Vaccine composition comprising porphyromonas gingivalis for preventing dementia, and preparation method therefor

Abstract

The present invention relates to the identification of a target material through which Porphyromonas gingivalis affects dementia, and a use of a Porphyromonas gingivalis vaccine for preventing or treating dementia. A composition comprising (Porphyromonas gingivalis, of the present invention, reduces the expression of IGFBP2, Aß and p-Tau and reduces the level of blood insulin, and thus can be used as an effective composition for a dementia vaccine or a composition for preventing, alleviating or treating dementia, and can be effectively used in a method for preventing or treating dementia.

Description

Dementia prevention vaccine composition containing Porphyromonas gingivalis and method for producing the same

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0010775, filed January 24, 2024, the entire disclosure of which is incorporated herein by reference.

[0002]

[0003] The present invention relates to the identification of a target substance that Porphyromonas gingivalis affects in dementia and the use of a Porphyromonas gingivalis vaccine for the prevention or treatment of dementia.

[0004]

[0005] This invention was completed with the support of the Ministry of Trade, Industry and Energy of the Republic of Korea under project number 20018138 (1415185039, 2410004785).

[0006]

[0007] Dementia is a syndrome characterized by a loss of intellectual ability and social functioning, resulting from various causes, and resulting in impairments in daily life. Dementia is a brain disease caused by damage and loss of nerve cells. It causes severe impairments in memory, concentration, language, and cognition that gradually progress over time, ultimately leading to death.

[0008] Known causes include Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

[0009] Current dementia treatments primarily alleviate early-stage symptoms, but their efficacy remains limited. New drugs based on their mechanism of action still require significant time for development and launch. Furthermore, candidate drugs often fail in clinical trials due to side effects and suboptimal therapeutic efficacy. Consequently, there is a growing need for new agents that exhibit both dementia prevention and improvement while minimizing side effects.

[0010] A recent study reported that Porphyromonas gingivalis (P. gingivalis) was found in autopsy brain samples from Alzheimer's disease patients and in the cerebrospinal fluid of people diagnosed with Alzheimer's disease.

[0011] P. gingivalis is a key pathogen in chronic periodontitis, an inflammatory disease characterized by oral bacterial imbalance and progressive destruction of tooth-supporting tissues. Furthermore, P. gingivalis can escape into the bloodstream and colonize various tissues outside the oral cavity, causing systemic inflammation and various associated diseases.

[0012] However, no studies have been published on the use of P. gingivalis as a dementia vaccine.

[0013]

[0014] [Prior Art Literature]

[0015] [Patent Document]

[0016] (Patent Document 1) KR 10-2009-0113998 (2009-11-24)

[0017] [Non-patent literature]

[0018] (Non-patent Document 1) Ha, Hyun-Su, et al. “Anti-atherosclerotic vaccination against Porphyromonas gingivalis as a potential comparator of statin in mice.” PeerJ9 (2021): e11293.

[0019]

[0020] The present inventors have made efforts to provide a novel composition for a dementia vaccine that has fewer side effects and exhibits effects in preventing and improving dementia, and as a result, have confirmed the relationship between Porphyromonas gingivalis and dementia and its potential as a dementia vaccine, thereby completing the present invention.

[0021] Accordingly, an object of the present invention is to provide a use of Porphyromonas gingivalis as a dementia vaccine.

[0022]

[0023] The present invention provides a vaccine composition for preventing dementia comprising Porphyromonas gingivalis.

[0024] According to a preferred embodiment of the present invention, the dementia is caused by at least one selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

[0025] According to a preferred embodiment of the present invention, the composition reduces the expression of IGFBP2, Aß and p-Tau.

[0026] According to a preferred embodiment of the present invention, the composition reduces insulin levels.

[0027] According to a preferred embodiment of the present invention, the composition comprises Porphyromonas gingivalis treated with heat shock and ultrasonic waves.

[0028] According to a preferred embodiment of the present invention, the composition additionally comprises poly I:C and / or PBS.

[0029] In addition, the present invention provides a pharmaceutical composition for preventing or treating dementia, comprising Porphyromonas gingivalis.

[0030] In addition, the present invention provides a pharmaceutical composition for improving cognitive ability comprising Porphyromonas gingivalis.

