Anti-abeta therapeutic vaccines

Abstract

A liposomal vaccine composition comprises a ?-amyloid (A?)-derived peptide antigen displayed on the surface of the liposome. The vaccine composition also comprises a peptide comprising a universal T-cell epitope encapsulated within the liposome. The vaccine composition also comprises an adjuvant, which may form part of the liposome and may be displayed at least in part on the surface of the liposome. These vaccine compositions are used for treating, preventing, inducing a protective immune response against or alleviating the symptoms associated with an amyloid-beta associated disease or condition or a condition characterised by, or associated with, loss of cognitive memory capacity in a subject. The vaccine compositions may be provided as a kit. Related methods of producing a liposomal vaccine composition are also provided.

Claims

1. A liposomal vaccine composition comprising: a. A ?-amyloid (A?)-derived peptide antigen displayed on the surface of a liposome; b. A peptide comprising a universal T-cell epitope encapsulated within the liposome that stimulates a helper T-cell response that enhances antibody production by B-cells, wherein the peptide comprises: i. the amino acid sequence of SEQ ID NO: 6 and the tetanus toxin domain of SEQ ID NO: 1; ii. the amino acid sequence of SEQ ID NO: 8, the tetanus toxin domain of SEQ ID NO: 2, and an immunogenic keyhole limpet hemocyanin (KLH) domain; iii. the amino acid sequence of SEQ ID NO: 8 and the tetanus toxin domain of SEQ ID NO: 3; or iv. an immunogenic influenza hemagglutinin domain, the diphtheria toxin domain of SEQ ID NO: 4, the tetanus toxin domain of SEQ ID NO: 4, and the Epstein Barr virus domain of SEQ ID NO: 4; and c. An adjuvant.

2. The liposomal vaccine composition of claim 1 wherein the peptide comprising a universal T-cell epitope comprises at least 30% hydrophobic amino acids.

3. The liposomal vaccine composition of claim 1 wherein the liposomal vaccine composition comprises at least two different universal T-cell epitopes encapsulated within the liposome.

4. The liposomal vaccine composition of claim 3 wherein each universal T-cell epitope is no more than 30 amino acids in length.

5. The liposomal vaccine composition of claim 1 wherein the liposomal vaccine composition comprises two, three or four different universal T-cell epitopes encapsulated within the liposome.

6. The liposomal vaccine composition of claim 1 wherein the peptide comprising a universal T-cell epitope comprises at least two different universal T-cell epitopes.

7. The liposomal vaccine composition of claim 1 wherein the peptide comprising a universal T-cell epitope comprises two, three or four universal T-cell epitopes.

8. The liposomal vaccine composition of claim 7 wherein at least two of the two, three or four universal T-cell epitopes are joined by a linker.

9. The liposomal vaccine composition of claim 8 wherein the linker comprises at least two amino acids.

10. The liposomal vaccine composition of claim 1 wherein the peptide comprising a universal T-cell epitope comprises an amino acid sequence selected from SEQ ID NO: 1 (SAT42), SEQ ID NO: 2 (SAT43), SEQ ID NO: 3 (SAT44), and SEQ ID NO: 4 (SAT47).

11. The liposomal vaccine composition of claim 1 wherein the adjuvant forms part of the liposome.

12. The liposomal vaccine composition of claim 1 wherein the adjuvant comprises monophosphoryl lipid A (MPLA), CpG, or both MPLA and CpG.

13. The liposomal vaccine composition of claim 1 wherein the peptide comprising a universal T-cell epitope is between 30 and 60 amino acids in length.

14. The liposomal vaccine composition of claim 6 wherein the peptide comprising a universal T-cell epitope is between 30 and 60 amino acids in length.

15. A kit for treating, preventing, inducing a protective immune response against or alleviating the symptoms associated with an amyloid-beta associated disease or condition in a subject comprising the liposomal vaccine composition of claim 1 together with instructions for administering the vaccine composition.

16. A liposomal vaccine composition comprising: a. A tetrapalmitolyated ?-amyloid (A?)-derived peptide antigen displayed on the surface of the liposome that comprises, consists essentially of or consists of amino acids 1-15 of A?; b. A peptide comprising a universal T-cell epitope encapsulated within the liposome wherein the peptide comprising a universal T-cell epitope comprises an amino acid sequence of SEQ ID NO: 3 (SAT44); and c. An adjuvant.

