Papillomavirus virus-like particle-based Anti-amyloid beta veterinary vaccine

Abstract

The present invention relates to a papillomavirus virus-like particle-based anti-amyloid beta Alzheimer's disease vaccine and to a vaccine for the purpose of preventing and treating amyloid beta-associated diseases in animals. Specifically, the present invention relates to an Alzheimer's disease vaccine in which an amyloid beta antigenic group is loaded onto virus-like particles of human papillomavirus (HPV). According to the present invention, an amyloid beta peptide fragment is linked to HPV VLPs having excellent structural and immunological properties, thereby greatly enhancing the effect of inducing antibodies against amyloid beta. In addition, it was confirmed that the vaccine prepared according to the present invention exhibits superior immunogenicity and seroconversion rate compared to conventional VLP-based Alzheimer's disease vaccines.

Description

Papillomavirus Virus-Like Particle-Based Anti-Amyloid Beta Animal Vaccine

[0001] The present invention relates to a vaccine comprising virus-like particles (VLPs) of papillomavirus loaded with an amyloid beta antigen group. Furthermore, the present invention relates to a vaccine for the prevention and treatment of amyloid beta-derived neurological diseases occurring in animals.

[0002] Dogs and cats kept as domestic pets suffer from many of the diseases commonly observed in humans. Like humans, these animals are susceptible to conditions such as cancer, aging, obesity, heart disease, diabetes, and kidney disease. Alzheimer's disease is an age-related illness characterized by entanglement of brain plaques, brain atrophy, and cognitive impairment. While Alzheimer's disease was previously thought to be unique to humans, this understanding had significant limitations as it was based on observations made within limited model systems. With advancements in medical and pharmaceutical technologies, human life expectancy has increased, accompanied by a corresponding extension of the lifespan of companion animals. Recently, with the extended lifespan of companion animals, approaches to understanding disease mechanisms and treatments in animals are being presented as highly effective models for understanding human aging. Given that amyloid beta, which constitutes the nervous systems of dogs, cats, and various other animals, exhibits high homology to human amyloid beta sequences, the development of vaccines and therapies targeting amyloid beta holds significant utility for treating and preventing neurological diseases in these animals as well as in humans.

[0003] 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 approximately 152 million) by 2050. Alzheimer's disease (AD) is one of the major causes of progressive dementia. Since Alzheimer's disease was first reported in 1907, numerous studies have been conducted on the mechanisms of onset and the progression of the disease. Recently, therapeutic antibodies utilizing passive immunity have shown positive effects in clinical trials, and while some candidate antibodies have received regulatory approval, the range of drugs capable of effectively treating Alzheimer's disease remains very limited.

[0004] Alzheimer's disease is a neurodegenerative disease characterized by pathological features such as the accumulation of amyloid beta, the formation of amyloid plaques, and the development of neurofibrillary tangles. Amyloid beta peptides, the main component of amyloid plaques, are formed from the proteolysis of a larger glycoprotein called amyloid precursor protein (APP). The amyloid precursor protein, a transmembrane protein, is first cleaved by α-secretase (non-amyloidogenesis pathway) or β-secretase (amyloidogenesis pathway) to produce membrane-bound α- / β- C-terminal fragments (CTFs). Subsequently, these α- / β- C-terminal fragments are degraded by γ-secretase into P3 (3 kDa) and amyloid beta peptides (4 kDa), respectively. Therefore, the process of amyloid formation in amyloid precursor proteins is driven by the sequential degradation of amyloid beta by β- and γ-secretases at the N and C terminals, respectively. Amyloid beta monomers generated from amyloid precursor proteins accumulate and self-assemble into soluble oligomers; however, because they are produced with different molecular weights and morphologies, they exhibit diverse characteristics. According to research, amyloid beta oligomers are known to be neurotoxic aggregates that induce functional neuronal death, cognitive impairment, and dementia, playing a significant role in the onset and development of Alzheimer's disease.

[0005] Therefore, considering that the accumulation of amyloid beta is a major cause of neuronal death in Alzheimer's disease, it has been believed that inhibiting the formation of amyloid beta oligomers or removing amyloid beta could contribute to the treatment of the disease. Various experimental models have confirmed that anti-amyloid beta immunotherapy is a key method for promoting amyloid beta removal. Anti-amyloid beta immunotherapy can be classified into active immunity (vaccines) and passive immunity (exogenous antibodies). Active immunity is a method that induces the body to generate endogenous antibodies against amyloid beta by administering amyloid beta or its fragments. 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 for passive immunotherapy can lower amyloid beta levels in the central nervous system (CNS), but caution is required regarding adverse reactions due to amyloid-related abnormal imaging findings (ARIA), which are the most common side effect of this class of drugs.

[0006] In a clinical trial administering AN-1792, the first active amyloid beta immunotherapy (vaccine), it was confirmed that brain amyloid beta plaques decreased and antibodies against amyloid beta were induced in some of the vaccinated patients. Even though AN-1972 was administered together with the adjuvant QS-21, antibodies were produced in only 20% of the vaccinated group. Additionally, meningoencephalitis was detected in 6% of the vaccinated individuals. These adverse effects are presumed to be caused by amyloid beta-specific T cell responses (Th1-type CD4). Therefore, to develop an effective Alzheimer's beta vaccine, strategies must be applied to induce high antibody titers while simultaneously avoiding amyloid beta-specific T cell responses.

[0007] VLPs have the advantage of being able to induce high levels of immune response and antibody titers. Because VLPs possess a tertiary structure unique to viruses, they are effectively recognized by immune cells, which can be utilized as a foundational technology for developing effective vaccines. Meanwhile, to avoid T cell responses, N-terminal peptide fragments containing short or fragmented amyloid beta or B cell epitopes can be used. CAD106, a second-generation amyloid beta vaccine, is a VLP-based vaccine composed of amyloid beta (1-6) peptide fragments bound to bacteriophage Q beta coat protein VLPs (hereinafter Qβ VLPs). In fact, CAD106 effectively induced antibodies against amyloid beta in animal models without activating amyloid beta-specific T cell responses.

[0008] Meanwhile, research on VLP-based vaccines with superior antibody induction capabilities is necessary for the development of vaccines for the prevention and treatment of Alzheimer's disease.

[0009] The objective of the present invention is to provide a vaccine composition for the prevention or treatment of Alzheimer's disease in animals comprising virus-like particles (VLPs) of papillomavirus attached with amyloid beta antigens as an active ingredient.

[0010] To achieve the above objective, the present invention provides a vaccine composition for preventing or treating Alzheimer's disease comprising VLPs of papillomavirus attached with amyloid beta antigen as an active ingredient, 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.

[0011] The present invention relates to a papillomavirus virus-like particle-based anti-amyloid beta Alzheimer's disease vaccine. Specifically, the present invention relates to an Alzheimer's disease vaccine in which an amyloid beta antigen group is loaded onto a virus-like particle of Human Papillomavirus (HPV). According to the present invention, by attaching an amyloid beta peptide fragment to HPV VLPs that have excellent structural and immunological properties, the antibody induction effect against amyloid beta can be significantly enhanced. Furthermore, it was confirmed that the vaccine produced according to the present invention exhibits superior immunogenicity and positivity compared to existing VLPs-based Alzheimer's disease vaccines.