[0031] In addition, the present invention comprises the steps of i) applying heat shock to Porphyromonas gingivalis; and

[0032] ii) a step of ultrasonic treatment of Porphyromonas gingivalis to which the above thermal shock has been applied;

[0033] A method for producing a vaccine composition for preventing dementia, which comprises:

[0034] According to a preferred embodiment of the present invention, the thermal shock in step i) is applied at 60 to 80°C for 0.5 to 2 hours.

[0035] According to a preferred embodiment of the present invention, the on-time of the ultrasonic treatment in step ii) is 3 to 7 seconds, the off-time is 1 to 5 seconds, and the process is repeated 4 to 8 times in 8 to 12 cycles.

[0036] In addition, the present invention provides a method for preventing or treating dementia, comprising administering heat-shock and ultrasound-treated Porphyromonas gingivalis to a subject in need thereof.

[0037]

[0038] Hereinafter, the present invention will be described in more detail.

[0039]

[0040] “Learning” in the present invention may mean the ability or behavior to perceive and change one’s own behavior, and may include spatial perception, cognitive ability, concentration, etc.

[0041] “Cognition” in the present invention may mean the ability to acquire, maintain, and utilize information, and may include cognitive abilities such as memory, spatiotemporal perception, judgment, language, and calculation abilities.

[0042] The "memory" of the present invention may mean the ability to acquire new information, learned experiences, or knowledge obtained from the surrounding environment, encode and store them in a specific part of the brain, and recall them.

[0043] The "vaccine" of the present invention may refer to an antigen used to automatically immunize humans or animals to prevent infectious diseases. The vaccine may include an attenuated live vaccine, an inactivated vaccine, an inactivated vaccine, a killed vaccine, a subunit vaccine, a synthetic vaccine, or a genetically engineered vaccine, and may refer to a vaccine containing a microorganism attenuated or killed by physical or chemical treatment.

[0044]

[0045] The present inventors identified target factors that induce cognitive impairment by infecting mice with Porphyromonas gingivalis (P. gingivalis), and completed a vaccine composition containing P. gingivalis that exhibits an effect of improving cognitive function impairment.

[0046] Specifically, in mice infected with P. gingivalis, P. gingivalis was expressed in the mouse brain and induced cognitive impairment, and p-Tau and Aß expression were confirmed to be upregulated in the mouse brain infected with P. gingivalis. In addition, by screening for target cytokines secreted from glial cells infected with P. gingivalis, IGFBP2, which regulates the IRS1 / AKT / GSK3ß pathway, was identified, and infected P. gingivalis was found in the pancreas and confirmed to decrease blood insulin levels. In addition, it was confirmed in a specific experiment that when P. gingivalis vaccine was administered before P. gingivalis infection, Alzheimer's disease-like pathology was improved.

[0047]

[0048] Accordingly, the present invention can provide a vaccine composition for preventing dementia comprising Porphyromonas gingivalis.

[0049] The strain of the above Porphyromonas gingivalis strain may be included in a strain lysate, a strain culture, a strain culture concentrate, a strain culture extract, or a dried form thereof. At this time, the "strain lysate" means something obtained by culturing the strain and mechanically or chemically disrupting it, and may include everything that has undergone additional processes such as extraction, dilution, concentration, and purification therefrom. The "strain culture" may mean a medium (solid, liquid, etc.) containing the strain, the medium (solid, liquid, etc.) itself from which the strain is separated after culturing the strain, or a supernatant thereof. The "strain culture concentrate" refers to something that is purified from the strain culture through ultrafiltration, ammonium sulfate treatment, column purification, concentration, etc., or a culture concentrate obtained through ultrafiltration, concentration, etc. "Strain culture extract" means an extract obtained from the culture or a concentrate thereof, and may include an extract, a diluted or concentrated extract, a dried product obtained by drying the extract, or a modified or purified product thereof, or a fraction obtained by fractionating the extract. The dried form may include a freeze-dried form.

[0050] According to a preferred embodiment of the present invention, the dementia may be caused by at least one selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

[0051] According to a preferred embodiment of the present invention, the composition may reduce the expression of IGFBP2, Aß and p-Tau.

[0052] According to a preferred embodiment of the present invention, the composition may reduce insulin levels.

[0053] According to a preferred embodiment of the present invention, the composition may include Porphyromonas gingivalis treated with heat shock and ultrasonic waves.

[0054] According to a preferred embodiment of the present invention, the composition may additionally comprise poly I:C and / or PBS.

[0055]

[0056] In addition, the present invention can provide a pharmaceutical composition for preventing or treating dementia, including Porphyromonas gingivalis.

