JP2009078995A - Immuno-vaccine for periodontal disease - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a vaccine for trans-mucosal administration for preventing or treating periodontal disease, highly safe to a human body. <P>SOLUTION: The vaccine for trans-mucosal administration is obtained by enabling an antigen capable of eliciting immunoresponse of P.gingivalis to an outer membrane protein to be exhibited on the surfaces of butyric acid bacteria. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to an immune vaccine for periodontal disease. More specifically, for prevention or treatment of periodontal disease, including lactic acid bacteria expressing an antigen capable of eliciting an immune response against the outer membrane protein of Porphyromonas gingivalis (P. gingivalis). The present invention relates to a vaccine for transmucosal administration.

  Conventional periodontal disease treatment has been mainly performed for surgical treatment for periodontal disease that has already progressed. However, in recent years, periodontopathic bacteria, pathogenic factors, and the like have been clarified, and new treatments and preventive methods for periodontal diseases targeting them have begun to be provided.

For example, P. cerevisiae is deeply involved in the onset and development of periodontal disease and is considered as one of the pathogenic bacteria of periodontal disease. In gingivalis, the capsule protein induces antibodies in mice, suggesting that it is effective as a vaccine (see, for example, Non-Patent Document 1).
In addition, the present inventors have disclosed P.I. Mucosal immune vaccine (for example, refer to Patent Document 1) or transdermal immune vaccine administered to mucosa together with an adjuvant using an outer membrane protein (hereinafter referred to as 40-k-OMP) having a molecular weight of 40-kDa specific to gingivalis (See, for example, Patent Document 2).

  However, since these vaccines use cholera toxin as an adjuvant, they cannot be applied to humans. Also, the use of detoxified cholera toxin has been studied, but safety to humans has not yet been confirmed, and it is not practical in actual use. Other adjuvants that can replace them have not been found at present, and the development of vaccines that are highly safe for the human body is desired.

As a method for enhancing immunity other than adding an adjuvant, a lactic acid bacteria display technique for presenting an antigen on the surface of lactic acid bacteria has been developed (see, for example, Patent Document 3). Since this technique can use lactic acid bacteria provided for food, it is considered safe and useful for the human body. However, there is no known example of providing a vaccine using this technique for the treatment and prevention of periodontal disease.
Harvey W, Guat-Chen F, Gordon D, Megji S, Evans A, Harris M. (1984) Evidence for fibroblasts as the major in prosthetic prosthesis. Arch. Oral. Biol. 29, 223-229 JP 2003-286191 A JP-A-2005-179301 JP 2007-131610 A

  An object of the present invention is to provide a vaccine for transmucosal administration for the prevention or treatment of periodontal disease that is highly safe for the human body.

As a result of intensive studies to solve the above problems, the present inventors have found that the surface of lactic acid bacteria has P.I. By presenting an antigen that can elicit an immune response against the outer membrane protein of gingivalis, it is possible to enhance the immunity to the antigen, and a highly safe vaccine for transmucosal administration that is effective in preventing or treating periodontal disease is obtained. As a result, the present invention has been completed.
The vaccine of the present invention maintains the same level of immune enhancement as a vaccine using cholera toxin as an adjuvant. The vaccine of the present invention can sufficiently secrete secretory IgA into saliva, which is said to be highly effective for oral diseases, and therefore immunizes against periodontal disease bacteria together with the secretion of IgG into the gingival crevicular fluid. Can be useful. In addition, the vaccine of the present invention is useful because it can be stored for a long time at room temperature.

