Composition for preventing and treating oral diseases or conditions, preparation method and use thereof

Abstract

The invention discloses a composition for preventing and treating oral diseases or conditions, a preparation method and application thereof. The external vesicles based on oral pathogenic bacteria are rich in nano-scale vesicles of immune activation and defense factors aiming at periodontitis, and after the external vesicles enter a body through oral mucosa administration, the external vesicles are passively targeted into lymph nodes related to the oral mucosa to induce mucosa immune specific antibody expression, so that the immune defense capacity to the pathogenic bacteria is improved. Preferably, a calcium phosphate shell is coated outside the oral bacterial outer vesicle, and the calcium phosphate shell can only be dissolved under an acidic condition, so that the engineered vesicle can be degraded after targeting to lymph nodes and being phagocytosed into dendritic cell lysosome, and the inflammatory reaction of a drug delivery area is avoided. The administration form of the sublingual disintegrating tablet can improve the bioavailability of the vaccine while local mucosal administration activates mucosal immunity, and the convenient administration form is also beneficial to the promotion and popularization of the periodontitis vaccine in clinical chair-side diagnosis and treatment or grassroots communities.

Description

Technical Field

[0001] This invention relates to the prevention and treatment of oral bacterial infectious diseases or conditions, and more specifically to compositions for the prevention and treatment of oral bacterial infectious diseases or conditions, their preparation methods and uses. Background Technology

[0002] Chronic periodontitis is a chronic infectious disease that leads to the slow loss of periodontal tissues such as the periodontal ligament and alveolar bone, eventually causing teeth to loosen and fall out. It is one of the leading oral diseases causing tooth loss in my country. As a long-term, persistent source of infection, periodontitis can affect overall health. Patients with periodontitis have a significantly increased risk of developing cardiovascular disease, myocardial infarction, diabetes, rheumatoid arthritis, lupus erythematosus, and Alzheimer's disease.

[0003] With global population growth and increased life expectancy, the prevention and treatment of periodontitis have gained increasing public attention. The initiating factor of periodontitis is the invasion of local plaque microorganisms and their toxic products, causing an inflammatory response in the local tissues, leading to damage and destruction of periodontal tissues. Currently, the main clinical treatments for periodontitis are basic treatments aimed at removing local plaque, such as supragingival scaling and subgingival curettage. However, due to the dysfunction of host immune cells caused by the local inflammatory microenvironment, although basic periodontal treatment can control plaque and restore the healthy color, shape, and texture of tissues, if there is a lack of immune defense against reinfection by pathogenic bacteria, periodontitis carries the risk of repeated relapses and even difficulty in tissue repair. Therefore, there is an urgent need to develop new clinical strategies and formulations for preventing the onset and recurrence of periodontitis.

[0004] The information in the background section is merely intended to illustrate the general background of the invention and should not be construed as an admission or implication in any way that such information constitutes prior art known to those skilled in the art. Summary of the Invention

[0005] Through large-scale clinical sample collection and correlation analysis, the applicant discovered that a lack of pathogen-specific sIgA antibodies in saliva is a key risk factor for poor treatment prognosis and high recurrence rate in patients with periodontitis, and that oromandibular lymphoid tissue, represented by the submandibular lymph nodes, is a key site for sIgA antibody production and expression. Based on this, the applicant further discovered that targeting and activating B cell-related functions in the submandibular lymph nodes and upregulating the level of pathogen-specific sIgA antibodies in saliva can effectively prevent and treat periodontitis. Specifically, this invention includes the following:

[0006] In a first aspect, the present invention provides a composition for preventing and treating oral bacterial infectious diseases or conditions, comprising bacterial exovesicles derived from oral pathogens such as periodontitis pathogens.

[0007] In some embodiments, the composition for preventing and treating oral bacterial infectious diseases or conditions according to the present invention includes pathogenic bacteria such as Porphyromonas gingivalis, Actinobacillus actinomycetii, Forsythorbium, Fusobacterium nucleatum, and Prevotella intermedius.

[0008] In some embodiments, the composition for preventing and treating oral bacterial infectious diseases or conditions according to the present invention is wherein the oral disease is an oral bacterial inflammatory disease, such as periodontitis.

[0009] In some embodiments, the composition for preventing or treating oral bacterial infections or conditions according to the present invention further comprises a calcium salt, preferably, the calcium salt coating the surface of the outer vesicle to form a shell.

