JPH0952846A - Periodontal disease preventive vaccine - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a vaccine useful for periodontal diseases. SOLUTION: This vaccine uses a synthetic peptide corresponding to a fragment derived from an amino acid sequence of the formula, constituting a fimbria of Actinobacillus acetinomycestemcomitan as one of bacteria to cause periodontal diseases or a substance obtained by bonding a polymer resin to the peptide as a vaccine antigen. In administering the vaccine, dermal administration, oral administration and administration through mucosa of oral cavity not by injection is preferable. In the case of administration especially through mucosa, an absorption promoter such as bile acid, surfactant, cholera toxin B subunit, etc., is preferably used. The amount of the antigen in the vaccine for one administration is preferably 0.005-2mg.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to Actinobacillus actinomycetemcomitans, which is one of the causative bacteria of periodontal disease.
The present invention relates to a periodontal disease preventive vaccine capable of preventing periodontal disease by suppressing the colonization of the oral cavity of (Ocycetemcomitans).

[0002]

2. Description of the Related Art Actinobacillus actinomycetemcomitance is considered to be the most influential causative agent of juvenile periodontitis. It has been detected and isolated from the periodontal pockets with high frequency, and has been attracting more attention as a bacterium involved in the onset and progress of periodontal disease.

In order to prevent periodontal disease, it is effective to suppress the colonization of causative bacteria in the oral cavity. Conventionally, as a method for preventing colonization of causative bacteria of periodontal disease and suppressing proliferation, Vaccines using all bacterial cells, cell surface substances or pili as antigens have been proposed (JP-A-59-128338, JP-A-61-140527, JP-A-5-132428). However, when whole cells or their extracts are used as antigens, there is a problem in terms of safety because a large number of components are contained as antigens. On the other hand, when fimbriae are used as antigens, the fimbriae antigens are proteins, and thus there is a drawback that the purification step is complicated to unify them to a desired degree of purification. Therefore, a highly safe and highly effective periodontal disease preventive vaccine has been demanded.

[0004]

Means for Solving the Problems and Modes for Carrying Out the Invention As a result of intensive studies to meet the above-mentioned demands, the present inventor has found that a fragment derived from an amino acid sequence constituting Actinobacillus actinomycetemcomitans pili. By using a synthetic peptide corresponding to the above or a polymer lysine bound thereto as a vaccine antigen, it is possible to effectively prevent oral colonization of Actinobacillus actinomycetemcomitans and effectively prevent periodontal disease. I found out that. In particular, the present inventor deciphered the amino acid sequence from the N-terminal to the 41st position of the pilus protein obtained from the bacterium of Actinobacillus actinomycetemcomitans, and found that the amino acid sequence was as follows: Found.

[0005]

[Sequence Table 1]

In this case, among the above sequences, the amino acid sequence from the N-terminal to the 18th to 38th amino acid, that is, the oligopeptide having the following amino acid sequence has strong antigenicity, and the synthetic peptide having such amino acid sequence is used as a vaccine antigen. The present inventors have found that periodontal diseases can be effectively prevented by using them, and have completed the present invention. [Amino acid sequence] Val Ala Val Phe Tyr Ser Asn Asn Gly Phe Ile Ala Asn Leu Gln Ser Lys Phe Phe Ser Leu

The present invention will be described in more detail below. The periodontal disease-preventing vaccine of the present invention is a synthetic peptide derived from the pilus protein of Actinobacillus actinomycetemcomitans, preferably this synthetic peptide is used as a polymer lysine. The bound one is used as a vaccine antigen.

As the synthetic peptide, the synthetic peptide having the above [amino acid sequence] is suitable.
In this case, the synthetic peptide is Pep according to a known method.
side synthesizer (430A, App
lied Biosystems Inc. , Fast
er City, CA).

[0009] Further, a method for binding with a polymer lysine in order to enhance the antigenicity of the above synthetic peptide is described by Narde.
Polymer lysine method (J. Immunol.,
148, 912-920, 1992).

In the present invention, the vaccine can be administered by subcutaneous injection, intramuscular injection, buccal injection, intravenous injection, nasal administration, oral administration, oral cavity mucosal administration, etc. From the viewpoint of resistance to A., nasal administration, non-injection administration, oral administration, oral cavity mucosal administration and the like are more preferable. In the case of a method of administration not by injection, that is, through a mucous membrane, a combined use of an absorption enhancer is particularly desirable. The amount of antigen in the vaccine administered once is 0.00
5 to 2 mg is preferable, and more preferably 0.01 to
It is 0.5 mg.

Examples of absorption enhancers used include bile acids or their derivatives or salts thereof (hereinafter collectively referred to as bile acid compounds), surfactants, cholera toxin B subunits and the like.

[0012] The bile acid compound is incorporated into the vaccine composition at 0.
It is preferable to add 002 to 2% by weight, and more preferably 0.02 to 0.5% by weight.

