JP2003286191A - Mucosal immune vaccine for periodontal disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a new mucosal immune vaccine for periodontal diseases. <P>SOLUTION: This mucosal vaccine for preventing or treating the periodontal diseases which is administered through mucosa contains an adventitial protein of P.gingivalis and a mucosal adjuvant as active ingredients. When the adventitial protein of the P.gingivalis which causes the periodontal diseases and the mucosal adjuvant are nasally administered, production of an antibody specific to the adventitial protein is induced in both the mucosal and systemic tissues, and the antibody significantly inhibits coagulation between the P. gingivalis and S.gordonii both of which deeply concern with onset and evolution of the periodontal diseases. Therefore, the adventitial protein is extremely effective as the vaccine for preventing or treating the periodontal diseases. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a mucosal immune vaccine for periodontal disease. More specifically, Porphyromonas ging
The present invention relates to a vaccine for transmucosal administration for preventing or treating periodontal disease, which comprises an outer membrane protein of ivalis and a mucosal adjuvant as active ingredients.

[0002]

2. Description of the Related Art Periodontal disease is one of the two major causes of tooth loss along with dental caries and is a major problem that modern dentistry must solve. Porphyromonas gingivalis, one of the black pigment-producing Gram-negative rods in the oral cavity, has been reported so far.
(P.gingivalis) has been suggested to be closely associated with the onset of periodontal disease (Speigel, CA et al., JP
eriodontal Res. 14: 376-382, 1979). That is, P.gi
ngivalis is frequently isolated from the periodontal pockets of adult periodontitis patients (Slots, J. Am. soc. Microbia
l, Washington DC: 27-45, 1982), a positive correlation was observed between the antibody titer against P. gingivalis in the serum of adult periodontitis patients and the disease progression (Mouton, C. et a
l., Infect Immun. 31: 182-192, 1981; Slots, J. et a
l., Scand. J. Dent. Res. 85: 114-121, 1977; Slots,
J. et al., J. Clinical. Periodontol. 6: 351-382,
1979; White, D et al., J. Periodontol. Res. 16: 25.
9-265, 1981; Tanner, ACR et al., J. Clinical.
Periodontol. 6: 278-307, 1979), and P.gingival
is has strong adhesion to epithelial cells (Okuda, K. et
al., cell surface morphology and adherence to eryt
hrocytes and human buccal epithelial cells 6: 7-12,
1981), producing a proteolytic enzyme (Toda, K. et.
al., J. Periodont. Res. 19: 372-381, 1984; Sorsa,
T. et al., J Periodont. Res. 22: 375-380, 1984; Abi
ko, Y. et al., J. Dent. Res. 64: 106-111, 1985; Miy
auchi, T. et al., Oral. Microbiol. Immunol. 4: 222-
226, 1989), having a lipopolysaccharide capable of bone resorption as a bacterial component (Y, Iino et al., Archs Oral Biol. 29: 59-6).
3, 1984) have been suggested to be the main causative factors of periodontal disease.

Further, a gene of a specific 40-kDa outer membrane protein of P. gingivalis has been cloned, and various pathogenicity of this protein has been reported (Abiko, Y.
et al., Arch. Oral. Biol. 35: 686-695, 1990). Streptococcus gor, which is thought to be involved in the transition of the gingival sulcus flora from Gram-positive cocci to Gram-negative bacilli
It was also reported that the 40-kDa outer membrane protein is involved in the aggregation of donii (S. gordonii) and P. gingivalis (Kamino, Y.
et al., J. Oral Biol. 40, 187-195, 1998). P.gingi
It has also been reported that 40-kDa outer membrane protein is involved in valis adhesion to gingival epithelial cells, hemagglutination, and protease activity (Katoh, M. et al., J periodont., 71,
368-375, 2000; Saitou, S. et al., J periodont. 7
0, 610-617, 1999; Shibata, Y. et al., JBio. Chem.
274, 5012-5020, 1999; Shibata, Y. et al., Inect. Im
mun. 66, 2207-2213, 1998; Abiko, Y. et al., Inect.
Immun. 56, 3966-3969, 1997; Saitou, S. et al., Ge
neral Pharmacol. 28, 675-680, 1997; Saitou, S. et
al., Biochem. Molecular Med. 58, 184-191, 1996; Hira
tsuka, K. et al., Arch. Oral. Biol 37, 717-724, 19
92; Hayakawa, M. et al., Int. J. Biochem. 24, 945-9.
50, 1992; Kawamoto, Y. et al., Int. J. Biochem. 23,
1053-1061, 1991). Thus, the 40-kDa outer membrane protein
It is considered to be an important protein deeply involved in the onset and progression of periodontal disease due to P. gingivalis. As mentioned above,
Despite the clarification of periodontopathic bacteria and pathogenic factors, the current treatment of periodontal disease is symptomatic treatment such as surgical treatment for advanced periodontal disease. is there.