[0012] Figure 1 shows the process of a method for manufacturing a virus-like particle-based vaccine for bacteriophage Q beta virus (hereinafter “QV-002”) and a method for manufacturing a virus-like particle-based vaccine for human papillomavirus (hereinafter “QV-002”). The manufacturing method for QV-002 and QV-002-S / PV-002 and PV-002-S is the same, and only the type of peptide [Aβ(1-6) / 3Aβ(1-6) peptide] used in the coupling process is different.

[0013] Figure 2 shows the transmission electron microscope results of QV-002 and PV-002, respectively, obtained by staining with negative staining (1% PTA) and observing at 100,000x magnification. Bar represents 50 nm. It shows that each sample maintains a spherical shape after purification and coupling are completed.

[0014] Figure 3 shows the results of comparing the amounts of amyloid beta peptides coupled to QV-002 and PV-002 via Western blot. For Western blot analysis, after performing protein quantification, QV-002 and PV-002 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 to amyloid beta antibodies was found to be significantly higher than that of QV-002. In the top panel of the results, M indicates the marker. The numbers in the left and right panels of the results indicate the molecular weight.

[0015] Figure 4 shows the results of comparing the amounts of amyloid beta peptides coupled to QV-002-S and PV-002-S via Western blot. For Western blot analysis, after performing protein quantification, 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 to the amyloid beta antibody was found to be significantly higher than that of QV-002-S.

[0016] Figure 5 shows the results of comparing the amounts of coupled amyloid beta peptides of QV-002-S, PV-002-S, QV-002, and PV-002 via Western blot. After performing 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, while HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, PV-002 showed the highest reactivity to the amyloid beta antibody.

[0017] Figure 6 relates to the measurement of the density of coupled amyloid beta peptides of QV-002-S / QV-002 / PV-002-S / PV-002, and is the result of performing a dot blot test using an anti-amyloid beta (1-10) monoclonal antibody. In the comparison between QV-002-S and QV-002, it was confirmed that QV-002, which is linked to the 3Aβ (1-6) peptide, reacted slightly higher, and PV-002-S and PV-002 showed much higher reactivity compared to QV-002-S and QV-002. Therefore, the results of Figure 6 show that the amount of peptides coupled to HPV VLPs is greater than the amount of peptides coupled to Qβ VLPs, regardless of the type of peptide.

[0018] Figure 7 shows the results of analyzing the anti-amyloid beta (1-10) monoclonal antibody reactivity of QV-002 and PV-002 using an enzyme-linked immunosorbent assay (ELISA). Antibody reactivity proportional to the coating amount was observed for QV-002 and PV-002. In addition, PV-002 showed higher antibody reactivity compared to QV-002.

[0019] Figures 8a and 8b show the results of analyzing immunogenicity after administering vaccine antigens at doses of 10 ng, 100 ng, and 1000 ng intramuscularly three times at 2-week intervals for each mouse group. Figure 8a shows the results of measuring serum anti-Aβ(1-42) IgG titers. In the 100 ng and 1000 ng administration groups, the antibody titer of the PV-002 group was measured to be significantly higher than that of the QV-002 group. Figure 8b shows the results of measuring serum anti-3Aβ(1-6) IgG titers. In the 100 ng administration group, the antibody titer of the PV-002 group was observed to be significantly higher than that of the QV-002 group.

[0020] Figures 9a and 9b show the positivity rates after the third intramuscular administration of vaccine antigens at concentrations of 10 ng, 100 ng, and 1000 ng for each mouse group. As can be seen in Figure 9a, the positivity rates of PV-002 in the 100 ng and 1000 ng administration groups were higher than those of the QV-002 group. Similarly, in Figure 9b, the positivity rate of PV-002 in the 100 ng administration group was higher than that of the QV-002 group.

[0021] Figures 10a and 10b show the results of analyzing immunogenicity after administering vaccine antigens of 10 ng, 100 ng, and 1000 ng intramuscularly four times to mouse groups. Figure 10a shows the results of measuring serum anti-Aβ(1-42) IgG titers. In the 100 ng administration group, the antibody titer of the PV-002 group was observed to be significantly higher than that of the QV-002 group. Figure 10b shows the results of measuring serum anti-3Aβ(1-6) IgG titers. In the 100 ng administration group, the antibody titer of the PV-002 group was observed to be significantly higher than that of the QV-002 group.

[0022] Figures 11a and 11b show the positivity rates after four intramuscular administrations of vaccine antigens at doses of 10 ng, 100 ng, and 1000 ng for each mouse group. As can be seen in Figure 11a, the positivity rate of the PV-002 group was higher than that of the QV-002 group in both the 100 ng and 1000 ng administration groups. Similarly, in Figure 11b, the positivity rate of PV-002 in the 100 ng administration group was higher than that of QV-002.

[0023] FIG. 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. The vector map of FIG. 12 is a plasmid prepared to express the Qβ coat protein, and is a schematic diagram prepared by inserting the sequence of the Qβ major coat protein (402 bp) into the pCDF-1b vector using restriction enzymes NcoI and AvrII.

[0024] FIG. 13 is the amyloid beta sequence used for coupling in the present invention. It is a peptide made by adding some amino acids to 1-6, that is, 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) included between the Aβ (1-6) repeat sequences and glycine-glycine-cysteine ​​(Gly-Gly-Cys) added to the C-terminus.

[0025] Figure 14 shows the results of analyzing immunogenicity after administering 100 ng of vaccine antigen intramuscularly three times at 2-week intervals for each mouse group. QV-002-S is a vaccine antigen in which Aβ(1-6) is coupled to the Qβ coat protein, and QV-002 is a form in which 3Aβ(1-6) is coupled to the Qβ coat protein. Similarly, PV-002-S is a vaccine antigen in which Aβ(1-6) is coupled to the HPV VLP, and PV-002 is a form in which 3Aβ(1-6) is coupled to the HPV VLP. Mice inoculated with PV-002 showed a significantly higher immune response compared to QV-002 (IQR 50: 1.0 vs 3.2; P < 0.00001). No antibody titers were observed in either the QV-002-S or PV-002-S inoculation groups (titers less than 100 were counted as 1.0).

[0026] Figure 15 shows the results of analyzing immunogenicity after administering 100 ng of vaccine antigen intramuscularly to the mice four times at 2-week intervals. Significantly higher antibody titers were observed in the PV-002 inoculation group compared to the QV-002 inoculation group (IQR 50: 1.5 vs 3.2; P < 0.0001).

[0027] Figure 16 shows the results of analyzing the area of ​​amyloid beta accumulated in the cerebral cortex through brain tissue analysis after 5 or 10 intramuscular administrations of the PV-002 vaccine at 80 μg / dose. A significant reduction was observed in the group administered the PV-002 vaccine 10 times compared to the PBS vaccination group.

[0028] Figure 17 shows the results of analyzing 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 vaccination group.

[0029] Figure 18 shows the results of analyzing 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 vaccination group.

[0030] Figure 19 shows the path length of the results confirming the cognitive improvement effect of mice vaccinated with the PV-002 vaccine using the Morris Water Maze experiment. 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. One-tenth of these doses were administered intramuscularly to the mice 10 times. Thus, the mice received the PV-002 vaccine at doses of 5 μg / dose, 10 μg / dose, 20 μg / dose, or 40 μg / dose. As a result, the path length was observed to decrease as the vaccine dosage increased.

