JP2018154574A - Eye drop vaccine and immune induction method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an eye drop vaccine for periodontal disease and an immune induction method for administering the eye drop vaccine to non-human mammals such as cats and dogs.SOLUTION: The eye drop vaccine comprises: (1) an antigenic agent derived from periodontal disease bacteria; and (2) liposomes. Note that formalin-inactivated disrupted microbial cells of Porphyromonas gingivalis can be given as an example of (1) the antigenic agent derived from periodontal disease bacteria. Furthermore, pH sensitive liposomes composed of a combination of dipalmitoylphosphatidylcholine, dioleylphosphatidylethanolamine, monophosphoryl lipid A, and 3-methylglutarylated polyglycidol can be given as an example of (2) the liposome. The immune induction method is a method of administering the eye drop vaccine to non-human mammals such as cats and dogs.SELECTED DRAWING: None

Description

  The present invention relates to an eye drop vaccine and an immunity induction method using the same.

  Periodontal disease is an oral disease with a very high morbidity rate in the veterinary small animal clinical field mainly targeting dogs and cats. Currently, the common preventive measure for periodontal disease is to give toothpaste and dental gum. However, these methods are burdensome for the owner because there are many dogs and cats that hesitate.

  In the case of periodontal disease, calculus removal performed under anesthesia is a common treatment method. However, periodontal disease is more common in relatively older domestic animals. For this reason, many owners hesitate to treat with anesthesia in consideration of the burden on the animals. Therefore, there is a demand for the development of preventive and therapeutic methods that place less burden on both owners and animals.

  Periodontal disease is caused by a group of anaerobic bacteria called black pigment producing bacteria. Among the anaerobic bacteria, Porphyromonas gingivalis is frequently detected in the lesions of periodontal disease in humans, and chronic dentition is caused by pathogenic factors of the fungus and host immune responsiveness. It has attracted the most attention as a pathogenic bacterium of periosis. In addition, Porphyromonas gingivalis proliferates in the gingival margin in primates other than mice, rats, and humans, and is involved in the onset and exacerbation of periodontal disease.

  In many infectious diseases, the pathogen invades and grows on the mucosal surface first. In periodontal disease, it is necessary for the initiation of infection that Porphyromonas gingivalis adheres to the surface of the periodontal epithelial cells, which is the mucosal surface. Therefore, in order to prevent and treat periodontal disease, it is considered effective to induce anti-porphyromonas gingivalis antibody on the mucosal surface (induction of mucosal immune response) (Non-patent Document 1). See).

  In order to induce an immune response, a vaccine is generally administered, and among them, a vaccine is generally administered from a systemic route represented by intramuscular injection. However, vaccination from the systemic route can induce a systemic immune response but not a mucosal immune response. For this reason, the periodontal disease prevention vaccine for dogs using the systemic route of administration, which was launched in the past, was withdrawn from the market soon after because it was unable to induce mucosal immune response and its protective effect was insufficient. .

  In order to induce a mucosal immune response, it is necessary to administer the vaccine via the transmucosal route rather than the systemic route. The main mucosal immune responses are secretory IgA antibodies and local IgG antibodies induced by vaccine administration and microbial invasion via the transmucosal route, and these antibodies prevent pathogens from adhering to mucosal cells. Stop effectively. Furthermore, unlike vaccination from the systemic route, vaccination by the transmucosal route can simultaneously induce not only antigen-specific mucosal immune responses but also systemic immune responses.

  In order to induce a mucosal immune response, a sufficient antigen-specific immune response cannot be induced even if only the antigen is administered to the mucosa. Therefore, establishment of an antigen administration method that effectively induces a mucosal immune response is required.

  By the way, liposomes are attracting attention as a vaccine carrier in recent years because they can be efficiently transported to a target place by encapsulating an antigen inside. In addition, according to conventional research, liposomes are known to have different pharmacokinetics depending on lipid composition, zeta potential, and particle size. By preparing the composition, antigens are stably transported to mucosal immunity-induced tissues. It has been shown that dendritic cells are actively released.

  Therefore, conventionally, transmucosal vaccines, especially eye drop vaccines, have been developed and put into practical use. Specifically, the following can be exemplified.

  First, “an inactivated vaccine containing an inactivated avian influenza virus as an antigen and not containing an oil adjuvant” can be mentioned (see Patent Document 1).

  In addition, “a poultry colibacterial vaccine characterized by containing a formalin-inactivated whole cell of Escherichia coli or a disrupted product thereof, and an adjuvant based on a liposome or a lipid having a liposome-like form” ( (See Patent Document 2).

  In addition, “an ophthalmic vaccine comprising the copolymer 1 without an adjuvant for conferring systemic neuroprotection and thereby conferring therapeutic immunity to glaucoma patients” (see Patent Document 3).

  Furthermore, “a vaccine comprising a viral vector containing a heterologous polynucleotide encoding feline herpesvirus gB antigen and the like and a pharmaceutically or veterinary acceptable carrier and can be administered by eye drops” (Patent Document 4). reference.).

  In addition, “an ophthalmic vaccine in which an immunogen of a peptide or protein to be administered for immunization is contained in a vaccine carrier composed of liposomes containing succinylated polyglycidol” (see Patent Document 5). .)

  However, an ophthalmic vaccine for periodontal disease in mammals such as dogs and cats has not been put to practical use.

JP 2013-1686 A Japanese Patent No. 4161736 Japanese Patent No. 5291878 Japanese Patent No. 5913316 Japanese Patent No. 5054546

Hiroyuki Azagami et al., “How Periodontopathic Bacteria Establish in the Mouth”, Chemistry and Biology Vol.35, No.12, 1997

  It is an object of the present invention to provide an eye drop vaccine for periodontal disease and an immunity induction method for administering the eye drop vaccine to non-human mammals such as dogs and cats.

  As a result of intensive studies, the inventors have instilled administration of an antigenic substance derived from periodontal disease bacteria to induce immunity against periodontal disease in small animals such as dogs and cats and to prevent and treat periodontal disease. The headline and the present invention were completed.

  That is, the eye drop vaccine of the present invention comprises (1) an antigenic substance derived from periodontal disease bacteria and (2) a liposome. In addition, (1) As an antigenic substance derived from periodontal disease bacteria, formalin-inactivated disrupted cells of Porphyromonas gingivalis can be exemplified. Examples of (2) liposomes include pH-sensitive liposomes composed of a combination of dipalmitoyl phosphatidylcholine, dioleyl phosphatidylethanolamine, monophosphoryl lipid A, and 3-methylglutarylated polyglycidol. Furthermore, the immunity induction method of the present invention is a method in which the eye drop vaccine of the present invention is administered to non-human mammals such as dogs and cats.