17. The liposomal vaccine composition of claim 16 wherein the adjuvant forms part of the liposome.

18. The liposomal vaccine composition of claim 16 wherein the adjuvant comprises monophosphoryl lipid A (MPLA), CpG, or both MPLA and CpG.

19. A liposomal vaccine composition comprising: a. A tetrapalmitolyated ?-amyloid (A?)-derived peptide antigen displayed on the surface of the liposome that comprises, consists essentially of or consists of amino acids 1-15 of A?; b. A peptide comprising a universal T-cell epitope encapsulated within the liposome wherein the peptide comprising a universal T-cell epitope comprises, consists essentially of or consists of an amino acid sequence of SEQ ID NO: 4 (SAT47); and c. An adjuvant.

20. The liposomal vaccine composition of claim 19 wherein the adjuvant forms part of the liposome.

21. The liposomal vaccine composition of claim 19 wherein the adjuvant comprises monophosphoryl lipid A (MPLA), CpG, or both MPLA and CpG.

22. The liposomal vaccine composition of claim 9, wherein the linker comprises, consists essentially of, or consists of the amino acids VVR or PMGAP (SEQ ID NO:11).

23. The liposomal vaccine composition of claim 3 wherein each universal T-cell epitope is no more than 20 amino acids in length.

Description

DESCRIPTION OF THE FIGURES

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

(2) FIG. 2A. Analysis of inhibition of A?1-42 self-association by ELISA of IgG antibodies in the plasma of C57BL/6 mice 21 or 7 days before (dotted lines) and 21 days after 1.sup.st immunization (bold lines) with indicated vaccines. Results are expressed as mean+/?standard deviation of 5 mice per group of the percentage of inhibition of A?1-42 self-association. The X axis indicates the serial dilutions of the plasma, while the Y axis indicates the percentage of inhibition of A?1-42 self-association. FIG. 2B. Inhibition of A?1-42 self-association shown as percentage (%) of inhibition at day 21 minus % of inhibition at day ?21 or ?7 (background-prebleeding) at 1/25 dilution of the plasma. The X axis indicates the groups treated with indicated vaccines, while the Y axis indicates the percentage of inhibition of A?1-42 self-association after subtraction of the background.

(3) FIG. 3. Analysis of A?1-42 oligomer-specific IgG antibodies by ELISA in plasma of C57BL/6 mice 21 (ACI-24.046) or 7 (ACI-24, ACI-24.043, ACI-24.044) days before and 21 days after 1st immunization with indicated vaccines. Results are expressed as geometric mean+/?95% CI of ng/mL with 5 mice per group. Statistical test among groups at day 21: Kruskal-Wallis test with Dunn's multiple comparisons. * p<0.05; ** p<0.01. The X axis indicates the groups immunized with indicated vaccines while the Y axis indicates the antibody titers expressed by ng/mL.

(4) FIG. 4. Analysis of A?1-42 avidity of IgG antibodies by ELISA in plasma of C57BL/6 mice 7 and 21 days after 1st immunization with indicated vaccines. Results are expressed as geometric mean+/?95% CI of avidity index with 5 mice per group. Statistical test: Mann-Whitney test between day 7 and day 21 for each group. * p<0.05; ** p<0.01. The X axis indicates the groups immunized with indicated vaccines while the Y axis indicates the avidity index.

(5) FIG. 5. Analysis of A? oligomer-specific IgG antibodies by MSD in serum of Cynomolgus monkeys before the first immunization (Day 1) and 1 week after the third immunization (Day 64) in ACI-24.046 (SAT44, n=8), ACI-24.045 (SAT43, n=4) or ACI-24.043 (SAT47, n=4) immunized monkeys. Results are expressed as geometric mean+/?95% CI of AU/mL. The X axis indicates the individual plasma from groups immunized with indicated vaccines while the Y axis indicates the antibody titers expressed by AU/mL.

(6) FIGS. 6A-6B. Analysis of A?1-42-specific IgG antibodies by ELISA in the plasma of C57BL/6 mice 7 days after the 3.sup.rd immunization (Day 36) with ACI-24 and ACI-24.046 (SAT44) vaccines (FIG. 6A) or with ACI-24 and ACI-24.043 (SAT47) vaccines (FIG. 6B). Results are expressed as geometric mean+/?95% CI of ng/mL with n=10 mice per group. The X axis indicates the vaccines used for immunization of each particular group, while the Y axis indicates antibody titers expressed in ng/mL. Statistical test: Mann-Whitney test between ACI-24 and the indicated vaccine. * p<0.05; ** p<0.01, ***p<0.001