On the other hand, in recent years, the immune mechanism on the mucosal surface has been elucidated one after another, and along with this, the development of a highly effective mucosal immune vaccine is under way. In the case of a mucosal immune vaccine, transmucosal administration of the antigen to the oral cavity, nasal cavity, etc. produces secretory IgA antibody in mucosal tissues such as saliva, while serum in systemic tissues such as gingival crevicular fluid. Derived IgG antibody is produced. Thus, transmucosal administration of an antigen can induce an immune response in both mucosal and systemic systems. One such attempt is Haem
It has been reported that intranasal administration to mice using the cell membrane protein of ophilus influenzae as an antigen induced IgA antibody in the nasal lavage fluid to produce IgG antibody in serum and induced immune responses in both mucosal and systemic systems. (Kuron
o, Y. et al., J. Immunol. 161: 4115-4121, 1998).
Furthermore, it was also reported that nasal administration of the outer membrane protein of Streptococcus mutans, a causative agent of caries, together with the mutant cholera toxin as an adjuvant induced an immune response in both mucosal and systemic systems. (Saito, M. et al., J. Infect. Dis. 183: 823-826, 2
001). Such a mucosal immune vaccine is highly expected as a therapeutic or prophylactic method as an alternative to symptomatic treatment such as surgical treatment of periodontal disease.

[0005]

However, no studies on mucosal immune vaccines for the prevention or treatment of periodontal disease have been reported so far. Therefore, an object of the present invention is to provide a mucosal immune vaccine effective for the prevention or treatment of periodontal disease.

[0006]

[Means for Solving the Problems] The present inventor has conducted extensive studies for the purpose of developing a new vaccine for the prevention or treatment of periodontal disease, and as a result, P. gi which is a causative bacterium of periodontal disease.
Nasal administration of ngivalis outer membrane protein to mice with cholera toxin as an adjuvant induces the production of outer membrane protein-specific antibody in both saliva and serum, and further, this specific antibody produced in serum was induced. Antibodies are closely associated with the onset and progression of periodontal disease. P. gingivalis and S. gord
Since it significantly inhibits the aggregation with onii, it was found that the nasal administration of this outer membrane protein is extremely effective as a mucosal immune vaccine for the prevention or treatment of periodontal disease, and completed the present invention. . Therefore, the present invention provides P. gingival
The present invention relates to a vaccine for transmucosal administration, which comprises an outer membrane protein of is and a mucosal adjuvant as active ingredients for the prevention or treatment of periodontal disease.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The antigen used as an active ingredient of a vaccine in the present invention is an outer membrane protein of P. gingivalis known as a major pathogenic bacterium of periodontal disease. Specifically, it is a protein consisting of the 22nd to 345th amino acid sequences shown in SEQ ID NO: 1 in the sequence listing. The amino acid sequence shown in SEQ ID NO: 1 is the amino acid sequence of the outer membrane protein of P. gingivalis revealed by cloning of the gene for the outer membrane protein of P. gingivalis (Abiko, Y. et al., Arch. O.
ral. Biol. 35: 689-695, 1990). The 1st to 21st amino acid sequences of SEQ ID NO: 1 correspond to the amino acid sequence of the signal peptide. In addition, in the present invention, as the antigen of the active ingredient, one or several amino acid residues in the 22nd to 345th amino acid sequence of SEQ ID NO: 1 are deleted,
A protein having a substituted and / or added amino acid sequence and having a function similar to that of the protein consisting of the 22nd to 345th amino acid sequences shown in SEQ ID NO: 1, that is, a mutant protein of the outer membrane protein of P. gingivalis is used. You can also do it. Here, a protein having a similar function means Pg consisting of the 22nd to 345th amino acid sequence shown in SEQ ID NO: 1.
A protein that has the same antigenicity as the outer membrane protein of ingivalis and induces the production of antibodies that are also effective as a vaccine for the prevention or treatment of periodontal disease.

