Papillomavirus-like particle-based Anti-amyloid beta alzheimer's disease vaccine

Abstract

The present invention relates to a papillomavirus-like particle-based anti-amyloid beta Alzheimer's disease vaccine. More specifically, the present invention relates to an Alzheimer's disease vaccine in which an amyloid beta epitope is loaded on virus-like particles of human papillomavirus (HPV). According to the present invention, an antibody-inducing effect against amyloid beta can be greatly improved by linking amyloid beta peptide fragments to HPV VLPs that have excellent structural characteristics and immune characteristics. In addition, it has been identified that the vaccine prepared in the present invention exhibits better immunogenicity and antibody conversion rate than a conventional VLP-based Alzheimer's disease vaccine.

Description

Papillomavirus virus-like particle-based anti-amyloid beta Alzheimer's disease vaccine

[0001] The present invention relates to a vaccine comprising amyloid beta antigens loaded onto virus-like particles (VLPs) of the papillomavirus. Furthermore, the present invention relates to a vaccine for the prevention and treatment of Alzheimer's disease (AD).

[0002] According to the World Health Organization (WHO), there are currently approximately 50 million people with dementia worldwide, and this number is expected to more than triple (to 152 million) by 2050. Alzheimer's disease (AD) is one of the leading causes of progressive dementia. Since the first report of AD in 1907, numerous studies have been conducted on the pathogenesis and progression of the disease. Recently, therapeutic antibodies using passive immunization have shown positive results in clinical trials, and some candidate antibodies have received approval. However, the range of drugs that can effectively treat AD is still very limited.

[0003] Alzheimer's disease is a neurodegenerative disease, and its pathological characteristics are known to be the accumulation of amyloid beta (Aβ), the formation of amyloid plaques, and the development of neurofibrillary tangles. Amyloid beta peptide, the main component of amyloid plaques, is formed from the proteolysis of a larger glycoprotein called amyloid precursor protein (APP). Amyloid precursor protein, a transmembrane protein, is first cleaved by α-secretase (non-amyloidogenic pathway) or β-secretase (amyloidogenic pathway), generating a membrane-bound α- / β- C-terminal fragment (CTF). Afterwards, the α- / β- C-terminal fragments are cleaved by γ-secretase into P3 (3 kDa) and amyloid beta peptide (4 kDa), respectively. Therefore, the process of amyloid formation from amyloid precursor protein is due to sequential degradation by β- and γ-secretase at the N and C termini of amyloid beta, respectively. Amyloid beta monomers generated from amyloid precursor protein accumulate and self-assemble into soluble oligomers, and various characteristics arise because they are generated in different molecular weights and shapes. Studies have shown that amyloid beta oligomers are neurotoxic aggregates and play an important role in the occurrence and development of Alzheimer's disease by causing functional neuronal cell death, cognitive impairment, and dementia.

[0004] Therefore, considering that amyloid beta accumulation is a major cause of neuronal cell death in Alzheimer's disease, it has been thought that inhibiting the formation of amyloid beta oligomers or removing amyloid beta could contribute to the treatment of Alzheimer's disease. Various experimental models have confirmed that anti-amyloid beta immunotherapy is a major method for promoting amyloid beta clearance. Anti-amyloid beta immunotherapy can be classified into active immunization (vaccine) and passive immunization (exogenous antibody). Active immunization is a method that administers amyloid beta or its fragments to induce endogenous antibodies against amyloid beta in the body. Although this method has the disadvantage of being difficult to predict immune responses and adverse reactions, unlike passive immunotherapy, it can induce a sustained polyclonal amyloid beta-specific response with short-term administration. On the other hand, drugs developed through passive immunotherapy can lower amyloid beta levels in the central nervous system (CNS), but caution is required regarding adverse reactions due to amyloid-associated abnormal imaging findings (ARIA), the most common side effect of this class of drugs.

[0005] In a clinical trial using AN-1792, the first amyloid beta active immunotherapy (vaccine), some vaccinated patients showed a reduction in brain amyloid beta plaques and the induction of antibodies against amyloid beta. Even though AN-1792 was administered together with the immune adjuvant QS-21, only 20% of the vaccinated group developed antibodies. Furthermore, meningoencephalitis was observed in 6% of the vaccinated subjects. These adverse events are believed to be due to amyloid beta-specific T cell responses (Th1-type CD4). Therefore, to develop an effective Alzheimer's beta vaccine, a strategy must be applied that can induce high antibody titers while simultaneously avoiding amyloid beta-specific T cell responses.

[0006] VLPs have the advantage of inducing high levels of immune responses and antibody titers. Because of their characteristic tertiary structure, VLPs are effectively recognized by immune cells, which can be utilized as a foundational technology for developing effective vaccines. Meanwhile, to avoid T cell responses, short or fragmented amyloid beta or N-terminal peptide fragments containing B cell epitopes can be used. CAD106, a second-generation amyloid beta vaccine, is a VLP-based vaccine composed of bacteriophage Q beta coat protein VLPs (Qβ VLPs) conjugated to amyloid beta (1-6) peptide fragments. Indeed, CAD106 effectively induced antibodies against amyloid beta without activating amyloid beta-specific T cell responses in animal models.

[0007] Meanwhile, research on VLP-based vaccines with superior antibody induction ability is needed to develop vaccines for the prevention and treatment of Alzheimer's disease.

[0008] The purpose of the present invention is to provide a vaccine composition for preventing or treating Alzheimer's disease, which comprises virus-like particles (VLPs) of papillomavirus to which amyloid beta antigen is attached as an active ingredient.

[0009] In order to achieve the above purpose, the present invention provides a vaccine composition for preventing Alzheimer's disease comprising, as an active ingredient, VLPs of papillomavirus to which amyloid beta antigen is attached, and specifically, a vaccine composition for preventing or treating Alzheimer's disease comprising VLPs of human papillomavirus (HPV) loaded with sequences 1-6 of amyloid beta.

[0010] The present invention relates to an anti-amyloid beta Alzheimer's disease vaccine based on a human papillomavirus (HPV) virus-like particle, and more particularly, to an Alzheimer's disease vaccine comprising an amyloid beta antigen group loaded onto a human papillomavirus (HPV) virus-like particle. According to the present invention, by linking an amyloid beta peptide fragment to HPV VLPs having excellent structural and immunological characteristics, the antibody induction effect against amyloid beta can be significantly enhanced. In addition, it was confirmed that the vaccine manufactured in the present invention exhibits superior immunogenicity and seroconversion rate than existing VLPs-based Alzheimer's disease vaccines.