[0057] According to a preferred embodiment of the present invention, the dementia may be caused by at least one selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

[0058] According to a preferred embodiment of the present invention, the composition may reduce the expression of IGFBP2, Aß and p-Tau.

[0059] According to a preferred embodiment of the present invention, the composition may reduce insulin levels.

[0060] According to a preferred embodiment of the present invention, the composition may include Porphyromonas gingivalis treated with heat shock and ultrasonic waves.

[0061] According to a preferred embodiment of the present invention, the composition may additionally comprise poly I:C and / or PBS.

[0062] The term 'prevention' in the present invention means any action that suppresses or delays the onset of dementia or a dementia-related disease due to Porphyromonas gingivalis of the present invention.

[0063] The term 'improvement' or 'treatment' of the present invention means any action that improves or benefits parameters related to dementia or dementia-related diseases, such as the degree of symptoms, due to Porphyromonas gingivalis of the present invention.

[0064] The pharmaceutical composition of the present invention may be administered orally or parenterally in various dosage forms. When formulating the composition, it may be prepared using one or more buffers (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, bulking agents, binders, adjuvants (e.g., aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrating agents, or surfactants, diluents, or excipients.

[0065] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing one or more compounds with at least one excipient, such as starch (including corn starch, wheat starch, rice starch, potato starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose or gelatin. For example, tablets or sugar-coated tablets can be obtained by mixing an active ingredient with a solid excipient, grinding the mixture, adding a suitable auxiliary agent, and then processing the mixture into a granule mixture.

[0066] In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, or preservatives may be included. In addition, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as disintegrants in some cases, and anticoagulants, flavoring agents, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, packaging agents, pigments, and preservatives may be additionally included.

[0067] Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, or suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0068] The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be formulated in the form of an injection for intraperitoneal, intravenous, subcutaneous or intracerebrovascular injection; or a nasal inhaler according to a method known in the art.

[0069] In the case of the above injection, it must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. Examples of suitable carriers for the injection include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), mixtures thereof, and / or vegetable oils. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. In order to protect the injection from microbial contamination, various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal may be additionally included. In addition, the injection may in most cases additionally include isotonic agents such as sugars or sodium chloride.

[0070] For inhalation dosage forms, the compounds used according to the present invention may conveniently be delivered in the form of an aerosol spray from a pressurized pack or nebulizer using a suitable propellant, such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or another suitable gas. For pressurized aerosols, the dosage unit may be determined by providing a valve to deliver a metered amount. For example, gelatin capsules and cartridges for use in inhalers or insufflators may be formulated to contain a powder mixture of the compound and a suitable powder base such as lactose or starch.

[0071] The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. That is, the total effective amount of the composition of the present invention can be administered to a patient as a single dose, or can be administered as a fractionated treatment protocol in which multiple doses are administered over a long period of time. It is important to consider all of the above factors and administer an amount that achieves the maximum effect with the minimum amount without causing side effects, and this can be easily determined by those skilled in the art.

[0072] The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease.

[0073] The composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.

[0074]

[0075] In addition, the present invention can provide a pharmaceutical composition for improving cognitive ability comprising Porphyromonas gingivalis.

[0076] According to a preferred embodiment of the present invention, improving cognitive ability may mean improving cognitive decline caused by dementia.

[0077] The above dementia may be caused by one or more of the following: Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

[0078] According to a preferred embodiment of the present invention, the composition may reduce the expression of IGFBP2, Aß and p-Tau.

[0079] According to a preferred embodiment of the present invention, the composition may reduce insulin levels.

[0080] According to a preferred embodiment of the present invention, the composition may include Porphyromonas gingivalis treated with heat shock and ultrasonic waves.

[0081] According to a preferred embodiment of the present invention, the composition may additionally comprise poly I:C and / or PBS.

[0082] Since the above pharmaceutical composition is identical to the concept used in the above pharmaceutical composition for preventing or treating dementia, the description is replaced with that description.

[0083]

[0084] In addition, the present invention comprises the steps of i) applying heat shock to Porphyromonas gingivalis; and

[0085] ii) a step of ultrasonic treatment of Porphyromonas gingivalis to which the above thermal shock has been applied;

[0086] A method for producing a vaccine composition for preventing dementia including the above can be provided.