The above-mentioned protein used as an active ingredient can be produced by a recombinant DNA method known per se using a gene encoding the protein. For example, the protein consisting of the 22nd to 345th amino acid sequence shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2 which is a gene encoding the protein.
Recombination using a gene consisting of nucleotides 64 to 1035
It can be produced by the DNA method. In addition, SEQ ID NO: 2
The 1st to 63rd base sequence of the above corresponds to the base sequence encoding the signal peptide. More specifically, a known recombinant plasmid into which a gene having the nucleotide sequence shown in SEQ ID NO: 2 is inserted, for example, Abiko, Y. et al., A
rch. Oral. Biol. 35: 689-695, 1990, pM.
Recombinant clone MD125 obtained by transforming Escherichia coli with D125 was cultured, and from the culture solution, P. gingivalis consisting of the 22nd to 345th amino acid sequences shown in SEQ ID NO: 1 was cultured.
Can be obtained. In addition, in SEQ ID NO: 1
A protein having an amino acid sequence in which one or several amino acid residues are deleted, substituted and / or added in the 22nd to 345th amino acid sequences,
The mutated protein, which is a protein having the same function as the outer membrane protein of P. gingivalis consisting of the 345th amino acid sequence, is
By applying, for example, the site-directed mutagenesis method to the gene having the nucleotide sequence of 64 to 1035 shown in SEQ ID NO: 2, the genes encoding those proteins are obtained, and the gene is used in the same manner. Can be produced by the recombinant DNA method well known in the art. These products are specifically manufactured by Molecular Clonin 2nd Edt., Cold Spring Harbor La.
It can be done easily with reference to basic books such as boratory Press (1989). Alternatively, the above-mentioned protein can be produced by chemically synthesizing it based on its amino acid sequence.

The mucosal adjuvant used in the present invention may be any adjuvant as long as it has a function as a regulatory factor for assisting the induction of mucosal immune response.
Examples of preferable mucosal adjuvants include cholera toxin, heat-labile toxin of toxigenic Escherichia coli, and the like. Among them, a detoxified cholera mutant toxin which is detoxified by diarrheagenic ADP-ribosyltransferase activity and detoxified and retains an adjuvant activity is preferable, and as such, for example, S61F, E112K (Yamamoto, S. et
al., J. Exp. Med., 185: 1203, 1997), R7K, S63K, R19
2G (Douce, G. et al., Proc. Natl. Acad. Sci. USA.,
92: 1644, 1995; Di Tommaso, A. Infect. Immun .., 64:
974, 1996; Dickinson, BL et al., Infct. Immun.,
63: 1617, 1995). These mucosal adjuvants can be produced by known methods.

The above-described vaccine for transmucosal administration containing the protein as an antigen and the mucosal adjuvant as active ingredients is usually administered by nasal administration or oral administration. It is particularly preferable to administer by nasal administration. The vaccine of the present invention is usually administered in the form of liquid or powder by dripping, spraying or spraying into the nasal cavity or the oral cavity. The dosage form of the vaccine of the present invention thus administered includes, for example, liquid preparations, suspension preparations and powder preparations. As a liquid agent,
Examples thereof include a protein as an active ingredient, which is an antigen, and a mucosal adjuvant dissolved in purified water or a buffer solution. As a suspending agent, the active ingredient is methyl cellulose,
Hydroxymethyl cellulose, polyvinylpyrrolidone,
Examples include gelatin, casein, etc., suspended in purified water, buffer, etc. Examples of the powder include those in which the active ingredient is well mixed with methyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose and the like. If necessary, an absorption promoter, a surfactant, a preservative, a stabilizer, a moisturizer, a humectant, a solubilizer and the like which are commonly used can be added to these preparations.