[0011] Figure 1 shows the process of a method for manufacturing a virus-like particle-based vaccine of bacteriophage Q beta virus (hereinafter “QV-002”) and a method for manufacturing a virus-like particle-based vaccine of human papillomavirus (hereinafter “PV-002”). QV-002 and QV-002-S / PV-002 and PV-002-S have the same manufacturing method, and differ only in the type of peptide [Aβ(1-6) / 3Aβ(1-6) peptide] used in the coupling process.

[0012] Figure 2 shows transmission electron microscopy images of QV-002 and PV-002, respectively, stained with negative staining (1% PTA) and observed at 100,000x magnification. The bar represents 50 nm. It shows that each purified and coupled sample maintained a spherical shape.

[0013] Figure 3 shows the results of a Western blot comparison of the amounts of amyloid beta peptide coupled to QV-002 and PV-002. After protein quantification for Western blot analysis, QV-002 and PV-002 were loaded at 200 ng, 100 ng, and 50 ng per well, respectively, and HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the reactivity of PV-002 to amyloid beta antibodies was significantly higher than that of QV-002. M in the upper panel of the results indicates marker. The numbers in the left and right panels of the results indicate molecular weights.

[0014] Figure 4 shows the results of a Western blot comparison of the amounts of amyloid beta peptide coupled to QV-002-S and PV-002-S. After protein quantification for Western blot analysis, QV-002-S and PV-002-S were loaded at 200 ng, 100 ng, and 50 ng per well, respectively, while HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the reactivity of PV-002-S against amyloid beta antibodies was significantly higher than that of QV-002-S.

[0015] Figure 5 shows the results of Western blot comparison of the amount of coupled amyloid beta peptide of QV-002-S, PV-002-S, QV-002, and PV-002. After protein quantification for Western blot analysis, QV-002-S, PV-002-S, QV-002, and PV-002 were loaded at 200 ng and 100 ng per well, respectively, and HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, PV-002 showed the highest reactivity to amyloid beta antibodies.

[0016] Figure 6 shows the results of the dot blot test using an anti-amyloid beta(1-10) monoclonal antibody to measure the coupled amyloid beta peptide density of QV-002-S / QV-002 / PV-002-S / PV-002. Comparison between QV-002-S and QV-002 confirmed that QV-002 with the 3Aβ(1-6) peptide linked showed a slightly higher reaction rate, and PV-002-S and PV-002 showed much higher reactivity than QV-002-S and QV-002. Therefore, the results in Figure 6 show that the amount of peptide coupled to HPV VLPs is greater than the amount of peptide coupled to Qβ VLPs, regardless of the type of peptide.

[0017] Figure 7 shows the results of enzyme-linked immunosorbent assay (ELISA) analysis of the anti-amyloid beta (1-10) monoclonal antibody reactivity of QV-002 and PV-002. Both QV-002 and PV-002 exhibited antibody reactivity proportional to the amount of coating. Furthermore, PV-002 exhibited a higher antibody reactivity than QV-002.

[0018] Figures 8a and 8b show the results of immunogenicity analysis after administering 10 ng, 100 ng, and 1000 ng of vaccine antigen intramuscularly three times at two-week intervals to each mouse group. Figure 8a shows the results of measuring the anti-Aβ(1-42) IgG titer in serum. In the 100 ng and 1000 ng administration groups, the antibody titer in the PV-002 group was measured to be significantly higher than that in the QV-002 group. Figure 8b shows the results of measuring the anti-3Aβ(1-6) IgG titer in serum. In the 100 ng administration group, the antibody titer in the PV-002 group was observed to be significantly higher than that in the QV-002 group.

[0019] Figures 9a and 9b show the positivity rates after the third intramuscular administration of 10 ng, 100 ng, and 1000 ng vaccine antigens to each mouse group. As shown in Figure 9a, the positivity rate of PV-002 in the 100 ng and 1000 ng administration groups was higher than that in the QV-002 group. Similarly, Figure 9b shows that the positivity rate of PV-002 in the 100 ng administration group was higher than that in the QV-002 group.

[0020] Figures 10a and 10b show the results of immunogenicity analysis after administering 10 ng, 100 ng, and 1000 ng of vaccine antigens intramuscularly four times to each mouse group. Figure 10a shows the results of measuring the anti-Aβ(1-42) IgG titer in serum. In the 100 ng administration group, the antibody titer in the PV-002 group was observed to be significantly higher than that in the QV-002 group. Figure 10b shows the results of measuring the anti-3Aβ(1-6) IgG titer in serum. In the 100 ng administration group, the antibody titer in the PV-002 group was observed to be significantly higher than that in the QV-002 group.

[0021] Figures 11a and 11b show the positivity rates after the fourth intramuscular administration of 10 ng, 100 ng, and 1000 ng vaccine antigens to each mouse group. As can be seen in Figure 11a, the positivity rates of the PV-002 group were higher than those of the QV-002 group in both the 100 ng and 1000 ng administration groups. Similarly, Figure 11b shows that the positivity rate of PV-002 in the 100 ng administration group was higher than that of QV-002.

[0022] Figure 12 is the sequence of the Qβ coat protein (133 amino acids) used in the present invention, and has the characteristic of self-assembling into VLPs under suitable conditions. In addition, the vector map of Figure 12 is a plasmid manufactured to express the Qβ coat protein, and is a schematic diagram manufactured by inserting the sequence (402 bp) of the Qβ major coat protein into the pCDF-1b vector using the restriction enzymes NcoI and AvrII.

[0023] Figure 13 is the amyloid beta sequence used for coupling in the present invention. It is a peptide created by adding some amino acids to 1-6, i.e. 6 amino acids, of the entire amyloid beta sequence (1-42). The Aβ(1-6) peptide has glycine-glycine-cysteine ​​(Gly-Gly-Cys) added to the C-terminus, and the 3Aβ(1-6) peptide has glycine-glycine-glycine (Gly-Gly-Gly) between the Aβ(1-6) repeat sequences and glycine-glycine-cysteine ​​(Gly-Gly-Cys) added to the C-terminus.

[0024] Figure 14 shows the results of immunogenicity analysis after administering 100 ng of vaccine antigen intramuscularly three times at two-week intervals to each mouse group. QV-002-S is a vaccine antigen in the form of Aβ(1-6) coupled to the Qβ coat protein, and QV-002 is a vaccine antigen in the form of 3Aβ(1-6) coupled to the Qβ coat protein. Similarly, PV-002-S is a vaccine antigen in the form of Aβ(1-6) coupled to HPV VLP, and PV-002 is a vaccine antigen in the form of 3Aβ(1-6) coupled to HPV VLP. Compared to QV-002, mice vaccinated with PV-002 showed a significantly higher immune response (IQR 50: 1.0 vs 3.2; P < 0.00001). No antibody titer was observed in either the QV-002-S or PV-002-S vaccinated groups (titers less than 100 were calculated as 1.0).