[0087] The term 'prevention' in the present invention means any action that suppresses or delays the onset of dementia or a dementia-related disease due to Porphyromonas gingivalis of the present invention.

[0088] According to a preferred embodiment of the present invention, the dementia may be caused by at least one selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

[0089] According to a preferred embodiment of the present invention, the composition may reduce the expression of IGFBP2, Aß and p-Tau.

[0090] According to a preferred embodiment of the present invention, the composition may reduce insulin levels.

[0091] According to a preferred embodiment of the present invention, the composition may additionally comprise poly I:C and / or PBS.

[0092] According to a preferred embodiment of the present invention, the thermal shock of step i) may be applied at 60 to 80°C for 0.5 to 2 hours.

[0093] According to a preferred embodiment of the present invention, the on-time of the ultrasonic treatment in step ii) may be 3 to 7 seconds, the off-time may be 1 to 5 seconds, and the ultrasonic treatment may be repeated 4 to 8 times in 8 to 12 cycles.

[0094]

[0095] In addition, the present invention provides a method for preventing or treating dementia, comprising administering heat-shock and ultrasound-treated Porphyromonas gingivalis to a subject in need thereof.

[0096] The term 'prevention' in the present invention means any action that suppresses or delays the onset of dementia or a dementia-related disease due to Porphyromonas gingivalis of the present invention.

[0097] The term 'improvement' or 'treatment' of the present invention means any action that improves or benefits parameters related to dementia or dementia-related diseases, such as the degree of symptoms, due to Porphyromonas gingivalis of the present invention.

[0098] According to a preferred embodiment of the present invention, the dementia may be caused by at least one selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

[0099] According to a preferred embodiment of the present invention, the Porphyromonas gingivalis may reduce the expression of IGFBP2, Aß and p-Tau.

[0100] According to a preferred embodiment of the present invention, the Porphyromonas gingivalis may reduce insulin levels.

[0101] According to a preferred embodiment of the present invention, the Porphyromonas gingivalis may be administered together with poly I:C and / or PBS.

[0102]

[0103] The composition comprising Porphyromonas gingivalis of the present invention reduces the expression of IGFBP2, Aß and p-Tau and reduces the level of insulin in the blood, and thus can be used as an effective composition for a dementia vaccine or a composition for preventing, improving or treating dementia, and can also be effectively used in a method for preventing or treating dementia.

[0104]

[0105] Figures 1a and 1b show the results of examining the level of P. gingivalis expression and brain cognitive function in the brain of mice infected with P. gingivalis.

[0106] Figures 2a and 2b show the results of confirming the expression levels of p-Tau and Aß in the mouse brain infected with P. gingivalis.

[0107] Figures 3a and 3b show the results of confirming the activation of glial cells in the mouse brain infected with P. gingivalis.

[0108] Figures 4a, 4b, and 4c show the results of confirming the relative expression of cytokines and chemokines in the mouse brain infected with P. gingivalis.

[0109] Figures 5a, 5b, and 5c show the results of identifying the relevant pathways of Alzheimer's disease in the mouse brain infected with Porphyromonas gingivalis (P. gingivalis).

[0110] Figure 6 shows the results of examining the effects of P. gingivalis infection on the pancreas and insulin levels in mice infected with P. gingivalis.

[0111] Figure 7 shows the results of examining the level of P. gingivalis expression and brain cognitive function in the brain of mice infected with P. gingivalis after administration of the P. gingivalis vaccine.

[0112] Figure 8 shows the results of confirming the expression levels of p-Tau and Aß in the brains of mice infected with P. gingivalis after administration of the P. gingivalis vaccine.

[0113]

[0114] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples.

[0115]

[0116] <Experimental Method>

[0117] 1. Animals

[0118] Male C57BL / 6 mice (7 weeks old) were purchased from Central Lab Animal Inc. (Seocho, Seoul, Republic of Korea) and housed under a 12-h light / dark cycle in a humidity- and temperature-controlled environment. Water was provided ad libitum throughout the experiment.

[0119] All animal experiments were performed in accordance with the guidelines and were approved by the Institutional Animal Care and Research Center of Yonsei University (IACUC approval number 20220258).

[0120]

[0121] 2. Bacterial infection

[0122] Eight-week-old C57BL / 6 mice were used for infection. Each mouse was injected with 1 × 10 using a sterile feeding needle. 12 Mice were orally inoculated with 100 μL of a bacterial suspension containing 10 CFU of Porphyromonas gingivalis. Control mice were treated with carboxymethyl cellulose (CMC) as a vehicle control.