[0031] 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 appeared similar to the latency of wild-type mice.

[0032] Figure 21 shows the results of ELISA analysis of the Aβ sequences to which antibodies induced in mice immunized with PV-002 react. In the ELISA reaction, a value of 0.2 or higher was considered a significant reaction. It was confirmed that the PV-002-induced antibodies recognize Aβ 1-42, Aβ 1-10, Aβ 2-11, and Aβ 3-12.

[0033] Figure 22 shows the results of confirming whether immune cells of mice immunized with PV-002 elicit a T-cell response using ELISPOT. It was confirmed that when splenocytes of mice immunized with PV-002 were stimulated with Aβ 1-42 or 3Aβ(1-6) peptides, there was no cellular response expressing interferon-gamma. On the other hand, it was confirmed that when stimulated with HPV VLP, a cellular response expressing interferon-gamma occurred. The Aβ(1-6) sequence loaded on PV-002 is a B-cell epitope, demonstrating that PV-002 immunization does not induce a T-cell response, which carries a risk of side effects, and selectively induces a B-cell-induced antibody response.

[0034] Figures 23a and 23b show the results of analyzing antibody reactions after administering 100 μg, 400 μg, and 800 μg of PV-002 antigen intramuscularly to Beagle dogs in each group, and collecting blood samples before immunization (0d), after the third immunization (56d), and after the fourth immunization (84d). Figure 23a shows the results of measuring the anti-Aβ(1-42) IgG titer in serum. In all groups, the antibody titer increased compared to before immunization, and in particular, in the 100 μg and 800 μg groups, the antibody titer after the fourth immunization (84d) was observed to be significantly higher than before immunization (0d). Figure 23b shows the results of measuring the anti-3Aβ(1-6) IgG titer in serum. In all groups, the antibody titer after the fourth immunization (84d) was observed to be significantly higher than before immunization (0d).

[0035] Figures 24a and 24b confirmed the seroconversion rates in the serum of beagle dogs by group in the above test. As can be seen in Figure 24a, the seroconversion rates of anti-Aβ(1-42) antibodies increased after immunization in all 100 μg, 400 μg, and 800 μg administration groups. Likewise, in Figure 24b, it was found that the seroconversion rates of anti-3Aβ(1-6) antibodies increased after immunization in all 100 μg, 400 μg, and 800 μg administration groups.

[0036] Figures 25a and 25b show the results of analyzing antibody responses in cerebrospinal fluid (CSF) collected before (0d) and after (84d) the fourth immunization, following intramuscular administration of 100 μg, 400 μg, and 800 μg of PV-002 antigen to Beagle dogs by group. Figure 25a shows the results of measuring the anti-3Aβ(1-6) IgG titer in the CSF. It was observed that the antibody titer increased in all groups compared to before immunization. In Figure 25b, the anti-3Aβ(1-6) IgG positivity rate in the CSF was measured, and it was found that the positivity rate increased in all groups compared to before immunization. This demonstrates that the antibody titer against anti-3Aβ(1-6) in the CSF increases when the PV-002 vaccine is immunized via the intramuscular route.

[0037] One of the VLP-based vaccines currently approved for use in humans is the Human Papillomavirus (HPV) vaccine. HPV VLP vaccines are known to induce a sustained and high-titer neutralizing antibody response against HPV. Therefore, it was expected that an anti-amyloid beta vaccine, in which an amyloid beta fragment is bound 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 that has excellent antibody induction ability when an amyloid beta (1-6) sequence or a peptide fragment in which the sequence is repeated three times is loaded onto HPV VLPs.

[0038] According to the general knowledge, peptides with short sequences alone are not suitable for inducing an antibody response. In particular, in the case of peptides derived from autoantigens such as amyloid beta, inducing an antibody response is even more difficult due to immune tolerance. To solve these problems, various types of delivery vehicles have been used in previously known methods to increase the immune response. The inventors of the present invention endeavored to develop a vaccine antigen loaded with amyloid beta sequences 1-6 onto HPV VLPs in order to develop an Alzheimer's disease vaccine targeting amyloid beta. As a result, it was discovered that the present vaccine (PV-002), in which amyloid beta 1-6 peptides are linked to HPV VLPs, enables the loading of peptide antigen groups at a higher level compared to Qβ VLP-based vaccines of the previously known method. Furthermore, it was discovered that this feature enables the induction of an enhanced level of anti-amyloid beta antibodies, thereby completing the present invention.

[0039]

[0040] The present invention provides a vaccine composition for the prevention or treatment of Alzheimer's disease comprising virus-like particles (VLPs) of papillomavirus attached with amyloid beta antigens as an active ingredient.

[0041] Preferably, the above papillomavirus VLPs may be human papillomavirus (HPV) VLPs, and more preferably, the above HPV VLPs may be HPV11 L1 VLPs purified from transformed host cells expressing the L1 protein of HPV type 11 (HPV11), but are not limited thereto.

[0042]

[0043] In the present invention, "HPV L1 protein" refers to a major protein that constitutes the capsid of HPV and is expressed from the L1 gene of HPV. The L1 protein has the characteristic of self-assembling into VLPs under suitable conditions, either together with the L2 protein, which is a minor protein constituting the capsid, or as the L1 protein alone.

[0044] 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.

[0045] According to a preferred embodiment of the present invention, the yeast cell may be Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces sp., Schizosaccharomyces pombe, Pichia Pastoris, or Hansenula polymorpha, but is not limited thereto.

[0046] In the present invention, a transformed host cell expressing HPV L1 protein refers to a host cell transformed with an expression vector that successfully expresses 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 HPV L1 protein according to the present invention can be easily prepared using methods known in the art.

[0047]

[0048] In the present invention, "Qβ coat protein" refers to a major protein that constitutes the capsid of Bacteriophage Qβ, expressed from the major coat protein gene of Qβ. The coat protein has a size of 133 amino acids and has the characteristic of self-assembling into VLPs under suitable conditions. This was used for comparison with the HPV VLPs of the present invention.

[0049]

[0050] 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 virus-like particles may completely lack a DNA or RNA genome, or, in the case of virus-like particles containing a viral capsid protein, may undergo spontaneous self-assembly.

[0051] The above-mentioned 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 specific host cell using a recombinant DNA molecule encoding the structural protein and the antigen recognition site, and then culturing it; the protein expressed within the cell can then be assembled on the cell surface and released into the culture supernatant. In this case, since the surface protein contained in the virus-like particles maintains its natural form without undergoing an immobilization process, it can induce a desired immune response against a specific pathogen within the organism.

[0052] The transformed host cells can be cultured under batch, feed-batch, or continuous fermentation conditions, and since the host cells can express the virus-like particles by transformation, the virus-like particle protein can be obtained from the cultured host cells.

[0053]

[0054] In the present invention, VLPs can be purified by performing chromatography on the lysate of the host cells. The chromatography available in the present invention is known in the art and is not limited thereto, but, for example, ion exchange chromatography such as cation exchange chromatography or anion exchange chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and affinity chromatography may be used. Since the substance to be separated and purified in the present invention is a protein, ion exchange chromatography, which 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, a type of cation exchange chromatography, and cation exchange chromatography.

[0055]

[0056] In the present invention, the amyloid beta antigen may be in the form of a peptide represented by the amino acid sequence of SEQ ID NO. 1 repeated 1 to 10 times, but is not limited thereto.