[0025] Figure 15 shows the results of immunogenicity analysis after administering 100 ng of vaccine antigen intramuscularly to the above mice four times at two-week intervals. Significantly higher antibody titers were observed in the PV-002 vaccination group compared to the QV-002 vaccination group (IQR 50: 1.5 vs 3.2; P < 0.0001).

[0026] Figure 16 shows the results of brain tissue analysis of the area of ​​amyloid beta accumulated in the cerebral cortex after five or ten intramuscular administrations of 80 μg / dose PV-002 vaccine. A significant decrease was observed in the group administered 10 doses of PV-002 vaccine compared to the PBS group.

[0027] Figure 17 shows the results of an analysis of the area of ​​amyloid beta accumulated in the hippocampus in the above experiment. It was confirmed that the area of ​​amyloid beta plaques was significantly reduced in the group that received 10 doses of the PV-002 vaccine compared to the PBS group.

[0028] Figure 18 shows the results of an analysis of the area of ​​amyloid beta accumulated in the thalamus in the above experiment. It was confirmed that the area of ​​amyloid beta plaques was significantly reduced in the group that received 10 doses of the PV-002 vaccine compared to the PBS group.

[0029] Figure 19 shows the path length of the Morris Water Maze experiment, which confirmed the cognitive improvement effect of PV-002 vaccine in mice. For immunization, the PV-002 vaccine was prepared at human doses of 50 μg / dose, 100 μg / dose, 200 μg / dose, or 400 μg / dose. Each mouse was administered 10 intramuscular doses of 1 / 10 of the dose. Therefore, the mice received PV-002 vaccine at doses of 5 μg / dose, 10 μg / dose, 20 μg / dose, or 40 μg / dose. The results showed that path lengths shortened as the vaccine dose increased.

[0030] Figure 20 shows the escape latency of the above experiment. The administration groups of 10 μg / dose, 20 μg / dose, and 40 μg / dose showed shortened escape latency, which was similar to the latency of wild type mice.

[0031] Figure 21 shows the results of ELISA analysis of Aβ sequences responsive to antibodies induced in PV-002-immunized mice. An ELISA value of 0.2 or higher was considered a significant response. PV-002-induced antibodies were confirmed to recognize Aβ 1-42, Aβ 1-10, Aβ 2-11, and Aβ 3-12.

[0032] Figure 22 shows the results of ELISPOT to confirm whether immune cells of PV-002-immunized mice induce T-cell responses. When splenocytes of PV-002-immunized mice were stimulated with Aβ 1-42 or 3Aβ(1-6) peptides, no interferon-gamma-expressing cell response was confirmed. On the other hand, when stimulated with HPV VLP, an interferon-gamma-expressing cell response was confirmed to occur. The Aβ(1-6) sequence loaded on PV-002 is a B-cell epitope, and it shows that PV-002 immunization does not induce a T-cell response with a risk of side effects and selectively induces a B-cell-induced antibody response.

[0033] One of the currently approved VLP-based vaccines for human use is the human papillomavirus (HPV) vaccine. HPV VLP vaccines are known to induce sustained, high-titer neutralizing antibody responses against HPV. Therefore, it was expected that an anti-amyloid beta vaccine comprising amyloid beta fragments conjugated to HPV VLPs would not only strongly induce amyloid beta antibodies but also avoid amyloid beta-specific T cell responses. Accordingly, the inventors of the present invention developed an anti-amyloid beta Alzheimer's disease vaccine with excellent antibody induction ability by loading the amyloid beta (1-6) sequence or a peptide fragment containing the sequence repeated three times onto HPV VLPs.

[0034] According to conventional wisdom, short peptide sequences alone are not suitable for inducing antibody responses. In particular, in the case of autoantigen-derived peptides such as amyloid beta, inducing antibody responses is even more difficult due to immune tolerance. To solve this problem, various types of carriers have been used in existing methods to increase the immune response. The present inventors have attempted to develop a vaccine antigen that loads amyloid beta sequences 1-6 onto HPV VLPs to develop an Alzheimer's disease vaccine targeting amyloid beta. As a result, the present vaccine (PV-002), which links amyloid beta 1-6 peptides to HPV VLPs, was found to be capable of loading a higher level of peptide antigen group than the previously known Qβ VLP-based vaccine. This feature enabled the induction of an improved level of anti-amyloid beta antibodies, thereby completing the present invention.

[0035]

[0036] The present invention provides a vaccine composition for preventing or treating Alzheimer's disease, which comprises virus-like particles (VLPs) of papillomavirus to which amyloid beta antigen is attached as an active ingredient.

[0037] Preferably, the VLPs of the papillomavirus may be VLPs of human papillomavirus (HPV), and more preferably, the VLPs of the HPV may be HPV11 L1 VLPs purified from a transformed host cell expressing the L1 protein of HPV type 11 (HPV11), but is not limited thereto.

[0038]

[0039] In the present invention, "HPV L1 protein" refers to a major protein that is expressed from the L1 gene of HPV and constitutes the capsid of HPV. The L1 protein has the property of self-assembling into VLPs under suitable conditions, either together with the L2 protein, a minor protein that constitutes the capsid, or alone.

[0040] The HPV type of the L1 protein used in the present invention is HPV type 11, and the cell used as the host cell is a yeast cell.

[0041] According to a preferred embodiment of the present invention, the yeast cell includes, but is not limited to, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces sp., Schizosaccharomyces pombe, Pichia pastoris, or Hansenula polymorpha.

[0042] In the present invention, a transformed host cell expressing the HPV L1 protein refers to a host cell transformed with an expression vector that successfully expresses the HPV L1 protein. The expression vector may include transcription or translation regulatory elements known in the art, or other marker genes. The transformed host cell expressing the HPV L1 protein of the present invention can be readily produced using methods known in the art.

[0043]

[0044] In the present invention, the "Qβ coat protein" refers to a major protein that constitutes the capsid of bacteriophage Qβ, which is expressed from the major coat protein gene of Qβ. The coat protein has a size of 133 amino acids and has the property of self-assembling into VLPs under suitable conditions. This was used for comparison with the HPV VLPs of the present invention.

[0045]

[0046] In the present invention, "Virus-Like Particles (VLPs)" refers to non-infectious viral subunits that may or may not contain viral proteins. For example, the VLPs may completely lack a DNA or RNA genome, or, in the case of VLPs containing viral capsid proteins, may undergo spontaneous self-assembly.

