxic T cells (CTLs), Th1 cells and a predominant lgG2a antibody response. For the avoidance of doubt reference to QS21 includes OPT-821 or PQS-21 (Ragupathi et al., 2010)Ref:

(Ragupathi G, Damani P, Deng K, Adams MM, Hang J, George C, Livingston PO, Gin DY. Preclinical evaluation of the synthetic adjuvant SQS-21 and its constituent isomeric saponins. Vaccine. 2010 Jun 11 ;28(26):4260-7.PubMedPMID: 20450868; PubMed Central PMCID: PMC2882175.

In a suitable form of the present invention, the compositions of the invention may comprise QS21 in substantially pure form, that is to say, the QS21 is at least 80%, at least 85%, at least 90% pure, for example at least 95% pure, or at least 98% pure. Compositions of the invention may comprise QS21 in an amount of between about 1 μg to about 100μg, for example between about 1 μς and about 60μg or between '^g and about 50μg, for example, about ^• ^g, about 12^g, about '^g, about 20μg, about 25μg, about 30μg, about 40μg or in particular about 50μg. In particular, QS21 is present in an amount between about 40μg and 60μg or between 45 and 55 μg or between 47 and 53μg or between 48 and 52 μg or between 49 and 51 or about 50μg. Alternatively QS21 may be present in an amount between 21 μg and 29 μg or between about 22μg and about 28μg or between about 23μg and about 27μg or between about 24μg and about 26 μg, or about 25 μg. In a further embodiment compositions of the invention may comprise QS21 in an amount of about 10 μg, for example between about 5μg and Ι δμς, about 6μg and about 14μς, about 7μg and about 13μς, about 8μg and about 12μg or about 9μg and about 1 1 μg, or about 10μς.

In a further embodiment, compositions of the invention may comprise QS21 in an amount of around about 5μg, for example between about 1 μς and 9μς, about 2μg and about 8μg, about 3μg and about 7μg, about 4μς and about 6μg, or about 5 μg.

A suitable amount of QS21 in the compositions of the invention is for example any of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg. Compositions of the invention comprising QS21 and a sterol, cholesterol in particular, show a decreased reactogenicity when compared to compositions in which the sterol is absent, while the adjuvant effect is maintained. Reactogenicity studies may be assessed according to the methods disclosed in WO 96/33739. Suitably the sterol is associated to the saponin adjuvant as described in WO 96/33739. In a particular embodiment, the cholesterol is present in excess to that of QS21 , for example, the ratio of QS21 :sterol will typically be in the order of 1 :100 to 1 :1 (w/w), suitably between 1 :10 to 1 :1 (w/w), and preferably 1 :5 to 1 :1 (w/w). In particular, the ratio of QS21 :sterol being at least 1 :2 (w/w). In a particular embodiment, the ratio of QS21 :sterol is 1 :5 (w/w). Suitable sterols include β-sitosterol, stigmasterol, ergosterol, ergocalciferol and cholesterol. In one particular embodiment, the compositions of the invention comprise cholesterol as sterol. These sterols are well known in the art, for example cholesterol is disclosed in the Merck Index, 11 th Edn., page 341 , as a naturally occurring sterol found in animal fat.

The compositions of the invention comprise QS21 in its less reactogenic composition where it is quenched with an exogenous sterol, such as cholesterol for example. As used herein, the term "detoxified QS21 " refers to QS21 in combination with a sterol. Several particular forms of less reactogenic compositions wherein QS21 is quenched with an exogenous cholesterol exist. In a specific embodiment, the saponin /sterol is in the form of a liposome structure (WO 96/337391 ).

The term "liposome(s)" generally refers to uni- or multilamellar (particularly 2, 3, 4, 5, 6, 7, 8, 9, or 10 lamellar depending on the number of lipid membranes formed) lipid structures enclosing an aqueous interior. Liposomes and liposome formulations are well known in the art. Lipids which are capable of forming liposomes include all substances having fatty or fat-like properties. Lipids which can make up the lipids in the liposomes may be selected from the group comprising glycerides, glycerophospholipides, glycerophosphinolipids, glycerophosphonolipids, sulfolipids, sphingolipids, phospholipids, isoprenolides, steroids, stearines, sterols, archeolipids, synthetic cationic lipids and carbohydrate containing lipids. In a particular embodiment of the invention the liposomes comprise a phospholipid. Suitable phospholipids include (but are not limited to): phosphocholine (PC) which is an intermediate in the synthesis of phosphatidylcholine; natural phospholipid derivates: egg phosphocholine, egg

phosphocholine, soy phosphocholine, hydrogenated soy phosphocholine, sphingomyelin as natural phospholipids; and synthetic phospholipid derivates: phosphocholine (didecanoyl-L-a-phosphatidylcholine [DDPC], dilauroylphosphatidylcholine [DLPC], dimyristoylphosphatidylcholine [DMPC], dipalmitoyl phosphatidylcholine [DPPC], Distearoyl phosphatidylcholine [DSPC], Dioleoyl phosphatidylcholine

[DOPC], 1-palmitoyl, 2-oleoylphosphatidylcholine [POPC], Dielaidoyl phosphatidylcholine [DEPC]), phosphoglycerol (1 ,2-Dimyristoyl-sn-glycero-3-phosphoglycerol [DMPG], 1 ,2-dipalmitoyl-sn-glycero-3- phosphoglycerol [DPPG], 1 ,2-distearoyl-sn-glycero-3-phosphoglycerol [DSPG], 1 -palmitoyl-2-oleoyl-sn- glycero-3-phosphoglycerol [POPG]), phosphatidic acid (1 ,2-dimyristoyl-sn-glycero-3-phosphatidic acid [DMPA], dipalmitoyl phosphatidic acid [DPPA], distearoyl-phosphatidic acid [DSPA]),

phosphoethanolamine (1 ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine [DMPE], 1 ,2-Dipalmitoyl-sn- glycero-3-phosphoethanolamine [DPPE], 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine DSPE 1 ,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine [DOPE]), phoshoserine, polyethylene glycol [PEG] phospholipid (mPEG-phospholipid, polyglycerin-phospholipid, funcitionilized-phospholipid, terminal activated-phosholipid). In one embodiment the liposomes comprise 1-palmitoyl-2-oleoyl-glycero-3- phosphoethanolamine. In one embodiment highly purified phosphatidylcholine is used and can be selected from the group comprising Phosphatidylcholine (EGG), Phosphatidylcholine Hydrogenated (EGG), Phosphatidylcholine (SOY) and Phosphatidylcholine Hydrogenated (SOY). In a further embodiment the liposomes comprise phosphatidylethanolamine [POPE] or a derivative thereof.

Liposome size may vary from 30 nm to several μιη depending on the phospholipid composition and the method used for their preparation. In particular embodiments of the invention, the liposome size will be in the range of 50 nm to 500 nm and in further embodiments 50 nm to 200 nm. Dynamic laser light scattering is a method used to measure the size of liposomes well known to those skilled in the art.

In particular liposomes of the invention may comprise dioleoyl phosphatidylcholine [DOPC] and a sterol, in particular cholesterol. Thus, in a particular embodiment, compositions of the invention comprise QS21 in any amount described herein in the form of a liposome, wherein said liposome comprises dioleoyl phosphatidylcholine [DOPC] and a sterol, in particular cholesterol. In a further particular embodiment, the compositions of the invention as described herein do not comprise an immunostimulatory oligonucleotide, particularly an immunostimulatory oligonucleotide comprising one or more CpG motifs. In one embodiment of the invention the TLR agonist described herein is delivered in the absence of an immunogen, such as in the absence of a polypeptide or polyscaacharide antigen. In one embodiment the TLR agonist described herein is provided in combination with a pharmaceutically acceptable excipient or additive. In one aspect the TLR agonist is able to induce the production of CD11 b+ phagocytic cells in the periphery.

In one aspect the TLR agonist is able to increase the phagocytic activity of CD1 1 b+ phagocytic cells, suitably as measured in blood or sera from the periphery of an animal.

Phagocytotic activity may be determined by the assay described herein, in which the uptake of a labelled antigen, such as amyloid beta fragment, is detected. One suitable assay for phagocytosis of

betacomprises incubation of lineage CD11 b+ monocytes with a labeled amyloid beta polypeptide and detection of the uptake of the labeled amyloid beta into the cells.

The Αβ mimotope is, in one aspect, delivered separately from the TLR agonist. In one aspect this separate delivery is delivery at a different time. The Αβ mimotope and TLR agonist may be delivered by different routes, or provided in different formulations. Separate delivery of the Αβ mimotope and TLR agonist may be achieved by delivery of an Αβ mimotope, optionally formulated with an adjuvant (which may be a TLR agonist), at a different time to the delivery of a composition comprising a TLR agonist. Separate delivery of the Αβ mimotope and TLR agonist may also be achieved by delivery of an Αβ mimotope not formulated with a TLR agonist at a different time to the delivery of a composition comprising a TLR agonist. In one embodiment, the Αβ mimotope is not formulated with a TLR agonist and may be delivered 1 , 2, 3, 4, 5, 6 or more times, suitably up to 3 or 4 times, before the first delivery of the TLR agonist.

Thus in one embodiment the TLR agonist is delivered after the Αβ mimotope, which may for example be 1 week, 2 weeks, 3 weeks, a month, 5 weeks, 6 weeks, 7 weeks or 2 months after the first delivery of means to generate the antibody , or even more than 2 months, for example where antibody titers take this long to develop. The Αβ mimotope may be delivered 1 , 2, 3, 4, 5, 6 or more times before the first delivery of the TLR agonist. The TLR agonist may be delivered single or multiple times such as 1 , 2, 3 or 4 times. For example, the delivery regimen may comprise delivery of an Αβ mimotope at day zero, followed by, if necessary, further delivery of Αβ mimotope in week 1 , 2, 3, or 4. Optionally delivery may be repeated with 1 or 2 or 3 or a 4 week intervals to induce an appropriate immune response, such as a suitable antibody response. A TLR agonist, alone or as part of a composition, may be delivered for example a week, 2 weeks, 3, weeks or 4 weeks after the initial antigen delivery, or 1 , 2, 3, or 4 weeks after a second or subsequent Αβ mimotope delivery. Αβ mimotopes may be combined with an adjuvant to enhance the specific immune response against the mimotope. For the avoidance of doubt, reference to adjuvant herein is generally made in the context of the component delivered with an Αβ mimotope to enhance the immunogenic response to that mimotope, noting that TLR agonists themselves may be used as adjuvants with antigens, and thus TLR agonists may play a role both in the generation of antibodies, and following the delivery or generation of an antibody or other immune response. The adjuvant used with the mimotope, where necessary, and the adjuvant employed after generation of antibodies may be the same adjuvant.

The adjuvant for use with an Αβ mimotope may be any suitable adjuvant which enhances the immune response against the mimotope. The adjuvant may be a TLR agonist, such as a TLR1 , TLR2, TLR 3, TLR 4, TLR 5, TLR 6, TLR 7, TLR 8, or TLR 9 agonist, such as a TLR2 or TLR4 or TLR9 agonist. The adjuvant may comprise a TLR agonist in combination with another adjuvant or pharmaceutically acceptable component. In one embodiment the Αβ mimotope is not combined with a TLR agonist.