[0123]

[0124] 3. Vaccination

[0125] 3.1. Vaccine Manufacturing

[0126] The vaccine antigen was produced by heat shocking and sonicating a P. gingivalis solution. The plates and broth used for preparation were placed in an anaerobic chamber and replaced 2–3 days prior to inoculation.

[0127] Specifically, 2-3 days after inoculating the vaccine antigen strain ATCC 53978 stock onto a blood agar plate (Tryptic sot broth 3%, Yeast extract 0.5%, 0.025% Resazurin 0.4%, Bacto agar 2%, Cystein HCL-H2O 0.05% Hemin solution 0.5 mg / ml, Vitamin K1 0.002 mg / ml, sheep blood 5%), a single colony was inoculated onto broth (Tryptic sot broth 3%, Yeast extract 0.5%, 0.025% Resazurin 0.4, Cystein HCL-H2O 0.05% Hemin solution 0.5 mg / ml, Vitamin K1 0.002 mg / ml) and cultured. The culture was performed at 37℃ by placing an AnaeroBag (KS-B2002) in an Anaerobic Jar (KS-SC3381) and maintaining anaerobic conditions with oxygen below 0.1% and carbon dioxide 7-15%. After the OD650nm value became 0.8, centrifugation was performed at 4℃, 100,000 rpm for 20 minutes. The supernatant was discarded, and the pellet was washed with PBS at 4℃, 100,000 rpm for 20 minutes, the supernatant was discarded, and the pellet was washed again (2 washes in total). After washing, 2 ml of PBS was added to the pellet and heat-shocked at 70℃ for 1 hour. After that, it was placed on ice and high frequency (Q125 (QSONICA) 70% amplitude, pulse on 5 sec, pulse off 3 sec, 10 cycles, 6 times) was performed. After centrifugation at 4°C (3000 rpm) for 10 minutes, the supernatant was collected. The protein concentration of the collected supernatant was measured using the MicroBCA™ Protein Assay Kit (Pierce, USA). The antigen was stored at -20°C.

[0128] A total of 10 ul of vaccine was prepared by mixing 10 ug (1 ul) of the antigen (10 ug / ul) prepared above, 10 ug (2.5 ul) of poly I:C (stock 4 ug / ul, Invitrogen), and 6.5 ul of PBS.

[0129] Quality Control of the vaccine antigen was performed by smearing the strain suspension on a blood agar plate again to check for contamination before applying heat shock during the vaccine antigen manufacturing process.

[0130]

[0131] 3.2 Vaccination

[0132] Mice were vaccinated intranasally two weeks prior to bacterial infection. The vaccine mixture was prepared in sterile PBS and administered by instillation using a pipette in a total volume of 12.5 μL (7.5 μL per nostril).

[0133]

[0134] 4. Behavioral Test

[0135] 4.1. Novel Object Recognition Test (NORT)

[0136] The novel object recognition test (NORT) is a widely used behavioral test to assess learning and memory functions in mice. The NORT was performed in a square arena (40 x 40 x 40 cm) over three days, comprising habituation, training, and exploration testing phases. Before testing, mice underwent a 30-minute acclimation period. During habituation, mice were removed from their home cages and placed in an empty arena for 5 minutes. During training sessions, identical objects were placed in opposite quadrants of the arena, and mice were allowed to explore both objects for 5 minutes. On the day of testing, one of the objects used during training was replaced with a novel object, and mice were allowed to freely explore both objects for 10 minutes. To minimize olfactory cues, the arena and objects were cleaned with 70% ethanol after each trial. Animal behavior was analyzed using video tracking software SMART 3.0 (PanLab, Holliston, MA, USA).

[0137]

[0138] 4.2. Y-maze test

[0139] The Y-maze test was conducted in a space with three arms arranged at a 120° angle. The spontaneous alternation test assessed spatial working memory based on the animals' natural tendency to explore a novel environment. Mice were placed in the center of the Y-maze, facing one of the arms. Mice were allowed to explore freely for 5–10 min, and the order of arm entries and the total number of arm entries were recorded. An alternation was defined as consecutive entries into three different arms. The percentage of spontaneous alternation was calculated using the following [Equation 1].

[0140] [Calculation Formula 1]

[0141] Voluntary shifts (%) = (number of shifts / total possible shifts) × 100.