[0047] The above virus-like particles can be manufactured by methods widely known in the art. For example, the virus-like particles can be manufactured by transforming a predetermined host cell with a recombinant DNA molecule encoding the structural protein and antigen recognition site, followed by culturing the transformed cell. The protein expressed within the cell can be assembled on the cell surface and then released into the culture supernatant. In this case, the surface protein contained in the virus-like particles maintains its natural form without undergoing an immobilization process, thereby inducing a desired immune response against a specific pathogen within the organism.

[0048] The transformed host cell can be cultured under batch, fed-batch or continuous fermentation conditions, and the host cell can express the virus-like particle by transformation, so that the virus-like particle protein can be obtained from the cultured host cell.

[0049]

[0050] In the present invention, the purification of VLPs can be accomplished by performing chromatography from the host cell lysate. Chromatography that can be used in the present invention is known in the art, and is not limited thereto, and examples thereof include, but are not limited to, ion exchange chromatography such as cation exchange chromatography or anion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and affinity chromatography. Since the material to be separated and purified in the present invention is a protein, ion exchange chromatography that is most suitable for the separation of proteins or peptides is preferred. In the following specific embodiment of the present invention, HPV L1 VLPs were successfully separated and purified using heparin resin chromatography and cation exchange chromatography, which are types of cation exchange chromatography.

[0051]

[0052] In the present invention, the amyloid beta antigen may be in the form of a peptide represented by the amino acid sequence of sequence number 1, which is linked by repeating 1 to 10 times, but is not limited thereto.

[0053] Preferably, the peptide (XXEFRHXX) represented by the amino acid sequence of SEQ ID NO: 1 may be, but is not limited to, the peptide (DAEFRH) represented by the amino acid sequence of SEQ ID NO: 2. The "X" described in SEQ ID NO: 1 does not mean a specific amino acid, but rather means that it does not contain any amino acid or may contain any amino acid.

[0054] More preferably, the amyloid beta antigen may additionally include a linker between the linked peptides, and the amyloid beta antigen may be a peptide represented by the amino acid sequence of SEQ ID NO: 3 or the amino acid sequence of SEQ ID NO: 4, but is not limited thereto.

[0055]

[0056] In the present invention, “Aβ(1-6)” is a peptide in which GGC is added to the C-terminus of 1-6 of the amyloid beta peptide sequence, and is a peptide described by the amino acid sequence represented by SEQ ID NO: 3 (Fig. 13). The term “3Aβ(1-6)” is a peptide in which the sequence in which GGG is added to the C-terminus of 1-6 of the amyloid beta peptide sequence is repeated twice, and GGC is added to the C-terminus of 1-6 of the amyloid beta peptide sequence, and is a peptide described by the amino acid sequence represented by SEQ ID NO: 4 (Fig. 13).

[0057]

[0058] In the present invention, “PV-002” is a substance that couples 3Aβ(1-6) to HPV VLPs, and “PV-002-S” is a substance that couples Aβ(1-6) to HPV VLPs. “QV-002” is a substance that couples 3Aβ(1-6) to Qβ VLPs, and “QV-002-S” is a substance that couples Aβ(1-6) to Qβ VLPs (same as CAD106) [J Neurosci. 2011 Jun 22;31(25):9323-31].

[0059]

[0060] In the present invention, the vaccine composition can covalently attach amyloid beta antigen onto VLPs of papillomavirus, and the covalent bond can be bonded through a chemical cross-linker. Preferably, the chemical cross-linker can be succinimidyl-6-(β-maleimidopropionamido) hexanoate (SMPH), but is not limited thereto.

[0061] The SMPH used in the present invention has an NHS (N-hydroxysuccinimide) ester, which allows it to bind to amines, and also has a maleimide, which is a linking group that allows it to bind to molecules containing a sulfhydryl group. The NHS ester reacts with primary amines at pH 7-9 to form an amide bond, and the maleimide reacts with sulfhydryl groups at pH 6.5-7.5 to form a thioether bond.

[0062]

[0063] In the present invention, the vaccine composition may additionally include an adjuvant, but is not limited thereto.

[0064]

[0065] In the present invention, the term "vaccine composition" refers to a composition capable of preventing subsequent diseases by administering a protein antigen having a vaccine effect. The vaccine composition may be a composition comprising papillomavirus VLPs attached to an amyloid beta antigen exhibiting antigenicity against Alzheimer's disease.

[0066] Specifically, the vaccine composition may comprise any one selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, immunostimulants, and combinations thereof.

[0067] The pharmaceutically acceptable carriers are known in the art and include proteins, sugars, and the like. The carriers may be aqueous or non-aqueous solutions, suspensions, and emulsions. Non-aqueous carriers include propylene glycol, polyethylene glycol, edible oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, including edible water and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous carriers include electrolyte replenishers, liquids, and nutritional supplements, such as those based on Ringer's dextrose. Preservatives and other additives may further include antimicrobial agents, antioxidants, chelating agents, inert gases, and the like. Preservatives may include, but are not limited to, formalin, thimerosal, neomycin, polymyxin B, and amphotericin B.

[0068] The above vaccine composition may further comprise an adjuvant. The adjuvant refers to a compound or mixture that enhances the immune response and / or promotes the rate of absorption after vaccination, and includes any absorption-promoting agent. Acceptable adjuvants include, but are not limited to, Freund's complete preservative, Freund's incomplete preservative, saponin, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, pluturon polyols, polyanions, peptides, oil or hydrocarbon emulsions, ketolimpet hemocyanin, dinitrophenol, and the like.

[0069] The above vaccine composition may additionally include an immunostimulant. The immunostimulant may include artificially synthesized levamisole, isoprenosine, and cytokines. Examples of cytokines include interferon-α, interleukin-2, GM-CSF, and GCSF. The vaccine composition may be used in a form comprising the virus-like particle or a concentrate thereof, or in the form of a dried powder of the transformed host cell itself or the transformed cell. In addition, the vaccine composition may be used in combination with other foods or food ingredients and may be used appropriately according to conventional methods.

[0070] The above vaccine composition may further comprise one or more second adjuvants selected from the group consisting of stabilizers, emulsifiers, aluminum hydroxide, aluminum phosphate, pH adjusters, surfactants, liposomes, iscom adjuvants, synthetic glycopeptides, bulking agents, carboxypolymethylene, bacterial cell walls, derivatives of bacterial cell walls, bacterial vaccines, animal poxvirus proteins, subviral particle adjuvants, cholera toxin, N,N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide, and mixtures thereof.

[0071] The above vaccine composition can be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, and sterile injectable solutions according to conventional methods. When formulating, diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants, which are commonly used, can be used together.

[0072] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations may be used together with the lecithin-like emulsifier and at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to the excipients, a lubricant, such as magnesium stearate or talc, may also be used.

[0073] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives can be used.