In one embodiment the adjuvant helps to promote an antibody response, such as a TH2 adjuvant, such as alum. In another embodiment the adjuvant is an oil-in-water emulsion. In another embodiment the adjuvant comprises a water-oil emulsion such as AS03 in combination with an aminoalkyl glucosaminide phosphate such as CRX601. In another embodiment the compositions of the invention comprise an adjuvant containing MPL, QS21 and liposome. In a particular aspect, the compositions of the invention comprise the adjuvant system AS01 B, which contains 50 μg MPL and 50 μg QS21 per human dose in a liposome formulation. In one aspect the adjuvant is Trehalose MPL from Sigma, which comprises monophosphoryl lipid A

(detoxified endotoxin) from S. minnesota (MPL) and synthetic trehalose dicorynomycolate (TDM) in 2% oil (squalene)-Tween 80-water.

For the avoidance of doubt the present invention also specifically relates to combinations of any adjuvant disclosed herein with any mimotope as disclosed herein, which may be used as immunogenic compositions and vaccines. Such combinations may be used without the need for delivery of a separate TLR agonist.

Such antigen plus adjuvant combinations may be delivered in single or multiple doses. In one embodiment the mimotope plus adjuvant combination is delivered to a subject 1 , 2, 3, 4, 5 or more times. The 2 ^nd , 3 ^rd , 4 ^th or subsequent delivery of the antigen plus adjuvant combination may act as a booster dose.

The compositions and methods of the various aspects of the invention may be used in the treatment or prevention of Alzheimer's disease. Treatment can be therapeutic or preventative. The subject will be one who is in need of such treatment, including individuals already suffering from a Alzheimer's, as well as individuals who are at risk of developing the disease or condition. As used herein, the term "treatment" encompasses the alleviation, reduction, prevention, or delay of onset, of at least one aspect or symptom of a disease or condition in a subject (compared to a subject who does not receive such treatment).

As used herein, the term "effective amount" means that amount of a composition or pharmaceutical agent that will elicit the biological or medical response of a tissue, system, animal or human that is being sought, for instance, by a researcher or clinician. As used herein a therapeutically effective amount is an amount effective to ameliorate or reduce one or more aspects or symptoms of a disease or condition in a subject. A prophylactically effective amount is an amount which prevents or delays the onset of one or more aspects or symptoms of a disease described herein. An amount may have both therapeutic (ameliorating) and prophylactic (preventing or delaying) effects.

As used herein, the ability of a treatment to prevent or reduce amyloid deposition is as compared to amyloid deposition in a comparable subject who has not received the treatment. As used herein, the ability of a treatment to stimlate the uptake of beta amyloid is as compared to uptake in a comparable subject who has not received the treatment. The methods described herein need not effect a complete cure or eradicate every symptom or manifestation of a disease or condition to constitute a useful treatment. As is recognised in the medical arts, drugs employed as therapeutic agents may reduce the severity of a given disease state, but need not abolish every manifestation of the disease to be regarded as useful therapeutic agents. Similarly, a prophylactically administered treatment need not be effective in preventing the onset of all aspects of a disease, or effective in all subjects treated, to constitute a viable prophylactic or preventative agent.

Simply reducing the impact of a disease (for example, by reducing the number or severity of its symptoms in an individual, or reducing the number of subjects affected), is sufficient.

Thus the invention relates to a method of prevention or treatment of Alzheimer's disease, the method comprising delivering to an individual an amyloid beta mimotope and an adjuvant. The invention also relates to a composition comprising an adjuvant and an amyloid beta mimotope for use in prevention or treatment of Alzheimer's disease.

The invention also relates to a TLR agonist and, separately, an amyloid beta mimotope (optionally combined with an adjuvant), for use in prevention or treatment of Alzheimer's disease.

In one embodiment the subject for prevention or treatment may have already been diagnosed with symptoms of Alzheimer's disease. In another aspect the subject for treatment has not already been diagnosed with Alzheimer's. For example, the subject may be suffering from mild cognitive impairment (MCI). Mild cognitive impairment is a syndrome defined as cognitive decline greater than that expected for an individual's age and education level but that does not interfere notably with activities of daily life. It is, thus, distinct from dementia, in which cognitive deficits are more severe and widespread and have a substantial effect on daily function. However, mild cognitive impairment with memory complaints and deficits (amnestic mild cognitive impairment) is consistently shown to have a high risk of progression to dementia, particularly of the Alzheimer type (Gauthier, S. et al. 2006 Lancet 367(9518): 1262-1270).

In one aspect the present invention relates to an effect on the deposits of amyloid protein, and in another aspect to an effect on behaviours that are associated with Alzheimer's disease, and in particular prevention or reduction of behaviours associated with Alzheimer's disease. In one aspect the methods and compositions of the invention have an effect both on amyloid protein deposition and behaviour associated with disease, such as behaviour associated with Alzheimer's disease, although in another aspect the methods and compositions of the invention have an effect either at the level of amyloid deposits or at the level of behaviour.

In one aspect the prevention or reduction in severity of Alzheimer's disease comprises prevention or reduction of loss of memory. In a further aspect the invention relates to relates to improvement in memory, for example spatial memory.

The invention also relates to any composition disclosed herein, such as (mimotope carrier), (mimotope, adjuvant, carrier) and (mimotope adjuvant) combinations, and to a method of treatment or prevention of Alzheimer's disease using said compositions.

In a particular embodiment, the present invention provides a composition comprising: · a mimotope comprising or consisting of any of the following sequences: DKELRI, SWEFRT,

GAEFRFT, DWEFRD, SLEFRF, GREFRN, SEFKHG, ILFRHG, TLHEFRH, , IRWDTP or HQKMIFA, optionally conjugated to CRM or KLH; and

• an adjuvant selected from 3DMPL, AS01 B, an AGP (such as CRX601 or CRX527) or an AGP combined with an oil in water emulsion (such as CRX601 with AS03). In one aspect, the composition of the invention comprises or consists essentially of a mimotope selected from DKELRI, SWEFRT, GAEFRFT, DWEFRD, SLEFRF, GREFRN, SEFKHG, ILFRHG, TLHEFRH, , IRWDTP or HQKMIFA , optionally conjugated to CRM or KLH, in combination with AS01 B.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence DKELRI, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease. According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence SWEFRT, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence GAEFRFT, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence DWEFRD, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease. According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence SLEFRF, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence GREFRN, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence SEFKHG, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence ILFRHG, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence TLHEFRH, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease. According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence IRWDTP, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease.

According to one embodiment, the invention provides a composition comprising an amyloid beta mimotope comprising the sequence HQKMIFA, wherein the mimotope is conjugated to CRM, and the adjuvant AS01 B, wherein the composition is suitable for treatment or prevention of Alzheimer's disease. The compositions and methods of the present invention may be used in one or more of preventing or reducing effect on deposits of amyloid protein, stimulation of innate immunity via microglia cells, increasing amyloid phagocytosis and preventing or reducing behaviours that are associated with Alzheimer's disease. The examples provided herein give suitable methods for assessing of these parameters.

Beta amyloid deposits may be measured as a function of the area of plaques in a brain section, or assessed by total protein concentration, as described in the attached Examples. Other suitable methods are disclosed in WO2009105641 .

Effects of the treatments and compositions of the invention on behaviour associated with Alzheimer's disease may be assessed in human patients, or in animal models, for example. Suitable animal models include the mouse APP model for Alzheimer's disease, the PS1 mouse model and the APP/PS1 model. See Richard, K.L. et al. J Neurosci 28, 5784-5793 (2008).

Suitable animal (rodent) tests include one or more of the T-water maze test, Passive avoidance test, or nesting behaviour tests as described herein (Filali M, et al Cognitive and non-cognitive behaviours in an APPswe/PS1 bigenic model of Alzheimer's disease. Genes Brain Behav. 2009 Mar;8(2): 143-8. Epub

2008 Dec 3. PubMed PMID: 19077180.). Other behavioural tests that may be employed are described in WO2009105641 , incorporated herein by reference.

Stimulation of the innate immune system may be effected by, and/or measured by, stimulation of microglia. In another aspect the innate immune response may be assessed by the triggering transcriptional activation of TLR2 in brain tissues, for example in appropriate animal mouse models.

The methods and compositions of the present invention may be used to protect or treat a mammal by means of administering via the systemic or mucosal route. These administrations may include injection via the intramuscular (i.m.), intraperitoneal (i.p.), intradermal or subcutaneous routes; or via mucosal administration to the oral/alimentary, sublingual, intranasal, respiratory, genitourinary tracts. The administration may include an ocular administration or administration by antigen loaded patches. The composition of the invention may be administered as a single dose, or multiple doses. In addition, the compositions of the invention may be administered by different routes for priming and boosting, for example, IM priming doses and IN for booster doses.

In one aspect intramuscular delivery of low doses of 3D MPL is not preferred. In one aspect the amount of 3D MPL used is equivalent to 50ug 3D MPL injected intraperitonealy In one aspect 3D MPL is delivered by intraperitoneal injection. The components of the present invention may be administered alone or with suitable pharmaceutical carriers, and can be in solid or liquid form, such as tablets, capsules, powders, solutions, suspensions, or emulsions.

The immunogenic compositions of the invention may be formulated into a "vaccine," and administered in free solution, or formulated with an excipient. Vaccine preparation is generally described in Vaccine Design ("The subunit and adjuvant approach" (eds Powell M. F. & Newman M.J.) (1995) Plenum Press New York). Encapsulation within liposomes is described by Fullerton, US Patent 4,235,877. The vaccines of the present invention may be stored in solution or lyophilized.

Effective doses of the components of the present invention, e.g. for the treatment of a subject having amyloid deposits or AD, vary depending upon many different factors, including means of administration, target site, physiological state of the patient, other medications administered, physical state of the patient relative to other medical complications, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. The amount of TLR agonist may depend on whether other components, such as an adjuvant are also administered. Subject doses of the TLR agonist described herein typically range from about 0. 1 μg to 50 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time there between. More typically mucosal or local doses range from about 10μg to 10mg per administration, and optionally from about 100μg to 1 mg, with 2-4 administrations being spaced days or weeks apart. More typically, immune stimulant doses range from ^g to 10 mg per administration, and most typically 10μg to 1 mg, with daily or weekly administrations. Doses of the compounds described herein for parenteral delivery e.g., for inducing an innate immune response, or in specialized delivery vehicles typically range from about 0.1 μg to 10 mg per administration, which depending on the application could be given daily, weekly, or monthly and any other amount of time therebetween. More typically parenteral doses for these purposes range from about 10μg to 5mg per administration, and most typically from about 100μg to 1 mg, with 2-4 administrations being spaced days or weeks apart. In some embodiments, however, parenteral doses for these purposes may be used in a range of 5 to 10,000 times higher than the typical doses described above. Suitable doses of antibody and antigen needed to demonstrate an effect in vivo, or to elicit an immune response in vivo respectively, are well known to the skilled person.