As the surface active agent, an anionic surface active agent, a cationic surface active agent, a nonionic surface active agent, an amphoteric surface active agent and the like are used, and the cationic surface active agent is particularly preferable. Further, these surfactants can be used together. In the case of the anionic surfactant and the nonionic surfactant, the amount of the surfactant is preferably 0.01 to 10% by weight, more preferably 0.1 to 5% by weight. In the case of a cationic surfactant, 0.001 to
It is 1.0% by weight, more preferably 0.01 to 0.5% by weight.

The cholera toxin B subunit is preferably contained in the vaccine composition in an amount of 0.001 to 1% by weight, more preferably 0.01 to 0.25% by weight. The cholera toxin B subunit may be simply blended in the vaccine composition, but by combining it with the above-mentioned vaccine antigen, the effect can be further enhanced.

As other components in the periodontal disease preventive vaccine of the present invention, adjuvants such as alum, aluminum hydroxide, aluminum phosphate, aluminum sulfate, and muramyl dipeptide; oleic acid, stearic acid, palmitic acid, etc. Adjuvant fat-soluble components of; humectants such as glycerin, sorbit, xylit, mannitol, lactide, maltite, polyethylene glycol; sorbic acid, chlorobutanol, benzoic acid, paraoxybenzoic acid ester, boric acid, dehydroacetic acid, thymol and other preservatives Agents: sodium polyacrylate, polyvinyl alcohol, polyvinylpyrrolidone, sodium carboxymethyl cellulose, methyl cellulose, hydroxyethyl cellulose, carrageenan, sodium alginate, gum arabic, xanthan Arm, montmorillonite, it can be blended kaolin, hydrated silica, magnesium aluminum silicate, a binder such as hectorite.

[0016]

EFFECT OF THE INVENTION The periodontal disease-preventing vaccine of the present invention is capable of safely and effectively producing an antibody titer against a causative bacterium by using, as an antigen, the above synthetic peptide or a polymer lysine-bonded product thereof. You can Therefore, the vaccine of the present invention can be effectively used for the prevention of periodontal disease.

[0017]

The present invention will be described below in more detail with reference to Examples, but the present invention is not limited to the following Examples.

[Example 1] Preparation of antigen Actinobacillus actinomycetemcomitans 31
The 0-a strain (hereinafter referred to as A.a.310-a strain) was cultured at 37 ° C. for 72 hours under CO ₂ conditions using Todd-Hebit medium. Next, the method of Inoue et al. (FEMS M
microbiol. Lett. , 69, 13-17, 1
990) to prepare pili antigens. The growing bacteria adhering to the glass wall were scraped off with policeman, suspended in phosphate buffered saline (PBS, pH 7.2), and centrifuged at 12,000 × g. The collected bacteria were treated with 0.15 M ethanolamine-hydrochloric acid buffer solution (pH 10.
5) and suspended in a blender on ice for 30 minutes. 20 minutes at 10,000 xg, then 25.0
Bacterial debris was removed by centrifugation at 00 xg for 30 minutes and the supernatant was saturated with saturated ammonium sulphate solution at 4 ° C to a final concentration of 10
It was added dropwise until the percentage was reached. The precipitate (coarse pili) was resuspended in an ethanolamine-hydrochloric acid buffer solution and centrifuged at 25,000 xg to remove components adhering to the cell membrane. This operation of salting out the supernatant was repeated under the same conditions. The precipitate was dissolved in 20 mM Tris-HCl buffer (pH 8.2) containing 5% sodium deoxycholate, and homogenized for 30 minutes at room temperature in a rolling stirrer. 10,000 × g,
The insoluble matter in the precipitate was removed by centrifugation for 30 minutes. 0.7% n
A similar operation was repeated using 20 mM Tris-hydrochloric acid buffer containing -octyl-β-glucopyranoside, and finally the precipitate was suspended in Tris-hydrochloric acid buffer. This suspension was used as a pili preparation. The prepared pili are SDS-PA
It was confirmed to be single-stranded by GE.

After removing the Tris-HCl buffer from the pilus preparation by filtration and freeze-drying, the purified pilus was subjected to a protein sequencer (PSQ-10, Shimadzu Corporation).
Edman reaction (J. Biol. C) in which the amino acid sequence from the N-terminal to the 41st position was improved by Hewick et al.
hem. , 256, 7990-7997, 1981). The molecular weight of the pili was about 54,000.

[0020]

[Sequence Table 2]

Next, based on the above amino acid sequence, several kinds of oligopeptides were peptide-synthesized.
r (430A, Applied Biosystems
Inc. , Foster City, CA). As a result of examining their antigenicity, the following sequences were found to have strong antigenicity. [Amino acid sequence] Val Ala Val Phe Tyr Ser Asn Asn Gly Phe Ile Ala Asn Leu Gln Ser Lys Phe Phe Ser Leu

Further, for the purpose of enhancing the antigenicity, the molecular weight of a synthetic peptide having this amino acid sequence (hereinafter, simply referred to as synthetic peptide) is determined by the polymer lysine method (J. Immunol., 148, 912-92 by Nardelli et al.
0, 1992). The branched lysine polymer resin was treated with a deprotecting reagent to remove the resin. The augmented polypeptide was extracted with 2N sodium acetate, dialyzed against distilled water and lyophilized.