[0074] Sterile aqueous solutions, non-aqueous preparations, suspensions, emulsions, and lyophilized preparations can be used for parenteral administration. Non-aqueous preparations and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0075] Below, the present invention is described in detail using examples that do not limit the scope of the invention. The following examples are intended to concretize the invention and do not limit or restrict the scope of the invention. Therefore, anything that a specialist in the technical field can easily infer from the detailed description and examples of the invention is interpreted as falling within the scope of the invention.

[0076]

[0077] <Experimental Example>

[0078] The following experimental examples are intended to provide experimental examples commonly applied to each embodiment according to the present invention.

[0079]

[0080] 1. Cell culture

[0081] In the present invention, in order to express bacteriophage Q beta (Qβ) coat protein (133 amino acids), Qβ coat protein DNA (402 bp) was synthesized and the synthesized DNA was cloned into the pCDF-1b plasmid vector (Fig. 12). The Qβ coat protein DNA was introduced into the plasmid using restriction enzymes NcoI and AvrII, and then transformed into Escherichia coli (competent cell BL21), and the transformed cells were selected using streptomycin. In the present invention, the transformed host cell expressing Qβ coat protein refers to a host cell that has been transformed and selected with an expression vector that successfully expresses Qβ coat protein. The selected cells were cultured in 10 mL of LB medium with shaking at 37°C for 16 hours, and the culture solution was inoculated into 500 mL LB for the main culture. After 4 hours of culture, 1 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) was added to the medium to express the lac operon, and the culture was completed by additional culture at 30°C for 6 hours.

[0082]

[0083] 2. Cell disruption

[0084] Cultured Qβ coat protein-expressing cells were mixed in lysis buffer [1X PBST (0.1% triton X-100), pH 7.2]. The cell mixture was again mixed with 0.5 mm glass beads (Biospec Product, USA) and vortexed to lyse the cells. Cell debris was removed by centrifugation at 12,000 g for 15 minutes.

[0085]

[0086] 3. Primary chromatography of Qβ VLPs

[0087] The primary purification of Qβ VLPs involved dialysis of the protein in the cell lysate followed by size exclusion chromatography. Superose 6 prep grade resin and a Tricorn 10 / 300 (Cytiva, USA) column were used for size exclusion chromatography. The lysate was dialyzed against dialysis buffer [phosphate buffer (pH 7.2) containing 0.325 M NaCl and 0.01% Tween 80] and passed through a resin equilibrated with the same mobile phase as the dialysis buffer.

[0088] The resin was washed by flowing a mobile phase five times the volume of the resin, and the proteins were eluted by separating them by size. Among the eluted fractions, the fraction containing the Qβ coat protein was selected and collected through SDS-PAGE analysis. The eluate containing the Qβ coat protein was concentrated using Amicon Ultra (Millipore, USA) and dialyzed against dialysis buffer for 12–18 hours.

[0089]

[0090] 4. Secondary chromatography of Qβ VLPs

[0091] After the first chromatography, the dialyzed solution was purified again through size exclusion chromatography. The resin used for the second chromatography was the same as that used in the first chromatography. The resin was equilibrated with the same mobile phase as the dialysis buffer before loading the sample. The resin was washed by flowing a volume of mobile phase five times the volume of the resin, and the proteins were eluted by separating them by size. Among the eluted fractions, the fraction containing the Qβ coat protein was selected and collected through SDS-PAGE analysis. The eluate containing the Qβ coat protein was concentrated to 1 mg / mL using Amicon Ultra (Millipore, USA).

[0092]

[0093] 5. Coupling

[0094] 5-1. Qβ VLPs-SMPH coupling

[0095] After dissolving SMPH (Sigma / USA) in DMSO, add it to coupling buffer-1 [phosphate-buffered saline (PBS) (pH 8.0) containing 2 mM EDTA] to make 10 mg / mL. Qβ VLPs and the prepared SMPH solution were reacted using a tube rotator at room temperature for 30–60 minutes in a dark-protected manner so that the volume was 10% of that of Qβ VLPs. The reaction solution was dialyzed against coupling buffer-2 [phosphate buffer (pH 7.2) containing 0.325 M NaCl and 0.01% Tween 80] for 12–18 hours.

[0096]

[0097] 5-2. Qβ VLPs - SMPH - Aβ(1-6) or 3Aβ(1-6) peptide coupling

[0098] Aβ(1-6) or 3Aβ(1-6) peptide (5 mg / mL) was mixed into the dialyzed solution to make up 10% of the volume of the completed solution. The reaction was performed at room temperature for 30–60 minutes in a light-protected manner using a tube rotator. The completed reaction solution was dialyzed against coupling buffer-2 for 12–18 hours. The dialyzed solution was filtered through a 0.22 μm PES syringe filter and stored at -60°C or lower.

[0099]

[0100] 5-3. HPV11 VLPs-SMPH coupling

[0101] HPV VLPs were prepared according to a previously published method. SMPH (Sigma / USA) was dissolved in DMSO and added to coupling buffer-1 to make 10 mg / mL. HPV11 VLPs and the prepared SMPH solution were reacted using a tube rotator at room temperature for 30–60 minutes, protected from light, until the volume was 10% of that of the HPV11 VLPs. The reaction solution was dialyzed against coupling buffer-2 for 12–18 hours.

[0102]

[0103] 5-4. HPV11 VLPs - SMPH - Aβ(1-6) or 3Aβ(1-6) peptide coupling

[0104] Aβ(1-6) or 3Aβ(1-6) peptide (5 mg / mL) was mixed into the dialyzed solution to make up 10% of the volume of the completed solution. The reaction was performed at room temperature for 30–60 minutes in a light-protected manner using a tube rotator. The completed reaction solution was dialyzed against coupling buffer-2 for 12–18 hours. The dialyzed solution was filtered through a 0.22 μm PES syringe filter and stored at -60°C or lower.

[0105]

[0106] 6. Third chromatography

[0107] 6-1. Chromatography of QV-002

[0108] The above-mentioned prepared solution was purified through size exclusion chromatography. The resin used for chromatography was the same as that used in the first chromatography. The resin was equilibrated with the same mobile phase as dialysis buffer before loading the sample. The resin was washed by flowing a volume of mobile phase five times the volume of the resin, and the proteins were separated by size and eluted. Among the eluted fractions, those containing QV-002 were selected and pooled through SDS-PAGE analysis.

[0109]

[0110] 6-2. Chromatography of PV-002

[0111] The above-prepared solution was purified through size exclusion chromatography. The resin was equilibrated with a mobile phase identical to dialysis buffer before loading the sample. The resin was washed by flowing a mobile phase five times its volume, and proteins were separated by size and eluted. Among the eluted fractions, those containing PV-002 were selected and pooled through SDS-PAGE analysis.