[0010] In some embodiments, the composition for preventing or treating oral bacterial infections or conditions according to the present invention is a vaccine, such as a periodontitis vaccine.

[0011] In some embodiments, the composition for preventing or treating oral bacterial infections or conditions according to the present invention is wherein the composition is a sublingual disintegrating tablet, oral lozenge, oral spray, or hydrogel formulation.

[0012] A second aspect of the present invention provides a method for preparing a composition for preventing and treating oral bacterial infectious diseases or conditions, comprising the steps of: providing exovesicles derived from oral pathogens and coating the surface of the exovesicles with calcium salts to obtain calcium-coated exovesicles.

[0013] In some embodiments, the method for preparing a composition for preventing or treating oral bacterial infections or conditions according to the present invention further includes the step of preparing a formulation, preferably a tablet, using calcium-coated exovesicles.

[0014] A third aspect of the invention provides the use of external vesicles derived from oral pathogens in the preparation of a medicament for increasing oral levels of specific IgG and / or sIgA antibodies against the source bacteria and for increasing serum levels of specific IgG and / or IgA antibodies against the source bacteria.

[0015] This invention discovers that the external vesicles of oral bacteria are rich in nanoscale vesicles containing immune-activating and defense factors against periodontitis. After administration via the oral mucosa, these vesicles passively target the oral mucosa-associated lymph nodes, inducing mucosal immune-specific antibody expression, thereby enhancing immune defense against pathogenic bacteria. In a preferred embodiment, the external vesicles are bacterial external vesicles of *Porphyromonas gingivalis*, *Actinomyces actinomycetes*, *Focusae*, *Fusobacterium nucleatum*, *Prevotella intermedius*, etc. In a preferred embodiment, the external vesicles of this invention are coated with a calcium phosphate shell, which is only soluble in acidic conditions. This allows the engineered vesicles to target the lymph nodes and be phagocytosed into dendritic cell lysosomes before degradation, avoiding inflammatory reactions at the administration site. In a preferred embodiment, the composition of this invention is prepared as a sublingual disintegrating tablet. This administration method allows for local administration to activate mucosal immunity while improving vaccine bioavailability. The convenient administration method also facilitates the promotion and popularization of periodontitis vaccines in chairside clinics or primary communities. Attached Figure Description

[0016] Figure 1 Application scenarios and mechanisms of action of sublingual disintegrating periodontitis vaccine based on Porphyromonas gingivalis external vesicles (OMV@CaP ODT) for the prevention of periodontitis.

[0017] Figure 2 Electron micrograph of Porphyromonas gingivalis external vesicles (OMV).

[0018] Figure 3 Protein composition of Porphyromonas gingivalis extravesicles (OMV).

[0019] Figure 4 Average particle size analysis results for OMV.

[0020] Figure 5 Average particle size analysis results for OMV and OMV@CaP.

[0021] Figure 6 Transmission electron microscopy (TEM) images and thermogravimetric (TGA) results. (A) TEM images of OMV and OMV@CaP; (B) TGA results of OMV@CaP.

[0022] Figure 7 The activation effect of OMV and OMV@CaP on mouse dendritic cells.

[0023] Figure 8 Disintegration of OMV@CaP ODT periodontitis vaccine under the tongue in mice.

[0024] Figure 9 Disintegration of OMV@CaP ODT periodontitis vaccine under the tongue of cynomolgus monkeys.

[0025] Figure 10 Lymph node targeting of OMV@CaP ODT periodontitis vaccine after intraoral administration in mice. (A) Small animal imaging results show that the fluorescently labeled OMV@CaP ODT periodontitis vaccine significantly accumulated in the submandibular lymph nodes of mice 12 hours after intraoral administration; (B) Small animal imaging observation of the submandibular lymph nodes of mice at different administration time points confirmed that both OMV@CaP and OMV@CaP ODT can effectively deliver vesicles to the submandibular lymph nodes of mice after administration, and OMV@CaP ODT has higher bioavailability.

[0026] Figure 11 Lymph node targeting of OMV@CaP ODT periodontitis vaccine after intraoral administration to cynomolgus monkeys. Photoacoustic results showed that the fluorescently labeled OMV@CaP ODT periodontitis vaccine was significantly enriched in the submandibular lymph nodes of cynomolgus monkeys 6 hours after intraoral administration.