The dose of the vaccine of the present invention varies depending on the subject to be administered, the method of administration, the mode of administration, etc., but the protein which is usually an antigen per adult is 100 μg to 1,
Dosages in the range of 000 μg, preferably in the range of 100 μg to 500 μg, are given two to three times over a period of several weeks to several months. In addition, the mucosal adjuvant is
In general, it ranges from 20 μg to 100 μg per adult,
It is preferably administered in the range of 20 μg to 50 μg. P.gingival
By administering the vaccine of the present invention comprising the outer membrane protein of is or its mutant protein and a mucosal adjuvant as an active ingredient, transmucosal administration such as nasal administration, the outer membrane protein is specific to both saliva, nasal fluid, etc. and serum. The production of antibodies is induced, and further, these specific antibodies produced are closely related to the onset and progression of periodontal disease.
Significantly inhibits aggregation with gordonii. Therefore, the vaccine of the present invention is extremely effective as a mucosal immune vaccine for preventing or treating periodontal disease.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples. Example 1 Systemic and mucosal set of vaccines for transmucosal administration of the present invention
Measurement of Immune Response Inducing Ability in Weave (1) Materials and Methods a) Mouse Mouse (BALB / C) 8-week-old female was purchased from Sankyo Lab Service Co., Ltd. b) Antigen and mucosal adjuvant In this experiment, P. gingivalis obtained by recombinant DNA method was used.
Outer membrane protein of E. coli was used as an antigen. That is, recombinant clone MD125 (Abiko, Y. et al., Arch. Ora obtained by transforming E. coli with the expression plasmid pMD125 into which the gene having the nucleotide sequence shown in SEQ ID NO: 2 was inserted.
L. Biol. 35: 689-695, 1990) and cultured in Kawamoto, Y.
According to the method described in et al., Int. J. Biochem. 57, 1053-1061, 1991, the recombinant clone MD125 was ultrasonically disrupted and purified through a column.
40-kDa of P. gingivalis consisting of the 345th amino acid sequence
An outer membrane protein (40-k-OMP) was obtained. Cholera Toxin (CT) used as a mucosal adjuvant is List Bio
Purchased from logical Laboratories. This cholera toxin is isolated from Vibrio cholerae Inaba 569B,
It is a wild-type toxin. c) Immunization method BALB / C mice were given 40-k-OMP (10 μg / 20 l) and CT (1 μg / l).
20μl) dissolved phosphate buffer solution into the nasal cavity of mice
Nasal administration was performed by dropping 0 μl each. The antigen and adjuvant were administered once a week for a total of 3 times.