  The eye drop vaccine and immunity induction method of the present invention can prevent and treat periodontal disease in domestic animals with less stress. Thereby, it becomes difficult for the domestic animal to suffer from periodontal disease, and the pain of the domestic animal and its owner can be reduced.

FIG. 1 is a graph showing changes in serum anti-OVA-IgG antibody titers before and after administration of an ophthalmic vaccine. FIG. 2 is a graph showing changes in the anti-OVA-IgA antibody titer in saliva after administration of an eye drop vaccine. FIG. 3 is a graph showing the results of examining immune induction in serum and saliva after administration of an eye drop vaccine. FIG. 4 is a graph showing the results of an agglutination test on serum and saliva using a living strain of Porphyromonas gingivalis. FIG. 5 is a graph showing the inhibitory activity of saliva against adhesion of Porphyromonas gingivalis to cultured cells. FIG. 6 is a graph showing the results of examining the saliva inhibitory activity against co-aggregation of Porphyromonas gingivalis and Actinomyces nesrundi. FIG. 7A is a graph showing the results of examining the salivary inhibitory activity against Porphyromonas gingivalis cytotoxicity. FIG. 7B is a photomicrograph of cultured cells. FIG. 8 is a graph showing the therapeutic effect of an ophthalmic vaccine for periodontal disease-affected dogs. FIG. 9 is a graph showing the therapeutic effect of an eye drop vaccine on periodontal disease-affected cats.

  The eye drop vaccine of the present invention comprises (1) an antigenic substance derived from periodontal disease bacteria and (2) a liposome. In addition, the immunity induction method of the present invention is a method in which the eye drop vaccine of the present invention is administered by eye drop to a non-human mammal. Therefore, these details will be described below.

1. Eyedrop vaccine (1) Antigenic substance derived from periodontal disease bacteria (a) Periodontal disease bacteria The periodontal bacteria targeted for the eyedrop vaccine of the present invention are not particularly limited as long as they are causative bacteria of periodontal disease. Specifically, Aggregatibacter (formerly known as Actinobacillus), Actinomyces, Porphyromonas, Prevotella, Fusobacterium , Periodontal disease bacteria contained in spirochetes, gram positive cocci, gram negative bacilli, anaerobic vibrio and the like.

  Among them, examples of periodontal disease bacteria included in the genus Aggregatibacter include Aggregatibacter actinomycetemcomitans (formerly known as Actinobacillus actinomycetemcomitans). .

  Examples of periodontal disease bacteria included in the genus Actinomyces include Actinomyces naeslundii.

  In addition, as periodontal disease bacteria contained in the genus Porphyromonas, Porphyromonas gingivalis, Porphyromonas sasaccharolytica, Porphyromonas endodontalis, etc. Is mentioned.

  Examples of periodontal disease bacteria included in the genus Prevotella include Prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, and the like.

  Examples of periodontal disease bacteria contained in the genus Fusobacterium include Fusobacterium nucleatum, Fusobacterium necrophorum, and Fusobacterium naviforme.

  Examples of periodontal disease bacteria contained in spirochetes include Treponema denticola.

  Furthermore, examples of periodontal disease bacteria contained in Gram-negative bacilli include Tannerella forsythia (formerly Tannerella forsythensis).

  Other examples include Bacteroides forsythus, Eikenella corrodens, Capnocytophaga gingivalis, and Wolinella recta.

  Among them, Porphyromonas gingivalis, Tanerella fauciacia, and Porphyro, which are pathogenic bacteria of chronic periodontal disease, are frequently detected in the affected area of periodontal disease and are pathogenic factors of the bacterial cells and host immune responsiveness. Actinomyces nesrundi and Treponema denticola, which co-aggregate with Monas gingivalis to form a biofilm, are preferred as periodontal pathogens that are subject to eye drop vaccines.

(B) Antigenic substance derived from periodontal disease bacteria As an antigenic substance derived from the periodontal disease bacteria of the present invention, disrupted periodontal bacteria or lysate of bacterial bodies, inactivated whole cells of periodontal bacteria, Examples include crushed cells or lysed cells. Here, inactivation means a state in which infectivity is lost by formalin or other chemical treatment, and functions as periodontal disease bacteria (functions such as infection and proliferation) are lost. In addition, the disrupted cells are those obtained by physically disrupting the cells by ultrasonic treatment or the like, and the solubilized cells are those obtained by solubilizing the cells with a surfactant or the like.

  Periodontal pathogen-derived antigenic substances include periodontal pathogens or flagella, periodontal pathogen lipopolysaccharide (LPS), periodontal pathogen outer membrane vesicles (OMVs), periodontal pathogen Proteolytic enzymes (such as gingipain) and periodontal disease outer membrane proteins.

  The content of the antigenic substance in the eye drop vaccine can be appropriately adjusted depending on the type of antigenic substance used, the subject to be administered, its weight, age, and the like. In addition, an antigenic substance can be used individually by 1 type or in combination of multiple types.

(2) Liposomes The liposomes of the present invention can be used without particular limitation as long as they are liposomes made of amphiphilic lipids that can be used in medicines and are harmless to living organisms. Examples of such liposomes include pH-responsive liposomes and positively charged liposomes.

  Here, pH-responsive liposomes are stable in a neutral environment, but in a weakly acidic environment, the liposome membrane becomes extremely unstable and induces membrane fusion, thereby enclosing a closed space surrounded by the liposome membrane. It is a liposome capable of releasing part or all of the substance encapsulated in the liposome membrane. Here, “under a weakly acidic environment” specifically refers to pH 6.9 or lower and pH 4 or higher in consideration of pH conditions that may occur in a living body.

  In addition, the release of the inclusion is due to either or both of the ability of the liposome to destabilize the liposome membrane itself and the increase in liposome membrane fusion by fusing the liposome membrane with another lipid bilayer membrane, etc. It is considered to be a thing.

  Specific examples of pH-responsive liposomes include those containing lipids and carboxyl group-modified polyglycidol as described in Patent Document 5.

  Examples of the lipid constituting the pH-responsive liposome include phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidic acids, long-chain alkyl phosphates, phosphatidylglycerols, cholesterol and the like. In addition, these lipids can be used individually by 1 type or in combination of multiple.

  Here, examples of the phosphatidylcholines include dimyristoyl phosphatidylcholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dioleyl phosphatidylcholine (DOPC), egg yolk lecithin (eggPC) and the like.

  Examples of phosphatidylethanolamines include dioleyl phosphatidylethanolamine (DOPE), dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine (DSPE), and the like.