Compositions of the invention may comprise a TLR4 agonist in an amount of between about 1 μg to about 100μg, for example between about ^g and about 60μg or between 10μg and about 50μg, for example, about 10μg, about 12^g, about 15μg, about 20μg, about 25μg, about 30μg, about 40μg or in particular about 50μg. In particular, QS21 is present in an amount between about 40μg and 60μg or between 45 and 55 μg or between 47 and 53μg or between 48 and 52 μg or between 49 and 51 or about 50μg. Alternatively QS21 is present in an amount between 21 μg and 29 μg or between about 22μg and about 28μg or between about 23μg and about 27μg or between about 24μg and about 26 μg, or about 25 μg. In a further embodiment compositions of the invention comprise a TLR4 agonist in an amount of about 10 μg, for example between about 5μg and 15μg, about 6μg and about 14μg, about 7μg and about 13μg, about 8μg and about 12μg or about 9μg and about 11 μg, or about 10μg. In a further embodiment, compositions of the invention comprise a TLR4 agonist in an amount of around about 5μg. A suitable amount of TLR4 agonist in the compositions of the invention is for example any of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50,55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 μg.

Any aspect or feature of the invention may be combinable with any other aspect or feature of the invention, even where disclosed in a specific example, except where obvious from the context. For example, antigens, adjuvants or TLR agonists disclosed in any aspect or feature are combinable with any other aspect or feature of the invention.

For the avoidance of doubt the terms 'comprising', 'comprise' and 'comprises' herein is intended by the inventors to be optionally substitutable with the terms 'consisting of, 'consist of, and 'consists of, respectively, in every instance. As used in this specification and claim(s), the words

"comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

The term "about" (or "around") in all numerical values allows for a 5% variation, i.e. a value of about 1.25% would mean from between 1.19%-1.31 %.

The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the measurement, the method being employed to determine the value, or the variation that exists among the study subjects.

The term "or combinations thereof" as used herein refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular

context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

As used herein, 'subject' includes human subjects. As used herein, 'individual' includes human individuals.

While the compositions and methods of this disclosure have been described in terms of suitable embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. All such similar

substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

All references or patent applications cited within this patent specification are herein incorporated by reference.

It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the disclosure. The principal features of this disclosure can be employed in various embodiments without departing from the scope of the disclosure. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this disclosure and are covered by the claims.. The disclosure will be further described by reference to the following, non-limiting, examples:

EXAMPLES

General: Methods and materials

All experiments with animals and related assays were performed in accordance with the Canadian Council on Animal Care (CCAC) guidelines for animal experimentation. Eight week old female C57BL/6 mice were obtained from Charles-Rivers laboratories (St-Constant, Quebec). The APP-PS1 mouse model was obtained from Jackson laboratories, stock 5866 (Savonenko et al., 2005 Savonenko A; Xu GM; Melnikova T; Morton JL; Gonzales V; Wong MP; Price DL; Tang F; Markowska AL; Borchelt DR. 2005. Episodic-like memory deficits in the APPswe/PS1dE9 mouse model of Alzheimer's disease:

relationships to beta-amyloid deposition and neurotransmitter abnormalities. Neurobiol Dis 18(3):602-17). Intramuscular injections in mice were performed on either the gastrocnemius anterior in 50 or 25 μΙ_ depending on the experiments. Intravenous injections (200 μΙ_) were performed in the tail vein.

The adjuvant compositions used were as follows:

Adjuvant composition: For AS01 B, AS03 and AS15 the full mouse dose is equal to 1 /10 of a human dose.

AS01 B is an Adjuvant System containing 3DMPL, QS21 and liposome (50 g 3DMPL and 50 μg QS21 human dose). The mouse dose of AS01 B contains 5ug of 3DMPLand 5ug of QS21. Those doses were injected using the intramuscular route (i.m.) 25 ul per mouse of AS01 B + 25 ul of PBS (phosphate buffer saline) or 25 ul of the appropriate peptide. AS03 is an Adjuvant System containing a-Tocopherol and squalene in an o/w emulsion. AS03 used in this study contained 23.72 mg/mL a-tocopherol, 21.38 mg/mL squalene and 9.72 mg/mL polysorbate-80 in PBS. The mean particle sizes of the adjuvant emulsions were determined by dynamic light scattering (Zeta-Nanosizer, Malvern Instruments). The final injection volumes (with or without vaccine) were 50 μΙ. PBS was used as diluents. A human dose of an AS03 _A -adjuvanted vaccine contains 1 1.86 mg a- tocopherol, 10.69 mg squalene and 4.86 mg polysorbate-80. Mice were therefore injected with an adjuvant dose no greater than 1/10th of an adult human dose.,

CRX601/AS03: 2 μg of CRX601 was diluted in PBS for a final volume of 25 μΙ. The 25 μΙ of CRX601 was added slowly to the 25 μΙ AS03 (2x concentrated). The vaccine was mixed by magnetic stirring at medium speed. Formulation was performed extemporaneously, the injections occurred within 60 min following the end of the formulation.

AS15 is an Adjuvant System containing 3DMPL, QS21 , CpG and liposome (50 μg 3DMPL, 50 μg QS21 and 420 μg CpG)

"3-0-desacyl-4'- monophosphoryl lipid A " 3D-MPL: is an immunostimulant derived from the

lipopolysaccharide (LPS) of the Gram-negative bacterium Salmonella minnesota. MPL has been deacylated and is lacking a phosphate group on the lipid A moiety. This chemical treatment dramatically reduces toxicity while preserving the immunostimulant properties. In the Figures and examples, all references to MPL are references to 3D-MPL)

QS21 : is a natural saponin molecule extracted from the bark of the South American tree Quillaja saponaria Molina. A purification technique developed to separate the individual saponins from the crude extracts of the bark, permitted the isolation of the particular saponin, QS21 , which is a triterpene glycoside demonstrating stronger adjuvant activity and lower toxicity as compared with the parent component. QS21 has been shown to activate MHC class I restricted CTLs to several subunit antigens, as well as to stimulate antigen specific lymphocytic proliferation.

CpG ODN 7909 is a synthetic single-stranded phosphorothioate oligodeoxy-nucleotide (ODN) of 24 bases length. Its base sequence is 5'-T CG T CG TTTTG-T CG TTTTGT CG TT-3' (SEQ ID NO: 9). Formulations were performed on the days of injections. The volume of injection for one mouse was 50 μΙ. A typical formulation contains as follows: 20 μg-25 μg antigen was diluted with H ₂ 0 and PBS pH 7.4 for isotonicity.

Conjugation of peptides:

Αβ1-6 peptide was purchased from 21 ^st Century Bio as a TFA salt. The mimotope peptides were purchased from Pichem or Anaspec. CRM-197 was produced by Eurogentec for bioconjugation purposes. The heterobifunctional crosslinking reagent GMBS was used to conjugate Αβ1-6 peptide to CRM197. Reaction of CRM197 with an excess of GMBS reagent at room temperature followed by removal of GMBS and associated by-products by ultrafiltration/diafiltration. Excess peptide was then added to the GMBS activated CRM197 and allowed to react for 2 hours. Final product was purified by ultrafiltration and characterized. In most of our experiment (in exception example 1-2-5), equivalent amount of 1 μg of Αβ1-6 peptide was injected per mouse when the Αβ1 -6 CRM vaccine was used. The amount of CRM conjugate used per mouse is in average of 5μg.

Example 1 (Figure 1 )

A dose-escalation study in which 3 dilutions of each adjuvant used herein (AS01 B or AS03) was performed in C57BL/6 mice. Injection of vaccines were performed at day 0-14-21 and sera collected at day 28. Seven groups of mice were used. All mice received 3μg peptide content of AB1 -6-CRM.

Adjuvants used were: none, AS01 B 1/25; AS01 B 1/5; AS01 B Full; AS03 1/25; AS03 1/5; and AS03 Full.

ΑηΙί-Αβ(1-42) antibody was measured using the method as described below. Results are shown in Figure

1 : Higher anti Αβ42 specific immunogenicity is observed with AS01 B compared to AS03 when used as an adjuvant in the presence of a stable dose of Αβ1-6 CRM conjugate (3 ug peptide content). Statistical comparison meaning is denoted (asterisk ( ^* ) indicates P less than 0.05) following ANOVA analysis of variance test combining with post-hoc Tukey-Kramer test.

Anti-amyloid beta (1 -42) antibodies were quantified in mice serum via ELISA, as the following:

Method: - Quantification of anti-amyloid beta 1-42 antibodies in mice serum using ELISA At terminal time point, whole blood is collected from mice and centrifuged on a vacutainer blood collection tube containing gel for serum separation. Serum samples are stored at -80°C. Streptavid in-coated plates (Greiner Bio-One, Germany) are first coated with beta-amyloid (1-42)-Lys(Biotin)-NH2 peptide (Anaspec, Inc.) at 0.5 g/mL, using 50 mM sodium carbonate buffer, overnight at 4°C. Plates are then washed 4 times using PBS / 0.05% Tween 20. Super Block (ScyTek laboratories) is added to the plates and incubated at 37°C for at least one hour. Serum samples and standard used herein were either: (3A5 monoclonal anti Αβ (AFFiRiS), anti Αβ42 antibody (6E10 antibody, Covance, Inc.) or anti Aβ1-6CRM sera from a quantified mouse polyclonal serum. Standards as well as the sera to analyze were plated in each plate and were serially diluted in the plates and incubated at 37°C for 2 hours. After a wash step, diluted peroxidase AffiniPure goat anti-mouse IgG, Fey fragment specific (Jackson ImmunoResearch

Laboratories Inc.) is added for 1 hour at 37°C. A last wash is performed before adding TMB substrate reagent (BD OptEIA™, BD Biosciences) for 30 min at RT. Immediately, plates are stopped using 2N sulfuric acid, and then read at 450 nm using SpectraMax microplate reader (Molecular Devices, Inc.).

Example 2 (Figure 2) A dose-escalation study in which few dilutions of each adjuvant used herein (CRX601 ; MPL; AS03;

AS03/CRX601 ; AS03/MPL; AS15) was performed in C57BL/6 mice. Few control groups were included such as ΑβΙ -6-CRM (3 [ig peptide dose) or a 4:1 mixture of Αβ1 -40/42 without adjuvant (4ug Αβ1-40; 1 ug+ Αβ1-40). Injection of vaccines using the intra muscular route were performed at day 0-14-28-42 and sera collected at day 56. The ΑβΙ-6-CRM containing group received 3μg based on peptide content. Adjuvants used herein at different doses were: CRX601 , MPL, AS03, AS03 + CRX601 , AS03 + MPL, AS15. ΑηΙί-Αβ(1-42) antibody response was measured using the method as described in example 1 .

Results shows that antibodies generated to amyloid beta 1-42 is highly promoted by different doses of TLR containing adjuvants such as MPL or CRX601 (TLR4 agonistic ligands), AS15 (a TLR4 and TLR9 agonistic ligand) in contrast to appropriate controls (non adjuvanted ΑβΙ-6-CRM or Αβ40/42 alone). Method:

The Αβ1 -40/ Αβ42 mix was prepared as previously described (Maier M, Seabrook TJ, Lemere CA.