[0027] Figure 12 After administration of OMV@CaP and OMV@CaP ODT, the submandibular lymph nodes of mice were significantly enlarged and the number of B cell follicles increased.

[0028] Figure 13 Activation of submandibular lymph nodes in cynomolgus monkeys after administration of OMV@CaP ODT periodontitis vaccine. (A) Ultrasound observation of submandibular lymph nodes in cynomolgus monkeys at different time points after administration showed enlargement of submandibular lymph nodes in the OMV@CaP ODT administration group, proving that the drug activated the immune response within the lymph nodes; (B) Gross observation showed that the submandibular lymph nodes in cynomolgus monkeys significantly enlarged after administration of OMV@CaP ODT periodontitis vaccine, consistent with the ultrasound results; (C) Histological staining results confirmed the enlargement of submandibular lymph nodes in cynomolgus monkeys after administration, consistent with the ultrasound results and gross observation.

[0029] Figure 14 The promoting effects of OMV@CaP and OMV@CaP ODT on the levels of antigen-specific antibodies in mouse saliva and serum. (A) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific sIgA antibodies in mouse saliva; (B) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibodies in mouse saliva; (C) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibodies in mouse serum; (D) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgA antibodies in mouse serum; (E) The promoting effect of OMV@CaP and OMV@CaP ODT on the level of antigen-specific sIgA antibodies in mouse saliva is long-lasting, and the effect of OMV@CaP ODT is better than that of OMV@CaP.

[0030] Figure 15 The promoting effects of OMV@CaP and OMV@CaP ODT on the levels of antigen-specific antibodies in the saliva and serum of cynomolgus monkeys. (A) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific sIgA antibody in the saliva of cynomolgus monkeys; (B) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibody in the saliva of cynomolgus monkeys; (C) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgG antibody in the serum of cynomolgus monkeys; (D) OMV@CaP and OMV@CaP ODT upregulated the level of antigen-specific IgA antibody in the serum of cynomolgus monkeys; (E) The promoting effect of OMV@CaP and OMV@CaP ODT on the level of antigen-specific sIgA antibody in the saliva of cynomolgus monkeys was long-lasting, and the effect of OMV@CaP ODT was better than that of OMV@CaP.

[0031] Figure 16 The protective effects of OMV@CaP and OMV@CaP ODT against periodontitis in mice. Histological staining results showed that OMV@CaP and OMV@CaP ODT effectively inhibited alveolar bone resorption and destruction caused by pathogenic bacterial infection, and also maintained a protective effect against periodontitis with long-term reinfection.

[0032] Figure 17 The protective effect of OMV@CaP ODT against periodontitis in cynomolgus monkeys in vivo. (A) Intraoral examination results showed that after modeling, the gingival redness, bleeding on probing, and probing depth of the affected teeth of cynomolgus monkeys were increased. The clinical manifestations of the affected teeth of cynomolgus monkeys in the OMV@CaP ODT group were significantly improved. (B) CBCT results showed that OMV@CaP ODT could effectively inhibit alveolar bone resorption and destruction caused by pathogenic bacterial infection, and it still had a protective effect against periodontitis that was reinfected in the future. (CD) Histological staining results showed that OMV@CaP ODT effectively improved irregular proliferation and inflammatory cell infiltration caused by local inflammatory stimulation in periodontal tissues. (E) Intraoral examination results showed that after modeling, the probing depth (PD), bleeding on probing index (BI), and positive rate of bleeding sites (BOP) of the affected teeth of cynomolgus monkeys were significantly increased. The disease indicators of the affected teeth of cynomolgus monkeys in the OMV@CaP ODT group were significantly improved, and it still had a protective effect against periodontitis that was reinfected in the future.

[0033] Figure 18The immune-activating effect of OMV@CaP ODT on clinical volunteers. (A) OMV@CaP ODT disintegrated under the tongue of volunteers within 1 minute; (B) After immunization with OMV@CaP ODT, the submandibular lymph nodes of all 10 volunteers were transiently enlarged, indicating local immune response activation; (C) OMV@CaP ODT immunization significantly increased the level of antigen-specific sIgA antibody in the saliva of all subjects. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] In this article, the term "oral bacterial infectious disease" refers to a disease in the oral cavity caused by bacterial imbalance or infection by pathogenic bacteria, preferably an inflammatory disease. Examples of such diseases include, but are not limited to, postoperative oral infections, refractory periapical diseases, gingivitis, and periodontitis.