  Examples of phosphatidyl serines include dioleyl phosphatidyl serine (DOPS) and dipalmitoyl phosphatidyl serine (DPPS).

  Examples of phosphatidic acids or long-chain alkyl phosphates include dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, and dicetyl phosphoric acid.

  Examples of the phosphatidyl glycerols include dimyristoyl phosphatidyl glycerol, dipalmitoyl phosphatidyl glycerol, and distearoyl phosphatidyl glycerol.

  Furthermore, examples of the carboxyl group-modified polyglycidol constituting the pH-responsive liposome include succinylated polyglycidol (SucPG) and 3-methylglutaryl-modified polyglycidol (MGluPG). These carboxyl group-modified polyglycidols can be used singly or in combination.

  Among these, pH-responsive liposomes containing DPPC, DOPE, MPL, and MGluPG are preferable because they can induce a high immune response.

  On the other hand, a positively charged liposome is a liposome that interacts with a negatively charged substance to form a stable complex. Specific examples of the positively charged liposome include those containing the lipid constituting the pH-responsive liposome and the positively charged lipid. Positively charged lipids include N- (α-trimethylammonioacetyl) -didodecyl-D-glutamate chloride (TMAG), 3β [N- (N ', N'-dimethylaminoethane) -carbamoyl] cholesterol ( DC-Chol) and didecyldimethylammonium bromide (DDAB). These lipids and positively charged lipids can be used alone or in combination.

  Generally, liposomes are multilamellar vesicles, dehydration-rehydration vesicles, large unilamellar vesicles or small unilamellar vesicles, depending on their structure or preparation method. The liposome of the present invention may be any of these.

  The liposome of the present invention can be prepared by a known liposome preparation method without any particular limitation. For example, as described in Patent Document 5, lipids are dissolved in an appropriate organic solvent (for example, chloroform, methanol), and these solvents are distilled off under reduced pressure to form a lipid thin film. A method of hydrating or swelling the thin film in water by a mechanical stirring means can be mentioned.

(3) Other components The ophthalmic vaccine of the present invention is a known solvent, adjuvant (antigenic reinforcing agent), thickener, buffer, isotonic agent, preservative, as long as the immune induction effect is not impaired. , Bactericides or antibacterial agents, stabilizers, chelating agents, preservatives, solubilizers, pH adjusters, surfactants and the like.

  Specifically, solvents such as water and ethanol, monophosphoryl lipid A (MPL), α-galactosylceramide, poly I: C, trehalose dimycolate, imidazoquinoline derivative (imiquimod or R-848), unmethylated CpG, cytokine , Lectins and other adjuvants, carboxyvinyl polymers, thickeners such as hydroxyethyl cellulose, sodium phosphate, sodium acetate, boric acid, borax, citric acid and other buffering agents, sodium chloride, concentrated glycerin and other isotonic agents Preservatives such as benzalkonium chloride and parabens, bactericides or antibacterial agents, stabilizers such as sodium citrate and sodium edetate, chelating agents such as ethylenediaminetriacetic acid and ethylenediaminetetraacetic acid, chlorobutanol and thimerosal Agents, solubilizing agents such as glycerin and propylene glycol, hydrochloric acid, pH adjusting agents such as sodium oxide, polyoxyethylene sorbitan monooleate, may contain surfactants such as polyoxyl stearate.

(4) Preparation Method of Eye Drop Vaccine The eye drop vaccine of the present invention can be prepared by a known method of including a drug or the like in a liposome. For example, the liposome components are mixed with a solvent and placed in a container. After removing the solvent and forming a lipid film on the inner wall of the container, a solution containing an antigenic substance or adjuvant is placed in the container. Examples include a method of encapsulating an antigenic substance or the like in a liposome membrane by stirring and mixing them.

2. Immunity induction method The immunity induction method of the present invention is a method in which the eye drop vaccine of the present invention is administered by eye drop to a non-human mammal. The method of the present invention can be applied without particular limitation as long as it is a non-human mammal, but considering the influence on the owner, it can be applied to domestic animals such as cattle, pigs, and horses, and pets such as dogs and cats. Preferably, application to dogs or cats is more preferable. Intraocular administration is an administration method in which the inactivated vaccine is administered to the eye socket and an antigenic substance is introduced into the body through the mucous membrane of the eye.

  As a specific eye drop administration method, for example, the eye drop vaccine of the present invention is filled in an eye drop container made of polyethylene terephthalate or the like, and the eye drop of the eye drop vaccine is placed on one eye of the non-human animal (preferably both eyes) from above. One method is to drop and administer it. The eye drop vaccine may be injected into the eye using a syringe, water gun, spray, or the like. Thereby, it can be administered to many non-human mammals at once.

  The dose of the eye drop vaccine may be appropriately adjusted according to the type, age, weight, etc. of the non-human mammal to be administered. Moreover, in the immunity induction method of the present invention, in order to confer sufficient immunity, it is preferable to administer twice or more at intervals of 2 weeks to 2 months or more.

  Hereinafter, the present invention will be described in more detail based on examples. The claims of the present invention are not limited in any way by the following examples.

I. Analysis of immune response by ophthalmic administration of pH-sensitive liposome-encapsulated antigen Whether mucosal immune response can be induced by encapsulating model antigen or Porphyromonas gingivalis antigen in pH-sensitive liposomes and instilling them into dogs Examined. Specifically, it investigated as follows.

I-1. Analysis of immune response by ophthalmic albumin-encapsulated pH-sensitive liposomes by ophthalmic administration Preparation of pH-sensitive liposomes encapsulating ovalbumin (OVA) as a model antigen was administered to dogs in serum and saliva. The immune response in was investigated. Specifically, it investigated as follows.

1. Materials and Methods (1) Test animals Animal experiments were in accordance with the guidelines for animal experiments at the Graduate School of Life and Environmental Sciences, Osaka Prefecture University. A 10-month-old beagle (female, approximately 10 kg in weight) was purchased from Kitayama Labes Co., Ltd. and reared and managed at the Animal Science Education Research Center. For the feeding, dry-type dog food was given once a day, and drinking water was freely consumed. The lighting was under 12 hours light period and 12 hours dark period.

(2) Preparation of OVA-encapsulated pH-sensitive liposomes
OVA-encapsulated pH-sensitive liposomes were prepared by the following procedure. DPPC (15 μmol, Sigma-Aldrich Co., LTd.), DOPE (15 μmol, Sigma-Aldrich Co., LTd.), MPL (60 μg, Sigma-Aldrich Co., LTd.), MGluPG (9.6 mg, lipids / polymer = 7/3, w / w) were each dissolved in an organic solvent. DPPC and DOPE were dissolved in chloroform: methanol = 2: 1 (v / v), MPL was dissolved in chloroform: methanol 1: 2 (v / v), and MGluPG was dissolved in methanol.