[0057] Preferably, the peptide (XXEFRHXX) represented by the amino acid sequence of SEQ ID NO. 1 may be the peptide (DAEFRH) represented by the amino acid sequence of SEQ ID NO. 2, but is not limited thereto. The "X" in SEQ ID NO. 1 does not refer to a specific amino acid, but rather means that it may not contain any amino acid or may contain any amino acid.

[0058] 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.

[0059]

[0060] In the present invention, “Aβ(1-6)” is a peptide in which GGC is added to the C-terminus of 1-6 of an amyloid beta peptide sequence, and is a peptide described by the amino acid sequence indicated 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 an amyloid beta peptide sequence is repeated twice, and GGC is added to the C-terminus of 1-6 of an amyloid beta peptide sequence, and is a peptide described by the amino acid sequence indicated by SEQ ID NO. 4 (Fig. 13).

[0061]

[0062] In the present invention, “PV-002” is a substance in which 3Aβ(1-6) is coupled to HPV VLPs, and “PV-002-S” is a substance in which Aβ(1-6) is coupled to HPV VLPs. “QV-002” is a substance in which 3Aβ(1-6) is coupled to Qβ VLPs, and “QV-002-S” is a substance in which Aβ(1-6) is coupled to Qβ VLPs (identical to CAD106) [J Neurosci. 2011 Jun 22;31(25):9323-31].

[0063]

[0064] In the present invention, the vaccine composition may attach an amyloid beta antigen to VLPs of papillomavirus by covalent bonding, and the covalent bonding may be formed through a chemical cross-linking linker, and preferably, the chemical cross-linking linker may be succinimidyl-6-(β-maleimidopropionamido) hexanoate [SMPH], but is not limited thereto.

[0065] The SMPH used in the present invention is a linker that has an NHS (N-hydroxysuccinimide) ester, which can bind to an amine, and also has a maleimide, which can bind to a molecule containing a sulfhydryl group. The NHS ester reacts with a primary amine at pH 7-9 to form an amide bond, and the maleimide reacts with a sulfhydryl group at pH 6.5-7.5 to form a thioether bond.

[0066]

[0067] In the present invention, the vaccine composition may additionally include an adjuvant, but is not limited thereto.

[0068]

[0069] 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 VLPs of Papillomavirus attached with an amyloid beta antigen exhibiting antigenicity against Alzheimer's disease.

[0070] Specifically, the vaccine composition may comprise any one selected from the group consisting of pharmaceutically acceptable carriers, adjuvants, immunostimulators, and combinations thereof.

[0071] The above-mentioned pharmaceutically acceptable carriers are known in the art and include proteins, sugars, etc. The carriers may be aqueous solutions 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 buffer media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactic acid-treated Ringer's, or fixative oils. Intravenous carriers include electrolyte supplements, liquids, and nutritional supplements, such as those based on Ringer's dextrose. They may additionally include antimicrobial agents, antioxidants, chelating agents, inert gases, etc., as preservatives and other additives. Preservatives may include, but are not limited to, formalin, thimerosal, neomycin, polymyxin B, and amphotericin B.

[0072] The above vaccine composition may further include 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, full Freund preservatives, incomplete Freund preservatives, saponins, mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, fluuron polyols, polyvalent anions, peptides, oil or hydrocarbon emulsions, keyhollympet hemocyanin, dinitrophenol, etc.

[0073] The above vaccine composition may further include an immunostimulant. The immunostimulant may include artificially synthesized levamisole, isoprenosine, and cytokines. Examples of cytokines include interferon-α, interleukin-2, GM-CSF, GCSF, etc. The above vaccine composition may be in a form containing the virus-like particles or a concentrate thereof, or may be used in the form of the transformed host cells themselves or a dried powder of the transformed cells. Additionally, the above vaccine composition may be used in combination with other foods or food ingredients and may be used appropriately according to conventional methods.

[0074] The above vaccine composition may further include 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, extenders, carboxypolymethylene, bacterial cell walls, derivatives of bacterial cell walls, bacterial vaccines, animal poxvirus proteins, viral subviral particle adjuvants, cholera toxins, N, N-dioctadecyl-N',N'-bis(2-hydroxyethyl)-propanediamine, monophosphoryl lipid A, dimethyldioctadecyl-ammonium bromide, and mixtures thereof.

[0075] The above vaccine composition may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, as well as sterile injectable solutions, according to conventional methods. When formulating, diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants may be used together.

[0076] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and in these solid dosage forms, at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., may be used together with the lecithin-like emulsifier. In addition, a lubricant such as magnesium styrate or talc may be used in addition to the excipients.

[0077] Liquid preparations for oral administration may include suspensions, liquid formulations, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients, such as humectants, sweeteners, flavorings, and preservatives, may be used.

[0078] Sterile aqueous solutions, water-insoluble preparations, suspensions, emulsions, and lyophilized preparations may be used for parenteral administration. Water-insoluble preparations and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleates.

[0079] The present invention is described in detail below according to embodiments that do not limit the invention. It should be understood that the following embodiments of the present invention are merely for the purpose of embodying the invention and do not limit or restrict the scope of the rights of the present invention. Accordingly, anything that can be easily inferred by a person skilled in the art to which the present invention pertains from the detailed description and embodiments of the present invention is interpreted as falling within the scope of the rights of the present invention.

[0080]

[0081] <Experimental Example>

[0082] The following experimental examples are intended to provide experimental examples that are commonly applied to each embodiment according to the present invention.

[0083]

[0084] 1. Cell culture

[0085] In the present invention, to express the bacteriophage Q-beta (Qβ) coat protein (133 amino acids), Qβ coat protein DNA (402 bp) was synthesized, and the synthesized DNA was cloned into a pCDF-1b plasmid vector (Fig. 12). After introducing the Qβ coat protein DNA into the plasmid using restriction enzymes NcoI and AvrII, it was transformed into Escherichia coli (Competent cell BL21), and the transformed cells were selected using streptomycin. In the present invention, the transformed host cell expressing the Qβ coat protein refers to a host cell selected after being transformed with an expression vector that successfully expresses the Qβ coat protein. The selected cells were cultured with shaking in 10 mL of LB medium at 37°C for 16 hours, and the culture medium was inoculated into 500 mL of LB for 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 additionally culturing at 30°C for 6 hours.

[0086]

[0087] 2. Cell disruption

[0088] Cultured Qβ coat protein-expressing cells were mixed with lysis buffer [1X PBST (0.1% Triton X-100) pH 7.2]. The cell mixture was then mixed with 0.5 mm glass beads (Biospec Product, USA) and vortexed to lyse the cells. Cell residue was removed by centrifugation at 12,000 g for 15 minutes.

[0089]

[0090] 3. Primary chromatography of Qβ VLPs

[0091] The primary purification of Qβ VLPs was performed by dialysis of proteins in the cell lysate followed by size exclusion chromatography. For size exclusion chromatography, Superose 6 prep grade resin and a Tricorn 10 / 300 (Cytiva, USA) column were used. The lysate was dialyzed with 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.

[0092] The resin was washed by flowing a mobile phase five times its volume, and proteins were separated and eluted by size. Among the eluted fractions, the fraction containing Qβ-coat protein was selected and collected via SDS-PAGE analysis. The eluent containing Qβ-coat protein was concentrated using an Amicon Ultra (Millipore, USA) and then dialyzed in dialysis buffer for 12 to 18 hours.