(7) TABLE-US-00001 Table of abbreviations ABTS 2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) A? Amyloid beta (abeta) Ac2O Acetic anhydride AD Alzheimer's Disease AP Alkaline phosphatase APC Antigen Presenting Cells BSA Bovine Calf Serum AU/mL Arbitrary Units per mL CI Confidence Interval DMF Dimethylformamide DMPC 1,2-Dimyristoyl-sn-glycero-3-phosphocholine DMPG 1,2-Dimyristoyl-sn-glycero-3-phosphorylglycerol DMSO Dimethyl sulfoxide ELISA Enzyme-linked immunosorbent assay HLA Human leukocyte antigen HPLC High-performance liquid chromatography HRP Horseradish peroxidase Ig Immunoglobulin KLH Keyhole limpet hemocyanin MPLA Monophosphoryl Lipid A MS Mass spectrometry MSD Meso Scale Discovery Pal1-15 Tetrapalmitoylated A?1-15 PBS Phosphate buffered saline PES Polyethersulfone pNPP p-nitrophenyl phosphate s.c. Subcutaneous TMB Tetramethylbenzidine TFA Trifluoroacetic acid TIS Triisopropylsilane TLR4 Toll-like receptor 4 Beta-OG n-Octyl-?-D-Glucopyranoside

(8) The invention will be further understood with reference to the following non-limiting examples:

Example 1. Design of New T-Cell Epitopes

(9) The ability of a T-cell epitope to activate T cells (immunogenicity score) is the result of two complementary properties: i) affinity to HLA and ii) capacity to bind different HLA haplotypes in a promiscuous manner. An in silico evaluation (Epivax) of several T-cell epitopes from different origins was performed with the objective of selecting the peptides with the highest immunogenicity score. In a preliminary phase, 10 different peptides from different origins (Keyhole limpet hemocyanin-KLH, Diphtheria toxin, Influenza virus, Epstein Barr virus and Herpes virus) were evaluated. Peptides with the best immunogenicity score (higher than 10) were selected due to their chance to be highly immunogenic in humans based on their predicted HLA affinity and HLA haplotype coverage (selected peptide sequences are shown in Table 1).

(10) TABLE-US-00002 TABLE1 Peptide Name Sequence origin SAT6 STLEYFLYDPIFFLHHSNTDRLWAIWQAL KLH QKYRGKPYNTANCAIVRHDTY (SEQIDNO:5) SAT13 VHHNTEEIVAQSIALSSLMV Diphtheria (SEQIDNO:6) Toxin SAT15 IDGVKLESMGVYQILAIYSTVASSL Influenza (SEQIDNO:7) hemagglutinin SAT17 VYGGSKTSLYNLRRGTALAI EpsteinBarr (SEQIDNO:8) Virus

(11) Following the screening results of the individual peptides, the combined promiscuous peptides composed of 2 or 3 immunogenic T-cell epitopes from different origins (named SAT42, SAT43 and SAT44) and the promiscuous peptides composed of trimmed peptides (e.g. SAT47 and SAT43) were designed (Table 2). The trimmed peptides were designed by selecting, in the sequence of each individual T-cell epitope, the most immunogenic 15-mer peptide sequence, based on the in silico predicted T-cell epitope hotspots. The goal was to increase the immunogenicity score without increasing the size of the final promiscuous peptide, due to peptide synthesis and vaccine encapsulation process constraints. In brief, the peptide synthesis yield and success rate is lowered upon increasing the length of the peptides, especially above 30 amino acids in length, and in addition they are composed mainly of hydrophobic residues, as for T-cell epitope peptides disclosed herein. In addition, the peptide encapsulation rate is lowered with the increasing length of the peptide, as the chances to accommodate it in the lumen of the liposomes are decreased as peptide length increases. The in silico immunogenicity score of these 4 promiscuous T-cell epitopes was very high and, importantly higher than that of the individual component peptides, therefore confirming that combining peptides from different origins can improve HLA affinity and HLA haplotype coverage (promiscuous T cell epitope sequences are shown in Table 2).