  These were mixed in a pear-shaped flask, the solvent was removed by a rotary evaporator, and a lipid film was prepared. After completely removing the organic solvent with a vacuum pump (room temperature for 30 minutes), add 1 ml of OVA phosphate buffered saline (PBS) solution (5 mg / ml) and let stand at room temperature for 3 minutes. Stir and encapsulate OVA in liposomes. After that, centrifugal washing was performed 3 times at 14,000 × g, 4 ° C. for 30 minutes. This liposome was suspended in PBS and used as an OVA-encapsulated pH-sensitive liposome.

  The amount of OVA encapsulated in the liposome was evaluated by the following method. An equal volume of isopropanol was added to 60 μl of OVA-encapsulated pH-sensitive liposome suspension and mixed well to release OVA in the liposomes, which was estimated from the protein concentration in the solution. The protein in the solution was quantified using Bio-Rad protein assay kit (Bio-Rad Laboratories, Inc.) using bovine serum albumin as a standard protein. In addition, PBS-encapsulated pH-sensitive liposomes encapsulating only PBS were prepared using the same lipid composition and procedure.

(3) Administration of OVA-encapsulated pH-sensitive liposomes to experimental animals Beagle was divided into two groups of 4 each, and the group administered with OVA-encapsulated pH-sensitive liposomes (1 mg OVA / 100 μl / eye) was designated as the OVA-encapsulated pH-sensitive liposome administration group. A group administered with PBS-encapsulated pH-sensitive liposomes (100 μl / eye) was used as a control group. Each liposome was administered twice in total every 2 weeks.

(4) Collection of serum and saliva Peripheral blood was collected from the cephalic vein before administration of each liposome and 2 weeks after the second administration. The collected peripheral blood was incubated at 37 ° C. for 60 minutes, and then centrifuged at 750 × g for 10 minutes to separate serum. Saliva was collected using a cotton swab two weeks after the second administration of each liposome. A cotton swab containing saliva was placed in a 15 ml plastic tube and centrifuged at 1,000 × g for 10 minutes to separate the saliva. The obtained serum and saliva were stored at −30 ° C. until analysis.

(5) Measurement of antibody titer in serum and saliva by ELISA
OVA solution (20 μg / ml in PBS) was added to the ELISA plate at 50 μl per well and allowed to stand at 4 ° C. overnight. Thereafter, the solution in the plate was removed, and 100 μl of 5% skim milk-added PBS was added per well, followed by incubation at 37 ° C. for 1 hour for blocking. After blocking, the plate was washed twice with PBS supplemented with 0.05% Tween 20, and 50 μl of 2-fold serially diluted sample (serum or saliva) was added per well and allowed to stand overnight at 4 ° C. for reaction.

  After the reaction, the plate was washed 3 times with PBS containing 0.1% Tween 20, and HRP-labeled anti-dog IgG antibody (10,000-fold diluted with PBS; Bethyl Laboratories) or HRP-labeled anti-dog IgA antibody (10,000-fold diluted with PBS; Bethyl Laboratories) was added as a secondary antibody at 50 μl per well and incubated at 37 ° C. for 1 hour.

  Thereafter, the plate was washed 5 times with PBS containing 0.1% Tween 20 and developed for 10 minutes using a peroxidase coloring kit ML-1130 O (Sumitomo Bakelite Co., LTd.). After the color development was stopped, the absorbance at a wavelength of 490 nm was measured using a microplate reader (Model 450, Bio-Rad Laboratories, Inc.). The highest dilution factor showing an absorbance of 2.5 times or more of the background was defined as the antibody titer of the sample.

(6) Statistical processing The average value ± standard deviation (SD) was calculated from the experimental data. Statistical significance was tested using Welch's t-test. Note that there was a significant difference when the p-value was 0.05 or less.

2. Results FIG. 1 shows the serum anti-OVA-IgG antibody titer before and after instillation administration. As shown in this figure, the serum anti-OVA-IgG antibody titer showed a slightly high value before administration of each liposome and showed an increasing tendency after administration, but there was no significant difference from the control group. It was. Thus, there was no difference in antigen-specific antibody titer with and without OVA administration.

  Moreover, the anti-OVA-IgA antibody titer in saliva after eye drop administration is shown in FIG. As shown in this figure, in the OVA-encapsulated pH-sensitive liposome-administered group at 2 weeks after the second administration, a significantly higher induction of anti-OVA-IgA antibody was confirmed compared with the control group (p <0.05). .

I-2. Analysis of immune response by administration of pH-sensitive liposomal ophthalmic vaccine encapsulating Porphyromonas gingivalis crushing antigen From the results of I-1, by administration of ophthalmic pH-sensitive liposomes, It became clear that there was a possibility of inducing an immune response. Therefore, a pH-sensitive liposome encapsulating an antigen obtained by inactivating and crushing periodontal disease bacteria, Porphyromonas gingivalis, was prepared, and this was instilled into dogs, and the immune response in serum and saliva was examined. Specifically, it investigated as follows.

1. Materials and Methods (1) Test animals Animal experiments were in accordance with the guidelines for animal experiments at the Graduate School of Life and Environmental Sciences, Osaka Prefecture University. A 10-month-old beagle (female, approximately 10 kg in weight) was purchased from Kitayama Labes Co., Ltd. and reared and managed at the Animal Science Education Research Center. For the feeding, dry-type dog food was given once a day, and drinking water was freely consumed. The lighting was under 12 hours light period and 12 hours dark period.

(2) Preparation of Porphyromonas gingivalis disrupted antigen Porphyromonas gingivalis (ATCC 33277) was obtained from The American Type Culture Collection. Porphyromonas gingivalis is a medium in which hemin (4 μg / ml) and menadione (0.4 μg / ml) are added to brain heart infusion medium (Nissui Pharmaceutical Co., LTd.), ANNSUBISHI GAS CHEMICAL COMPANY , INC) was used under anaerobic conditions of 37 ° C., 80% N ₂ , 10% H ₂ and 10% CO ₂ .