D) Detection of 40-k-OMP-specific antibody Antigen-specific IgA antibody titer in saliva and nasal lavage fluid of mice immunized with 40-k-OMP, and serum-specific antigen-specific IgA and IgG antibodies Value of Enzyme-linked immunosorben
It measured using the t assay (ELISA) method. The antibody titer was determined by serially diluting the immunized group and the control group and comparing the absorbances of the respective wells. The number of antigen-specific antibody-producing cells is Enzyme
-Measured using the linked Immunospot (ELISPOT) method. The number of spots in each well was calculated with a microscope (OLIMPUS SZH10). e) Separation and preparation of lymphocytes from spleen, salivary gland and nasal mucosa Spleen cells were separated from the spleen of immunized mice using a mesh, and RPMI164 containing 2% bovine neonatal serum (hereinafter 2% NBS).
It was suspended in 0 (Immune and Biological Laboratories) and centrifuged at 400 × g for 8 minutes. Then, after destroying the red blood cells in the splenocytes, 40
It was washed at 0 × g for 8 minutes. Trypan blue (Life Technologies, Inc) was added to the obtained lymphocyte suspension, and the number of lymphocytes was measured with a hemocytometer. In addition, salivary glands extracted from mice were treated with RPMS containing collagenase (0.3 mg / ml).
The cells were treated with I 1640 three times at 37 ° C. for 20 minutes, and the resulting cell suspension was centrifuged at 400 × g for 8 minutes. Next, 100% Percoll solution (Pharmacia) was diluted with RPMI1640 containing 2% NBS, and
The cells were suspended by adjusting to a% Percoll solution. Also, 75
% Percoll solution adjusted to 50% in 75% Percoll solution
The Percoll solution was overlaid and centrifuged at 20 ° C. at 600 × g for 20 minutes. Then, lymphocytes were separated from the layer between the 75% Percoll solution and the 50% Percoll solution, washed, trypan blue was added, and the lymphocytes were measured with a hemocytometer. The nasal mucosa lymphocytes were isolated by scraping the jawbone from the mouse and scraping the tissue of the nasal cavity. The obtained lymphocytes were suspended in RPMI 1640 containing 2% NBS and centrifuged at 400 xg for 8 minutes.
After washing, the number of lymphocytes was measured with a hemocytometer. f) Measurement of 40-k-OMP-specific cell proliferating activity Lymphocytes were isolated from spleen and cervical lymph nodes of nasally immunized mice (5 × 10 ⁵ / ml) and 40-k-OMP (5 μg / ml) The cells were cultured together with 37 ° C and 5% CO ₂ for 96 hours. End of culture
[ ³ H] thymidine (0.5 μCi) was added 18 hours before, and the cell proliferation activity due to [ ³ H] thymidine incorporation was measured (Yamamo
to, M. et al., J. Immunol. 161: 4115-4121, 1998).

(2) Results a) 40-k-OMP specific antibody-producing ability 40-k-OMP nasal immunization effectively induces a 40-k-OMP specific immune response in the mucosal system. In order to measure swelling, 40-k-OMP was administered intranasally once a week for a total of 3 times, and 1 week after the final administration, 40-k-OMP-specific IgA antibody titers in nasal wash and saliva The results of measurement by using the ELISA method are shown in FIG. 1A. As can be seen from FIG. 1A, in the group in which 40-k-OMP alone was intranasally immunized in the mouse, no increase in antibody titer was observed in the nasal wash and saliva. However, 40-kO
In the group immunized with MP together with CT as a mucosal adjuvant, remarkable 40-k-OMP-specific antibody titers were detected in nasal wash and saliva. In addition, 40-kO in serum of mice immunized intranasally
The measurement result of MP-specific antibody titer is shown in FIG. 1B. As can be seen from FIG. 1B, an increase in antigen-specific IgM, IgG, and IgA antibody titers was observed in the serum. 1A and 1B
Shows the average value and standard deviation when the test was carried out three times in total using four mice in one group, and ND is below the detection limit.

B) Measurement of the number of 40-k-OMP-specific antibody-producing cells The IgA antibody in saliva obtained by nasal immunization with 40-k-OMP was derived from salivary gland-derived antibody-producing cells. In order to clarify whether it is the contamination from serum, 40-k-OMP was intranasally administered once a week for a total of 3 times, and one week after the final administration, salivary glands, nasal tissues and spleen The results of measuring the number of 40-k-OMP-specific antibody-producing cells by the ELISPOT method are shown in FIG. As can be seen from FIG. 2A, a significant number of antigen-specific IgA antibody-producing cells were detected in the salivary glands and nasal tissues of the group nasally immunized with 40-k-OMP together with CT. Further, as can be seen from FIG. 2B, IgG and IgA antibody-producing cells were detected in the splenocytes. 2A
And FIG. 2B shows the average value and standard deviation when a total of 3 times was performed using 4 mice per group.