Modulation of the humoral and cellular immune response in Abeta immunotherapy by the adjuvants monophosphoryl lipid A (MPL), cholera toxin B subunit (CTB) and E. coli enterotoxin LT(R192G). Vaccine. 2005. Oct 25;23(44):5149-59. PubMed PMID: 16054274. Twenty-two groups of mice were used. As shown on Figure 2 (from left to right) the treatments given were: PBS; Mix 4:1 AB1 -40/42; 3 [ig AB(1 -6)-CRM; 3 ig AB(1-6)-CRM with 0.2 [ig CRX601 ; 3 [ig AB(1- 6)-CRM with 2.0 [ig CRX601 ; 3 [ig AB(1-6)-CRM with 5.0 [ig CRX601 ; 3 [ig AB(1-6)-CRM with 25 [ig CRX601 ; 3 [ig AB(1-6)-CRM and 5.0ug 3DMPL; 3 [ig AB(1-6)-CRM and 23.7 [ig MPL; 3 [ig AB(1 -6)-CRM and50 MPL; 3 μg AB(1-6)-CRM and AS03 1/25; 3 μg AB(1-6)-CRM and AS03 1/5; 3 μg AB(1 -6)-CRM and AS03 full; 3 μg AB(1 -6)-CRM and AS03 and 0.2 ig CRX601 ; 3 μg AB(1 -6)-CRM and AS03 and 2.0 μg CRX601 ; 3 μg AB(1-6)-CRM and AS03 and 5.0 μg CRX601 ; 3 μg AB(1 -6)-CRM and AS03 and 25 μg CRX601 ; 3 μg AB(1-6)-CRM and AS03 and 5.0 μg 3DMPL; 3 μg AB(1 -6)-CRM and AS03 and 23.7 μg 3DMPL; 3 μg AB(1-6)-CRM and 1/25 AS15; 3 μg AB(1-6)-CRM and 1/5 AS15; 3 μg AB(1 -6)-CRM and Full dose of AS15.

For the AS01 B, AS03 and AS 15. The mouse dose (full dose) is equal of the 1 /10 human dose. The mouse dose of AS01 B contains 5ug of MPL 3D co-formulated in neutral liposome, 5ug of QS21. Those doses are per mouse and were injected using the intramuscular route (i.m.) 25 ul per mouse of AS01 B + 25 ul of PBS (phosphate buffer saline) or 25 ul of the appropriate peptide.

AS03: The mouse dose of AS03 contains 25ug of SB62 tocopherol-base water-oil emulsion. The preparation of the SB62 emulsion is made by mixing under strong agitation of an oil phase composed of hydrophobic components (a-tocopherol and squalene) and an aqueous phase containing the water soluble components (Tween 80 and PBS mod (modified), pH 6.8). While stirring, the oil phase (1/10 total volume) is transferred to the aqueous phase (9/10 total volume), and the mixture is stirred for 15 minutes at room temperature. The resulting mixture then subjected to shear, impact and cavitation forces in the interaction chamber of a microfluidizer (15000 PSI-8 cycles) to produce submicron droplets (distribution between 100 and 200 nm). The resulting pH is between 6.8±0.1. The SB62 emulsion is then sterilised by filtration through a 0.22 μιη membrane and the sterile bulk emulsion is stored refrigerated in Cupac containers at 2 to 8° C. Sterile inert gas (nitrogen or argon) is flushed into the dead volume of the SB62 emulsion final bulk container for at least 15 seconds. The final composition of the SB62 emulsion is as follows: Tween 80:1.8% (v/v) 19.4 mg/ml; Squalene: 5% (v/v) 42.8 mg/ml; a-tocopherol: 5% (v/v) 47.5 mg/ml; PBS-mod: NaCI 121 mM, KCI 2.38 mM, Na2HP04 7.14 mM, KH2P04 1.3 mM; pH 6.8±0.1. 25 ul per mouse of the AS03 was used per mouse. CRX601/AS03: 2 μg of CRX601 was diluted in PBS for a final volume of 25 μΙ. The 25 μΙ of CRX601 was added slowly to the 25 μΙ AS03 (2x concentrated). The vaccine was mixed by magnetic stirring at medium speed. Formulation was performed extemporaneously, the injections occurred within 60 min following the end of the formulation.

AS15: As described WO 00/62800. AS15 is a combination of the two adjuvant systems, AS01 B and AS07A. AS07A is composed of CpG 7909 (also known as CpG 2006) in phosphate buffer saline.

Formulations: Formulations were performed on the days of injections. The volume of injection for one mouse was 50 μΙ. A typical formulation contains as follows: 20 ig-25 μg antigen was diluted with H ₂ 0 and PBS pH 7.4 for isotonicity.

Results are shown in Figure 2, where the Y axis is concentration of anti-A (1 -42) antibodies in ng/ml. Higher anti Αβ42 specific immunogenicity was observed with CRX601 co-formulated with AS03 adjuvant compared non-adjuvant control or AS03 adjuvanted peptide.

Example 3 (Figure 3)

The combination of TLR4 containing agonist and Αβ42 specific antibodies synergistically up-regulates the Abeta phagocytosis. The experiment looked at ex vivo uptake of Αβ 1 -42 HiLyte Fluor 488 within 2 hours by CD1 1 b+ peripheral blood monocytes. The PBMCs were prepared from C57BI/6 mice following injection of AS01 B or PBS as control, then, pre incubated in vitro, with a mouse monoclonal anti Αβ 2E7or irrelevant mouse IgG as control. A flow cytometry analysis shows a higher uptake of the Αβ by CD1 1 b+ cells from mice treated with AS01 B than those who received PBS. In addition the presence of the ABeta-specific mAb increase the uptake of ABeta compared to IgG control. The combination of TLR4 agonist and Αβ specific antibodies up-regulate the Αβ phagocytosis more than each agent separately (synergy). Therefore, the uptake is synergistically enhanced when the mAb 2E7 was combined with monocytes from AS01 B- treated mice compared to PBS control groups.

Method: - Ex vivo uptake assay of AB1-42 and monocytes analysis:

Preparation of cells: Peripheral blood was drawn from vaccinated C57BL/6 mice via cardiac puncture with lithium-heparin as anticoagulant, 24-hours after injection of the adjuvants used herein. Red blood cell lysis was performed twice on pooled blood with Ammonium Chloride-based Buffer (Sigma, Steinheim, Germany) and cells were counted with the EasyCount™ System (Immunicon). Ex-vivo Αβ phagocytosis: cells were seeded at 10 ⁶ cells/mL onto a 24-well tissue culture plate and stimulated for 2h or 24 h in the presence or absence of 1 μg ml of Αβ1 -42 HiLyte Fluor™488 (Anaspec, Fremont, CA), which was pre-incubated or not for 1 h with 1 μg ml of anti-amyloid β antibodies (e.g. anti- Αβ1 -17 lgG1 , clone 6E10, Signet Laboratories, Dedham, MA) or with purified IgG from mouse serum (Sigma), as control. FACS analysis: cells were harvested after incubation with fluorescent Αβ peptide with Trypsin/EDTA and cold PBS and washed three times. 500,000 cells were incubated in 96-well plate for 10 min. on ice in the presence of Rat anti- Mouse CD16/CD32 (clone 2.4G2 - BD Fc Block™) and further stained for 30 min. with a combination of the following directly conjugated antibodies at their pre-determined optimal concentration: PE- Hamster anti-Mouse CD3 (clone 145-2C11 ), Rat anti-Mouse CD45R/B220 (clone RA3-6B2), Rat anti-Mouse Ly-6G (clone 1A8), Mouse anti-Mouse NK1 .1 (clone PK136), APC-conjugated Rat anti-Mouse CD11 b (clone M1/70), PE-Cy7-conjugated Hamster anti-Mouse CD11 c (clone HL3), (all from BD PharMingen). Cells were finally washed twice and fixed for 15 min. with a 2% paraformaldehyde solution in PBS. FMOs controls were always included in the assays.

Samples were acquired on a flow cytometer (BD FACSCanto II) and data analyzed with the FACSDiva software (BD Biosciences). Monocytes were identified by their Side/Forward scatter properties, excluding debris and gated as CD3- /CD45R/B220-/Ly-6G-/NK1 .1-(Lineage-)/CD11 b+ cells. Αβ uptake was assessed by reporting the percentage and Mean Fluorescence Intensity (GeoMean) of positive HiLyte fluor488 Αβ1-42 cells among gated monocytes.

Example 4 (Figure 4) Ex vivo uptake of Αβ 1-42 HiLyte Fluor 488 pre incubated or not with polyclonal anti Αβ 1 -6 antibodies (mouse sera) by blood live monocytes obtained from C57BI/6 mice following a single injection of AS01 B. The in vitro incubation with fluorescent Αβ1-42 +/- different concentration of anti Αβ1 -6 sera was performed as described in example 3. On Figure 4, white bars represent mice receiving PBS (adjuvant control); black bars represent AS01 B treatment. We show that monocytes coming from AS01 B-injected animals had an increased activity of Αβ phagocytosis. Moreover, the phagocytosis was promoted when Αβ1-6 specific sera were used. As shown on Figure 4, Preincubation with 1 .0 ug/mL anti-AB1-6 sera resulted in a 2.7 fold increase in uptake in monocytes from mice receiving AS01 B, compared to mice receiving PBS. This Αβ uptake phenomenon promoted by antibody and adjuvant could be observed starting at low the 0.1 ug/ml dose of anti Αβ-specific antibody. The dose of 1 ug/ml of anti Αβ sera on monocytes coming from AS01 B injected animals resulted to the highest level of Αβ uptake as described in the last column on the graph. Using the developed flow cytometry readout described herein, we are able to measure the efficacy of the Αβ-specific antibodies from adjuvanted immunization.

Example 5 (Figure 5)

In order to better evaluate the effect of adjuvanted vaccine on Αβ uptake or phagocytosis in a

physiological context rather than just Ex vivo or In vitro as previously described. We therefore developed an In vivo phagocytosis assay in which we could inject the fluorescent Αβ within the bloodstream (intra vascular=i.v.) of animals and track or measure the rapid uptake within just 2 hours post i.v. injection. The active immunization (vaccine) model used herein was ΑβΙ -eCRM (3 μg dose based on peptide content) + CRX601/AS03. The passive immunization group received the anti Αβ antibody (2E7). Negative control was the PBS injected group. The animals were injected at day 0-14-21 (Figure 5A). At day 22, the mice received i.v. the HiLyte Fluor 488 Αβ as described below. Two hours later, the peripheral blood was analyzed for their Α υρΐ8ΐ<β activity. In figure 5, we demonstrate that the Αβ uptake in peripheral blood is promoted by antibody from both active and passive immunization. In addition, we demonstrate that the active immunization by the combination of Αβ antigen + AS01 B can be more efficient than passive immunization using the simple injection of 2E7 in the same schedule of immunization. Figure 5B shows that the % of HiLyte Fluor 488 positive monocytes (indicating phagocytosis of Αβ 1-42) was higher in the active immunization group, (Αβ1-6 CRM + AS03/CRX601 ,a synthetic TLR4 co-formulated with AS03 emulsion) compared to the PBS control or the passive immunization ( intravenous injection of anti AB monoclonal antibody (2E7)).

Method:

- In vivo uptake assay of Αβ42 after Immunization or passive immunotherapy.

Mice: C57BL/6 female mice were purchased from Charles River. Three immunization groups were: 1-PBS injected via the intra muscular route; 2- AS03/CRX601 (the full mouse dose of AS03=1/10 human dose of AS03) co-formulated with 2μg of CRX601 per mouse plus Αβ1-6 conjugated to CRM (3ug peptide dose); 3: The passive transfer in the tail vein of 150ug of the 2E7 anti Αβ antibody.