  Porphyromonas gingivalis disrupted antigen was prepared by the following procedure. Porphyromonas gingivalis was cultured under anaerobic conditions at 37 ° C. for 72 hours. Formalin was added to the medium to a final concentration of 0.5% and inactivated by leaving overnight. The resulting bacterial suspension was centrifuged at 10,000 × g for 30 minutes to remove the supernatant, and then washed with PBS three times in total to remove formalin. The obtained inactivated bacteria were subjected to ultrasonic treatment (20 kHz, 50 W) for 15 minutes using an ultrasonic generator (BRANSON Sonifier 250, Emerson Japan) to obtain a pulverized Porphyromonas gingivalis. The main contents of the pulverized Porphyromonas gingivalis include cell walls of bacteria including pili, lipopolysaccharide (LPS), capsule, proteolytic enzyme (mainly gindipain), hemagglutinin, bacterial outer membrane protein, etc. It is.

(3) Preparation of pH-sensitive liposome vaccine encapsulating Porphyromonas gingivalis antigen Encapsulating Porphyromonas gingivalis crushed material in the same manner as that for preparing OVA-encapsulated pH-sensitive liposomes in 1-1 A pH-sensitive liposomal vaccine encapsulating Romonas gingivalis antigen was prepared. PBS-encapsulated pH-sensitive liposomes were also prepared in the same manner as I-1.

(4) Vaccine administration to experimental animals Beagle was divided into 2 groups of 4 animals, and the group administered with Porphyromonas gingivalis crushed antigen-encapsulated pH-sensitive liposome vaccine (1 mg protein / 100 μl / eye) was designated as the vaccine administration group . The vaccine was administered 3 times in total every 2 weeks. A group administered with PBS-encapsulated pH-sensitive liposomes (100 μl / eye) was used as a control group.

(5) Collection of serum and saliva Serum and saliva were collected a total of 4 times before vaccine administration, 2 weeks after the first vaccine administration, 2 weeks after the second vaccine administration, and 2 weeks after the third vaccine administration. Serum and saliva were collected by the same method as I-1.

(6) Antibody titer determination in serum and saliva by ELISA method Add 50 μl per well of Porphyromonas gingivalis antigen solution (20 μg / ml in PBS) and leave at 4 ° C overnight. did. Thereafter, the solution in the plate was removed, and 100 μl of 5% skim milk-added PBS was added per well, followed by incubation at 37 ° C. for 1 hour for blocking. After blocking, the plate was washed twice with PBS supplemented with 0.05% Tween 20, and 50 μl of 2-fold serially diluted sample (serum or saliva) was added per well and allowed to stand overnight at 4 ° C. for reaction.

  After the reaction, the plate was washed 3 times with PBS containing 0.1% Tween 20 and diluted with HRP-labeled anti-dog IgG antibody (10,000-fold diluted with PBS; Bethyl Laboratories) or HRP-labeled anti-dog IgA antibody IgA (10,000-fold diluted with PBS). ; Bethyl Laboratories) was added as a secondary antibody at 50 μl per well and incubated at 37 ° C. for 1 hour.

  Thereafter, the plate was washed 5 times with PBS containing 0.1% Tween 20 and developed for 10 minutes using a peroxidase coloring kit ML-1130 O (Sumitomo Bakelite Co., Ltd.). After the color development was stopped, the absorbance at a wavelength of 490 nm was measured using a microplate reader (Model 450, Bio-Rad Laboratories, Inc.). The highest dilution factor showing an absorbance of 2.5 times or more of the background was defined as the antibody titer of the sample.

(7) Measurement of agglomeration titer by agglutination test
In order to clarify whether the antigen-specific antibody induced by administration of the pH-sensitive liposome vaccine by instillation route can recognize not only the Porphyromonas gingivalis disrupted antigen but also the surface of Porphyromonas gingivalis cells, Agglutination tests were used. Specifically, it investigated as follows.

A viable Porphyromonas gingivalis that has not been inactivated and sonicated is washed twice with PBS and diluted to OD ₆₀₀ = 1 (corresponding to 1x10 ⁹ CFU / ml), and Porphyromonas gingivalis suspension A suspension was prepared. Thereafter, 50 μl of serum or saliva diluted at various magnifications and a suspension of Porphyromonas gingivalis (50 μl) were added to a 96-well V-bottom plate (AGC Techno Glass Co., LTd.) And stirred well. Aggregation was observed after standing at 37 ° C. for 1 hour in a humid environment, and the maximum dilution factor at which aggregation occurred was defined as the aggregation titer.

(8) Statistical processing The average value ± standard deviation (SD) was calculated from the experimental data. Statistical significance was tested using Welch's t-test. Note that there was a significant difference when the p-value was 0.05 or less.

2. Results (1) Antibody induction in whole body (serum) and oral mucosa (saliva)
The results of examining immune induction (induction of antigen-specific IgG antibody and IgA antibody) in serum and saliva after instillation of pH-sensitive liposome vaccine are shown in FIG. 3A shows the result of serum anti-porphyromonas gingivalis-IgG antibody titer, and FIG. 3B shows the result of salivary anti-porphyromonas gingivalis-IgA antibody titer.

  As shown in these figures, in serum, the anti-Porphyromonas gingivalis-IgG antibody titer increased significantly compared with the control group from 2 weeks after the second administration in the vaccine administration group (p <0.05). In saliva, the anti-porphyromonas gingivalis-IgA antibody titer increased significantly compared to the control group from the second week after the second administration in the vaccine administration group (p <0.05). On the other hand, in the control group, neither anti-Porphyromonas gingivalis-IgA nor anti-Porphyromonas gingivalis-IgG showed any change in antibody titer.

(2) Recognition of Porphyromonas gingivalis cell surface In order to clarify whether the surface of Porphyromonas gingivalis cell body can be recognized, the result of agglutination test using live bacteria is shown in FIG. 4A shows the results relating to the serum anti-porphyromonas gingivalis agglutinin titer, and FIG. 4B shows the salivary anti-porphyromonas gingivalis agglutinin titer.

  As shown in FIG. 4A, the serum showed a tendency to increase the agglutinin titer with time in the vaccine administration group, and the increase was significant compared to the control group after 2 weeks from the second administration (p <0.05). . In addition, as shown in FIG. 4B, in saliva, as in serum, an increasing tendency of the agglutinin titer was observed in the vaccine administration group from 2 weeks after the second administration, and the agglutinin was observed from 2 weeks after the third vaccine administration. The titer increased significantly compared with the control group (p <0.05). On the other hand, in the control group, no change in the agglutinin titer was observed in both serum and saliva.

II. Analysis of the pathogenic factor neutralization activity induced in saliva of dogs instilled with pH-sensitive liposomal vaccine containing Porphyromonas gingivalis antigen (in vitro)
Porphyromonas gingivalis is known to develop and exacerbate periodontal disease due to multiple factors. Specifically, there are the following factors. First, Porphyromonas gingivalis attaches to oral epithelial cells at the stage of infection establishment, directly injuring cells and causing inflammation. As a result, leakage of components that promote the growth of other causative bacteria such as tissue fluid and blood components starts with tissue destruction, and periodontal disease worsens.