[0038] In this article, the term "condition" refers to the symptoms and signs caused by oral bacterial infections. This article primarily focuses on gingival redness and swelling, pain, abscesses, which may be accompanied by bleeding, alveolar bone resorption, tooth loosening, wound pain, purulent discharge, hyperplasia, ulceration, recurrent periapical abscesses accompanied by discomfort when biting, and bad breath.

[0039] In this text, the term "prevention and treatment" refers to therapeutic treatments and preventive or preventative measures aimed at preventing or mitigating (reducing) undesirable physiological changes or disorders, such as the onset, progression, or recurrence of inflammatory diseases. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction of disease severity, decrease in susceptibility to disease, stabilization of the disease state (i.e., no worsening), delay or slowing of disease progression, improvement or mitigation of the disease state, and ablation. Those requiring treatment include individuals who already have the condition or disorder, those susceptible to the condition or disorder, or those who require prevention of the condition or disorder.

[0040] [Composition] In a first aspect, the invention provides a composition for preventing or treating oral bacterial infectious diseases or conditions, sometimes referred to herein as a "vaccine", comprising exovesicles derived from oral pathogens.

[0041] In this invention, the pathogenic bacteria are microorganisms present in the oral cavity that can cause, but are not limited to, periodontitis or other infectious diseases. Examples include, but are not limited to, *Porphyromonas gingivalis*, *Actinomyces actinomycete*, *Fusobacterium nucleatum*, and *Prevotella intermedius*. Preferably, the periodontitis pathogen of this invention is *Porphyromonas gingivalis*. In this invention, the oral pathogenic bacteria used to produce external vesicles can be one of the above-mentioned bacteria, or a combination of two or more. The bacteria of this invention can be natural bacteria or engineered bacteria obtained through genetic engineering.

[0042] In this invention, external vesicles refer to bacterial outer membrane vesicles (OMVs) secreted by bacteria, which have a vesicle-like structure with a lipid bilayer. Preferably, the external vesicles are naturally secreted bacterial nanoparticles, more preferably nanostructures secreted by the outer membrane of Gram-negative bacteria, with an average diameter generally 1-1000 nm, preferably 5-800 nm, more preferably 10-700 nm, further preferably 15-600 nm, and most preferably 20-250 nm, such as 20 nm, 50 nm, 80 nm, 100 nm, 120 nm, 150 nm, 180 nm, 200 nm, 250 nm, 300 nm, 350 nm, 400 nm, 450 nm, 500 nm, 550 nm, etc. In some embodiments, the particle size of the external vesicles of this invention is, for example, between 20-250 nm, thereby allowing them to passively accumulate in lymph nodes and activate antigen-specific immune responses. OMVs contain various bacterial-derived components, such as enzymes, virulence factors, and bacterial-specific antigens, which can effectively promote the recognition and activation of immune cells, giving them a unique advantage in the development of adjuvants and vaccines. The form of the exovesicles in the composition is not limited; they can be in dry powder, solution, or suspension form, etc.

[0043] In some embodiments, the exovesicles of the present invention include a shell layer on their outer surface, said shell layer being, for example, bound to or encapsulated on the outer surface of the exovesicle by mineralization. Exemplarily, the shell layer comprises a calcium salt. Examples of calcium salts are not limited, but are generally slightly soluble salts; preferably, the calcium salt is soluble in acid but slightly soluble or insoluble in water, thereby ensuring that the exovesicles are only degraded after targeting lymph nodes and being phagocytosed into the lysosomes of dendritic cells, avoiding inflammatory reactions at the administration site. Examples of such calcium salts include, but are not limited to, calcium phosphate and calcium carbonate.

[0044] In some embodiments, the compositions of the present invention are vaccines used to treat or prevent oral infectious diseases or conditions, particularly periodontal disease, including improving or reducing the severity of inflammation, swelling, and improving or reducing bone resorption, bone loss, etc. caused by periodontal disease.

[0045] The compositions of the present invention can be in any suitable dosage form, such as injections, suspensions, emulsifiers, tablets, etc. The compositions of the present invention can be administered into the body in known ways, such as by intramuscular injection to the tissue of interest, or alternatively by intravenous, percutaneous, intranasal, oral, mucosal, or other delivery methods. Such administration can be performed via single or multiple doses. Those skilled in the art will understand that the actual dose to be administered herein can vary considerably depending on a variety of factors, such as the target cells, biological type or tissue thereof, the general condition of the subject to be treated, the route of administration, the manner of administration, etc.