[0142]

[0143] 5. Brain sample preparation

[0144] Mice were randomly assigned to Western blot (WB) or immunohistochemistry (IHC) groups. Mice were deeply anesthetized with an intraperitoneal injection of a mixture of zoletil (100 mg / kg, Virbac, Carros, Alpes-Maritimes, France) and xylazine (Rompun; 10 mg / kg, Elanco, Greenfield, IN, USA). For WB, mice were perfused intracardially with saline (Dai Han Pham Co.). Whole brains were dissected, and the cortex (CTX) and hippocampus (HIPP) were isolated, frozen in liquid nitrogen, and stored at -80°C until further experiments. For IHC, mice were perfused transcardially with 1X phosphate-buffered saline (PBS; Biosesang, Yongin-si, Gyeonggi-do, Republic of Korea) plus saline (Dai Han Pham Co.) and 4% paraformaldehyde (PFA; Sigma-Aldrich). Brains were extracted and incubated in 4% PFA (Sigma-Aldrich) at 4°C for 24 h. Fixed brain tissues were placed in 30% sucrose at 4°C for 1–3 days and stored at -80°C until further experiments.

[0145]

[0146] 6. Western Blot (WB)

[0147] CTX and HIPP tissues were lysed in ice-cold radioimmunoprecipitation assay buffer (Tech & Innovation, Chuncheon-si, Gangwon-do, Republic of Korea) containing a phosphatase and protease inhibitor cocktail solution (GenDEPOT, Baker, TX, USA) using a homogenizer. Protein concentrations were determined using the bicinchoninic acid assay. Each sample containing 50 μg of protein was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoresed onto a polyvinylidene difluoride membrane (Millipore, Burlington, MA, USA). The membrane was blocked with 5% bovineserum albumin (BSA, GenDEPOT) for 1 h at room temperature to prevent nonspecific binding and then incubated overnight at 4°C with primary antibodies. After washing three times with tris-buffered saline (TBS) containing 0.5% Tween 20 (TBS-T, GenDEPOT), the membrane was incubated with horseradish peroxidase (HPR)-conjugated IgG (H+L) secondary antibody for 2 h at room temperature. After washing three times with 0.5% TBS-T (GenDEPOT), signals were detected using ECL reagent (GenDEPOT) and visualized using LAS 4000 mini (Fujifilm Life Science USA, Stamford, CT, USA).

[0148]

[0149] 7. Immunohistochemistry (IHC)

[0150] Brain hemispheres were freshly embedded in Tissue-Tek® OCT compound (Sakura Finetek, Chuo-ku, Tokyo, Japan) in a cryomold and then frozen. The frozen tissues were cut into 14-μm-thick slices. The sections were fixed in cold methanol at -20°C for 15 min. After washing three times with 1X PBS (Biosesang), the sections were permeabilized with 1X PBS (Biosesang) containing 0.25% Triton X-100 (Sigma-Aldrich) for 30 min. After washing three times with 1X PBS (Biosesang), the sections were blocked with 5% BSA (GenDEPOT) for 1 h at room temperature to block nonspecific binding. The sections were then incubated with primary antibodies overnight at 4°C. After incubation, the sections were washed three times with 1X PBS (Biosesang) and incubated with the appropriate secondary antibody for 2 h at room temperature in a dark chamber. The sections were washed three times with 1X PBS (Biosesang), stained with 6-diamidino-2-phenylindole (DAPI, Thermo Fisher Scientific, Waltham, MA, USA) for 5 min at room temperature in a dark chamber, and then washed again three times with 1X PBS (Biosesang). Images were acquired using an LSM 710 microscope (Carl Zeiss, Oberkochen, Baden-Württemberg, Germany).

[0151]

[0152] 8. Primary cell culture

[0153] Brain tissue was dissociated into single-cell suspensions using the Adult Brain Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) according to the manufacturer's instructions. After enzymatic digestion, mechanical dissociation was performed using the gentleMACS™ Dissociator with Heaters (Miltenyi Biotec). The resulting cell suspension was treated with Debris Removal Solution to remove myelin and cell debris. Red blood cells were then removed using Red Blood Cell Removal Solution.

[0154] Astrocytes were isolated using the Anti-ACSA-2 MicroBead Kit (Miltenyi Biotec). Cell suspensions were incubated with ACSA-2 MicroBeads, and labeled cells were isolated using an autoMACS® Pro Separator (Miltenyi Biotec) in positive selection mode.