[0093]

[0094] 4. Secondary chromatography of Qβ VLPs

[0095] The solution obtained after completing dialyzing following the above primary chromatography was subjected to secondary purification via size exclusion chromatography. The resin used for the secondary chromatography was the same as that used in the primary chromatography. The resin was equilibrated with a mobile phase identical to the dialysis buffer before loading the sample. The resin was washed by flowing a mobile phase five times its volume, and proteins were eluted by separating them by size. Among the eluted fractions, the fraction containing Qβ-coat protein was selected and collected via SDS-PAGE analysis. The eluent containing Qβ-coat protein was concentrated to a concentration of 1 mg / mL using an Amicon Ultra (Millipore, USA).

[0096]

[0097] 5. Coupling

[0098] 5-1. Qβ VLPs-SMPH coupling

[0099] SMPH (Sigma / USA) was dissolved in DMSO and added to coupling buffer-1 [phosphate-buffered saline (PBS) containing 2 mM EDTA (pH 8.0)] to a concentration of 10 mg / mL. The Qβ VLPs and the prepared SMPH solution were reacted using a tube rotator at room temperature under light protection for 30 to 60 minutes to reach 10% of the volume of the Qβ VLPs. The reaction-completed solution was dialyzed in coupling buffer-2 [phosphate buffer containing 0.325 M NaCl and 0.01% tween 80 (pH 7.2)] for 12 to 18 hours.

[0100]

[0101] 5-2. Qβ VLPs - SMPH - Aβ(1-6) or 3Aβ(1-6) peptide coupling

[0102] Aβ(1-6) or 3Aβ(1-6) peptide (5 mg / mL) was mixed into the above-mentioned dialyzed solution to a volume of 10% of the completed solution. The reaction was carried out using a tube rotator while shielding from light at room temperature for 30 to 60 minutes. The completed solution was dialyzed in coupling buffer-2 for 12 to 18 hours. The dialyzed solution was filtered through a 0.22 μm PES syringe filter and stored at -60℃ or below.

[0103]

[0104] 5-3. HPV11 VLPs-SMPH coupling

[0105] HPV VLPs are prepared according to previously disclosed methods. SMPH (Sigma / USA) is dissolved in DMSO and added to coupling buffer-1 to a concentration of 10 mg / mL. HPV11 VLPs and the prepared SMPH solution are reacted using a tube rotator at room temperature in the dark for 30 to 60 minutes to reach 10% of the volume of HPV11 VLPs. The reaction-completed solution is dialyzed in coupling buffer-2 for 12 to 18 hours.

[0106]

[0107] 5-4. HPV11 VLPs - SMPH - Aβ(1-6) or 3Aβ(1-6) peptide coupling

[0108] Aβ(1-6) or 3Aβ(1-6) peptide (5 mg / mL) was mixed into the above-mentioned dialyzed solution to a volume of 10% of the completed solution. The reaction was carried out using a tube rotator while shielding from light at room temperature for 30 to 60 minutes. The completed solution was dialyzed in coupling buffer-2 for 12 to 18 hours. The dialyzed solution was filtered through a 0.22 μm PES syringe filter and stored at -60℃ or below.

[0109]

[0110] 6. Tertiary Chromatography

[0111] 6-1. Chromatography of QV-002

[0112] The solution prepared above was purified by size exclusion chromatography. The resin used for chromatography was the same as the resin 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 mobile phase with a volume five times that of the resin, and proteins were eluted by separating them by size. Among the eluted fractions, the fraction containing QV-002 was selected and collected through SDS-PAGE analysis.

[0113]

[0114] 6-2. Chromatography of PV-002

[0115] The solution prepared above was purified by size exclusion 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 mobile phase with a volume five times that of the resin, and proteins were eluted by separating them by size. Among the eluted fractions, the fraction containing PV-002 was selected and collected through SDS-PAGE analysis.

[0116]

[0117] 7. Transmission Electron Microscopy Analysis

[0118] QV-002 and PV-002 were adsorbed onto a carbon-coated grid and stained with 1% phosphotungstic acid for about 1 minute. Transmission electron microscope images were taken at 100K magnification using TEM 80 kV (JEM1010, Japan).

[0119]

[0120] 8. Western blot

[0121] SDS-PAGE was performed according to Laemmli's method, and proteins developed on the SDS-PAGE gel were transferred to a PVDF membrane. Amyloid beta peptides were 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 HRP-conjugated anti-mouse IgG polyclonal antibody (HRP-conjugated goat anti-mouse IgG, Bethyl, USA) as the secondary antibody.

[0122]

[0123] 9. Dot Blot

[0124] QV-002-S, QV-002, PV-002-S, and PV-002 were prepared by diluting them to different concentrations. After immersing the PVDF membrane in 100% methanol for 1-2 minutes, 2 μL of the prepared samples were spotted. Once the PVDF membrane dried, it was immersed in 5% skim milk and blocked at room temperature for 60 minutes. After washing once with TBST (0.1% tween 20) solution, it was immersed in the primary antibody solution and subjected to an antigen-antibody reaction for 150 minutes. The anti-amyloid beta (1-10) monoclonal antibody of the above mouse was used as the primary antibody. After the primary antibody reaction was completed, the PVDF membrane was washed three times with TBST (0.1% tween 20) solution for 10 minutes each. After reacting in the secondary antibody solution (goat HRP-conjugated anti-mouse IgG polyclonal antibody (HRP-conjugated goat anti-mouse IgG, Bethyl, USA) for 60 minutes, the sample was washed 4 times for 10 minutes with TBST (0.1% tween 20) solution.

[0125]

[0126] 10. Protein Quantification

[0127] Protein concentration was measured using a BCA protein quantification kit (Bio-Rad Laboratories, USA) with bovine serum albumin (BSA; Pierce, USA) as the standard.

[0128]

[0129] 11. Antibody Reactivity Analysis

[0130] 96-well plates were coated with QV-002 or PV-002 diluted twofold starting from 1 ng / well; the amount of each sample was confirmed via protein quantification prior to coating. For antigen coating, the plates were incubated at 37°C for 120 minutes, washed three times with PBS-T, and then coated with PBS-T containing 5% skim milk (BD Difco, USA). 20 Blocked for 2 hours at room temperature with PBS (containing 0.05% tween 20). The anti-amyloid beta (1-10) monoclonal antibody of the above mouse was blocked with PBS-T containing 0.5% skim milk. 20 After diluting to 1 / 5000 using [method], the coated QV-002-S, QV-002, PV-002-S, and PV-002 were reacted at 37°C for 120 minutes. After washing three times with PBS-T, the goat HRP-conjugated anti-mouse IgG polyclonal antibody (HRP-conjugated goat anti-mouse IgG, Bethyl, USA) was reacted with PBS-T containing 0.5% skim milk. 20 It was diluted to a ratio of 1:5000 and reacted on a plate at 37°C for 60 minutes. The plate was then treated with PBS-T 20 After washing five times, a color reaction was performed. Color development was carried out using o-phenylenediamine (Sigma, USA), and absorbance was measured at 492 nm.