Mice were immunized three times (at Day 0, 14 and 21 ) intramuscularly with (AS03-CRX601 + Αβ1 -

6CRM; 1 μg per mouse based on peptide content) or by the passive immunotherapy using anti Αβ42 monoclonal antibody (150ug of 2E7 antibody per mouse). At day 22, 5 μg per mouse of fluorescent

HiLyte Fluor™ 488-labeled Amyloid β 1-42 (Anaspec, Fremont, CA) was injected in the tail vein.

Preparation of cells: Peripheral blood was drawn from immunized C57BL/6 mice via cardiac puncture with lithium-heparin as anticoagulant, 2 hours after i.v. injection of the Αβ1-42 HiLyte Fluor™ 488 (Day 22).

Plasma of pooled blood was decanted and saved for measurement of antibody titres by ELISA. Red blood cell lysis was performed twice with Ammonium Chloride-based Buffer (Sigma, Steinheim, Germany) and cells were counted with the EasyCount™ System (Immunicon).

FACS analysis: 500,000 cells were aliquot in a 96-well plate, washed once, incubated for 10 min. on ice in the presence of Rat anti- Mouse CD16/CD32 (clone 2.4G2 - BD Fc Block™) and further stained for 30 min. with a combination of the following directly conjugated antibodies at their pre-determined optimal concentration: PE- Hamster anti-Mouse CD3 (clone 145-2C11 ), Rat anti-Mouse CD45R/B220 (clone RA3-6B2), Rat anti-Mouse Ly-6G (clone 1A8), Mouse anti-Mouse NK1 .1 (clone PK136), APC-conjugated Rat anti-Mouse CD11 b (clone M1/70), PE-Cy7-conjugated Hamster anti-Mouse CD11 c (clone HL3), (all from BD PharMingen). Cells were finally washed twice and fixed for 15 min. with a 2% paraformaldehyde solution in PBS. FMOs controls were always included in the assays. Samples were acquired on a flow cytometer (BD FACSCanto II) and data analyzed with the FACSDiva software (BD Biosciences).

Monocytes were identified by their Side/Forward scatter properties, excluding debris and gated as CD3- /CD45R/B220-/Ly-6G-/NK1 .1-(Lineage-)/CD11 b+ cells. Αβ uptake was assessed by reporting the percentage and Mean Fluorescence Intensity (GeoMean) of positive HiLyte fluor™ 488 Αβ1 -42 cells among gated monocytes.

Example 6 (Figure 6)

Up regulation of circulating monocytes numbers following the injection of compositions comprising TLR4 agonists such as 3D MPL, AS01 B, AS15, CRX527 or CRX601 via the i.m. or i.p. routes. Eight groups of C57BL/6 mice were treated with a single injection with the following: PBS i.m. (control); AS01 B Full Dose (i.m.); AS15 Full Dose i.m.; CRX527 (20ug/mouse i.p.); CRX601 (20 ug/mouse i.p.); CRX601 (1 ug/mouse i.m.); 3D MPL (50ug/mouse, i.p.); 3D MPL (5ug/mouse, i.m.). Total peripheral blood was harvested and the monocyte number was conducted as previously described (Example 3). As shown in Figure 6, all TLR4-containing adjuvant used herein triggers higher number of monocytes.

Especially the AS01 B group denote the highest increase of monocytes among the groups. Up to 5 fold of total monocytes could be increased by AS01 B vs the PBS group.

Example 7 (Figure 7)

A single intramuscular injection of different doses of AS01 B (1/20 vs. 1/5 vs. mouse full dose) was carried out. Four groups of C57BL/6 mice were treated with the following (single i.m. injection): PBS i.m. (control); AS01 B 1/20 dose; AS01 B 1/5 dose; and AS01 B full mouse dose. Monocyte analysis was conducted as described in Example 3. The results show that a dilution of 1/20 of AS01 B was enough to trigger an increase of the monocyte count within the peripheral blood compared to PBS. A constant increase is noted until the mouse full dose, i.e. the AS01 B mouse full dose is containing 5 μg of 3D MPL and 5 μg of QS21. Example 8 (Figure 8)

To examine the function of the increase of monocytes from AS01 B or CRX601 in the peripheral blood, we examined the capacity of those monocytes to uptake Αβ42 in a test tube as previously described

(Example 3 under the method section: Ex-vivo Αβ phagocytosis). Three groups of C57BL/6 mice were treated with the following: PBS i.m.; AS01 B i.m. (5ug/mouse); CRX601 i.m. (2ug/mouse). Results are shown in Figure 8. Flow cytometry analysis demonstrated that the intramuscular injection of AS01 B (mouse full dose, 5 ug per mouse) or CRX601 ^g dose) trigger the monocytes to be able to uptake a higher amount of Αβ42 compared to a non adjuvanted mouse monocytes (PBS group). Example 9 (Figure 9)

Phagocytosis of beta-amyloid 1 -42 peptide by the human microglial cell line CHME was observed after the addition of TLR2 agonist containing adjuvants Protollin and Pam3CysLip peptide. In this experiment, the highest increase of phagocytosis (86% of cells) was observed with the purified Pam3CysLip peptide from Neisseria meningitides strain 8047. The synthetic peptide sequence of the Pam3Cys Lip peptide is described herein: Pam3Cys - SQ EPAAPAAEAT PAAEAP (SEQ ID NO: 6; WO 2010/028246). Protollin was used at concentration 1 ug per ml based on the LPS content from Shigella flexneri. Pam3Cys lip peptide was used at concentration of 5ug per ml of the pure tri-palmitoylated peptide.

As shown in Figure 9, phagocytosis of Αβ1-42 by human microglia cell line (CHME) is increased by the pre-incubation (18 hrs.) of the cells with these TLR2 adjuvants in the presence of Abeta. . Additionally, cells were imaged using 42 HiLyte 488 Fluo (Anaspec, Inc.) at 2 ug per ml in DMEM complete media (Invitrogen). Lysotracker red staining was performed and slides were mounted and co-stained with DAPI to show the nucleus (blue). Fluorescence microscopy of human microglia cell line showed the localization of Abeta1 -42 within the lysosome after each treatment (results not shown). Lysotracker red reagent was purchased from Invitrogen and used as manufacturer recommendations.

Human microglia cell line CHME had a higher amyloid phagocytic activity following stimulation with TLR2 pure agonist Pam3CysLip peptide compared to PBS and to medium extend Protollin, which was previously shown to increase the phagocytic activity of Abeta1-42 (Hjorth et al Int J Alzheimers Dis. 2010 May 20;2010. pii: 798424). Example 10 (Figure 10)

A dissection of the major components within AS01 B shows that QS21 combined with liposome is important for these three major factors: monocytic activity (Figure 10A-B); Αβ uptake capacity (Figure 10C); and for Αβ-specific immunogenicity (Figure 10D).

To better compare the liposomal formulation of QS21 compared to the single component separately in animals, we performed innate immunity analysis by counting the monocytes in the peripheral blood from injected mice. A single injection of QS21 + liposome promotes after 24hrs an increase in the number of monocytes that could reach up to 13% of total peripheral white blood cells (Figure 10A) compared to QS21 alone group (9.1 %). Moreover, we observed an increased number of activated Ly6C+ monocytes (86%) after QS21 + liposome treatment compared to PBS (69%) or QS21 alone (75%) (Figure 10B). The method of monocyte analysis by flow cytometry is described in Example 3. To measure whether those monocytes were able to clear Αβ amyloid, we performed an Αβ42 uptake assay and observed that QS21 + liposome injection was promoting the Αβ42 uptake by peripheral blood monocytes (Figure 10C) as efficient as AS01 B could do. Flow cytometry analysis demonstrated that intramuscular injection of DQ (QS21 + liposome) triggers the monocytes to uptake an higher amount (2 fold) of Αβ42 compared to PBS or QS21 (5 μg) injected mice (Figure 10C). This suggests that QS21 alone does not promote Αβ uptake and the QS21 combined with liposome is necessary to promote this effect. Therefore, the role of using AS01 B in the following experiments is justified. Furthermore, the liposomal QS21 formulation was assessed for its ability to enhance the humoral immune response by the ELISA method as described in Example 1. Seven groups of mice received the following treatments: Αβ1-6 CRM + saline (PBS); Αβ1-6 CRM + QS21 ; Αβ1-6 CRM + QS21 + liposomes; Αβ1-6 CRM + MPL + liposomes; Αβ1-6 CRM + AS01 B; Αβ1-6 CRM + liposomes; Αβ1-6 CRM + MPL (Figure 10D). The liposomal QS21 formulation and its separate components were combined with an Αβ antigen (native Αβ1 -6 conjugated to CRM197) to analyse their effect on antibody response. Immunization at day 0-14 and 21 was performed and sera were collected at day 28, and the anti-A i-42 IgG specific titers measured. Statistical analysis using ANOVA and Tukey's multiple comparison tests show that the liposome + QS21 + Αβ1-6 CRM197 provided a significantly higher anti^1 -42 antibody titre than QS21 + Αβ1-6 CRM197. QS21 + Αβ1 -6 CRM197 group is not different to the liposome + Αβ1 -6 CRM197 group, suggesting that the combination of liposome and QS21 provides an improved antibody response toward Αβ42 compared to QS21 alone.

Statistical analysis was done using SAS and Unistat platform. Results of the multiple comparison analysis are shown in the following table:

Method:

ELISA method is described in Example 1. Monocyte were assessed used the methods described in Example 3. An equivalent amount of 1 μg of Αβ1 -6 peptide was injected per mouse when the Αβ1 -6 CRM197 peptide was used. The amount of CRM197 conjugate used per mouse was 4^g. An equivalent of 5 μg of QS21 molecule per mouse was used when QS21 was injected. For the DOPC-cholesterol liposome formulation, 10C^g of DOPC and 25 μg cholesterol were injected together per mouse.

Intramuscular injection of liposomal 3DMPL (5 ug of SUV MPL) or intramuscular injection 3DMPL itself at the 5 ug dose was used herein.

Example 11 (Figure 11 )

Figure 11 shows that a 2 month regimen vaccination in amyloid transgenic animals using AS01 B combined with mimotope (e.g., p1252) or native related amyloid peptide (Αβ1-6) in presence of Αβ- specific antibodies triggers a rapid (2hr) and higher amyloid uptake capacity Ex vivo. APP/PS1 bearing mutation mice (TASTPM) were injected at day 0, 14, 21 , 28, 42, 56 with these 5 formulations using the intra muscular route:

At day 57, blood was harvested as described in Example 3.