C) 40-k-OMP-specific cell proliferation activity To analyze the 40-k-OMP-specific T helper cell response of systemic and mucosal immune tissues, 40-k-OMP was administered once a week. Total 3
40-k- splenocytes (SP) and cervical lymph node cells (CLN) collected from mice were administered intranasally and one week after the final administration.
The results of measuring cell proliferation activity by restimulation with OMP and incorporation of [ ³ H] thymidine are shown in FIG. As can be seen from FIG. 3, remarkable cell proliferation activity was observed in the group immunized with 40-k-OMP together with CT. In addition, FIG. 3 shows the average value and the standard deviation when a total of 3 times was performed using 4 mice per group. These results indicate that nasal immunization with 40-k-OMP induced a 40-k-OMP-specific T cell-dependent antibody response in both the mucosal and systemic immune systems. It was

Example 2 Antibody P. gingivalis induced in nasally immunized mice
Confirmation of inhibitory effect on aggregation of S. gordonii with S. gordonii (1) Materials and methods a) Purified vesicles P.gingivalis381 strain was anaerobic in BHI medium supplemented with hemin (5 μg / ml) and vitamin K (0.5 μg / ml). (80% N ₂ ,
The cells were cultured in 10% H ₂ and 10% CO ₂ . Purification of vesicles from the culture solution is described in Mayrand, D. et al., Can. J. Microbiol.
35, 607-613, 1989. The culture solution was centrifuged at 100,000 xg for 30 minutes and the resulting supernatant was ultraraf
It concentrated to 250 ml using the iltration system (Millipore). After concentration, 50 mM T containing 0.5 mM dithiothreitol
Dialysis was performed with ris-HCl (pH 7.5) at 4 ° C. overnight. After dialysis,
Centrifuge at 90,000 xg for 2 hours and precipitate the resulting precipitate with PBS.
And dialysis at 4 ° C. overnight to give a vesicle suspension. Vesicles were stored at -20 ° C until used.

B) Production of S. gordonii S. gordonii was cultured in an anaerobic state of 80% nitrogen, 10% hydrogen and 10% carbon dioxide at 37 ° C for 2 days. After culturing, the cells were suspended in PBS and the absorbance was adjusted to 1.0 at a wavelength of 600 nm. c) Purification of antibody Serum was collected from mice immunized nasally with 40-k-OMP, and HiTr
ap ^TM Protein G HP (Amersham Pharmacia Biotech AB,
Uppsala, Sweden.). d) Aggregation test An aggregation inhibition test was performed according to the method of Hiratsuka et al. (Infect. Immun. 57: 1618-1620, 1989). Use 50 μl of 50-fold diluted vesicles (60 μg / ml) and purified antibody (50 μl).
S. gordon after shaking culture for 30 minutes at ℃, 60rev / min
ii Activating 100 μl of the adjustment solution for 5 minutes to make Kolenbrander (Infe
ct. Immun. 33: 95-102, 1981) and the like. The judgment was made according to the following criteria. +4: Large aggregates are formed immediately after addition and the suspension is transparent. +3: Large agglomerates, but the suspension is transparent. +2: Agglomerates are formed, but it takes time to form them. +1: The suspended liquid is turbid and the small aggregates are scattered. 0: No aggregate is observed in the suspension.

(2) Results The results of the aggregation test are shown in FIG. As can be seen from FIG. 4, vesicles treated with 40-k-OMP and IgG antibody purified from the serum of mice nasally immunized with CT showed a score of +1 after 5 minutes of mixing (D). On the other hand, in the group which was intranasally immunized with 40-k-OMP alone, the score was +3 (C). Sg
A score of +3 was obtained for vesicles treated with antibody purified from serum of mice not immunized with ordonii (B).
From the above results, the antibodies induced by nasal immunization of 40-k-OMP and CT were found to be vesicles of P. gingivalis and S. gordonii.
It was revealed to significantly inhibit aggregation with S. gordonii.