That is, the present invention relates to the following transmucosal vaccine for prevention or treatment of periodontal disease (1) to (12).
(1) A vaccine for transmucosal administration for the prevention or treatment of periodontal disease containing lactic acid bacteria expressing an antigen, wherein the antigen is capable of inducing an immune response against the outer membrane protein of Porphyromonas gingivalis. A vaccine for transmucosal administration for the prevention or treatment of periodontal disease.
(2) The vaccine according to (1), wherein the antigen is the protein according to any one of 1) to 3) below.
1) Porphyromonas gingivalis outer membrane protein with a molecular weight of 40-kDa (40-k-OMP)
2) a protein comprising the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing 3) consisting of an amino acid sequence having 90% or more identity with the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing The vaccine according to (1) or (2) above, wherein the protein is a protein that can elicit an immune response against the outer membrane protein of Porphyromonas gingivalis (3) The lactic acid bacterium is Lactobacillus casei.
(4) The vaccine according to any one of (1) to (3) above, wherein the lactic acid bacterium expressing the antigen is NITE P-418.
(5) The vaccine according to any one of (1) to (4) above, wherein the lactic acid bacterium expressing the antigen is dead or live.
(6) The vaccine according to any one of (1) to (5) above, which inhibits the hemagglutination activity of Porphyromonas gingivalis and prevents or treats periodontal disease.
(7) The vaccine according to any one of (1) to (6), which is for nasal administration.
(8) A method for preventing or treating periodontal disease using the vaccine according to any one of (1) to (7) above.
(9) A shuttle vector comprising a base sequence encoding the protein according to any one of 1) to 3) below.
1) Porphyromonas gingivalis outer membrane protein with a molecular weight of 40-kDa (40-k-OMP)
2) a protein comprising the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing 3) consisting of an amino acid sequence having 90% or more identity with the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing A protein that is capable of inducing an immune response against an outer membrane protein of Porphyromonas gingivalis (10) one or more of genes pgsB, pgsC, and pgsA constituting a poly-γ-glutamate synthetase complex The shuttle vector according to (9) above, comprising a vector for surface expression comprising
(11) A shuttle vector pKT1 / pgsA-pg40 comprising a base sequence encoding an outer membrane protein (40-k-OMP) having a molecular weight of 40-kDa of Porphyromonas gingivalis.
(12) The shuttle vector pKT1 / pgsA-pg40 according to (11) above, comprising the base sequence set forth in SEQ ID NO: 2 in the sequence listing.

  The vaccine obtained by the present invention is useful because it has higher safety to the human body than the vaccine using cholera toxin as a conventional adjuvant and maintains the same level of immune enhancement effect.

The “vaccine” of the present invention refers to P.I. It refers to a vaccine for transmucosal administration for the prevention or treatment of periodontal disease that can elicit an immune response to the outer membrane protein of gingivalis.
In the present invention, the vaccine is P.I. Any antigen can be used so long as it contains an antigen capable of eliciting an immune response against the outer membrane protein of gingivalis. The immunity induced by the present invention includes both cellular immunity and humoral immunity.

  P. Examples of antigens that can elicit an immune response to the outer membrane protein of gingivalis include P. gingivalis. 40-k-OMP which is an outer membrane protein of gingivalis. A protein having the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or a protein comprising an amino acid sequence having 90% or more identity with this amino acid sequence, Examples thereof include proteins capable of eliciting an immune response to the outer membrane protein of gingivalis. Furthermore, a protein comprising an amino acid sequence having 95% or more homology with the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing, or one or several amino acid residues in the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing Is a protein having an amino acid sequence deleted, substituted and / or added, Examples thereof include proteins capable of eliciting an immune response to the outer membrane protein of gingivalis.

  In order to obtain an antigen in lactic acid bacteria in order to obtain the “vaccine” of the present invention, it is preferable to use lactic acid bacteria display technology. Lactic acid bacteria display technology refers to the expression of foreign proteins on the surface of lactic acid bacteria using a vector for surface expression containing one or more of the genes pgsB, pgsC and pgsA constituting the poly-γ-glutamate synthetase complex. It is a technology that makes it manifest.

The surface expression vector of the present invention may be a surface expression vector containing any one or more of pgsB, pgsC, and pgsA among the genes constituting the poly-γ-glutamate synthetase complex. Any of them can be used. It preferably contains pgsA, and it is particularly preferred to use a transformation vector pKT1 containing pgsA.
In addition, in this surface expression vector, P.I. A shuttle vector was constructed by inserting a gene encoding a protein capable of eliciting an immune response against the outer membrane protein of gingivalis, and an antigen of the present invention was expressed by culturing a transformed lactic acid bacterium using this gene. Lactic acid bacteria can be obtained.

As the shuttle vector of the present invention, any shuttle vector that can express an antigen on the surface of lactic acid bacteria can be used. an outer membrane protein (40-k-OMP) having a molecular weight of 40-kDa of gingivalis, a protein comprising the 22-344th amino acid sequence shown in SEQ ID NO: 1 of the sequence listing, or the 22-344th sequence shown in SEQ ID NO: 1 of the sequence listing A protein comprising an amino acid sequence having 90% or more identity with the amino acid sequence of A shuttle vector containing a base sequence encoding any protein that can elicit an immune response against the outer membrane protein of gingivalis is preferred.
Furthermore, a shuttle vector comprising a surface expression vector comprising one or more of the genes pgsB, pgsC and pgsA constituting the poly-γ-glutamate synthetase complex is preferred. . A shuttle vector pKT1 / pgsA-pg40 containing a base sequence encoding an outer membrane protein (40-k-OMP) having a molecular weight of 40-kDa of gingivalis is preferable. The base sequence of shuttle vector pKT1 / pgsA-pg40 is shown in SEQ ID NO: 2 in the sequence listing.