[0112]

[0113] 7. Transmission electron microscope analysis

[0114] QV-002 and PV-002 were adsorbed onto carbon-coated grids and then stained with 1% phosphotungstic acid for approximately 1 minute. Transmission electron micrographs were taken using a TEM 80 kV (JEM1010, Japan) at 100K magnification.

[0115]

[0116] 8. Western blot

[0117] SDS-PAGE was performed according to the method of Laemmli, and the proteins developed on the SDS-PAGE gel were transferred to a PVDF membrane. Amyloid beta peptide was detected using a mouse anti-human amyloid beta (1-10) monoclonal antibody (BIOLEGEND clone 20.1; Cat no. 806201) as the primary antibody and a goat anti-mouse IgG polyclonal antibody (HRP-conjugated goat anti-mouse IgG, Bethyl, USA) as the secondary antibody.

[0118]

[0119] 9. Dot Blot

[0120] QV-002-S, QV-002, PV-002-S, and PV-002 were diluted to different concentrations and prepared. The PVDF membrane was soaked in 100% methanol for 1-2 minutes, and 2 μL of the prepared sample was spotted. When the PVDF membrane was dry, it was soaked in 5% skim milk and blocked at room temperature for 60 minutes. After washing once with TBST (0.1% Tween 20) solution, it was soaked in primary antibody solution and antigen-antibody reaction was performed for 150 minutes. The mouse anti-amyloid beta (1-10) monoclonal antibody was used as the primary antibody. After the primary antibody reaction, the PVDF membrane was washed three times with TBST (0.1% Tween 20) solution for 10 minutes each. After immersing in a secondary antibody solution (goat HRP-conjugated anti-mouse IgG polyclonal antibody (HRP-conjugated goat anti-mouse IgG, Bethyl, USA) for 60 minutes, the cells were washed four times for 10 minutes each with TBST (0.1% tween 20) solution.

[0121]

[0122] 10. Protein quantification

[0123] The protein concentration was measured using a BCA protein quantitation kit (Bio-Rad Laboratories, USA) using bovine serum albumin (BSA; Pierce, USA) as a standard.

[0124]

[0125] 11. Antibody reactivity analysis

[0126] A 96-well plate was coated with QV-002 or PV-002 at two-fold dilutions starting from 1 ng / well. The amount of each sample was confirmed through protein quantitative analysis before coating. For antigen coating, the plate was incubated at 37°C for 120 minutes, washed three times with PBS-T, and then resuspended in PBS-T containing 5% skim milk (BD Difco, USA). 20 (PBS containing 0.05% Tween 20) was blocked for 2 hours at room temperature. Anti-amyloid beta (1-10) monoclonal antibody of the above mouse was added to PBS-T containing 0.5% Skim milk. 20 After diluting to 1 / 5000 using , it was reacted with coated QV-002-S, QV-002, PV-002-S, PV-002 at 37℃ for 120 minutes. After washing three times with PBS-T, goat HRP-conjugated anti-mouse IgG polyclonal antibody (HRP-conjugated goat anti-mouse IgG, Bethyl, USA) was added to PBS-T containing 0.5% skim milk. 20 Diluted in a ratio of 1:5000 and reacted on a plate at 37°C for 60 minutes. The plate was washed with PBS-T 20 After washing five times, a color development reaction was performed. Color development was performed using o-phenylenediamine (Sigma, USA), and absorbance was measured at 492 nm.

[0127]

[0128] 12. Mouse Immunity and Immunogenicity Evaluation of QV-002 and PV-002

[0129] To evaluate the immunogenicity of QV-002 and PV-002, C3H / He J Yok Slc (TLR4 gene mutant; resistant to LPS) mice were used. To immunize mice with QV-002 and PV-002, the purity and concentration of the proteins were confirmed according to the protein quantitative analysis and SDS-PAGE methods described above. Mice were immunized via intramuscular injection and were immunized four times at two-week intervals. For each immunization, 10 ng, 100 ng, and 1000 ng of protein were administered intramuscularly for each group. Blood was collected through the tail vein 10 to 14 days after the third and fourth immunizations. To recover serum, mouse blood was centrifuged at 13,500 g for 10 minutes, and the supernatant was collected and stored below -60°C until antibody titer measurement. Anti-3Aβ(1-6) antibody titers and anti-Aβ(1-42) antibody titers in mouse blood were measured using enzyme-linked immunosorbent assay (ELISA) according to a published method.

[0130] The response of PV-002-immunized mice to peptide sequences was confirmed by ELISA. The peptides were prepared and confirmed by sequences of Aβ 1-42, 1-10, 2-11, 3-12, 4-13, 5-14, or 19-28. The presence or absence of a T-cell response was confirmed in splenocytes of PV-002-immunized mice. After splenocytes were treated with Aβ 1-42, 3Aβ(1-6), or HPV VLP, cells expressing interferon-gamma were measured.

[0131]

[0132] 13. Brain tissue examination and behavioral test evaluation using TG mice

[0133] APP / PS1 (ARTE10, Taconic) mice were used in this experiment. APP / PS1 mice received intramuscular injections of PBS, VLPs (or VV-002), or PV-002. The experiment began with mice aged 1–2 months, and the mice received 10 injections over approximately 9 months. Afterwards, memory improvement was confirmed in the Morris Water Maze, and the area of ​​amyloid plaques was determined through brain tissue staining. The results of the Water Maze experiment were expressed as path length and escape latency. To determine the area of ​​amyloid plaques, the mouse brains were analyzed in sagittal sections. The analyzed areas were the hippocampus, thalamus, or cortex.

[0134]

[0135] 14. Statistical Analysis

[0136] In the evaluation of immunogenicity of antibody responses, the statistical significance of differences between groups was determined using the Mann-Whitney U-test. P < 0.05 was considered a significant difference (P * < 0.05, P ** < 0.01, P *** < 0.001, P **** < 0.0001). In other experiments, the significance between groups was determined using the Student's t-test or the Kruskal-Wallis test. The statistical significance of each experiment is described in detail in the examples below.

[0137]

[0138] <Example 1> Transmission electron microscope analysis of QV-002 and PV-002

[0139] Figure 2 shows the results of staining QV-002 and PV-002 with negative staining (1% Phosphotungstic acid, PTA) and analyzing them using a transmission electron microscope at 100,000x magnification. The size of QV-002 was confirmed to be approximately 20 nm, and the size of PV-002 was confirmed to be approximately 55 nm, and both were observed to have a spherical shape. Therefore, the test results demonstrate that each sample maintained its spherical shape even after coupling and purification.