[0020]

As described above, by intranasally administering 40-kDa outer membrane protein of P. gingivalis, which is a causative bacterium of periodontal disease, and a mucosal adjuvant to mice, both mucosal and systemic tissues can be obtained. The production of antibody specific to the outer membrane protein is induced in Escherichia coli, and this antibody significantly inhibits the aggregation of P. gingivalis and S. gordonii, which are closely related to the onset and progression of periodontal disease. Therefore, this outer membrane protein is extremely effective as a mucosal immune vaccine for the prevention or treatment of periodontal disease.

[0021]

[Sequence list]                             SEQUENCE LISTING <110> Nihon University    <120> Mucosal Immune Vaccine for Periodontal Disease    <130> DA-03294    <160> 2       <210> 1 <211> 345 <212> PRT <213> Porphyromonas gingivalis    <400> 1 Met Lys Arg Leu Leu Leu Ser Ala Ala Ile Leu Ser Ser Met Ala Leu   1 5 10 15 Phe Asn Val Asn Ala Gln Glu Leu Lys Thr Ser Ala Asp Met Lys Gly              20 25 30 Ser Phe Lys Lys Asn Val Val Leu Glu Val Phe Thr Ala Glu Trp Cys          35 40 45 Gly Tyr Cys Pro Gly Gly Lys Glu Arg Ile Ala Lys Ala Ile Glu Met      50 55 60 Leu Asp Asp Glu Tyr Lys Glu Arg Val Phe Gln Thr Phe Val His Tyr  65 70 75 80 Asn Asp Gly Ile Ser Lys Lys Trp Pro Arg Val Gly Gln Leu Phe Ile                  85 90 95 Ala Leu Asp Gln Thr Leu Gly Ile Pro Gly Phe Pro Thr Phe Ser Val             100 105 110 Cys Arg Met Glu Lys Lys Gly Glu Asn Leu Ser Ile Gly Ala Pro Ile         115 120 125 Ala Ile Lys Asn Lys Ile Met Lys Gly Phe Gly Asp Gly Thr Ala Pro     130 135 140 Ala Glu Val Asn Leu Lys Leu Thr Lys Gly Ala Thr Pro Glu Asp Val 145 150 155 160 Cys Thr Ala Thr Phe Thr Gly Lys Val Asp Ala Asp Leu Ile Gly Lys                  165 170 175 Pro Leu Met Leu Thr Ala Tyr Val Leu Lys Asn Asn Met Lys Pro Ile             180 185 190 Asn Pro Gln Asn Gly Ala Gly Asp Gly Tyr Leu His Gln His Thr Val         195 200 205 Leu Met Ile Leu Ser Thr Asp Val Lys Gly Asp Ala Leu Asn Ile Ala     210 215 220 Ala Asp Gly Ser Phe Thr Ile Lys Lys Glu Phe Lys Leu Asp Gly Phe 225 230 235 240 Glu Ile Lys Asp Thr Asp Val Leu Ala Phe Val His His Pro Met Ser                 245 250 255 Asn Ala Glu Asn His Ser Ile Ile Asn Ala Gly Gln Glu Ser Leu Asp             260 265 270 Lys Ala Glu Pro Thr Ala Thr Glu Gln Ile Val Ala Thr Pro Ser Val         275 280 285 Lys Ala Tyr Val Gln Asn Gly Lys Ile Val Val Glu Glu Glu Tyr Ser     290 295 300 Lys Met Glu Val Phe Asn Ala Thr Gly Gln Leu Val Lys Asn Glu Ser 305 310 315 320 Leu Val Pro Gly Val Tyr Val Val Arg Ile Thr Ala Asn Gly Val Met             325 330 335 His Phe Leu Lys Val Leu Val Pro         340 345       <210> 2 <211> 972 <212> DNA <213> Porphyromonas gingivalis    <400> 2 atgaaaagat tattactctc tgctgctatc ctaagtagta tggctttgtt taatgtcaat 60 gcacaagagt tgaaaacctc tgctgacatg aaaggttctt ttaagaagaa tgtggtattg 120 gaggtattta ctgccgaatg gtgcggttac tgtccaggtg gtaaagagcg cattgcaaaa 180 gcaattgaaa tgttggatga tgaatataag gagcgtgttt ttcagacatt tgttcattat 240 aatgatggga tctcaaaaaa atggcctcgt gttggccaac ttttcattgc attggatcaa 300 acattgggca ttccgggttt tccgactttt tcagtttgcc gtatggagaa aaaaggtgaa 360 aatctttcaa taggtgctcc aatagcaatt aaaaataaga ttatgaaagg ttttggtgat 420 ggtacagccc ctgcagaggt aaaccttaaa ttgaccaaag gtgcaacacc ggaagatgta 480 tgtacagcta catttactgg taaagtcgat gcagacctca tagggaaacc tcttatgttg 540 actgcatatg tattgaaaaa caatatgaag cctattaatc cgcaaaatgg agctggggat 600 ggatatctcc accaacatac tgtgttaatg attctctcca cagatgtaaa aggagacgct 660 ttaaatattg cagccgatgg aagttttacc atcaagaaag aatttaagtt ggatggcttt 720 gaaataaaag atacagatgt tcttgctttc gtacaccatc caatgtccaa tgcggaaaac 780 cattctatta tcaatgccgg gcaagaaagc cttgataaag cagagcctac agctacagaa 840 caaattgttg ctaccccctc tgtcaaagca tatgttcaga atggcaaaat tgttgtagag 900 gaagagtatt ccaagatgga agtattcaat gcaactggtc aacttgtcaa aaatgaatcc 960 cttgtccccg gtgtctatgt tgtccgtata acggcaaacg gtgtaatgca tttccttaaa 1020 gtcttagttc cttga 1035