  Porphyromonas gingivalis forms a biofilm by coaggregating with a kind of oral bacteria, Actinomyces nesrundi, and is resistant to antibacterial innate immunity substances and drugs such as saliva in the oral environment To win. In addition, epithelial cell damage is promoted by stimulation of bacterial factors.

  Therefore, antibodies induced by a pH-sensitive liposome vaccine encapsulating Porphyromonas gingivalis-encapsulated antigen were attached to Porphyromonas gingivalis (II-1) epithelial cells, (II-2) Actinomyces nesrundi (II-3) whether or not damage to epithelial cells can be suppressed was analyzed. Specifically, it investigated as follows.

II-1. Analysis of inhibitory activity of Porphyromonas gingivalis on adhesion to HeLa cells Periodontal disease is caused by adhesion and proliferation of Porphyromonas gingivalis to gingival epithelial cells. It is thought to lead to the suppression of the onset of the disease. Thus, human epithelial cell line HeLa cells were used as a model of gingival epithelial cells, and it was analyzed whether antibodies induced in saliva can suppress the adhesion of Porphyromonas gingivalis to HeLa cells. Specifically, it investigated as follows.

1. Materials and methods (1) Cell lines used
HeLa cells were cultured in Dulbecco's Modified Eagle medium (Nissui PHARMACEUTICAL Co., LTd.) Supplemented with 10% fetal bovine serum (FBS). Porphyromonas gingivalis was obtained and cultured in the same manner as in Example 1.

(2) Test for inhibiting adhesion of Porphyromonas gingivalis to epithelial cells by saliva The adhesion inhibition test to epithelial cells by Porphyromonas gingivalis was carried out by partially modifying the method of Nakagawa et al. Specifically, it investigated as follows.

  Nakagawa, I., Inaba, H., Yamamura, T., Kato, T., Kawai, S., Ooshima, T. and Amano, A. 2006. InvasionofepithelialcellsandproteolysisofcellularfocaladhesioncomponentsbydistincttypesofPorphyromonasgingivalisfimbriae.Infect.Immun2.74:

First, HeLa cells were cultured on a 24-well plate (1 × 10 ⁴ cells / well, AGC Techno Glass Co., LTd.) Under conditions of 37 ° C., 5% CO ₂ , 95% air. Cultured overnight. Subsequently, the cells were washed 3 times with PBS. The saliva obtained in the experiment of I-2 in Example 1 was diluted to final concentrations of 1: 2, 1: 4, 1: 8, 1:16 and mixed with Porphyromonas gingivalis. The layered HeLa cells were added so that the multiplicity of infection of Porphyromonas gingivalis was 500. In addition, what added PBS instead of saliva to Porphyromonas gingivalis was made into the control group.

After standing at 37 ° C. for 90 minutes in the presence of 5% CO _2, Porphyromonas gingivalis not attached to the HeLa cells was removed by washing 3 times with PBS. These were stained with Giemsa, and the number of HeLa cells was observed under an optical microscope, and the number of cells to which bacteria were attached was counted. The inhibition rate was determined based on the following calculation formula.

(3) Statistical processing The mean value ± standard deviation (SD) was calculated from the experimental data. Statistical significance was tested using Welch's t-test. Note that there was a significant difference when the p-value was 0.05 or less.

2. Results FIG. 5 shows the activity of inhibiting the adhesion of Porphyromonas gingivalis to cultured cells of saliva. As shown in this figure, in the vaccine administration group, attachment of Porphyromonas gingivalis to HeLa cells was suppressed depending on the saliva concentration. On the other hand, almost no inhibitory activity was observed in the control group. More specifically, in the saliva of the vaccine administration group, 35.8 ± 5.6% adhesion suppression was observed at the final dilution ratio of 1: 2, and 28.0 ± 5.8% adhesion suppression was observed even at the final dilution ratio of 1: 4 (both There is a significant difference at p <0.05 compared to the control group.)

II-2. Analysis of inhibitory activity against the co-aggregation of Porphyromonas gingivalis and Actinomyces nesrundi Porphyromonas gingivalis forms a biofilm by coaggregating with Actinomyces nesrundi, a kind of oral bacteria, Gain resistance to drugs in the oral environment. In addition, it promotes periodontal disease by stimulating various pathogenic factors produced by bacteria that coexist in itself and in biofilm. Therefore, suppression of biofilm is thought to lead to prevention of periodontal disease progression.

  Therefore, we analyzed whether saliva obtained by administration of Porphyromonas gingivalis crushed antigen-encapsulated pH-sensitive liposome vaccine can suppress coaggregation of Porphyromonas gingivalis and Actinomyces nesrundi. Specifically, it investigated as follows.

1. Materials and Methods (1) Bacterial strain used Porphyromonas gingivalis was obtained and cultured in the same manner as in Example 1. Actinomyces Nesrundi (ATCC 12104) was obtained from The American Type Culture Collection. Actinomyces Nesrundi in the medium with a partial modification of ATCC medium 1490 (Grilled Beef changed to Beef extract powder (Sigma-Aldrich Co., Ltd.)) Aneropac Kenki (MITSUBISHI GAS CHEMICAL COMPANY, INC) Was cultured under conditions of 37 ° C., 80% N ₂ , 10% H ₂ and 10% CO ₂ .

(2) Coaggregation inhibition test of Porphyromonas gingivalis and Actinomyces nesrundi by saliva The coaggregation inhibition test was carried out by partially modifying the following methods reported so far. Specifically, it investigated as follows.

  Cisar, J.O., Kolenbrander, P.E.andMcIntire, F.C.1979.SpecificityofcoaggregationreactionbetweenhumanoralstreptococciandstrainsofActinomycesviscosusandActinomycesnaeslundii.Infect.Immun.24: 742-752.

First, the cultured Porphyromonas gingivalis and Actinomyces nesrundi were each washed three times with PBS and diluted to an OD ₆₀₀ = 1 to obtain a suspension. Next, add 50 μl of the Porphyromonas gingivalis suspension to the 96-well V-bottom plate, add the same volume of 1: 2 to 2-fold diluted saliva solution, stir, and then in a humid environment at 37 ° C for 1 hour. Cultured. Further, 50 μl of Actinomyces nesrundi suspension was added and further cultured for 1 hour in a humid environment at 37 ° C., and then the coaggregation inhibitory activity was observed under an optical microscope. The coaggregation-inhibiting antibody titer was the reciprocal of the highest dilution factor that inhibited coaggregation.