(12) TABLE-US-00003 TABLE2 Name Sequence Peptidedesign Peptideorigin SAT42 VHHNTEEIVAQSIALSSLMVPMGA SAT13+PMGAP+ Diphtheria PQYIKANSKFIGITEL Tetanustoxin Toxin+Tetanus (SEQIDNO:1) toxin SAT43 VYGGSKTSLYNLRRGTALAIVVRQ SAT17+VVR+Tet EpsteinBarr+ YIKANSKFIGITELVVRPIFFLHHSN anustoxin+VVR+ Tetanus+KLH TDRLWAI SAT6 (SEQIDNO:2) SAT44 VYGGSKTSLYNLRRGTALAIVVRQ SAT17+VVR+Tet EpsteinBarr+ YIKANSKFIGITEL anustoxin Tetanus (SEQIDNO:3) SAT47 SMGVYQILAIYSTVVRIVAQSIALSS SAT15+VVR+SAT Influenza VVRYIKANSKFIGVVRLYNLRRGTA 13+VVR+Tetanus+ hemagglutinin+ L VVR+SAT17 Difteria+Tetanus+ (SEQIDNO:4) EpsteinBarr

Example 2. Vaccine Synthesis and Formulation

(13) General Method of Universal T-Cell Epitope Peptide Synthesis and Purification

(14) T-cell peptides were manufactured by linear solid phase peptide synthesis (SPPS) on 2-Chlorotrityl resin using standard Fmoc chemistry. Standard coupling procedure was performed using 3.0 equivalent of amino acid and coupling reagent in the presence of 3.0 equivalent of base in DMF for 1 hour at room temperature. For difficult coupling sequences double coupling was implemented with extended reaction time. After the completion of the amino acid coupling, an acetylation capping step was introduced using 5.0 equivalent of Ac2O in pyridine to abort the undesired peptide chain elongation. The resin was washed with DMF and Fmoc group was removed by using 20% piperidine in DMF for 5 min. After finishing the SPPS, global deprotection and peptide cleavage from the resin was done using standard cleavage cocktail (TFA/TIS/water) for 2 hours at room temperature. The resin was filtered off and washed with TFA. The crude product was subsequently precipitated with 10-fold excess volume of cold isopropyl ether/hexane and the solid was filtered off by using a glass frit and dried under vacuum. The crude peptide was purified on reversed phase C18 column using a gradient of solvent A (water, 0.1% TFA) and solvent B (acetonitrile, 0.1% TFA) on a preparative HPLC system. The HPLC fractions containing desired peptide with purity above 90% were pooled together diluted in water and performed an ion exchange. The desired ion exchange fractions were lyophilized. The identity and purity of final peptide was characterized and confirmed by HPLC-MS analysis.

(15) Preparation of the ACI-24.043/ACI-24.044/ACI-24.045/ACI-24.046/Vaccines (Thin Lipid Film)

(16) The vaccines containing encapsulated T-cell epitopes peptide were produced by thin-lipid film technology followed by homogenization and extrusion. First, by solubilizing DMPC, DMPG (Lipoid, Germany), cholesterol and monophosphoryl hexa-acyl Lipid A, 3-deacyl synthetic or 3D-(6-acyl) PHAD? (Avanti Polar Lipids, USA) at molar ratios 9:1:7:0.05 in ethanol at 60? C., respectively. Ethanol was evaporated under vacuum rotavapor in order to obtain thin lipid film.

(17) Lipid film was rehydrated with one of these buffers (depending on T-cell epitope peptide to be encapsulated): 20 mM sodium acetate pH 4 (Fluka), 5% DMSO (Sigma Aldrich) in MilliQ water containing 0.8 mg/mL T-cell epitope peptide SAT42, or 0.1?PBS pH 7.4, 5% DMSO (all Sigma-Aldrich) in MilliQ water containing 0.3-0.4 mg/mL T-cell epitope peptide SAT43, SAT44 or SAT47.

(18) Solution was gently stirred for 15 minutes. Sample was further vigorously vortexed in the presence of glass beads. Resulting multilamellar vesicles were subjected to 10 freeze-thaw cycles (liquid N2 and waterbath at 37? C.) and submitted to homogenization followed by sequential extrusion through polycarbonate membranes (Whatman, UK) with a pore size of 0.1/0.08 ?m. Both homogenization and extrusion steps were performed using EmulsiFlex-C5 (Avestin, Canada). Extruded liposomes were concentrated by ultrafiltration and buffer was exchanged to PBS pH 7.4 by diafiltration (10 times exchange). The resulting liposomes were diluted in PBS pH 7.4 and heated to 30? C. prior to Pal1-15 addition.