[0155] Microglia were isolated using CD11b (Microglia) MicroBeads (Miltenyi Biotec). CD11b-labeled cells were isolated using an autoMACS®Pro Separator in positive selection mode.

[0156]

[0157] 9. Proteome profiler mouse cytokine array

[0158] To assess the relative expression of cytokines and chemokines in mouse brain, mouse cytokine array analysis was performed using the Mouse XL Cytokine Array Kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturer's instructions. Signals were detected using a chemi-reagent mixture (R&D Systems) and visualized using a LAS 4000 Mini (Fujifilm Life Science USA). Positive signals were quantified using QuickSpots (Ideal Eyes Systems, Bountiful, UT, USA).

[0159]

[0160] 10. Insulin ELISA

[0161] Mouse plasma was collected and centrifuged at 6,000 rpm for 10 minutes at 4°C to obtain clear plasma. Mouse insulin levels were measured using the Mouse Insulin ELISA Kit (Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. Absorbance was measured at 450 nm using a microplate reader.

[0162]

[0163] 11. Statistical Analysis

[0164] All statistical analyses were performed using GraphPad Prism version 8 (Dotmatics, San Diego, CA, USA). Statistical significance was determined using repeated-measures one-way analysis of variance (ANOVA) and one-way analysis of variance (ANOVA), and the unpaired t-test was used to compare the means of two groups. All values ​​are expressed as the mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

[0165]

[0166] [Example 1]

[0167] Effects of P. gingivalis infection on learning and memory

[0168] We aimed to confirm the expression of Porphyromonas gingivalis (P. gingivalis) in the mouse brain infected with P. gingivalis using immunohistochemical analysis and Western blot.

[0169] As a result, as shown in [Figure 1a] and [Figure 1b], P. gingivalis expression was confirmed in the brain of mice infected with P. gingivalis, and in particular, in the Western blot, the P. gingivalis infection group showed an increase of approximately 2 times compared to the control group.

[0170] Additionally, P. gingivalis infection worsened brain cognitive function in the NORT and Y-maze tests.

[0171]

[0172] [Example 2]

[0173] Upregulation of pTau and Aß levels by P. gingivalis infection in mouse brain

[0174] We aimed to determine the expression levels of p-Tau and Aß in the cortex (CTX) and hippocampus (HIPP) isolated from the brains of mice infected with P. gingivalis.

[0175] As a result, as shown in [Fig. 2a] and [Fig. 2b], p-Tau and Aß expression were upregulated in MPA2-positive cells of CTX and HIP infected with P. gingivalis. Compared with the control group, p-Tau levels increased approximately 3-fold in CTX infected with P. gingivalis and 2.3-fold in HIP, and Aß levels increased approximately 2.5-fold in CTX infected with P. gingivalis and 2.4-fold in HIP, compared with the control group.

[0176]

[0177] [Example 3]

[0178] Glial cell activation by P. gingivalis infection in the mouse brain

[0179] We aimed to confirm the activation of glial cells in the brains of mice infected with P. gingivalis.

[0180] As a result, as shown in [Figure 3a] and [Figure 3b], the immunohistochemical test results showed that CD86 and GBP2 positive cells were highly expressed in the brain infected with P. gingivalis compared to the control group.

[0181] Additionally, P. gingivalis infection was able to induce a pro-inflammatory phenotype (CD86) in microglia, and P. gingivalis-infected astrocytes were judged to be converted to a pro-inflammatory phenotype (GBP2).

[0182]

[0183] [Example 4]

[0184] Screening of target cytokines released from glial cells activated by P. gingivalis infection

[0185] To determine the relative expression of cytokines and chemokines in the mouse brain, a proteome profiler cytokine array analysis was performed.

[0186] As a result, as shown in [Figure 4a] to [Figure 4c], many cytokines were expressed in microglia and astrocytes infected with P. gingivalis compared to the control group.

[0187] The present inventors identified six candidate cytokines, including chitinase 3-like 1, CX3CL1, cystatin C, IGFBP-2, osteopontin, and serpin E1. Among the cytokines, we focused on IGFBP2 because it prevents IGF-1 from binding to its receptor, thereby inducing Alzheimer's disease.

[0188]

[0189] [Example 5]

[0190] Impact of IGFBP2 Overexpression on the IRS1 / AKT / GSK3ß Pathway Associated with Alzheimer's Disease

[0191] We aimed to identify the pathway involved in Alzheimer's disease caused by P. gingivalis infection.