[0046] In this invention, the composition further includes a pharmaceutically acceptable carrier, which is well known in the art and can be determined by those skilled in the art to meet clinical standards. A pharmaceutically acceptable carrier includes diluents and excipients, a pharmaceutically acceptable material, composition, or carrier, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, and such pharmaceutically acceptable material, composition, or carrier participates in the delivery or transport of a drug from one organ or part of the body to another organ or part of the body. Each carrier must be "acceptable," meaning it is compatible with other components of the formulation and does not harm the patient. Some examples of pharmaceutically acceptable carriers include: sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives and analogs such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth gum powder; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols such as propylene glycol; polyols such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters such as ethyl oleate and ethyl lauryl ester; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginate; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer; and other non-toxic and compatible substances used in pharmaceutical preparations. Wetting agents, emulsifiers and lubricants, such as sodium dodecyl sulfonate, magnesium stearate, and polyoxyethylene-polypropylene copolymers, as well as colorants, release agents, coating agents, sweeteners, flavorings and fragrances, preservatives and antioxidants may also be present in the composition.

[0047] In some embodiments, the compositions of the present invention are solid dosage forms for oral administration, including tablets, pills, powders, granules, or capsules. In these solid dosage forms, the active ingredient is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with: (a) fillers or compatibilizers, such as starch, lactose, sucrose, glucose, mannitol, and silica; (b) binders, such as hydroxymethyl cellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose, and gum arabic; (c) humectants, such as glycerin; (d) disintegrants, such as agar, calcium carbonate, potato starch or cassava starch, alginate, certain complex silicates, and sodium carbonate; (e) slowing agents, such as paraffin; (f) absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl monostearate; (h) adsorbents, such as kaolin; and (i) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium dodecyl sulfate, or mixtures thereof. Buffers may also be included in capsules, tablets, and pills.

[0048] In some embodiments, the formulations of the present invention are substances suitable for rapid disintegration into fine particles in the oral mucosa region, thereby enabling the functional ingredients to dissolve and be absorbed rapidly to exert their effects, such as sublingual disintegrating tablets, oral lozenges, oral sprays, hydrogel formulations, etc. Their components include at least one of the following: dry starch, sodium carboxymethyl starch, low-substituted hydroxypropyl methylcellulose, croscarmellose, croscarmellose sodium, micronized silica gel, microcrystalline cellulose, and effervescent disintegrants.

[0049] In some embodiments, the formulation of the present invention is a sublingual disintegrating tablet comprising microcrystalline cellulose, croscarmellose sodium, starch, and micronized silica. Preferably, based on weight, the amount of microcrystalline cellulose is generally 50-80%, more preferably 60-70%, and more preferably 60-65%. The amount of starch is generally 10-40%, preferably 10-30%, and more preferably 10-20%. The amount of croscarmellose sodium is generally 10-40%, preferably 10-30%, and more preferably 15-25%. The amount of micronized silica is generally 1-5%, preferably 1-4%, and even more preferably 1-3%. In the present invention, the protective agent prepared by comprising exovesicles coated with calcium phosphate shells and sublingual disintegrating tablet excipients is in tablet form, which can be stably stored and transported.

[0050] In some embodiments, the composition of the present invention disintegrates under the tongue after being taken orally, releasing calcium phosphate-coated engineered Porphyromonas gingivalis exovesicles which target the oral mucosa-associated lymphoid tissue (OMA) into the oral cavity. After being phagocytosed by dendritic cells, the OMA presents the antigenic components of the pathogen and activates T cells and B cells, promoting the expression of high-affinity, specific, long-acting IgG and sIgA antibodies in saliva, and inhibiting the colonization and infection of the pathogen, thereby achieving an effective, efficient, and long-lasting preventive effect against periodontitis.

[0051] [Preparation Method] A second aspect of the present invention provides a method for preparing a composition for preventing or treating oral bacterial infectious diseases or conditions, comprising the steps of providing exovesicles derived from oral pathogens and coating the surface of the exovesicles with calcium salts to obtain calcium-coated exovesicles.