(19) A tetrapalmitoylated human peptide Pal1-15 (Bachem AG, Switzerland) was dissolved in 10 mM Na2HPO4, pH 11.4 in MilliQ water with 1% ?-OG (Sigma-Aldrich, USA), injected in the liposomal solution at 30? C. and stirred for 30 minutes followed by concentration steps through ultrafiltration and dilution in PBS pH 7.4 by diafiltration. The resulting liposomes were then sterile filtered by passing through 0.2 ?m polyethersulfone (PES) membrane syringe filters and stored at 5? C.

(20) Preparation of the ACI-24.043 Vaccine (Crossflow Injection)

(21) The lipids (DMPG, DMPC, cholesterol and 3D-(6-acyl) PHAD? (Avanti Polar Lipids, USA)) were dissolved in 96% EtOH in a heating cabinet at 60? C. After complete dissolution of the lipids, the solution was filtered through a 0.2 ?m pore size filter into the injection system which was heated to 60? C. In detail, the appropriate amount of ACI-24.043 (SAT47) was dispersed in EtOH at room temperature by the aid of sonication (EtOH concentration is typically 2% v/v of final SAT47 solution). After complete dispersion of the peptide, His-Sucrose buffer (10 mM Histidine, 250 mM Sucrose) was added to achieve a drug to lipid ratio of 1/50 by mass. The SAT47 solution was filtered through a 0.2 ?m pore size filter (Sartoscale filter) into the injection buffer bottle which was then heated up to 40? C. Liposomes are formed at the site of injection when the lipid/EtOH solution and the injection buffer mixes. Immediately after liposome formation there was an online dilution step with 10 mM Histidine, 250 mM Sucrose in order to decrease the EtOH concentration. The intermediate liposomes were extruded through 100 nm pore size polycarbonate membranes (1 pass) at RT. Ultra-/diafiltration (UDF) using a hollow fiber membrane (MWCO: 500 kD) was performed to remove EtOH and the buffer was exchanged to PBS pH 6.9 (10 volume exchanges). SAT47 liposomes were then diluted using the dispersion buffer (PBS pH 6.9) to a total lipid concentration of 1 mg/mL and warmed up to 35? C. The Pal1-15 was dissolved in a 10% w/v solution of beta-OG in 10 mM Na2HPO4 pH 11.4 buffer at 60? C. and was further diluted with the same buffer to a final concentration of 1 mg/mL. The pH was adjusted to 11.4. After mixing of these two solutions using a crossflow injection module, the liposomal suspension was further incubated at 35? C. for 30 minutes under stirring to allow complete insertion of Pal1-15. A second UDF step using a hollow fiber membrane (MWCO: 500 kD) was performed to remove beta-OG and to exchange buffer to 10 mM Histidine, 250 mM Sucrose (10 volume exchanges). The product was concentrated in its final volume and filtered through a 0.2 ?m Acrodisc mPES syringe filters.

(22) Preparation of the ACI-24.046 Vaccine (Cross Flow Injection)

(23) The lipids (DMPG, DMPC, cholesterol and 3D-(6-acyl) PHAD? (Avanti Polar Lipids, USA)) were dissolved in 96% EtOH in a heating cabinet at 60? C. After complete dissolution of the lipids, the solution was filtered through a 0.2 ?m pore size filter into the injection system which was heated to 60? C. In parallel, ACI-24.046 (SAT44) was dissolved in the injection buffer (10 mM Histidine, 250 mM Sucrose) at 40? C. After complete dissolution of the SAT44, the solution was filtered through a 0.2 ?m pore size filter (Sartoscale) into the injection buffer bottle which was heated to 40? C. Liposomes are formed at the site of injection when the lipid/EtOH solution and the injection buffer mixes. Immediately after liposome formation there was an online dilution step with 10 mM Histidine, 250 mM Sucrose in order to decrease the EtOH concentration. The intermediate liposomes were extruded through 100 nm pore size polycarbonate membranes (1 pass) at room temperature. Ultra-/diafiltration (UDF) using a hollow fiber membrane (MWCO: 500 kD) was performed to remove EtOH and the buffer was exchanged to PBS pH 6.9 (10 volume exchanges). SAT44 liposomes were then diluted using the dispersion buffer (PBS pH 6.9) to a total lipid concentration of 1 mg/mL and warmed up to 35? C. The Pal1-15 was dissolved in a 10% w/v solution of beta-OG in 10 mM Na2HPO4 pH 11.4 buffer at 60? C. and was further diluted with the same buffer to a final concentration of 1 mg/mL. The pH was checked and carefully adjusted back to 11.4. After mixing of these two solutions using an injection module, the liposomal suspension was further incubated at 35? C. for 30 minutes under stirring to allow complete insertion of Pal1-15. A second UDF step using a hollow fiber membrane (MWCO: 500 kD) was performed to remove beta-OG and to exchange buffer to 10 mM Histidine, 250 mM Sucrose (10 volume exchanges). The product was concentrated in its final volume and finally filtered through a 0.2 ?m Acrodisc mPES syringe filters.