Blood live monocytes were obtained 24 hours following the last injection of the vaccines. The flow cytometry readout (as described in Example 3) showed a higher efficacy of the Αβ-specific uptake promoted by anti Αβ antibodies (either monoclonal or polyclonal sera) when used on monocytes from animal treated with AS01 B-containing adjuvant. The antibodies used herein were either polyclonal anti Αβ1-6 sera or Αβ1 -7 specific monoclonal antibody (2E7). That AS01 B + antibody synergistic effect was observed either in Αβ1-6 CRM + AS01 B vaccine group (over 80% of the monocytes contain Αβ) or in p1252-CRM + AS01 B group (over 70% of the monocytes contain Αβ). In particular, we show herein that monocytes from Αβ1 -6 CRM + AS01 B vaccinated amyloid mouse model (TASTPM mice) are increased compared to non-immunized animals in the presence of homologous polyclonal anti Aβ1-6CRM sera (diluted 1 in 10) in vitro from vaccination or either by the adding of a constant concentration (1 ug per ml) of anti Αβ monoclonal antibody (2E7). In contrast, that synergistic effect was not observed with the non- TLR containing adjuvant such as AS03 emulsion used herein. The level of antibody from a pool of plasma from each mouse group was performed and is presented as follow:

Interestingly, we were able to perform a rescue experiment in which the in vitro addition of only 1 ug per ml of anti Αβ antibody (2E7) on monocytes from AS01 B alone injected group was able to rescue the Αβ uptake activity, suggesting that a low antibody titer such as ^g ml of Αβ specific antibody in presence of AS01 B adjuvant could promote the amyloid clearance within the blood.

Example 12 (Figure 12)

To measure whether AS01 B CRM (CRM197 used herein) with or without N-terminus of amyloid sequence conjugated peptides affect the endogenous Αβ level in the peripheral blood of amyloid accumulating mouse model. We therefore started a long prophylactic treatment in a mouse model that possess a high and fast amyloid burden such as the TASTPM mouse as described by Howlett et al., 2004 (Howlett DR, Richardson JC, Austin A, Parsons AA, Bate ST, Davies DC, Gonzalez Ml. Cognitive correlates of Abeta deposition in male and female mice bearing amyloid precursor protein and presenilin-1 mutant transgenes. Brain Res. 2004;1017(1-2):130-6. PubMed PMID: 15261108).

A schedule of immunisation over 9 months was used and was considered a prophylactic model because we started the treatment before the Αβ accumulation and extended that treatment until the mice were 1 year old. Mice were immunized ten times starting at 3 months old. 25 mice per group were used at starting time for statistical reason based on power calculation from the previous experiment and from the fact that we could lose 10% of the mice because of high incidence of natural death in that transgenic model. Intramuscular injections starting at 3 months were called for logical reason: day 0, day 28, 56, 84, 112, 140, 168, 196, 224 and day 252. The amyloid accumulating mouse model (TASTPM) was used herein. Immunization groups were the following:

1- CRM + AS01 B 2- Api-6 CRM + AS01 B

3- A 3-8CRM + AS01 B

4- A (p)E-8CRM + AS01 B

5- PBS

The Αβ fragments conjugated to CRM were used at 1 ug per mouse based on peptide content. At day 253, peripheral blood was drawn from mice via cardiac puncture and monocytes were analyzed by flow cytometry to measure the amount left of amyloid β in the monocytes after that long prophylactic treatment. The methodology is derived from the method described in example 3 except that a fluorescent Alexa 488 labeled Αβ specifc antibody was used to detect the endogenous amyloid within monocytes instead of using exogenous HiLyte Flur Αβ peptides as used in example 5. The intra cellular staining of Αβ using 6E10 antibody (Covance Inc.) was performed by the usage of a saponin containing buffer, e.g., PermWash, as described by the manufacturer (BD Biosciences, Canada). The results showed that most of the AS01 B containing groups do exhibit less Αβ in the peripheral blood cells such as the monocytes (CD11 b+, Lin- and Αβ+) compared to the PBS treated group which showed higher amount of Αβ in the peripheral monocytes (Figure 12A). More important, the Aβ1 -6CRM + AS01 B group exhibit less amyloid within the monocyte among all groups. This result suggests that AS01 B with the help of antibodies promotes the Αβ clearance in the long term since the Αβ level is low in the AS01 B treated group compared to the placebo control (PBS). Furthermore, the impact of such treatment was evaluated on brain homogenates (Figure 12ABand 12C). Interestingly, the fine quantification of soluble Αβ1 -40 and Αβ1-42 peptides, which are the most abundant Αβ isoforms in Alzheimer's and are known to be toxic in their soluble form were quantified by ELISA as previously described by the manufacturer (Millipore, USA). The quantification showed that Αβ1-40 and Αβ1 -42 were down modulated by AS01 B-CRM and AS01 B + ΑβΙ-eCRM groups. In particular, AS01 B + ΑβΙ-eCRM exhibit the lowest amount of the toxic soluble Αβ1- 42 form (Figure 12C).

Example 13 (Figure 13)

To better evaluate an optimal dose of Αβ-derived peptide or antigen to use in mice, we performed a dose/range experiment in which the native amyloid peptides and their derived mimotopes peptides conjugated to the CRM197 were tested at 3 different doses. The doses used were based on peptide content after calculation excluding the amount of protein from the CRM197 carrier (notified by CRM abbreviation). Doses used herein were: 0.01 μg or O. ^g or ^g. All vaccines were adjuvanted with a constant dose of AS01 B formulation (i.e., 5μg MPL, 5μg QS21 in cholesterol liposome). Mice were injected at day 0, 14 and 21 and terminal sera were collected at day 28.

Results are shown in Figure 13. Results show that mimotopes can induce a potent, in a dose-dependent manner, Αβ-specific antibody response when formulated with AS01 B. The potency of this antibody response is in the comparable range level of the response obtained with the native N-terminal Abeta peptide (Abeta1-6) formulated with AS01 B (Figure 13A+B). The ELISA readouts to measure the specific anti Αβ1-42 antibody response show that 1 μg dose exhibit most of the time the highest antibody response and the statistical analysis result was the following:

Statistical analysis from immunogenicity data to evaluate pairwise comparison after one-way analysis of Variance (ANOVA) followed by the Tukey post-test.

In conclusion, therefore, the following experiments described herein in (Figure 13C-13D) using amyloid peptide and their mimotopes conjugated to CRM97 were using the 1 μg dose of peptide per mouse as the optimal dose for injection using the intramuscular route in mice. A particularly noteworthy observation is that mimotopes formulated with AS01 B induce a comparable antibody response directed against the formulated vaccine itself as measured by anti CRM197 carrier antibody level (Figure 13D). Figure 13D demonstrates anti-carrier antibody response in vivo in C57BL/6 showing that an immune response against the vaccine itself is elicited. Example 14 (Figure 14)

High Αβ42 specific Immunogenicity promoted by the TLR2 containing adjuvant Pam3CysLip peptide fused with an amyloid the A β fragment model (Αβ1 -6). The sequence of the TLR2 agonist used herein (Pam3CysLip Αβ1 -6) is: Pam3Cys - SQ EPAAPAAEAT PAAEAPDAEFRH (SEQ ID NO:7). The result shows that a TLR2 ligand fused to amyloid peptide could elicit an anti Αβ42 specific immunogenicity in a single peptide formulation in the amyloid deposition mouse model (TASTPM). The immunogenicity results following 12 injections of 10ug of the Pam3Cys - SQ EPAAPAAEAT PAAEAP DAEFRH (SEQ ID NO:7) using the intramuscular route is presented in Figure 14.

Example 15 (Figure 15) We show that a TLR2 agonist containing formulation could improve the working memory in amyloid deposition model (TASTPM). To determine whether a TLR2 agonist will improve the cognitive impairment in TASTPM mice, the Pam3CysLip adjuvant fused to the Αβ1-6 peptide was injected 12 times at weekly basis using the intramuscular route and the memory was assessed using the T-water maze test. The comparison with the PBS injected group shows that a trend of improvement in the right-left discrimination tests is observed after the 12 injections of Pam3CysLip Αβ1-6 peptide. TASTPM mice have received once a week for a period of 12 weeks the following treatment: PBS or Pam3CysLip Αβ1-6 peptide.

Example 16 (Figure 16)

The use of the TLR2 agonist, i.e. Pam3CysLip Αβ1-6 peptide, improves the survival rate of TASTPM mouse model. 23 out of 25 mice survived after weekly injections of Pam3CysLip A β1-6 peptide compared to PBS injected animals in which only 13 out 25 TASTPM mice survived (followed for 112 days). The Kaplan-Meier survival curve is shown in Figure 16.

Example 17 (Figure 17)

To better valuate the impact of TLR4-containing adjuvants, i.e. AS01 B and CRX601/AS03, on the in vivo amyloid Αβ1-42 uptake capacity. We designed an experiment in which C57BL/6 mice were immunized 4 times with different vaccines formulation and/or adjuvant formulation followed by a tail vain injection of fluorescent Αβ peptide at the last day of the experiment to measure the Αβ uptake in a 2hr time point.

Immunization schedule:

The In vivo Αβ uptake method is described as the following protocol:

C57BL/6 female mice were purchased from Charles River laboratories (St-Constant, Qc, Canada). Mice were immunized at day 0, 14, 21 and 28 using the intramuscular route as described herein in the Figure 17 with different immunomodulators (AS03 or AS01 B or CRX601/AS03). The AS03 and AS01 B mouse dose (=1 :10 human dose) have been described previously. 2 μg per mouse was used for CRX601 and was combined with the mouse dose of AS03 before injection using the intramuscular route. At day 29, 24hr later following the last injection, 5 μg per mouse of fluorescent HiLyte Fluor™ 488-labeled Amyloid β 1-42 (Anaspec, Fremont, CA) was injected in the tail vein. 2 hrs. Later, peripheral blood was drawn from immunized C57BL/6 mice via cardiac puncture with lithium-heparin as anticoagulant. Plasma of pooled blood was decanted and saved for measurement of antibody titers by ELISA. Red blood cell lysis was performed twice with Ammonium Chloride-based Buffer (Sigma, Steinheim, Germany) and cells were counted with the EasyCount™ System (Immunicon).