[0131]

[0132] 12. Evaluation of mouse immunology and immunogenicity of QV-002 and PV-002

[0133] C3H / He J Yok Slc (TLR 4 gene mutation; possessing resistance to LPS) mice were used to evaluate the immunogenicity of QV-002 and PV-002. To immunize mice with QV-002 and PV-002, the purity and concentration of the proteins were verified according to the protein quantification and SDS-PAGE methods known above. Mice were immunized via intramuscular injection, administered four times at 2-week intervals. For the first immunization, 10 ng, 100 ng, and 1000 ng of protein were administered intramuscularly for each group. Blood was collected via the tail veins of mice 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 at -60°C or below until antibody titer measurement. Anti-3Aβ (1-6) antibody titers and anti-Aβ (1-42) antibody titers in mouse blood were measured using an enzyme-linked immunosorbent assay (ELISA) according to a known method.

[0134] The response of PV-002 immune mice to peptide sequences was confirmed by ELISA. The peptides prepared and tested were Aβ sequences 1-42, 1-10, 2-11, 3-12, 4-13, 5-14, or 19-28. The presence or absence of T-cell response was confirmed in the splenocytes of PV-002 immune mice. After treating splenocytes with Aβ 1-42, 3Aβ(1-6), or HPV VLP, cells expressing interferon-gamma were measured.

[0135]

[0136] 13. Evaluation of Brain Tissue Examination and Behavioral Experiments Using TG Mice

[0137] APP / PS1 (ARTE10, Taconic) mice were used in this experiment. APP / PS1 mice were administered PBS, VLP (or VV-002), or PV-002 via the intramuscular route. The experiment began with mice aged 1 to 2 months, who received 10 administrations over approximately 9 months. Subsequently, the effect on memory improvement was assessed using the Morris Water Maze, followed by the determination of amyloid plaque area through brain tissue staining. The results of the Water Maze experiment were expressed as path length and escape latency. To determine the amyloid plaque area, the mouse brains were analyzed in sagittal sections. The regions analyzed were the hippocampus, thalamus, or cortex.

[0138]

[0139] 14. Evaluation of Immunity and Immunogenicity of PV-002 in Beagle Dogs

[0140] Beagle dogs were used to evaluate the immunogenicity of PV-002. To immunize the beagle dogs with PV-002, the purity and concentration of the protein were confirmed according to the protein quantification analysis and SDS-PAGE methods known above. The beagle dogs were immunized via intramuscular injection and administered four times at three-week intervals. For each group, 0.1 mg, 0.4 mg, and 0.8 mg of protein were administered intramuscularly. Blood samples were collected three times: before administration (Day 0), two weeks after the third administration, and at the end of the study (Day 56 and Day 84), and cerebrospinal fluid (CSF) was collected twice: before administration (Day 0) and at the end of the study (Day 84). To collect blood, approximately 2 mL was collected from the jugular vein into a vacutainer tube containing a clot activator, left at room temperature for about 15 to 20 minutes to coagulate, and then centrifuged at 3,000 rpm for 15 minutes. To collect cerebrospinal fluid, the animal was anesthetized, and the area to be collected was extensively shaved. The area was then thoroughly disinfected using 70% ethanol and povidone, and a needle was inserted into the spinal canal to collect more than 150 μL. The serum and cerebrospinal fluid were stored in a deep freezer set to -80°C until analysis. The anti-3Aβ(1-6) antibody titers and anti-Aβ(1-42) antibody titers in the Beagle dog blood and cerebrospinal fluid were measured using an enzyme-linked immunosorbent assay (ELISA) according to known methods.

[0141]

[0142] 15. Statistical Analysis

[0143] The statistical significance of differences between groups in the evaluation of the immunogenicity of antibody responses was determined using the Mann-Whitney U-test. A P < 0.05 difference was considered significant (P * < 0.05, P ** < 0.01, P *** < 0.001, P**** < 0.0001). For other experiments, significance between groups was determined using the Student t-test or the Kruskal-Wallis test. The statistical significance of each experiment is explained in detail in the examples below.

[0144]

[0145] <Example 1> Transmission Electron Microscopy Analysis of QV-002 and PV-002

[0146] Figure 2 shows the results of analyzing QV-002 and PV-002 using a transmission electron microscope after staining with negative staining (1% phosphotungstic acid, PTA) and magnifying each by 100,000x. 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; both were observed to have a spherical shape. Therefore, these test results demonstrate that each sample maintains a spherical shape even after coupling and purification are completed.

[0147]

[0148] <Example 2> Comparative analysis of amyloid beta antibody reactivity according to VLP types and coupled peptide types (Western blot)

[0149] The manufacturing process of QV-002-S, QV-002, PV-002-S, and PV-002 is shown in Fig. 1. Loading samples were prepared by dividing them into QV-002-S, QV-002, PV-002-S, and PV-002 that underwent a coupling process, and Qβ VLPs and HPV 11 VLPs that did not undergo a coupling process. Figs. 3 and 4 show the results of a comparative analysis of amyloid beta antibody reactivity according to the type of VLPs. Fig. 3 shows the results of comparing the amount of coupled amyloid beta peptides of QV-002 and PV-002, which were coupled to Qβ VLPs and HPV11 VLPs, respectively, via 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, while HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the intensity of the PV-002 band against the anti-amyloid beta (1-10) antibody was higher than that of QV-002. Figure 4 shows the results of comparing the amounts of coupled amyloid beta peptides in QV-002-S and PV-002-S, which coupled Aβ (1-6) to Qβ VLPs and HPV11 VLPs, respectively, via 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, respectively, while HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the reactivity of PV-002-S to the anti-amyloid beta (1-10) antibody was found to be higher than that of QV-002-S.

[0150] Figure 5 shows the final results of comparing the amyloid beta peptide concentrations according to the types of VLPs and the types of peptides coupled thereto using Western blot. QV-002-S, PV-002-S, QV-002, and PV-002 were loaded at 200 ng and 100 ng per well, respectively, while HPV11 VLPs and Qβ VLPs were loaded at 100 ng per well. As a result, the reactivity to the anti-amyloid beta (1-10) antibody was significantly higher in PV-002 compared to QV-002, and stronger reactions were observed in the order of QV-002-S < QV-002 < PV-002-S < PV-002. Therefore, it was confirmed that the concentration of amyloid beta peptide in PV-002, which coupled 3Aβ (1-6) peptide to HPV11 VLPs in the same amount of sample, was the highest.

[0151]

[0152] <Example 3> Comparative analysis (dot blot) of amyloid beta antibody reactivity according to VLP types and coupled peptide types

[0153] This relates to the quantitative analysis of amyloid beta peptide coupled to QV-002-S / QV-002 / PV-002-S / PV-002. The results are based on a dot blot test performed 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 the comparison between QV-002-S and QV-002, it was confirmed that QV-002, which is coupled with the 3Aβ (1-6) peptide, showed a slightly higher reaction, while no difference was observed between PV-002-S and PV-002 (Fig. 6). Overall, PV-002-S and PV-002 showed much higher reactivity compared to QV-002-S and QV-002 (Fig. 6). Thus, the results of 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 type of peptide, and confirmed that for Qβ VLPs, the drug coupled to the 3Aβ(1-6) peptide has higher antibody reactivity compared to the drug coupled to the Aβ(1-6) peptide.