Example 3. Proof-of-Concept (PoC) In Vivo Immunogenicity Studies of Vaccines with Encapsulated T-Cell Epitopes

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

(25) Plates were coated with 10 ?g/ml of human A?1-42 peptide film (Bachem, Switzerland) overnight at 4? C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween/PBS, serial dilutions of plasma were added to the plates and incubated at 37? C. for 2 hours. After washing, plates were incubated with alkaline phosphatase (AP) conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, PA, USA) for 2 hours at 37? C. After final washing, plates were incubated for 2.5 hours with AP substrate (pNPP) and read at 405 nm using an ELISA plate reader. Results are expressed by reference to serial dilutions of a commercially available antibody (6E10, Biolegend, UK, Cat. 803002). FIG. 1A shows the A?1-42-specific IgG titers induced by ACI-24 vaccine with or without encapsulated T-cell epitope over time. Even though ACI-24 vaccine showed the highest A?1-42-specific IgG titers at Day 7, after the 1st immunization, an increase in antibody titers was observed after the 2.sup.nd and 3.sup.rd immunization when a T-cell epitope was encapsulated in ACI-24 vaccine. The results in FIG. 1B show that immunization with ACI-24 vaccines comprising encapsulated T-cell epitopes induced an increase of A?-specific antibody titers as compared to ACI-24, which reached statistical significance for the group immunized with ACI-24.043 (with encapsulated SAT47) vaccine.

(26) Vaccines with encapsulated SAT42, SAT43, SAT44 or SAT47 were tested in a Cynomolgus monkey study. Four monkeys per group received three monthly s.c. immunizations (Day 1, 29 and 57) with ACI-24.044 (encapsulated SAT 42two groups with a total of 8 monkeys), ACI-24.046 vaccine (encapsulated SAT 442 groups with a total of 8 monkeys), ACI-24.045 vaccine (encapsulated SAT 434 monkeys) or ACI-24.043 vaccine (encapsulated SAT 474 monkeys). Blood was collected before the first immunization (Day 1) and 1 and 3 weeks after each immunization (Day 8, 22, 36, 50, 64 and 78) to measure A?1-42-specific IgG titers by ELISA.

(27) Plates were coated with 10 ?g/ml of human A?1-42 peptide film (Bachem, Switzerland) overnight at 4? C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween 20/PBS, 8 two-fold serial dilutions of sera were added to the plates and incubated at 37? C. for 2 hours. After the washing, plates were incubated with a horseradish peroxidase (HRP)-conjugated anti-monkey-IgG antibody (KPL, Cat. No. 074 11 021) for 2 hours at 37? C. After washing, plates were incubated with 50 ?l of ABTS/H2O2 (2,2-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (HRP substrate) and read at 405 nm after one hour using an ELISA plate reader. Results are expressed by reference to serial dilutions of a positive monkey pool used as standard.

(28) The immunogenicity of vaccines with different T-cell epitopes was compared with ACI-24 vaccine. Table 3 shows the A?-specific antibody titer fold increase as compared to ACI-24 vaccine 1 week after the third immunization. All tested vaccines, ACI-24.046 (SAT44), ACI-24.043 (SAT47) ACI-24.045 (SAT43) and ACI-24.044 (SAT42) induced an increase of the antibody titers of at least 7 fold (ACI-24.044 with encapsulated SAT42), as compared to the titers induced by ACI-24 vaccine. ACI-24.043 vaccine (with encapsulated SAT47) and ACI-24.046 (with encapsulated SAT44) induced significantly higher A?-specific antibody titers as compared to ACI-24 1 week after the third immunization (Table 3). ACI-24.043 vaccine (with encapsulated SAT47) and ACI-24.046 (with encapsulated SAT44) each have high Epivax scores (142.89 and 57.2 respectively).