(3) Statistical processing The mean value ± standard deviation (SD) was calculated from the experimental data. Statistical significance was tested using Welch's t-test. Note that there was a significant difference when the p-value was 0.05 or less.

2. Results The results of examining the coaggregation inhibitory activity of Porphyromonas gingivalis and Actinomyces nesrundi in the obtained saliva are shown in FIG. As shown in this figure, in the Porphyromonas gingivalis disrupted antigen-encapsulated pH-sensitive liposome vaccine administration group, an increase in the coaggregation-inhibiting antibody titer was observed at 4 weeks, 2 weeks after the second administration (in the control group). On the other hand, there was a significant difference at p <0.05), and a further increasing tendency of the coaggregation inhibitory antibody titer was observed even at 6 weeks, 2 weeks after the third vaccine administration (p <0.05 compared with the control group). . On the other hand, no increase in coaggregation inhibitory antibody titer was observed in the control group.

II-3. Analysis of the inhibitory effect of Porphyromonas gingivalis on oral epithelial cell injury Various pathological conditions related to periodontal disease are caused by injury of oral epithelial cells by proteolytic enzymes etc. produced by Porphyromonas gingivalis . For this reason, suppression of cytotoxicity of Porphyromonas gingivalis on oral epithelial cells is thought to lead to prevention of periodontal disease and improvement of the disease state.

  Therefore, using FaDu cells (human oral epithelial cells), which are human oral epithelial cells, as a model of gingival cells, saliva obtained by administration of a pH-sensitive liposome vaccine encapsulating Porphyromonas gingivalis crush antigen was obtained as Porphyromonas. -It was analyzed whether gingivalis can suppress cytotoxicity of FaDu cells. Specifically, it investigated as follows.

1. Materials and methods (1) Cell lines and bacterial strains used
FaDu cells were cultured in Eagle's minimum essential medium (Nissui PHARMACEUTICAL CO., Ltd.) with 10% FBS. Porphyromonas gingivalis was obtained and cultured in the same manner as in Example 1.

(2) Cytotoxicity test The cytotoxicity test was carried out by partially modifying the following technique reported so far. Specifically, it investigated as follows.

  Eick, S., Rodel, J., EinaxJ.W.andPfister, W.2002.InteractionofPorphyromonasgingivaliswithKBcells: comparisonofdifferentclinicalisolates.OralMicrobiol.Immunol.17: 201-208.

First, FaDu cells were seeded in a 24-well culture plate (AGC Techno Glass Co., LTd.) At 5 × 10 ³ cells / well and cultured for 24 hours before the experiment. Next, a medium containing Porphyromonas gingivalis mixed with various concentrations of saliva samples or untreated Porphyromonas gingivalis (1x10 ⁴ cells / well) is added to the wells, and the mixture is incubated at 37 ° C in a humid environment. Incubate for hours. Furthermore, the plate was washed 3 times with PBS to remove cells detached from the plate, and the cells remaining attached to the plate were counted as viable cells. A control group was prepared by adding only PBS without adding Porphyromonas gingivalis to FaDu cells. Further, the ratio of the number of viable cells to the number of cells in the control group (cell viability) was calculated according to the following formula.

(3) Statistical processing The mean value ± standard deviation (SD) was calculated from the experimental data. Statistical significance was tested using Welch's t-test. Note that there was a significant difference when the p-value was 0.05 or less.

2. Results FIG. 7A shows the results of examining the porphyromonas gingivalis cytotoxicity-inhibiting activity of saliva obtained by vaccine administration. As shown in this figure, the survival rate of FaDu cells treated with saliva in the vaccine administration group was significantly higher than that of the control group at 1: 2 and 1: 4, as compared to the control group (p <0.05). FIG. 7B shows a micrograph (400 times) of cultured cells at a saliva dilution ratio of 1: 2. As shown in FIG. 7B, compared to the control group, many live cells could be observed in the FaDu cells treated with the saliva of the vaccine administration group.

III. Evaluation of therapeutic effect by administration of pH-sensitive liposome vaccine encapsulating Porphyromonas gingivalis antigen in periodontal disease affected animals In Example 2, in the saliva of dogs administered with pH-sensitive liposome vaccine encapsulating Porphyromonas gingivalis antigen The induced pathogenic factor neutralizing activity was analyzed. As a result, antibodies in saliva suppress factors related to the onset of periodontal disease of Porphyromonas gingivalis (adhesion to epithelial cells, co-aggregation with Actinomyces nesrundi, cytotoxicity to oral epithelial cells) It was shown that. Therefore, the vaccine was administered to an animal suffering from periodontal disease, and the therapeutic effect of periodontal disease by the vaccine administration was evaluated. Specifically, it investigated as follows.

III-1. Evaluation of therapeutic effects on periodontal disease dogs Porphyromonas gingivalis crushed antigen-encapsulated pH-sensitive liposome vaccine was administered to dogs suffering from periodontal disease, and the therapeutic effect of the vaccine administration was evaluated. Specifically, it investigated as follows.

1. Materials and Methods (1) Test animals Of dogs with periodontal disease who have visited the Noda Hanshin Animal Hospital and have given sufficient informed consent to their owners, select dogs with a halitosis of moderate (apparently smell) or higher It is. The selected dog was a miniature dachshund or chihuahua, the age was 7 to 15 years or the age was unknown (breeding history of 5 years or more), and the body weight was 3.7 to 5.2 kg.

(2) Preparation of Porphyromonas gingivalis crushed antigen-encapsulated pH sensitive liposome vaccine Porphyromonas gingivalis crushed antigen and Porphyromonas gingivalis crushed antigen encapsulating pH sensitive liposome vaccine were prepared by the method described in Example 1.

(3) Vaccine administration to periodontal disease dogs Periodontal disease dogs are divided into 2 groups of 4 each, and a pH-sensitive liposomal vaccine (1 mg protein / 100 μl / eye) encapsulating Porphyromonas gingivalis crush antigen is administered by eye drops. The treated group was designated as a vaccine administration group, and the untreated group was designated as a control group. The vaccine was administered twice every two weeks.

(4) Evaluation of clinical score The clinical score of periodontal disease was determined according to the criteria described in the following literature. Details of the criteria are listed in Table 1. The overall clinical score was obtained by integrating the scores of each item.

  Immunology Research Institute in Gifu. `` Efficacy and safety evaluation test for periodontal disease dogs by administration of dry pet food supplemented with anti-gingipain antibody for 8 weeks '' http://www.igy-research.com/Data3 (PETPG). pdf [Accessed 29 December 2016].