[0154]

[0155] <Example 4> Comparative analysis of amyloid beta antibody reactivity of QV-002 / PV-002 at different concentrations (ELISA)

[0156] The results of analyzing the anti-amyloid beta (1-10) monoclonal antibody reactivity of QV-002 and PV-002 using an enzyme-linked immunosorbent assay (ELISA) are shown in Fig. 7. QV-002 and PV-002 were diluted 10 times in doubling increments starting from 1 ng and coated on each well. As a result of comparing ELISA reactivity, it was confirmed that the reactivity of PV-002 was higher than that of QV-002.

[0157]

[0158] <Example 5> Comparison of Immunogenicity between QV-002 and PV-002

[0159] 1. Comparison of anti-Aβ(1-42) IgG titers of QV-002 and PV-002

[0160] The results of measuring serum anti-Aβ(1-42) IgG titers after performing tertiary and quaternary immunizations are shown in Figures 8a and 10a. In the 10 ng administration group, no antibody titers were observed for either QV-002 or PV-002. In the group immunized with 100 ng after the tertiary immunization, a significant difference (P ** A < 0.01) was confirmed, and a significant difference (P < 0.01) between QV-002 and PV-002 in the group immunized with 1000 ng was observed (P * A difference (P < 0.05) was confirmed. After the fourth immunization, a significant difference (P < 0.05) was observed in the group immunized with 100 ng. ** It was confirmed that < 0.01). Therefore, it was confirmed that PV-002 can induce anti-Aβ(1-42) IgG antibody titers with a smaller amount than QV-002, and that it induces high antibody titers more rapidly than QV-002.

[0161] After the third immunization, the mouse positivity rate for anti-Aβ(1-42) IgG was measured, and in the QV-002 administration group, the positivity rate was 8% for the 100 ng group and 46% for the 1000 ng group. In contrast, in the PV-002 administration group, a relatively high positivity rate was observed, with 69% for the 100 ng group and 92% for the 1000 ng group (Fig. 9a). After the fourth immunization, the mouse positivity rate for anti-Aβ(1-42) IgG was measured, and in the QV-002 administration group, the positivity rate was 8% for the 100 ng group and 69% for the 1000 ng group. In contrast, in the PV-002 administration group, a relatively high positivity rate was observed, with 62% for the 100 ng group and 92% for the 1000 ng group [Fig. 11a]. Therefore, it was confirmed that PV-002 induces a higher positivity rate for anti-Aβ(1-42) IgG than QV-002.

[0162]

[0163] 2. Comparison of anti-3Aβ(1-6) IgG titers of QV-002 and PV-002

[0164] The results of measuring serum anti-3Aβ(1-6) IgG titers after performing the 3rd and 4th immunizations are shown in Figures 8b and 10b. In the 10 ng administration group, no antibody titers were observed for either QV-002 or PV-002. In the group immunized with 100 ng after the 3rd and 4th immunizations, a significant difference (P **** It was confirmed that < 0.0001). Therefore, it was confirmed that PV-002 induces high anti-3Aβ(1-6) IgG antibody titers with a smaller amount than QV-002.

[0165] After the third immunization, the mouse positivity rate for anti-3Aβ(1-6) IgG was measured, and a positivity rate of 8% was confirmed in the QV-002 100 ng administration group and an positivity rate of 85% was confirmed in the PV-002 100 ng administration group (Fig. 9b). After the fourth immunization, the mouse positivity rate for anti-3Aβ(1-6) IgG was measured, and a positivity rate of 15% was confirmed in the QV-002 100 ng administration group and a positivity rate of 92% was confirmed in the PV-002 100 ng administration group (Fig. 11b). Therefore, it was confirmed that PV-002 induces a much higher positivity rate for anti-Aβ(1-42) IgG than QV-002.

[0166]

[0167] 3. Comparison of Immunogenicity of QV-002-S, QV-002, PV-002-S, and PV-002

[0168] QV-002-S is a form in which Aβ(1-6) is coupled to the Qβ coat protein VLP, and QV-002 is a form in which 3Aβ(1-6) is coupled to the Qβ coat protein VLP. PV-002-S is a form in which Aβ(1-6) is coupled to the HPV VLP, and PV-002 is a form in which 3Aβ(1-6) is coupled to the HPV VLP. The antibody titers of mice immunized three times via the intramuscular route at a dose of 100 ng / dose of each antigen form are shown in Fig. 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 of less than 100 (antibody titers of less than 100 are indicated as 1.0 in the Log10 value). PV-002 showed significantly higher antibody titers compared to the QV-002 group (P < 0.00001).

[0169] Figure 15 shows the results of measuring antibody titers after four immunizations in the above experiment. Similarly, the PV-002 immunization group showed significantly higher antibody titers compared to the QV-002 immunization group (P < 0.0001). These results demonstrate that the 3Aβ(1-6) sequence can induce a higher level of antibody titer against Aβ 1-42 compared to Aβ(1-6). Furthermore, it shows that HPV VLP is a superior form of VLP for inducing antibody titers against Aβ 1-42 compared to Qβ VLP.

[0170]

[0171] 4. Effect of PV-002 vaccine on reducing Aβ plaques in brain tissue

[0172] After immunizing with PV-002 5 or 10 times via intramuscular injection at 80 μg / dose, the area of ​​Aβ plaques in brain tissue was stained in sagittal sections and compared. WT refers to the wild-type mouse group. PEV-002 is the group administered an adjuvant, receiving aluminum hydroxide via the same method. VV-002 is the group administered HPV VLP. Figures 16, 17, and 18 show the area of ​​Aβ plaques in the cortex, hippocampus, and thalamus. It was confirmed that the area of ​​Aβ plaques was significantly reduced in the group immunized with PV-002 10 times compared to the PBS immunization group (Kruskal-Wallis).

[0173]

[0174] 5. Memory-improving effects of the PV-002 vaccine

[0175] The memory-improving effect of the PV-002 vaccine administration was confirmed through the Morris Water Maze experiment. WT refers to the wild-type mouse group. For administration, vials were prepared at 50 μg / dose, 100 μg / dose, 200 μg / dose, or 400 μg / dose based on human vaccination standards, and 1 / 10 of these doses were administered to mice. Therefore, mice received doses of 5 μg / dose, 10 μg / dose, 20 μg / dose, or 40 μg / dose. Immunization with the antigen was performed via intramuscular administration. VV-002 refers to the group administered HPV VLP, which was administered at a dose of 40 μg / dose per mouse. PEV-002 refers to the group administered only the aluminum hydroxide adjuvant.

[0176] Figure 19 presents the path length results according to the Morris Water Maze experiment. It was confirmed that the path length decreased as the antigen dosage increased. The path length of the PV-002 vaccine administration group was compared with that of the PEV-002 and VV-002 groups [student t-test; P ** <0.01 (vs PEV-002), P ## [< 0.01 (vs VV-002)]. The arrow indicates the path length on day 5 for the PV-002 inoculation group. Figure 20 shows the escape latency results. Similarly, the escape latency of the PV-002 inoculation group was compared with that of the PEV-002 group or the VV-002 group [student t-test; P * <0.05 (vs PEV-002), P # < 0.05 (vs VV-002)].

[0177]

[0178] 6. Aβ recognition by PV-002-induced antibodies

[0179] Seven types of peptides were synthesized to identify the Aβ recognition sites of serum antibodies from mice immunized with PV-002. In the ELISA reaction, a value of 0.2 or higher was considered a significant reaction. As shown in Figure 21, the serum antibodies from mice immunized with PV-002 were found to significantly recognize Aβ sites 1-42, 1-10, 2-11, and 3-12.