[0192] As a result, as shown in [Figure 5a] to [Figure 5c], it was confirmed that P. gingivalis infection induced IGFBP2 overexpression in CTX and HIP compared to the control group. IGFBP2 overexpression is associated with insulin resistance, which induces the regulation of the IRS1 / Akt / GSK3β pathway in Alzheimer's disease pathology.

[0193] Additionally, phosphorylation of IRS1 / AKT / GSK3ß was decreased in P. gingivalis infection compared to the control group.

[0194]

[0195] [Example 6]

[0196] Insulin deficiency due to destruction of the pancreas by P. gingivalis infection

[0197] Because P. gingivalis infection affects insulin as well as IGFBP-2, which influences the IRS1 / AKT / GSK3β cell signaling pathway involving p-tau, we sought to determine the effects of P. gingivalis infection on pancreatic and insulin levels.

[0198] As a result, as shown in [Figure 6], infected P. gingivalis was found in the pancreas and TUNEL-positive cells increased, indicating that infected P. gingivalis induced an increase in TUNEL-positive cells in pancreatic islets. In addition, when insulin was measured in the blood, it was confirmed that the insulin level decreased in the P. gingivalis group compared to the control group.

[0199]

[0200] [Example 7]

[0201] Effects of P. gingivalis vaccine on learning and memory

[0202] We aimed to determine the effects of administering the P. gingivalis vaccine before P. gingivalis infection on learning and memory in mice.

[0203] As a result, as shown in [Figure 7], the P. gingivalis vaccine suppressed P. gingivalis expression in CTX and HIPP and improved learning and memory functions of mice.

[0204]

[0205] [Example 8]

[0206] Effect of P. gingivalis vaccine on IGFBP2, Aß, and pTau

[0207] We aimed to determine the expression levels of p-Tau and Aß in the cortex (CTX) and hippocampus (HIPP) isolated from the brains of mice administered the P. gingivalis vaccine before P. gingivalis infection.

[0208] As a result, as shown in [Figure 8], the P. gingivalis vaccine inhibited IGFBP2 expression in the mouse brain. In addition, Aß and p-Tau expression were reduced in CTX and HIP treated with the P. gingivalis vaccine.

[0209]

[0210] The composition comprising Porphyromonas gingivalis of the present invention reduces the expression of IGFBP2, Aß and p-Tau and reduces the level of insulin in the blood, and thus can be utilized as an effective composition for a dementia vaccine or a composition for preventing, improving or treating dementia, and can also be effectively utilized in a method for preventing or treating dementia, and thus has industrial applicability.

Claims

1. A vaccine composition for preventing dementia containing Porphyromonas gingivalis.

2. A composition according to claim 1, characterized in that the dementia is caused by at least one selected from the group consisting of Alzheimer's disease, vascular dementia, Parkinson's disease, Lewy body dementia, Huntington's disease, Creutzfeldt-Jacob disease, and Pick's disease.

3. A composition according to claim 1, characterized in that the composition reduces the expression of IGFBP2, Aß and p-Tau.

4. A composition according to claim 1, characterized in that the composition reduces insulin levels.

5. A composition according to claim 1, characterized in that the composition comprises Porphyromonas gingivalis treated with heat shock and ultrasonic waves.

6. A composition according to claim 1, characterized in that the composition additionally comprises poly I:C and / or PBS.

7. A pharmaceutical composition for preventing or treating dementia containing Porphyromonas gingivalis.

8. A pharmaceutical composition for improving cognitive ability containing Porphyromonas gingivalis. 9.i) a step of applying heat shock to Porphyromonas gingivalis; and ii) a step of ultrasonic treatment of Porphyromonas gingivalis to which the above thermal shock has been applied; A method for producing a vaccine composition for preventing dementia comprising:

10. A manufacturing method characterized in that in the 9th paragraph, the thermal shock of step i) is applied at 60 to 80°C for 0.5 to 2 hours.

11. A manufacturing method characterized in that in the 9th paragraph, the on-time of the ultrasonic treatment in step ii) is 3 to 7 seconds, the off-time is 1 to 5 seconds, and the process is repeated 4 to 8 times in 8 to 12 cycles.

12. A method for preventing or treating dementia, comprising administering heat-shocked and ultrasonic-treated Porphyromonas gingivalis to a subject in need thereof.

Images