The flow cytometry (FACS) experiment is performed as the following: 500,000 cells were aliquoted in a 96-well plate, washed once, incubated for 10 min. on ice in the presence of Rat anti- Mouse CD16/CD32 (clone 2.4G2 - BD Fc Block™) and further stained for 30 min. with a combination of the following directly conjugated antibodies at their pre-determined optimal concentration: PE- Hamster anti-Mouse CD3 (clone 145-2C11 ), Rat anti-Mouse CD45R/B220 (clone RA3-6B2), Rat anti-Mouse Ly-6G (clone 1A8), Mouse anti-Mouse NK1.1 (clone PK136), APC-conjugated Rat anti-Mouse CD11 b (clone M1/70), PE-Cy7- conjugated Hamster anti-Mouse CD1 1c (clone HL3), (all from BD PharMingen). Cells were finally washed twice and fixed for 15 min. with a 2% paraformaldehyde solution in PBS. FMOs controls were always included in the assays. Samples were acquired on a flow cytometer (BD FACSCanto II) and data analyzed with the FACSDiva software (BD Biosciences). Monocytes were identified and counted by their Side/Forward scatter properties, excluding debris and gated as CD3-/CD45R/B220-/Ly-6G-/NK1 .1 - (Lineage-)/CD1 1 b+ cells. Αβ uptake was assessed by reporting the percentage and Mean Fluorescence Intensity (GeoMean) of positive HiLyte fluor™ 488 Αβ1-42 cells among gated monocytes or total live cells. Overall, these results (Figure 17) demonstrate that TLR4-containing adjuvants AS01 B and CRX601 in AS03 emulsion enhance the monocyte number (Figure 17A). Moreover, we observe that the final boost at day 29 with TLR4-containing adjuvant promotes the Αβ uptake (Figure 17B). In particular, this phenomenon of high amyloid uptake is higher in the groups that exhibit high antibody response as shown in Figure 17C. The results show that 4 consecutive immunizations with AS01 B + Αβ1-6 CRM induce the highest in vivo Αβ uptake. The results show that the stimulation of the innate immune system is the trigger for this enhanced antibody-mediated phagocytosis since the mice that were immunized with 3 consecutive injections of AS03 + Αβ1-6 CRM and, then, a final boost of AS01 B (TRL4-containing AS) alone (without antigen) before the phagocytic assay show a higher Αβ1 -42 uptake compared to animals injected 3x with Αβ1-6 CRM followed with the boost of AS03 alone (without TRL4). Similarly, a higher Αβ1-42 uptake is observed in the groups immunized 3 times with AS03 + Αβ1 -6, then, CRX601 /AS03 vs. AS03 + Αβ1 -6, then, AS03 alone, which confirms the importance of the TLR4 agonists in this formulation to activate the innate immune system leading to an increased phagocytosis. In contrast, the AS03 alone injected group (group 4) do not show monocyte number increase and did not promote Αβ uptake even after 4 injections. The contribution of AS01 B in the increased antibody-mediated phagocytosis was significant since all groups with antigen that did not contain AS01 B showed a lower clearance than those with AS01 B (Figure 17B), despite an antibody response of the same magnitude in the different groups (Figure 17C). Moreover, the AS01 B group that did not contain antigen only modestly stimulate phagocystosis vs. the group AS01 B + antigen, which confirms the importance of the presence of anti Αβ antibody induction in this synergistic phagocytosis process. In conclusion, this in vivo methodology developed herein shows that the Αβ uptake is promoted during or after the induction of the polyclonal Αβ-specific antibody response. More, that phenomenon is synergised with adjuvant containing formulations such as AS01 B or CRX601 , suggesting the presence in the peripheral blood of a mode of action that is anti Αβ antibody-mediated phagocytosis. Example 18 (Figure 18)

To better compare AS01 B (MPL + QS21 in liposome) to the benchmark adjuvant, which is the aluminium hydroxide in combination with the same amyloid conjugated peptide (Aβ1-6CRM) we performed an experiment in which the Αβ specific titer (Figure 18A) and Αβ uptake capacity (Figure 18B) was measured. In brief, C57BL/6 mice were immunized with the product listed herein using the intramuscular route (50μΙ per animal) at day 0-14-21 -28. At day 29, sera were collected in each individual mouse (n=6 mice per group).

The dose of peptide was 1 μg of Αβ1-6 peptide (DAEFRHC). The content of CRM197 in our formulations having 1 μg of Αβ1-6 peptide is 5 μg of CRM197 protein. More details are as the following:

The results described in Figure 18A show that immunization with AS01 B + ΑβΙ-eCRM triggers the highest Αβ1-42 specific immunogenicity. The comparison with the aluminium hydroxide group shows that AS01 B + ΑβΙ-eCRM vaccine triggers a 5 fold higher antibody titer compared to aluminium hydroxide + ΑβΙ-eCRM vaccine. The negative controls herein such as PBS and AS01 B group alone did not trigger Αβ1-42 specific antibody response as expected because no antigen were used in these formulations.

Aβ1-6CRM alone induces a very weak to undetectable Αβ1-42 specific antibody response, implying that vaccine adjuvants are also necessary to induce the appropriate immunogenicity specific for amyloid.

Furthermore, to evaluate whether the same animals possess functional antibodies to target the uptake of Αβ, we performed an in vivo phagocytosis using HiLyte Fluor Αβ injected passively in the blood stream for a period of 2hr as described previously. The results in Figure 18B show the requirement to have both components, AS01 B and the antigen, to induce a robust capture and clearance of peripheral Abeta by the peripheral CD11 b+ monocytes in vivo compared to Alum + Ag, AS01 B alone or Ag alone.

Example 19 (Figure 19)

To better compare the model of mimotope such as p1252 with AS01 B or AS03 with appropriate controls in amyloid uptake capacity test such as the in vivo phagocytosis test as previously described (Example 5 or Example 17), we then performed an immunotherapy experiment in 3 month-old TASTPM mice, which were injected four times intramuscularly (at Day 0, 14, 21 , 28) with various vaccine formulations. At Day 29, mice received 5 μg of fluorescent Αβ in the tail vein and two hours post-injection, whole blood was collected from 7 mice per group, pooled and the isolated peripheral blood cells were immunostained after red blood cells lysis, with a lineage cocktail of fluorochrome-conjugated antibodies for the selection of peripheral blood monocytes (CD3e- NK1 .1 - B220- Ly-6G- /CD11 b+) among other cell types by flow cytometry. The level of Αβ-uptake was assessed by the percentage and Mean Fluorescence Intensity of Αβ positive cells among monocytes. The result showed in Figure 19A that four intramuscular injections of p1252-CRM combined to either - AS01 _B or at a lesser extent to -AS03, can trigger an increase in Αβ uptake by blood monocytes of 3 month-old TASTPM transgenic mice, comparable to what is observed with Aβ1-6-CRM-AS01 _B (Fig. 19 A), showing that the uptake of Αβ induced by AS01 _B alone is synergised by the presence of even low levels of specific anti-Αβ antibodies (Fig. 19B). Once again, as described in Figure 1 1 in another setting, we have also been able to observe that a mimotope vaccination could also trigger a high amount of amyloid uptake within the peripheral blood despite their lower antibody titers. The mimotope-induced immune response seems relatively more efficient (comparison of titer vs number of CD11 + cells and % of Αβ positive cells) compared to native peptide vaccine. These results could suggest a surprisingly better suitability of mimotope vaccines compared to native peptide vaccines. This phenomenon is driven by the presence of the appropriate adjuvant as described herein.

Example 20 (Figure 20)

To better evaluate 4 different mimotopes in a same experiment in amyloid mouse model, we performed a 2-months treatment window schedule in amyloid transgenic model schedule in which the monocytes counting (20A), the amyloid uptake capacity (20B) and the immunogenicity was measured (20C). In brief, 3 month-old TASTPM mice were injected six times intramuscularly over a 56 days period (at Day 0, 14, 21 , 28, 42, 56) with various vaccine formulations. At Day 57, mice received 5 μg of fluorescent Αβ in the tail vein and two hours post-injection, whole blood was collected from 7 mice per group, pooled and the isolated peripheral blood cells were immunostained after red blood cells lysis, with a lineage cocktail of fluorochrome-conjugated antibodies for the selection of peripheral blood monocytes (CD3e- NK1 .1 - B220- Ly-6G- /CD1 1 b+) among other cell types by flow cytometry. The level of Αβ-uptake was assessed by the percentage and Mean Fluorescence Intensity of Αβ positive cells among monocytes.

AS01 B + Αβ1 -6 native peptide or related mimotope, such as p1252, p4381 , p4390 and p4715, were all showed tendency to increase the monocyte counting in peripheral blood (Figure 20A). Αβ1-6 native peptide or related mimotope, such as p4381 , p4390 and in lower extent p4715 and p1252, showed a clear up-regulation of exogenous amyloid uptake capacity (Figure 20B). These results show that Αβ based vaccines formulated with AS01 B drastically improve the phagocytosis of Amyoid beta by circulating CD1 1 b positive monocytes. Interestingly, despite the fact that the titer of the Αβ1-42 specific antibody were 50-fold lower (Figure 20C) in the p1252-CRM AS01 _B group compared to the Αβ1-60ΡΜ + AS01 B group, the amyloid uptake was similar in both groups. In addition, other mimotopes showed a similar trend to the p1252 group. Again, the results may suggest surprisingly better suitability of mimotopes in comparison to native peptide vaccines.

Example 21 (Figure21)

The same animals described in example 20 were investigated for their amyloid brain content using western blot technology to quantify their soluble amyloid oligomers. On day 57, animals were

anaesthetized, dissected and complete brain were collected and snap frozen in liquid nitrogen. Brain samples were then stored at -80°C.

Homogenization of mouse brain samples

Frozen brain samples were homogenized similarly to procedures described previously (Englund H., Sehlin D., Johansson A.-S., Nilsson L. N. G.; Gellerfors P., Paulie S., Lannfelt L. and Pettersson F. E.,

2007 Sensitive ELISA detection of amyloid-β protofibrils in biological samples, Journal of Neurochemistry, 103: 334-345; Frenkel D., Puckett L, Petrovic S., Xia W., Chen G., Vega J., Dembinsky-Vaknin A., Shen J., Plante M., Burt D. S., Weiner H. L, 2008 A Nasal Proteosome Adjuvant Activates Microglia and Prevents Amyloid Deposition, Ann. Neurol., 63: 591-601 ). Specifically, brain hemispheres were transferred in 300μΙ in PBS 1X (Invitrogen) supplemented with Complete protease inhibitor (Roche) and were homogenized using a Kontes Motor Cordless homogenizer (749540-0000) and plastic pestles (749521-1590). Brain homogenate was then centrifuged at 75 000 X g for 30 minutes at 4°C and supernatant, referred to as soluble brain extract, was collected and stored at -80°C.

Measurement of protein concentration and analysis by Western blot Protein concentration was measured using the Bradford method (Coomassie Plus - The better Bradford assay reagent, 23238, Thermoscientific) accordingly to the manufacturer instructions. For total Αβ detection, 50 μg of soluble brain extract were separated on a precast 10-20% SDS polyacrylamide Tris- Tricine gel (Bio-Rad, Canada). Separated proteins were then transferred onto polyvinylidene fluoride (PVDF) membranes (PerkinElmer, Canada). Membrane were incubated in DPBS (Gibco, USA) at 90°C for 10 min. Membrane were then incubated for 1 hour in blocking solution, which consists in TBS-T (50 mM Tris-HCI pH 8,0; 150 mM NaCI; 0.05% Tween 20) supplemented with 1 %BSA (Sigma, Canada) and 5% non-fat dry milk, and probed for 16 hours at 4°C with a mouse anti-amyloid beta protein monoclonal antibody clone 6E10 (Covance, USA) diluted to 1 :1000 in TBS-T supplemented with 5% BSA and 0.02% sodium azide. Membranes were washed once for 15 min and three times for 5 min in TBS-T at room temperature. Between each washing step, membranes were rinsed three times with TBS-T. Membranes were then incubated for 1 hour at room temperature in blocking solution supplemented with goat anti- mouse IgG HRP (Jackson, USA) diluted at 1 :10000. Membranes were washed and rinsed as described above before being washed for an additional 5 min in TBS (50 mM Tris-HCI pH 8,0; 150 mM NaCI) and rinsed four times with Milli-Q water. Localization of the HRP signal on the membrane was revealed using Super Signal West Dura Extended Duration Substrate (Thermo Scientific, USA) accordingly to the manufacturer instructions and detected on Amersham Hyperfilm (GE, USA). To detect β-actin, membranes were stripped in Reblot Plus Strong solution (Millipore, USA) accordingly to the manufacturer recommendation and incubated in blocking solution for 1 hour. Membrane were then incubated for 16 hours at 4°C in anti^-actin rabbit monoclonal antibody (13E5, Cell Signaling Technology, USA) diluted at 1 :2000 in TBS-T supplemented with 5% BSA and 0.02% sodium azide. Membranes were washed, incubated with goat anti-rabbit IgG HRP (H+L) (Jackson, USA) diluted at 1 :5000 in blocking buffer, washed and revealed as described above. Films were digitalized using a HP Scanjet 4370 (HP) and signal intensity was measured using ImageJ (version 1.44p, NIH Image).