[0180]

[0181] 7. Confirmation of the absence of a T-cell response in PV-002 immunity

[0182] T-cell responses to Aβ are known to be a cause of meningoencephalitis. To determine whether the PV-002 antigen induces a T-cell response, splenocytes of PV-002-immunized mice were stimulated with specific antigens to check for the presence of T-cell responses, the results of which are presented in Fig. 22. When stimulated with Aβ 1-42 or 3Aβ(1-6), no cells expressing interferon-gamma were observed, confirming that no T-cell response occurred. In contrast, when stimulated with HPV VLP, cells expressing interferon-gamma were significantly observed (student t-test; P ** <0.01). Therefore, it was confirmed that T-cell responses to HPV VLP are induced by PV-002 immunity, but T-cell responses to Aβ are not.

[0183]

[0184] <Example 6> Confirmation of Antibody Inducing Ability of PV-002 in Beagle Dogs

[0185] 1. Measurement of anti-Aβ(1-42) IgG titer of PV-002 in serum of Beagle dogs

[0186] Figure 23a shows the results of measuring anti-Aβ(1-42) IgG titers in serum before (0 d), after the third immunization (56 d), and after the fourth immunization (84 d). In the 100 μg administration group, a significant difference in titers (P** < 0.01) was observed between the serum before administration and the serum after the fourth immunization, while in the 400 μg administration group, no significant difference in titers was observed between the serum before administration and the serum after the fourth immunization. In the 800 μg administration group, a significant difference in titers (P** < 0.01) was observed between the serum before administration and the serum after the fourth immunization. The reason there was no statistically significant difference in titers between the serum before administration and the serum after immunization in the 400 μg administration group is that some individuals in the test group seroconverted to antibodies, while others did not. Therefore, it was confirmed that PV-002 can induce anti-Aβ(1-42) IgG antibody titers in most beagle dogs and induce high antibody titers even with a relatively small amount (100 μg).

[0187] As a result of measuring the positivity of anti-Aβ(1-42) IgG in serum before (0 d), after the third immunization (56 d), and after the fourth immunization (84 d), a positivity rate of 100% after the third immunization and 100% after the fourth immunization was confirmed in the 100 μg administration group. On the other hand, a positivity rate of 100% after the third immunization and 50% after the fourth immunization was confirmed in the 400 μg administration group, and a positivity rate of 100% after the third immunization and 100% after the fourth immunization was confirmed in the 800 μg administration group (Fig. 24a). Therefore, it was confirmed that PV-002 induces a high positivity rate of anti-Aβ(1-42) IgG in Beagle dogs when administered at a dose of 100 μg or more, and it was confirmed that the antibody positivity rates after the third and fourth immunizations were similar in most Beagle dogs.

[0188]

[0189] 2. Measurement of anti-3Aβ(1-6) IgG titer of PV-002 in Beagle serum

[0190] Figure 23b shows the results of measuring anti-3Aβ(1-6) IgG titers in serum before (0 d), after the third immunization (56 d), and after the fourth immunization (84 d). A significant difference in titers (P** < 0.01) was confirmed in the serum before administration and after the fourth immunization for all 100 μg, 400 μg, and 800 μg administration groups. Therefore, it was confirmed that PV-002 can induce anti-3Aβ(1-6) IgG antibody titers in Beagle dogs and induce high antibody titers even with a relatively small amount (100 μg).

[0191] As a result of measuring the positivity rate of anti-3Aβ(1-6) IgG in serum before (0 d), after the third immunization (56 d), and after the fourth immunization (84 d), it was confirmed that the positivity rates of the 100 μg, 400 μg, and 800 μg administration groups were all 100% after the third immunization and 100% after the fourth immunization (Fig. 24b). Therefore, it was confirmed that PV-002 induces a high positivity rate of anti-3Aβ(1-6) IgG in Beagle dogs when administered at a dose of 100 μg or more, and that the antibody positivity rates in the serum after the third immunization and after the fourth immunization were similar.

[0192]

[0193] 3. Measurement of anti-3Aβ(1-6) IgG titer of PV-002 in Beagle cerebrospinal fluid

[0194] Figure 25a shows the results of measuring anti-3Aβ(1-6) IgG titers in cerebrospinal fluid (CSF) before (0 d) and after (84 d) the fourth immunization. It was confirmed that the titers in the CSF after the fourth immunization increased compared to the CSF before administration for all groups: the 100 μg, 400 μg, and 800 μg administration groups (for visual convenience in Figure 25a, the titer results were not confirmed as accurate values ​​when the minimum dilution factor was less than 10, so the graph was constructed by determining the dilution factor as 5 (Log5 = 0.70), which is half of 10). Therefore, it was confirmed that PV-002 can induce anti-3Aβ(1-6) IgG antibody titers in the CSF of beagle dogs and that the titers tend to increase in proportion to the dosage.

[0195] As a result of measuring the positivity rate for anti-3Aβ(1-6) IgG in CSF before (0 d) and after the fourth immunization (84 d), the 100 μg, 400 μg, and 800 μg administration groups all showed a positivity rate of 100% after the fourth immunization (Fig. 25b). Therefore, it was confirmed that PV-002 induces a high positivity rate for anti-3Aβ(1-6) IgG in the CSF of Beagle dogs when administered at a dose of 100 μg or more.

[0196]

[0197] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A vaccine composition for the prevention or treatment of Alzheimer's disease comprising virus-like particles (VLPs) of papillomavirus attached with amyloid beta antigens as an active ingredient.

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

3. A vaccine composition for the prevention or treatment of Alzheimer's disease, characterized in that, in paragraph 2, the HPV VLPs are HPV11 L1 VLPs purified from transformed host cells expressing the L1 protein of HPV type 11 (HPV11).

4. A vaccine composition for preventing or treating Alzheimer's disease according to claim 1, characterized in that the amyloid beta antigen is in a form in which a peptide represented by the amino acid sequence of SEQ ID NO. 1 is repeated 1 to 10 times.

5. A vaccine composition for preventing or treating Alzheimer's disease, characterized in that, in claim 4, the peptide represented by the amino acid sequence of SEQ ID NO. 1 is a peptide represented by the amino acid sequence of SEQ ID NO.

2.

6. A vaccine composition for the prevention or treatment of Alzheimer's disease according to claim 4, characterized in that the amyloid beta antigen additionally comprises a linker between the linked peptides.

7. A vaccine composition for the prevention or treatment of Alzheimer's disease, characterized in that, in claim 6, the amyloid beta antigen is a peptide represented by the amino acid sequence of SEQ ID NO. 3 or the amino acid sequence of SEQ ID NO.

4.

8. A vaccine composition for the prevention or treatment of Alzheimer's disease according to claim 1, characterized in that the vaccine composition has an amyloid beta antigen attached to VLPs of Papillomavirus by covalent bonding.

9. A vaccine composition for the prevention or treatment of Alzheimer's disease, characterized in that, in claim 8, the covalent bond is formed through a chemical cross-linking linker.

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

11. A vaccine composition for the prevention or treatment of Alzheimer's disease according to claim 1, characterized in that the vaccine composition additionally comprises an adjuvant.

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