  As the lactic acid bacterium for expressing the antigen, any lactic acid bacterium that is safe for the human body can be used. Examples include the genus Lactococcus, Streptococcus, Lactobacillus, Pediococcus, and Leuconostoc, and specific species include Lactobacillus acidophilus, Lactobacillus plantus, Lactobacillus, Lactobacillus, and Lactobacillus. Among these, it is particularly preferable to use Lactobacillus casei which is generally widely used for food fermentation and has high safety.

Specific examples of lactic acid bacteria expressing the antigen of the present invention include NITE P-418.
As the lactic acid bacterium expressing the antigen, either live or dead bacteria can be used. These lactic acid bacteria are useful because they can impart the immunostimulatory ability inherent to the lactic acid bacteria and the effect on the living body in addition to the effect as a vaccine.
Furthermore, dead bacteria do not have to worry about the effects of recombinants because the recombinants are inactivated while exhibiting the original functions of such lactic acid bacteria. In addition, since it is not regarded as a recombinant organism, it does not fall under the category of Cartagena Act and is easy to handle.
Thus, since the lactic acid bacterium expressing the antigen of the present invention is a bacterium that is safe for the human body and works well, it can be used as a probiotic lactic acid bacterium for foods and the like.

  In the vaccine of the present invention, lactic acid bacteria expressing an antigen can be used as they are. Moreover, what was formulated into the liquid agent, the suspension agent, the powder agent etc. can also be used as a vaccine of this invention as needed. Examples of the solution include those obtained by dissolving lactic acid bacteria expressing an antigen in purified water, a buffer solution or the like. Examples of the suspending agent include suspending lactic acid bacteria expressing an antigen in purified water, buffer solution, etc. together with methylcellulose, hydroxymethylcellulose, polyvinylpyrrolidone, gelatin, casein and the like. Examples of the powder include those obtained by thoroughly mixing lactic acid bacteria expressing an antigen together with methylcellulose, hydroxymethylcellulose, hydroxypropylmethylcellulose and the like. In these preparations, absorption promoters, surfactants, preservatives, stabilizers, moisture-proofing agents, moisturizing agents, solubilizing agents and the like that are usually used can be added as necessary.

The vaccine of the present invention is for transmucosal administration, and is preferably administered nasally or orally. Nasal or oral administration is preferable because it has the following merits 1) to 3) compared to conventional injection immunization. 1) In addition to inducing an antibody response in serum, an antibody response is also induced in secretions such as saliva. Considering that the invasion route of pathogenic microorganisms is mucosal tissue producing secretory fluid, it is useful because it is possible to prevent the invasion of microorganisms at the entrance. 2) A needle stick accident and serious side effects that may occur in the case of injection immunization can be avoided. 3) Injectable vaccines require preparation of sterile syringes, injection needles, etc., but vaccination is simpler than that.
Furthermore, nasal administration is preferable in terms of high reactivity and sensitivity of the living body, and oral administration is preferable in terms of high convenience. The vaccine of the present invention can be administered by dropping, spraying or spraying in the nasal cavity or oral cavity, usually in liquid or powder form.

The vaccine of the present invention varies depending on the subject to be administered, the administration method, the dosage form, etc., but usually in the range of 100 mg to 5000 mg, preferably in the range of 100 mg to 1000 mg once per adult, for several weeks to several months. It is preferable to administer 12 to 15 times.
The transmucosal administration of the vaccine of the present invention induces the production of outer membrane protein-specific antibodies in both saliva, nasal fluid, etc. and serum, and these specific antibodies produced are P. is deeply involved in the onset and progression of the disease. significantly inhibits the hemagglutination activity of gingivalis. Therefore, the vaccine of the present invention is extremely effective as a mucosal immunization vaccine for prevention or treatment of periodontal disease.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.

<Preparation of vaccine for transmucosal administration>
By the following steps 1-5 or 1-6, P.I. A lactic acid bacterium expressing an antigen capable of eliciting an immune response against the outer membrane protein of gingivalis was obtained, and a vaccine comprising this as an active ingredient was prepared.