As they are aging, TASTPM transgenic mice produce soluble Αβ peptides, which are able to form oligomers as well as fibril giving rise to plaque. This genetic model was found to accumulate amyloid deposition as early as three months. In addition, at later age, for example at 10 months, these mice form numerous plaques correlating with cognitive impairment (Cheng Z, Zhang J, Liu H, Li Y, Zhao Y, Yang E. 2010 Central nervous system penetration for small molecule therapeutic agents does not increase in multiple sclerosis- and Alzheimer's disease-related animal models despite reported blood-brain barrier disruption. Drug Metab Dispos. 38(8):1355-1361 ; Howlett DR, Richardson JC, Austin A, Parsons AA, Bate ST, Davies DC, Gonzalez Ml. 2004 Cognitive correlates of Abeta deposition in male and female mice bearing amyloid precursor protein and presenilin-1 mutant transgenes. Brain Res., 1017(1-2): 130- 6). As observed for PBS-treated animals, these mice produce oligomers such as trimers in the soluble fraction of their brain. Interestingly, animals treated with p4381-CRM + AS01 b had a slight, but not significant reduction of soluble trimers in their brain. Importantly, brain from animals treated with p4390- CRM + AS01 b or with p4715-CRM + AS01 b had significantly less trimeric Αβ than the one of control (PBS) animals. These data illustrate that administration of p4390-CRM or p4715-CRM with AS01 b contributes to diminish the accumulation of soluble oligomeric forms of Αβ in the brain of transgenic mice.

Example 26 (Figures 26-30)

Material and Methods: Peptide ELISA:

In order to perform ELISAs for detecting the immune responses in vaccinated animals, peripheral blood was drawn from mice using heparin as anticoagulant and plasma was prepared from these samples. The diluted plasma was then used for ELISA analysis. For this purpose, the wells of the ELISA plates (Nunc Maxisorb) were coated with peptide-BSA conjugates. Subsequently, diluted plasma was added and the detection of peptide specific antibodies was performed with biotinylated anti-mouse IgG (Southern Biotech) and subsequent colour reaction using Streptavidin-POD (Roche) and ABTS. For IgG subtype analyses, specific biotinylated anti murine lgG1 , lgG2a, lgG2b and lgG3 antibodies (Southern Biotech) were used .

Cytokine determination:

To determine the concentration of MCP-1 (CCL-2) in the circulation of vaccinated animals, blood was collected from animals 3 hours after i.m. injection of AS03 containing vaccines. Subsequently, plasma was prepared from blood samples and MCP-1 concentration in individual samples was defined using the FlowCytomix bead array system (eBioscience) and flow cytometric analysis.

Results:

In vivo characterisation of MIMOTOPE-vaccine candidates: f. Effect of MIMOTOPE-CRM197 conjugates using different adjuvant systems

1A: Oil in water emulsions elicit stronger immune responses than the classical adjuvant Aluminium Hydroxide (Alum)

Female C57/bl6 mice, 11 mice per group, were intramuscularly immunized with 10 μg peptide coupled to CRM-197. The mimotope peptides used were p1 122 (DKELRI) and p1252 (SWEFT). As adjuvant Alum or a squalene-based, oil in water emulsion containing squalene, DL-a-tocopherol and polysorbate 80 was used. Animals were vaccinated 3 times in biweekly intervals and plasma samples were taken biweekly as well. The in vitro ELISA assay to determine the antibody titer following immunisation was performed with plasma of single mice (see Fig. 26, respectively).

Figure 26A shows representative examples for assays used to characterise MIMOTOPEMIMOTOPEs in vivo. Immune responses elicited by MIMOTOPEMIMOTOPE-CRM197 adjuvanted with the oil in water emulsion AS03 show higher anti injected peptide titers (anti-p1122 and anti-p1252 levels depicted in Fig.26A) as well as higher titers against amyloid beta peptide (Αβ1-10, see Fig. 26B) as the ALUM adjuvanted conjugates applied in parallel.

In an alternative experiment, hAPP overexpressing mice (as suitable AD model; hPrP-hAPP695

(KM670/671 NL); Hsiao, K et al. Science 274:99-102, Oct 4, 1996.)), 6 (control group)-1 1 mice (vaccine group) per group, were subcutaneously immunized with 30 μg peptide coupled to CRM-197 to assess whether similar titers can be obtained in a system displaying human amyloid beta as target of the MIMOTOPEMIMOTOPE-conjugate vaccine. As adjuvant a squalene-based, oil in water emulsion containing squalene, DL-a-tocopherol and polysorbate 80 was used. Animals were vaccinated 3 times in biweekly intervals and then boosted at 4 months after the last injection and plasma samples were taken biweekly as well. The in vitro ELISA assay to determine the antibody titer following immunisation was performed with plasma of single mice (see Fig. 27, respectively).

Figure 27 shows representative examples for assays used to characterise MIMOTOPEs in vivo. Immune responses elicited by MIMOTOPEMIMOTOPE-CRM197 adjuvanted with the oil in water emulsion AS03 show comparable anti injected peptide titers (anti-p1252 levels depicted in Fig.27A)as well as titers against amyloid beta peptide Αβ1-10 (anti-p1252 levels depicted in Fig.27A, Αβ1 -10 data not shown) as wildtype animals. Titers against amyloid beta peptide (Αβ1 -42, see Fig. 27B) were high as well and anti irrelevant peptide titers (see Fig. 27C), determining the reactivity against non-mimotope/non-amyloid beta peptides and peptide conjugates, was very low as well indicating the induction of a highly specific immune response. Anti amyloid titers were significantly higher than values obtained by CRM197-AS03 alone.

The plasma samples obtained in this experiment were also assessed for the presence of peptide specific IgG subtypes induced by the conjugate vaccine. This analysis (see Fig.27D, anti-injected peptide response) revealed a predominant lgG1 response present in treated animals. This indicates a IL-4 driven Th2-mediated humoral immune response. 1B: Oil in water emulsions elicit a significant change in peripheral monocytes/macrophages upon immunisation

In order to analyse whether MIMOTOPEMIMOTOPE-CRM197 adjuvanted with an oil in water emulsion (e.g.: AS03) was able to change the cytokine milieu and thus influence peripheral monocyte/macrophage activation, we determined the levels of the Chemokine (C-C motif) ligand 2 (CCL2) protein also known as monocyte chemotactic protein-1 (MCP-1 ). MCP1 is known to activate monocytes/macrophages and is further produced by those activated monocytes/macrophages. MCP1 levels were determined in plasma from treated animals 3 hours after i.m. injection of AS03 containing vaccines following immunization two, three and four, respectively.

The mean MCP-1 concentration was determined after immunizations two, three, and four in plasma of MIMOTOPEMIMOTOPE-CRM197-AS03 treated animals (Figure 27E and data not shown). The relative differences of the measurements obtained were similar. The mean MCP-1 concentration determined after injection three in the MIMOTOPEMIMOTOPE-CRM-AS03 treated group was about 7600 pg/ml whereas in CRM197-AS03 treated animals the MCP-1 concentration was found to be significantly lower (roughly 2 times). Both values were significantly higher than the levels obtained in PBS treated control animals. These results indicate that vaccines containing AS03 lead to a profound change in activated peripheral macrophages/monocytes and that this effect is even increased in the presence of

MIMOTOPEMIMOTOPE induced antibodies directed against amyloid beta peptides. 1C: Adjuvants containing TLR4 agonists elicit stronger immune responses than the classical adjuvant Aluminium Hydroxide (Alum)

Female BALB/c mice, 1 1 mice per group, were intramuscularly immunized with 10 μg peptide coupled to CRM-197. As adjuvant Alum or a mix of Alum and the TLR4 agonist MPL (adjuvant used: AS04) was used. Animals were vaccinated 3 times in biweekly intervals and plasma samples were taken biweekly as well. The in vitro ELISA assay to determine the antibody titer following immunisation was performed with plasma of single mice (see Fig. 47 respectively).

Figure 28 show representative examples for assays used to characterise MIMOTOPEMIMOTOPEs in vivo. Immune responses elicited by MIMOTOPE-CRM197 adjuvanted with the TLR4 agonist containing adjuvant show higher anti injected peptide titers (anti-ρβΐ 122 and anti-p1252 levels depicted in Fig.28A) as well as higher titers against amyloid beta peptide (Αβ1-10, see Fig. 28B) as the ALUM adjuvanted conjugates applied in parallel.

In two additional experiments using additional MIMOTOPE-CRM197 conjugates as well as combinations of such conjugates formulated with alternative adjuvants containing TLR4 agonists vaccine induced immunogenicity was assessed. In the first experiment, female C57/bl6 mice, 5 mice per group, were intramuscularly immunized with 1 μg peptide coupled to CRM-197. The mimotope peptides used were p4381 (SEFKHG), p1252 (SWEFRT), p4390 (ILFRHG) and p4715 (TLHEFRH). As adjuvant a mix of the saponin QS21 , of liposomes and of the TLR4 agonist 3D-MPL was used (referred to as AS01 B). Animals were vaccinated 3 times in biweekly intervals and plasma samples were taken biweekly as well. The in vitro ELISA assay to determine the antibody titer following immunisation was performed with plasma of single mice (see Fig. 29 respectively).

Figure 29 shows representative examples for assays used to characterise MIMOTOPEs in vivo. Immune responses elicited by MIMOTOPE-CRM197 adjuvanted with the TLR4 agonist containing adjuvant show comparably high anti injected peptide titers (anti-p4381 , p4390, p4715 and anti-p1252 levels depicted in Fig.29 A)as well as comparable titers against amyloid beta peptide (Αβ1-10, see Fig. 29B). Titers induced are significantly higher than control treated animals receiving PBS applied in parallel.

In the parallel experiment, hAPP over-expressing mice (Tg2576 animals used as suitable AD model), 10 mice per vaccine group, n=5 for control treated animals, were intramuscularly immunized with 1 μg peptide coupled to CRM-197. As adjuvant a mix of the saponin QS21 , of liposomes and of the TLR4 agonist MPL was used (referred to as AS01 B). Animals were vaccinated 3 times in biweekly intervals and plasma samples were taken biweekly as well. The in vitro ELISA assay to determine the antibody titer following immunisation was performed with plasma of single mice (see Fig. 29 respectively). Figure 30 shows representative examples for assays used to characterise MIMOTOPEs in vivo. The mimotope peptides used were p4381 (SEFKHG), p4390 (ILFRHG) and p4715 (TLHEFRH). Immune responses elicited by MIMOTOPE-CRM197 adjuvanted with the TLR4 agonist containing adjuvant show comparably high anti injected peptide titers (anti-p4381 , p4390 and p4715 levels depicted in Fig.30A) as well as comparable titers against amyloid beta peptide (Αβ1 -10, see Fig. 30B)as in the wildtype experiment. In addition similar anti ΑβΙ-42 responses could be induced in both experiments as well (see Fig. 29C and 30C). Titers induced are significantly higher than titers observed in control treated animals receiving either CRM197 adjuvanted with a TLR4 agonist containing formulation or animals receiving PBS alone.