[0140]

[0141] <Example 2> Comparative analysis of amyloid beta antibody reactivity according to the type of VLPs and the type of coupled peptide (Western blot)

[0142] The manufacturing process of QV-002-S, QV-002, PV-002-S, and PV-002 is shown in Figure 1. The loading samples were prepared by dividing them into QV-002-S, QV-002, PV-002-S, and PV-002 that went through the coupling process and Qβ VLPs and HPV 11 VLPs that did not go through the coupling process. Figures 3 and 4 show the results of comparative analysis of amyloid beta antibody reactivity according to the type of VLPs. Figure 3 shows the results of comparing the amount of coupled amyloid beta peptide of QV-002 and PV-002, which are 3Aβ(1-6) coupled to Qβ VLPs and HPV11 VLPs, respectively, through Western blot. After protein quantification for Western blot analysis, QV-002 and PV-002 were loaded at 200 ng, 100 ng, and 50 ng per well, respectively, and HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the intensity of the PV-002 band for the anti-amyloid beta(1-10) antibody was higher than that of QV-002. Figure 4 shows the results of comparing the amount of coupled amyloid beta peptide of QV-002-S and PV-002-S, which coupled Aβ(1-6) to Qβ VLPs and HPV11 VLPs, respectively, through Western blot. After protein quantification for Western blot analysis, QV-002-S and PV-002-S were loaded at 200 ng, 100 ng, and 50 ng per well, and HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the reactivity of PV-002-S to anti-amyloid beta (1-10) antibody was higher than that of QV-002-S.

[0143] Figure 5 shows the final results of Western blot comparison of the amyloid beta peptide concentration according to the type of VLPs and the type of coupled peptide. QV-002-S, PV-002-S, QV-002, and PV-002 were loaded at 200 ng and 100 ng per well, respectively, and HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the reactivity to anti-amyloid beta(1-10) antibody was significantly higher in PV-002 than in QV-002, and the reaction was stronger in the order of QV-002-S < QV-002 < PV-002-S < PV-002. Therefore, it was confirmed that PV-002, which coupled 3Aβ(1-6) peptide to HPV11 VLPs in the same amount of sample, had the highest concentration of amyloid beta peptide.

[0144]

[0145] <Example 3> Comparative analysis of amyloid beta antibody reactivity according to the type of VLPs and the type of coupled peptide (dot blot)

[0146] This is the result of a dot blot test performed on the quantitative analysis of amyloid beta peptide coupled to QV-002-S / QV-002 / PV-002-S / PV-002 using a mouse anti-amyloid beta(1-10) monoclonal antibody as the primary antibody and a secondary antibody solution (goat HRP-conjugated anti-mouse IgG polyclonal antibody (HRP-conjugated goat anti-mouse IgG, Bethyl, USA)). In a comparison between QV-002-S and QV-002, it was confirmed that QV-002 linked to 3Aβ(1-6) peptide reacted slightly more highly, and no difference was observed between PV-002-S and PV-002 (Fig. 6). Overall, PV-002-S and PV-002 showed much higher reactivity than QV-002-S and QV-002 (Fig. 6). Therefore, the results in Fig. 6 indicate that the amount of peptide coupled to HPV VLPs is greater than the amount of peptide coupled to Qβ VLPs, regardless of the peptide type, and in the case of Qβ VLPs, it was confirmed that drugs coupled to 3Aβ(1-6) peptides had higher antibody reactivity than drugs coupled to Aβ(1-6) peptides.

[0147]

[0148] <Example 4> Comparative analysis of amyloid beta antibody reactivity according to concentration of QV-002 / PV-002 (ELISA)

[0149] The results of enzyme-linked immunosorbent assay (ELISA) analysis of the anti-amyloid beta (1-10) monoclonal antibody reactivity of QV-002 and PV-002 (Fig. 7). QV-002 and PV-002 were diluted 10-fold, starting from 1 ng, and coated onto each well. The ELISA reactivity comparison confirmed that PV-002 had a higher reactivity than QV-002.

[0150]

[0151] <Example 5> Comparison of immunogenicity of QV-002 and PV-002

[0152] 1. Comparison of anti-Aβ(1-42)IgG titers of QV-002 and PV-002

[0153] The results of measuring the anti-Aβ(1-42) IgG titer in serum after the third and fourth immunizations are shown in Fig. 8a and Fig. 10a. In the 10 ng administration group, no antibody titer was observed for both QV-002 and PV-002. In the group immunized with 100 ng after the third immunization, there was no significant difference (P) between QV-002 and PV-002. ** < 0.01) was confirmed, and there was a significant difference (P) between QV-002 and PV-002 in the group immunized with 1000 ng * < 0.05) was confirmed. After the fourth immunization, there was a significant difference (P ** < 0.01) was confirmed. Therefore, it was confirmed that PV-002 can induce anti-Aβ(1-42) IgG antibody titer even at a lower dose than QV-002, and that it induces high antibody titer more quickly than QV-002.

[0154] After the third immunization, the mouse seroconversion rate for anti-Aβ(1-42) IgG was measured. In the QV-002 administration group, the 100 ng administration group had a seroconversion rate of 8%, and in the 1000 ng administration group, the seroconversion rate was 46%. In contrast, in the PV-002 administration group, the 100 ng administration group had a relatively high seroconversion rate of 69%, and in the 1000 ng administration group, the seroconversion rate was 92% (Fig. 9a). After the fourth immunization, the mouse seroconversion rate for anti-Aβ(1-42) IgG was measured. In contrast, in the PV-002 administration group, the 100 ng administration group had a relatively high seroconversion rate of 62%, and in the 1000 ng administration group, the 100 ng administration group had a relatively high seroconversion rate of 92% [Fig. 11a]. Therefore, PV-002 was confirmed to induce a higher positivity for anti-Aβ(1-42) IgG than QV-002.

[0155]

[0156] 2. Comparison of anti-3Aβ(1-6)IgG titers of QV-002 and PV-002

[0157] The results of measuring the anti-3Aβ(1-6) IgG titer in serum after the third and fourth immunizations are shown in Fig. 8b and Fig. 10b. In the 10 ng administration group, no antibody titer was observed for both QV-002 and PV-002. In the group immunized with 100 ng after the third and fourth immunizations, there was no significant difference (P) between QV-002 and PV-002. **** < 0.0001) was confirmed. Therefore, PV-002 was confirmed to induce high anti-3Aβ(1-6) IgG antibody titers at a lower dose than QV-002.