(29) TABLE-US-00004 TABLE 3 A?-specific antibody titer fold increase as compared to ACI-24 (1 week after the third immunization, Day 64) ACI-24.046 ACI-24.043 ACI-24.045 ACI-24.044 Vaccine (encapsulated SAT44) (encapsulated SAT47) (encapsulated SAT43) (encapsulated SAT42) A?-specific IgG 40 144 17 7 titer fold increase p = 0.0027 (**) p = 0.0003 (***) p = 0.1408 (ns) p = 0.6003 (ns) over ACI-24 Statistical test: Kruskal-Wallis test with Dunn's multiple comparisons. * p < 0.05; (**) p < 0.01; (***) p < 0.001; ns: non significant.

(30) Following the results obtained in vivo (FIG. 1) with the vaccines ACI-24.046 (encapsulated SAT44) and ACI-24.043 (encapsulated SAT47) manufactured according to thin-lipid film technology, we tested the in vivo immunogenicity of the same vaccines manufactured according to a crossflow injection method. Wild type C57BL/6 mice received a total of three subcutaneous (s.c.) immunizations at days 0, 14 and 28 of ACI-24, ACI-24.046 (with encapsulated SAT44) or ACI-24.043 (with encapsulated SAT47) vaccines. Blood samples were collected at days ?7, 7, 21 and 35 to measure A?1-42-specific IgG titers by ELISA. The results in FIG. 6 show that immunization with ACI-24 vaccines comprising encapsulated T-cell epitopes induced a significant increase of A?-specific antibody titers as compared to ACI-24.

Example 4. Quality of Induced A?-Specific Antibodies

(31) 4.1 In Vitro Inhibition of Human Apt-42 Self-Association

(32) The quality of induced A?-specific antibodies was tested in vitro by measuring inhibition of A?1-42 self-association/aggregation. This assay is based on the ability of mouse pre- and post-immunization plasma to impair the natural predisposition of human A?1-42 to self-associate.

(33) Standard ELISA plates were coated with 1 ?g/mL A?1-42 overnight at 4? C. Plates were washed 4 times with 300 ?L of 0.05% Tween 20/PBS. Saturation was achieved by adding 0.5% BSA/PBS and incubating for 1 hour at 37? C. After washing, four 2-fold serial dilutions of plasma were added to the plates for 20 minutes at room temperature with agitation. Biotinylated A?1-42 was added to each well to a final concentration of 0.1 ?g/mL and incubated at room temperature for 2 hours with agitation. Biotinylated A?1-42 without plasma was used as positive control for A?1-42 self-association (considered as 100% of self-association, 0% of inhibition). After a washing step, plates were incubated with a horseradish peroxidase (HRP)-conjugated to streptavidin (R&D Systems, Canada, Ref. 890803) at 1/200 dilution in 0.5% BSA/0.05% Tween 20/PBS for 1 hour at room temperature with agitation. After washing, the plates were incubated with Sure Blue Reserve TMB substrate (Seracare, Cat. 5120-0081) for 10 minutes. The reaction was stopped with Bethyl stop solution (Bethyl Laboratories, Inc, Cat. E115) and plates were read at 450 nm using an ELISA plate reader. The percentage inhibition of self-association was calculated using as reference the biotinylated A?1-42 without plasma as positive control (0% inhibition).

(34) The results showed that the A?-specific antibodies generated after 2 immunizations with all vaccines containing a T-cell epitope impaired A?1-42 self-association more efficiently than antibodies induced by ACI-24 (FIG. 2A). Since the pre-bleeding plasma induces a background inhibition of self-association, the percentage at day 21 was normalized by subtracting the background of the pre-bleeding plasma. The A?1-42 specific antibodies generated by immunization with all ACI-24 vaccines containing a T-cell epitope showed higher inhibition of A?1-42 self-association as compared to ACI-24; this inhibition reached statistical significance in the group immunized with ACI-24.046 (with encapsulated SAT44) (FIG. 2B).

(35) 4.2 Generation of Antibodies Recognizing A? Oligomers

(36) In order to evaluate the specificity of induced antibodies in C57BL/6 mice to bind the pathological A?, A?1-42-oligomers specific IgG responses were determined by ELISA. Plates were coated with 10 ?g/ml of oligomers prepared as previously described (Adolfsson, 2012) overnight at 4? C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween 20/PBS, serial dilutions of plasma were added to the plates and incubated at 37? C. for 2 hours. After washing, plates were incubated with alkaline phosphatase (AP) conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, Cat: 115-055-164, PA, USA) for 2 hours at 37? C. After final washing, plates were incubated for 2.5 hours with AP substrate (pNPP) and read at 405 nm using an ELISA plate reader. Results are expressed by reference to serial dilutions of a commercial available antibody (6E10, Biolegend, UK, Cat. 803002).