[0052] In this invention, the external vesicles derived from oral pathogenic bacteria can be obtained by directly purchasing existing external vesicles or by preparing them using known methods. The known methods for preparing the external vesicles are not particularly limited, and any method can be used. Exemplarily, they are obtained by culturing bacteria such as *Porphyromonas gingivalis* into a culture medium, removing the bacterial precipitate by centrifugation, and then ultracentrifuging. Further preferably, a step of filtration after bacterial removal to remove residual bacteria is performed.

[0053] In some embodiments of this invention, the calcium salt used to coat the surface of the calcium-coated exovesicle is generally a slightly soluble or insoluble salt, preferably an acid-soluble salt. The step of coating the exovesicle surface with calcium salt to obtain calcium-coated exovesicles is not limited, but preferably includes a step of contacting and reacting a soluble calcium salt solution with the exovesicle, for example, mineralizing it, thereby making the calcium salt on the exovesicle surface a slightly soluble or insoluble salt.

[0054] In this invention, the mineralization step includes first adding OMVs to DMEM medium and equilibrating overnight at a low temperature (e.g., below 10°C, preferably 4°C). Then, CaCl2 (preferably 0.1-5 M, more preferably 0.5-2 M) is added to the reaction system to initiate the mineralization reaction. The reaction time is preferably 1-5 hours, and the reaction temperature is generally 20-45°C. An exemplary reaction, for example, involves adding OMVs containing 1 mg of protein to DMEM medium and equilibrating overnight at 4°C. Then, 10 µL of 1M CaCl2 is added to the reaction system to initiate the mineralization reaction. The reaction time is preferably 1-5 hours. The reaction temperature is generally 20-45°C, preferably 25-38°C, such as 30°C, 32°C, 34°C, 36°C, 37°C, etc. Under these conditions, the outer vesicles react with a solution of a soluble calcium salt, such as calcium chloride. Calcium ions bind to the negatively charged outer vesicles, and then calcium phosphate crystallizes to form nuclei, thereby forming outer vesicles coated with a calcium phosphate shell. Furthermore, preferably, the preparation method of the present invention further includes a step of centrifugation to collect the precipitate.

[0055] In some embodiments, the preparation method of the present invention further includes the step of freeze-drying the exovesicles coated with calcium salts.

[0056] [use] A third aspect of the invention provides the use of external vesicles derived from oral pathogens in the preparation of medicaments for increasing oral IgG and / or sIgA antibody levels or for increasing serum IgG and / or IgA antibody levels.

[0057] This invention discovers that pathogenic bacteria such as Porphyromonas gingivalis contain a large number of nanoscale vesicles of periodontitis-related immune activation and defense factors in their outer vesicles. Through formulation, these vesicles can be effectively targeted to the mucosa-associated lymph nodes to activate the body's mucosal immunity, thereby generating a large number of specific IgG and sIgA antibodies in the oral cavity.

[0058] Example I. Preparation This embodiment is an exemplary example of tablet preparation, specifically including: 1. Isolation and acquisition of Porphyromonas gingivalis exovesicles (OMV) *Porphyromonas gingivalis* was cultured in a special medium (3.7% brain heart extract powder, 1% heme chloride, 0.1% vitamin K) for 48 h. The bacterial precipitate was removed by centrifugation, and residual bacteria in the supernatant were removed using a 0.22 μm filter. The collected supernatant was centrifuged at 110,000 g for 4 h. The precipitate was collected and resuspended in PBS to obtain *Porphyromonas gingivalis* exovesicles, which can be stored for short periods at 4°C or long periods at -20°C.

[0059] 2. Preparation of *Porphyromonas gingivalis* exovesicles coated with calcium phosphate shells (OMV@CaP) In the preferred embodiment, 1 mg of protein OMV is first added to DMEM medium and equilibrated overnight at 4 °C. Then, 10 μL of 1M CaCl2 is added to the reaction system to initiate the mineralization reaction. After the reaction, the precipitate is collected by centrifugation at 14000 g for 15 min and washed twice with ultrapure water to obtain *Porphyromonas gingivalis* exovesicles coated with a calcium phosphate shell (OMV@CaP). These can be stored for short-term preservation at 4 °C or freeze-dried and then stored long-term at -80 °C.