[0158] After the third immunization, the mouse seroconversion rate for anti-3Aβ(1-6) IgG was measured. An 8% seroconversion rate was observed in the QV-002 100 ng group and an 85% seroconversion rate was observed in the PV-002 100 ng group (Fig. 9b). After the fourth immunization, the mouse seroconversion rate for anti-3Aβ(1-6) IgG was measured. A 15% seroconversion rate was observed in the QV-002 100 ng group and a 92% seroconversion rate was observed in the PV-002 100 ng group (Fig. 11b). Therefore, it was confirmed that PV-002 induces a much higher seroconversion rate for anti-Aβ(1-42) IgG than QV-002.

[0159]

[0160] 3. Comparison of immunogenicity of QV-002-S, QV-002, PV-002-S, and PV-002

[0161] QV-002-S is a form in which Aβ(1-6) is coupled to Qβ coat protein VLP, and QV-002 is a form in which 3Aβ(1-6) is coupled to Qβ coat protein VLP. PV-002-S is a form in which Aβ(1-6) is coupled to HPV VLP, and PV-002 is a form in which 3Aβ(1-6) is coupled to HPV VLP. The antibody titers of mice immunized three times via the intramuscular route with 100 ng / dose of each type of antigen are shown in Figure 14. The antibody titers are for Aβ 1-42, and are expressed as IQR 25%, IQR 50%, and IQR 75%. Both QV-002-S and PV-002-S showed antibody titers less than 100 (antibody titers less than 100 are expressed as 1.0 in Log10 values). PV-002 showed significantly higher antibody titers compared to the QV-002 group (P < 0.00001).

[0162] Figure 15 shows the results of antibody titer measurement after four immunizations in the above experiment. Similarly, the PV-002 immunized group showed significantly higher antibody titer than the QV-002 immunized group (P < 0.0001). These results demonstrate that the 3Aβ(1-6) sequence can induce higher levels of antibody titer against Aβ 1-42 than Aβ(1-6). Furthermore, it demonstrates that HPV VLP is a superior form of VLP for inducing antibody titer against Aβ 1-42 compared to Qβ VLP.

[0163]

[0164] 4. PV-002 vaccine reduces brain tissue Aβ plaques

[0165] After 5 or 10 immunizations with PV-002 via intramuscular injection at 80 μg / dose, Aβ plaques in sagittal sections of brain tissue were stained and compared for area. WT represents the wild-type mouse group. PEV-002 is the group administered an immune adjuvant and received aluminum hydroxide in the same manner. VV-002 is the group administered HPV VLP. Figures 16, 17, and 18 show the Aβ plaque areas in the cortex, hippocampus, and thalamus. Compared to the PBS immunization group, the group that received 10 immunizations with PV-002 showed a significant decrease in the area of ​​Aβ plaques (Kruskal-Wallis).

[0166]

[0167] 5. Memory-improving effect of PV-002 vaccine

[0168] The memory-enhancing effect of PV-002 vaccine administration was confirmed through the Morris Water Maze experiment. WT refers to the wild-type mouse group. For administration, vials containing 50 μg / dose, 100 μg / dose, 200 μg / dose, or 400 μg / dose were prepared based on human inoculation standards, and 1 / 10 of these doses were administered to mice. Accordingly, mice received doses of 5 μg / dose, 10 μg / dose, 20 μg / dose, or 40 μg / dose. Antigen immunization was administered intramuscularly. VV-002 refers to the group that received HPV VLPs at a dose of 40 μg / dose per mouse. PEV-002 refers to the group that received only aluminum hydroxide adjuvant.

[0169] Figure 19 presents the path length results from the Morris Water Maze experiment. It was confirmed that the path length decreased as the antigen dose increased. The path length of the PV-002 vaccine group was compared with the path lengths of the PEV-002 group and the VV-002 group [student t-test; P ** <0.01 (vs PEV-002), P ## < 0.01 (vs VV-002)]. The arrow indicates the path length on day 5 of the PV-002 vaccination group. Figure 20 shows the escape latency results. Similarly, the escape latency of the PV-002 vaccination group was compared with the PEV-002 group or the VV-002 group [student t-test; P * <0.05 (vs PEV-002), P # < 0.05 (vs VV-002)].

[0170]

[0171] 6. Aβ recognition by PV-002-induced antibodies

[0172] To identify the Aβ recognition sites of serum antibodies from PV-002-immunized mice, seven peptides were synthesized. A value of 0.2 or higher in the ELISA reaction was considered significant. As shown in Figure 21, serum antibodies from PV-002-immunized mice significantly recognized Aβ residues 1-42, 1-10, 2-11, and 3-12.

[0173]

[0174] 7. Confirmation of absence of T-cell response in PV-002 immunity

[0175] T-cell response to Aβ is known to cause meningoencephalitis. To confirm whether PV-002 antigen induces a T-cell response, splenocytes of PV-002-immunized mice were stimulated with each antigen to determine whether a T-cell response was present, and the results are presented in Figure 22. When stimulated with Aβ 1-42 or 3Aβ (1-6), cells expressing interferon-gamma were not observed, confirming that a T-cell response did not occur. On the other hand, when stimulated with HPV VLP, cells expressing interferon-gamma were significantly observed (Student's t-test; P ** <0.01). Therefore, it was confirmed that T-cell responses to HPV VLPs were induced by PV-002 immunization, but T-cell responses to Aβ were not induced.

[0176]

[0177] While specific aspects of the present invention have been described in detail above, it will be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and are not intended to limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A vaccine composition for preventing or treating Alzheimer's disease, comprising virus-like particles (VLPs) of papillomavirus with amyloid beta antigen attached as an active ingredient.

2. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that in paragraph 1, the VLPs of the papillomavirus are VLPs of human papillomavirus (HPV).

3. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that in the second paragraph, the HPV VLPs are HPV11 L1 VLPs purified from a transformed host cell expressing the L1 protein of HPV type 11 (HPV11).

4. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that in the first paragraph, the amyloid beta antigen is in the form of a peptide represented by the amino acid sequence of sequence number 1, which is repeated 1 to 10 times.

5. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that the peptide represented by the amino acid sequence of sequence number 1 in paragraph 4 is a peptide represented by the amino acid sequence of sequence number 2.

6. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that in paragraph 4, the amyloid beta antigen additionally includes a linker between the linked peptides.

7. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that in paragraph 6, the amyloid beta antigen is a peptide represented by the amino acid sequence of sequence number 3 or the amino acid sequence of sequence number 4.

8. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that the vaccine composition in paragraph 1 covalently attaches amyloid beta antigen to VLPs of papillomavirus.

9. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that the covalent bond in paragraph 8 is bonded through a chemical cross-linker.

10. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that in claim 9, the chemical cross-linker is succinimidyl-6-(β-maleimidopropionamido) hexanoate [succinimidyl-6-(β-maleimidopropionamido) hexanoate; SMPH].

11. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that the vaccine composition additionally comprises an adjuvant in the first paragraph.

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