(37) Each sample was tested in eight or four 2-fold serial dilutions, starting at 1/100, 1/400, 1/800 or 1/1600 dilution, based on A?1-42 antibody titers. The results in FIG. 3 show that immunization with all ACI-24 vaccines containing a T-cell epitope induced an increase of A?1-42 oligomer-specific antibody titers as compared to ACI-24, which reached statistical significance for the group immunized with ACI-24.043 (with encapsulated SAT47) vaccine. The avidity index of induced antibodies in C57BL/6 mice, 7 and 21 days after immunization was determined by ELISA assay. One half of a standard ELISA plate was coated with 10 ?g/mL of A?1-42 peptide film and the other half with 1 ?g/mL of A?1-42 peptide film overnight at 4? C. After washing with 0.05% Tween 20/PBS and blocking with 1% BSA/0.05% Tween 20/PBS, eight 2-fold serial dilutions of plasma were added to both coating conditions and incubated at 37? C. for two hours. After a washing step, plates were incubated with alkaline phosphatase (AP)-conjugated anti-mouse IgG antibody (Jackson ImmunoResearch, Cat: 115-055-164, PA, USA) for 2 hours at 37? C. After final washing, plates were incubated for 2.5 hours with AP substrate (pNPP) and read at 405 nm using an ELISA plate reader. Results are expressed by reference to serial dilutions of a commercially available antibody (6E10, Biolegend, UK, Cat. 803002).

(38) For the determination of the avidity index, AU/mL were calculated for each sample on both coatings using the standard curve obtained on 10 ?g/mL of A?1-42 peptide. O.D. values between 0.6 and 2.8 were used for the back-calculation of the concentration. The avidity index is calculated as a ratio between the antibody concentration on the lower coating concentration (1 ?g/mL of A?1-42 peptide) and the saturated coating (10 ?g/mL of A?1-42 peptide).

(39) The results in FIG. 4 show that immunization with all ACI-24 vaccines containing a T-cell epitope induced an A?1-42-specific antibody avidity maturation between the 1st and the 2.sup.nd immunization (day 7 and day 21 respectively), which reached statistical significance in the groups immunized with ACI-24.044 (with encapsulated SAT42) and ACI-24.043 (with encapsulated SAT47).

(40) In order to evaluate the specificity of induced antibodies in Cynomolgus monkeys to bind the pathological A? A?1-42 oligomer-specific IgG titers were measured by Meso Scale Discovery (MSD) technology at Day 64 (1 week after the third immunization) in sera of Cynomolgus monkeys immunized with ACI-24.046 (with encapsulated SAT442 groups with a total of 8 monkeys), ACI-24.045 vaccine (encapsulated SAT 434 monkeys) or ACI-24.043 vaccine (encapsulated SAT 474 monkeys). MSD streptavidin plates were saturated over night with 5% of Blocker A (MSD, Ref. R93BA-4) at 4? C. The day after, plates were washed 4 times with 0.05% Tween 20/PBS and coated with 25 ?l of capturing antibody biotinylated 6E10 (Biolegend, Ref. 803008) in PBS at 0.5 ?g/ml for 1 hour at 37? C. on a shaker. After washing, plates were incubated with 25 ?l of A?1-42 oligomers (Adolfsson, 2012) at 10 ?g/ml in PBS for 1 hour at 37? C. on a shaker. Plates were washed and incubated with eight 2-fold dilutions of monkey sera (starting dilution 1/50 in 1% Skim milk/0.05% Tween/PBS). Samples were incubated 2 hours at 37? C. on a shaker. Plates were washed 4 times and anti-human IgG detection antibody labeled with SULFO-TAG (Jackson, Ref. 109-005-098) was added, diluted in 1% Skim milk/0.05% Tween 20/PBS for 1 hour at 37? C. on a shaker. After 4 washes, MSD read buffer T 2X (MSD, Ref. R92TC-2) was added and plates were read within 5 minutes. Results are expressed by reference to serial dilutions of monkey pool used as standard.

(41) The results showed that all tested vaccines ACI-24.046 (encapsulated SAT44), ACI-24.043 (encapsulated SAT47) and ACI-24.045 (encapsulated SAT43) induced an increase of antibodies able to recognize A? oligomers at day 64 (1 week after third immunization) compared with day 1 (prior to first immunization); see FIG. 5.

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(43) Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications and patents specifically mentioned herein are incorporated by reference in their entirety for all purposes in connection with the invention.

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