[Brief description of drawings]

FIG. 1 is a graph showing the ability to induce an outer membrane protein-specific antibody in saliva, nasal lavage fluid, and serum of mice intranasally administered with P. gingivalis 40-kDa outer membrane protein and cholera toxin. .

FIG. 2 is a graph showing the number of outer membrane protein-specific antibody-producing cells in salivary glands, nasal tissues, and spleen of mice intranasally administered with P. gingivalis 40-kDa outer membrane protein and cholera toxin. .

FIG. 3 is a graph showing an outer membrane protein-specific cell proliferative activity in splenocytes and cervical lymph nodes of mice intranasally administered with P. gingivalis 40-kDa outer membrane protein and cholera toxin.

FIG. 4 shows inhibition of P. gingivalis vesicle-S. Gordonii aggregation when treated with sera of mice nasally administered with P. gingivalis 40-kDa outer membrane protein and cholera toxin. FIG.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Masafumi Yamamoto             4-8-24, Kudan-minami 4-chome, Chiyoda-ku, Tokyo             Incorporated in Nihon University (72) Inventor Mitsuo Hayakawa             4-8-24, Kudan-minami 4-chome, Chiyoda-ku, Tokyo             Incorporated in Nihon University F term (reference) 4C085 AA03 AA38 BA20 BB11 CC07                       CC32 DD62 FF19 GG08 GG10                 4H045 AA30 BA10 CA11 DA86 EA31

Claims (5)

[Claims] 1. A vaccine for transmucosal administration for preventing or treating periodontal disease, which comprises an outer membrane protein of Porphyromonas gingivalis and a mucosal adjuvant as active ingredients. 2. The outer membrane protein is a protein consisting of the 22nd to 345th amino acid sequence shown in SEQ ID NO: 1 of the Sequence Listing, or one or several amino acids in the 22nd to 345th amino acid sequence of SEQ ID NO: 1. Residues deleted, substituted and /
Alternatively, the vaccine according to claim 1, which is a protein having an added amino acid sequence and having a function similar to that of the protein consisting of the 22nd to 345th amino acid sequences shown in SEQ ID NO: 1. 3. The vaccine according to claim 1, which is for intranasal administration. 4. The vaccine according to claim 1, wherein the mucosal adjuvant is a detoxified cholera mutant toxin. 5. Porphyromonas gi in the gingival sulcus cell plexus
The vaccine according to any one of claims 1 to 4, which inhibits aggregation of ngivalis and Streptococcus gordonii to prevent or treat periodontal disease.