(5) Statistical processing The mean ± standard deviation (SD) was calculated from the clinical score. Statistical significance was tested using Welch's t-test. Note that there was a significant difference when the p-value was 0.05 or less.

2. Results Gingival congestion, gingival bleeding, gingival swelling and bad breath, which are clinical symptoms of periodontal disease, are evaluated based on the individual clinical score criteria for each individual, and the total clinical score obtained by integrating the individual clinical scores is also evaluated. did. The result is shown in FIG.

  As shown in FIG. 8, in the vaccine administration group, the gingival redness, gingival swelling, and bad breath scores were significantly improved in the second week after the second administration (p <0.05), but the gingival bleeding score was significantly improved. Was not seen. On the other hand, no improvement was observed in any of the clinical scores in the control group, and the gingival bleeding score was significantly worsened (p <0.05).

  Similarly, as shown in FIG. 8, the overall clinical score also showed a significant improvement in the vaccine administration group 2 weeks after the second administration (p <0.05). On the other hand, there was no improvement in the control group, but there was a tendency to get worse.

  From these results, it was shown that the Porphyromonas gingivalis crushed antigen-encapsulated pH-sensitive liposome ophthalmic vaccine has a therapeutic effect on dog periodontal disease or an effect of suppressing the progression of symptoms.

III-2. Evaluation of therapeutic effects on periodontal diseased cats An inactivated Porphyromonas gingivalis crush antigen-encapsulated pH-sensitive liposome vaccine was instilled into periodontal diseased cats, and the therapeutic effect of the vaccine administration was evaluated.

1. Materials and Methods (1) Test animals Among cats with periodontal disease who have visited the Noda Hanshin Animal Hospital and have given sufficient informed consent to their owners, choose a cat with a moderate (or clearly smelling) halitosis It is. The cats selected were hybrids, ages 2-15 years, and weights 2.6-5.2 kg.

(2) Preparation of inactivated Porphyromonas gingivalis crushed antigen-encapsulated pH-sensitive liposome vaccine Preparation of Porphyromonas gingivalis crushed product and Porphyromonas gingivalis crushed antigen-encapsulated pH-sensitive liposome vaccine by the method described in Example 1 did.

(3) Vaccine administration to periodontal disease affected cats The periodontal disease affected cats were divided into 2 groups, and the group treated with instilled pH-sensitive liposomal vaccine (0.5mg protein / 50μl / eye) containing Porphyromonas gingivalis crush antigen. The vaccine administration group (4 animals) was used, and the untreated group was the control group (9 animals). The vaccine was administered a total of 2 times every 2 weeks.

(4) Evaluation of clinical score The clinical score of periodontal disease was determined according to the criteria shown in Table 1. The overall clinical score was obtained by integrating the scores of each item.

(5) Statistical processing The mean ± standard deviation (SD) was calculated from the clinical score. Statistical significance was tested using Welch's t-test. Note that there was a significant difference when the p-value was 0.05 or less.

2. Results Gingival congestion, gingival bleeding, gingival swelling and bad breath, which are clinical symptoms of periodontal disease, are evaluated based on the individual clinical score criteria for each individual, and the total clinical score obtained by integrating the individual clinical scores is also evaluated. did. The result is shown in FIG.

  As shown in FIG. 9, in the vaccine administration group, the gingival redness score was significantly improved in the second week after the first immunization, and the gingival bleeding and halitosis scores were significantly improved in the second week after the second immunization (p <0.05). However, there was no significant improvement in the gingival swelling score. On the other hand, no improvement was observed in any of the clinical scores in the control group, and the bad breath score was significantly worse (p <0.05).

  Similarly, as shown in FIG. 9, the overall clinical score also showed a significant improvement in the vaccine administration group at 2 weeks after the first immunization (p <0.01). On the other hand, in the control group, there was no improvement, but rather a tendency to worsen.

  From these results, it was shown that the Porphyromonas gingivalis crushed antigen-encapsulated pH-sensitive liposome ophthalmic vaccine has a therapeutic effect or an effect of suppressing the progression of symptoms against periodontal disease in cats.

Claims (10)

  An ophthalmic vaccine comprising (1) an antigenic substance derived from periodontal disease bacteria and (2) a liposome.   Periodontal disease bacteria are Aggregate bacter actinomycetemcomitans, actinomyces neslundi, porphyromonas gingivalis, porphyromonas assaccarolytica, porphyromonas endontalis, prevotella intermedia, Prevotella nigrescens, Prevotella melaninogenica, Fusobacterium nucleatum, Fusobacterium necroform, Fusobacterium naviform, Treponema denticola, Tanerella forsaicia, Bacteroides forsinsus, Aikenella colodens, Capnocytorepha fondari The ophthalmic vaccine according to claim 1, which is at least one bacterium selected from the group consisting of:   The ophthalmic vaccine according to claim 2, wherein the periodontal disease bacteria is Actinomyces nesrundi or Porphyromonas gingivalis.   Antigenic substances include periodontal bacteria crushed cells or lysates, periodontal bacteria inactivated whole cells, crushed cells or lysates, periodontal pathogens or flagella, teeth 2. The substance according to claim 1, which is at least one substance selected from the group consisting of lipopolysaccharides of periodontal disease bacteria, outer membrane vesicles of periodontal disease bacteria, proteolytic enzymes of periodontal disease bacteria, and outer membrane proteins of periodontal disease bacteria. Eye drop vaccine.   The ophthalmic vaccine according to claim 4, wherein the antigenic substance is formalin-inactivated disrupted cells of periodontal disease bacteria.   The ophthalmic vaccine according to claim 1, wherein the liposome is a pH-sensitive liposome. Liposomes
Dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleylphosphatidylcholine, egg yolk lecithin, dioleylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleylphosphatidylserine A group consisting of phosphatidylserine, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dicetylphosphate, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, cholesterol At least with one or more lipids selected,
At least one carboxyl group-modified polyglycidol selected from the group consisting of succinylated polyglycidol and 3-methylglutaryl polyglycidol;
An ophthalmic vaccine according to claim 6 comprising:   The ophthalmic vaccine according to claim 7, wherein the liposome is composed of a combination of dipalmitoyl phosphatidylcholine, dioleyl phosphatidylethanolamine, monophosphoryl lipid A, and 3-methylglutarylated polyglycidol.   A method for inducing immunity, comprising administering the eye drop vaccine according to claim 1 to 8 to a non-human mammal.   The immunity induction method according to claim 9, wherein the non-human mammal is a dog or a cat.