[0060] 3. Preparation of periodontitis vaccine based on engineered Porphyromonas gingivalis exovesicles in sublingual disintegrating tablets In a preferred embodiment, after preparing *Porphyromonas gingivalis* exovesicles coated with a calcium phosphate shell (OMV@CaP), 10 μg (for tablets with a 2 mm diameter) or 100 μg (for tablets with a 6 mm diameter) of OMV@CaP is lyophilized and then thoroughly mixed with tablet excipients (based on weight: 62% microcrystalline cellulose, 20% croscarmellose sodium, 16% starch, and 2% micronized silica gel). The mixture is then further compressed using a 2 mm or 6 mm tableting machine to obtain the corresponding sublingual disintegrating tablets (OMV@CaP ODT). These tablets can be stored for short periods at 4°C or for long periods at -80°C.

[0061] The following experiments used OMV solution, OMV@CaP solution, and OMV@CaP ODT tablets as subjects.

[0062] II. Results 1. Isolation and characterization of Porphyromonas gingivalis exovesicles In this embodiment, *Porphyromonas gingivalis* exovesicles (OMVs) were isolated and collected by ultracentrifugation, and their morphology was observed using transmission electron microscopy. The results are as follows: Figure 2 As shown in the figure. Next, the protein composition of *Porphyromonas gingivalis* and its external vesicles (OMV) was analyzed using a proteomics chip, and the results are as follows. Figure 3 As shown, OMV contains multiple pathogenic antigens and outer membrane components of *Porphyromonas gingivalis*. The average particle size analysis results for OMV are as follows: Figure 4As shown, the average particle size of OMV is approximately 50.75 nm.

[0063] 2. Preparation of periodontitis vaccine using sublingual disintegrating tablets of Porphyromonas gingivalis exovesicles and its activation effect on immune cells. Following the method described in Part 1, *Porphyromonas gingivalis* exovesicles coated with a calcium phosphate shell were prepared (OMV@CaP). Figure 5 As shown, the average particle size of OMV@CaP coated with a calcium phosphate shell is approximately 105.7 nm. Transmission electron microscopy (TEM) images are shown below. Figure 6 As shown, its outer shell is coated with a calcium phosphate shell, and its overall particle size is larger than that of OMV. Mouse dendritic cells were isolated and cultured in vitro, and then treated with 1 μg / mL LPS, OMV, and OMV@CaP for 48 hours, respectively. The activation of dendritic cells was detected by flow cytometry. The results are as follows: Figure 7 As shown, OMV and OMV@CaP significantly upregulated the expression of antigen presentation-related markers MHC-I, MHC-II, CD40, CD80 and CD86 in dendritic cells, and the promoting effect of OMV@CaP was more significant than that of OMV.

[0064] Following the methods described in Part I, sublingually disintegrating periodontitis vaccines for mice and cynomolgus monkeys were prepared, and the intraoral disintegration of the sublingually disintegrating periodontitis vaccine (OMV@CaP ODT) was observed. Figure 8 (mice) and Figure 9 As shown in the (cynomolgus monkey) illustration, the OMV@CaP ODT disintegrates rapidly within 1 minute after being placed in the mouth. In vivo tracing techniques, including photoacoustic and small animal imaging, confirmed that OMV@CaP can target the submandibular lymph nodes of mice and cynomolgus monkeys after administration. Figure 10 (mice) and Figure 11 As shown in the image (cynomolgus monkey). Gross observation and histological staining of the submandibular lymph nodes in mice and cynomolgus monkeys after administration also confirmed that the OMV@CaP ODT periodontitis vaccine activated a local immune response, resulting in increased lymph node volume, as shown in the image. Figure 12 and Figure 13 As shown.

[0065] 3. The promoting effect of OMV@CaP and OMV@CaP ODT on the level of antigen-specific antibodies in saliva and serum. After drug administration, saliva and serum were continuously collected from mice and cynomolgus monkeys in each group to detect the titers of anti-Porphyromonas gingivalis-specific IgG and sIgA antibodies. Figure 14 (mice) and Figure 15As shown in the (cynomolgus monkey) data, OMV@CaP and OMV@CaP ODT significantly promoted the expression of anti-Porphyromonas gingivalis-specific IgG and sIgA antibodies in the samples, and their effects were long-lasting.