Example 2 An immunization experiment was conducted using Japanese white rabbits. The antigens shown below were injected subcutaneously with Freund's complete adjuvant. After the first immunization, the mice were immunized every 2 weeks for a total of 6 times. Immune antigen A: non-immunized group (saline) B: A. a. 310-a strain formalin-treated cells C: Synthetic peptide obtained in Example 1 D: Conjugate of the synthetic peptide and polymer lysine

Next, one week after the final immunization, blood and saliva were collected, and the antibody titers in the serum and saliva were measured by the ELISA method. The antibody titer was measured by the ELISA method as follows. 96 various cells prepared to 100 μg / ml
0.1% after coating by adding to the well microplate
% Gelatin solution was added for blocking. Appropriately diluted serum or saliva was added to the washed wells for reaction. After washing, alkaline phosphatase-labeled sheep anti-rabbit immunoglobulin (H + L) antibody was added and reacted. After washing, p-nitrophenyl phosphate disodium was added, and the absorbance at 405 nm was measured. The results are shown in Tables 1 and 2.

[0025]

[Table 1]

[0026]

[Table 2]

As is clear from the results in Tables 1 and 2,
When a synthetic peptide is immunized, antibodies produced in serum and saliva have high reactivity with the bacterium, and Streptococcus sanguis (SS
Anguis), Streptococcus salivarius (S. salivarus) and the like, and thus the reaction specificity was extremely high.

[Example 3] The inhibitory effect of the antigen vaccine prepared in Example 1 on the oral colonization of the bacterium was examined.

An immunization experiment was carried out using rats (ODU rats) in which periodontitis frequently occurs in the lower anterior gum. 10 for each group
50 μl each of the antigens shown in Table 3 prepared at 200 μg / ml was subcutaneously injected into one ODU rat to immunize it. After the first immunization, the same immunization was performed once a week for a total of 6 immunizations.

Four weeks after the first immunization, three days after A. a. 3
10-a strain culture broth (OD550 nm = 0.1) 0.1
ml into the oral cavity of the rat to give A. a. Infected with fungus. Three weeks after bacterial infection, rat gum plaque was collected, and A. a. The numbers of bacteria and total anaerobic bacteria were measured. A. to total anaerobic bacterial count a. The ratio of the number of bacteria was calculated, and a. It was defined as the bacterial colonization rate. The results are shown in Table 3.

[0031]

[Table 3]

As is clear from the results of Table 3, the group immunized with the synthetic peptide which is the vaccine antigen of the present invention was compared with the control group by A. a. The bacterial colonization rate is significantly low,
Therefore, it was confirmed that the vaccine of the present invention can remarkably suppress the colonization of the bacterium in the oral cavity.

Examples of prescriptions are shown below. [Example 4] Injectable vaccine Synthetic peptide 0.1% Aluminum phosphate 0.1% Gelatin 0.02% Thimerosal 0.01% Purified water Residual amount ──────────────── ──────────────────── Total 100%

[Example 5] Injectable vaccine Synthetic peptide 0.05% Lactose 2.5% Glucose 2.5% Human serum albumin 0.2% Gelatin 0.5% Purified water Residual amount ─────── ──────────────────────────── Total 100%

[Example 6] Oral vaccine Synthetic peptide 0.1% Sucrose 35.0% Gelatin 0.5% Purified water Residual amount ──────────────────── ──────────────── Total 100%

Example 7 Nasal Vaccine Synthetic Peptide 0.05% Benzethonium Chloride 0.1% Glycerin 0.8% Hydroxyethyl Cellulose 0.8% Purified Water Residual Content ──────────── ──────────────────────── Total 100%

[Example 8] Nasal vaccine Synthetic peptide 0.02% Sodium glycocholate 0.1% Glycerin 2.0% Cholera toxin B subunit 0.05% Purified water Residual amount ────── ───────────────────────────── Total 100%

Example 9 Nasal Vaccine Synthetic Peptide 0.01% Benzalkonium Chloride 0.05% Glycerin Fatty Acid Ester 0.8% Sorbit 0.6% Methylcellulose 0.6% Purified Water Residual Volume ─── ──────────────────────────────── Total 100%

Example 10 Nasal Vaccine Synthetic Peptide-Cholera Toxin B Subunit 0.02% Cholic Acid 0.2% Polyoxyethylenesorbitan Fatty Acid Ether 1.0% Sorbit 2.0% Butylparaben HCl 0. 1% Purified water Residual amount ─────────────────────────────────── Total 100%

Claims (3)

[Claims] 1. A periodontal disease preventive vaccine comprising a synthetic peptide as an antigen, which is a synthetic peptide corresponding to a fragment derived from the amino acid sequence constituting the pili of Actinobacillus actinomycetemcomitans. 2. The periodontal disease preventive vaccine according to claim 1, wherein the synthetic peptide bound to polymer lysine is used as an antigen. 3. The periodontal disease preventive vaccine according to claim 1 or 2, wherein the synthetic peptide has the following amino acid sequence. [Amino acid sequence] Val Ala Val Phe Tyr Ser Asn Asn Gly Phe Ile Ala Asn Leu Gln Ser Lys Phe Phe Ser Leu