[0066] 4. Protective effects of OMV@CaP and OMV@CaP ODT on periodontitis in mice in vivo. A mouse model of periodontitis was established using suture ligation and bacterial smear. The protective effects of prophylactic administration of OMV@CaP and OMV@CaP ODT on mouse periodontitis were investigated in vivo. Results are as follows: Figure 16 As shown in the results, both OMV@CaP and OMV@CaP ODT effectively inhibited alveolar bone resorption and destruction caused by pathogenic bacteria in primary periodontitis. OMV@CaP ODT was more effective than OMV@CaP, and there was no significant difference compared with the clinically commonly used antibiotic minocycline hydrochloride. However, because OMV@CaP and OMV@CaP ODT activated the long-term expression of the key marker sIgA antibody in saliva, they still had a protective effect against long-term reinfection of periodontitis. However, the antibiotic group mice did not show any advantage in protecting against the periodontitis recurrence model, and exhibited severe alveolar bone resorption upon reinfection with pathogenic bacteria.

[0067] 5. The protective effect of OMV@CaP ODT against periodontitis in cynomolgus monkeys in vivo. A periodontitis model in cynomolgus monkeys was established using silk ligation and bacterial smear. The protective effect of OMV@CaP ODT on periodontitis in cynomolgus monkeys was investigated. The results are as follows: Figure 17 As shown in the results, similar to the results in the mouse in vivo model, OMV@CaP ODT not only effectively inhibited alveolar bone resorption and destruction caused by primary infection with pathogenic bacteria, but also showed a long-term protective effect against periodontal pathogen infection, and still had a protective effect on the periodontal relapse model of long-term reinfection.

[0068] 6. The antibody-boosting effect of OMV@CaP ODT on the saliva of healthy subjects in vivo. Ten healthy subjects were recruited to investigate the effect of OMV@CaP ODT on enhancing human salivary antibodies. The results are as follows: Figure 18 As shown in the figure. The results indicate that OMV@CaP ODT can effectively activate the immune response of the submandibular lymph nodes. All subjects showed a significant increase in sIgA antibodies specific to the pathogenic periodontitis bacteria in their saliva, and the OMV@CaP ODT showed a significant in vitro antibacterial effect against the pathogenic bacteria.

[0069] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions.

Claims

1. A composition for preventing and treating oral bacterial infections or conditions, characterized in that, This includes external vesicles derived from oral pathogens, such as periodontitis pathogens.

2. The composition for preventing and treating oral bacterial infections or conditions according to claim 1, characterized in that, The pathogenic bacteria include Porphyromonas gingivalis, Actinobacillus actinomycetii, Fuseltia, Fusobacterium nucleatum, and Prevotella intermedius.

3. The composition for preventing and treating oral bacterial infections or conditions according to claim 1, characterized in that, The oral diseases mentioned are inflammatory diseases caused by oral bacterial infections, such as periodontitis.

4. The composition for preventing and treating oral bacterial infections or conditions according to claim 1, characterized in that, It further includes calcium salts. Preferably, the surface of the outer vesicle is bound with calcium salts. More preferably, the calcium salts coat the surface of the outer vesicle to form a shell. The calcium salts on the surface of the outer vesicles are slightly soluble or insoluble salts. Preferably, they are acid-soluble salts.

5. The composition for preventing and treating oral bacterial infections or conditions according to claim 1, characterized in that, The composition is a vaccine, such as a periodontitis vaccine.

6. The composition for preventing and treating oral bacterial infections or conditions according to claim 1, characterized in that, The composition is in the form of a sublingual disintegrating tablet, oral lozenge, oral spray, or hydrogel.

7. A method for preparing a composition for preventing and treating oral bacterial infections or conditions, characterized in that, include: The method provides external vesicles derived from oral pathogens and brings calcium salts into contact with the external vesicles so that the surface of the external vesicles is bound with calcium salts. Preferably, the calcium salts are coated on the surface of the external vesicles to obtain calcium-coated external vesicles.

8. The method according to claim 7, characterized in that, include: An external vesicle derived from oral pathogens is provided, and a soluble calcium salt is brought into contact with the external vesicle for reaction, such as mineralization, so that the surface of the external vesicle is bound with calcium salt. Preferably, the calcium salt is coated on the surface of the external vesicle to obtain calcium-coated external vesicles. The calcium salt on the surface of the external vesicle is a slightly soluble salt or an insoluble salt, preferably an acid-soluble salt.

9. The method according to claim 7, characterized in that, The method further includes the step of using calcium-coated exovesicles to form a formulation, preferably a tablet.

10. Use of external vesicles derived from oral pathogens in the preparation of medicaments for increasing oral levels of specific IgG and / or sIgA antibodies against the source bacteria and for increasing serum levels of specific IgG and / or IgA antibodies against the source bacteria.

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