1. Preparation of E. coli-lactic acid bacteria shuttle vector pLCE3 For the construction of the E. coli / lactic acid bacteria shuttle vector, a region containing E. coli replicon pMβ1 and erythromycin resistance gene (Erm) was derived from pLC494 (Japanese Patent Application No. 2005-278830: Applicant Okayama University, Geno) (Lack BL) and the repA gene.
The constructed E. coli / lactic acid bacteria shuttle vector was transformed into E. coli (DH5α) by the heat shock method, and then isolated by the alkaline lysis method. This E. coli / lactic acid bacteria shuttle vector was named pLCE3, and FIG. It was shown to. The base sequence of the pLCE3 vector is shown in SEQ ID NO: 3 in the sequence listing.

2. Construction of Transformation Vector Using pgsA (Japanese Translation of PCT International Publication No. 2005-050001) of the extracellular membrane protein gene (pgsBCA) involved in the synthesis of polyγ-glutamic acid derived from Bacillus sp. A transformation vector capable of surface expression was constructed.
First, an expression cassette P-ldh UTLS (distributed from Kobe University) -pgsA-linker region containing the pgsA gene, which is one of the poly-gamma glutamate synthesis genes, and the oligonucleotide described in SEQ ID NO: 4 in the Sequence Listing PCR reaction using the oligonucleotide shown in SEQ ID NO: 5 as each primer [95 ° C, 5 minutes / (95 ° C, 1 minute / 55 ° C, 30 seconds / 72 ° C, 30 seconds: 30 cycles) / 72 ° C. 1 minute / 15 ° C]. Both ends of the amplified gene region were configured to contain restriction enzyme HindIII and SacI recognition sequences with both primers. After completion of the reaction, the amplified fragment was purified using Nucleospin Exact Kit (MACHEREY-NAGEL) and digested with restriction enzymes HindIII and SacI. Thereafter, it was introduced into pLCE3 treated with the same restriction enzyme and transformed into E. coli DH5α. This transformation vector was named pKT1, and FIG. It was shown to.

3. Construction of Shuttle Vector pKT1 / pgsA-pg40 Next, using the pg40 gene encoding 40-k-OMP as a template, the oligonucleotide shown in SEQ ID NO: 6 and the oligonucleotide shown in SEQ ID NO: 7 were used. PCR reaction [95 ° C., 5 minutes / (95 ° C., 1 minute / 55 ° C., 30 seconds / 72 ° C., 2 minutes: 30 cycles) / 72 ° C., 2 minutes / 15 ° C.] used as each primer was performed. The base sequence of the pg40 gene encoding 40-k-OMP used as a template is shown in SEQ ID NO: 8 in Sequence Listing.
Both ends of the amplified gene region were configured to contain restriction enzyme BamHI and XbaI recognition sequences with both primers. After completion of the reaction, the amplified fragment was purified using Nucleospin Exact Kit (MACHEREY-NAGEL) and digested with restriction enzymes BamHI and XbaI. Thereafter, the vector was introduced into pKT1 treated with the same restriction enzyme to construct a shuttle vector. The constructed shuttle vector was named pKT1 / pgsA-pg40 and is shown in FIG. The base sequence of this shuttle vector pKT1 / pgsA-pg40 is shown in SEQ ID NO: 2 in the Sequence Listing.
The constructed shuttle vector pKT1 / pgsA-pg40 was transformed into E. coli DH5α by the heat shock method. The obtained transformant was cultured in an LB liquid medium, and the cells were collected, and then the shuttle vector pKT1 / pgsA-pg40 was extracted with Wizard (registered trademark) Plus SV Miniprepsp (Promega).

4). Transformation of lactic acid bacteria 3. The shuttle vector pKT1 / pgsA-pg40 extracted in (1) was electroporated according to various protocols (Bertier, F. et al., Coffey, A. et al., GASS ON, MJ et al., Serror, P. et al.). Lb. Casei L-49 was transformed.
That is, DNA and competent lactic acid bacteria were mixed and maintained on ice for 30 seconds. Cell samples to be transformed were transferred to a 0.1 cm cuvette (Bio-Rad) and transformed with a gene pulser (peak voltage 1.5 kV; capacitance 25 uF; resistance 200Ω; Bio-Rad). Subsequently, the transformed lactic acid bacteria were seeded on an MRS agar medium containing 20 ug / ml erythromycin, and viable cells were selected. The introduction of the shuttle vector pKT1 / pgsA-pg40 was confirmed in the colonies of lactic acid bacteria obtained by transformation by colony PCR.

5). Confirmation of expression of pgsA-pg40 Western blotting was performed to confirm protein expression of the pKT1 / pgsA-pg40 transformant.
First, the above 4. The transformant obtained in the above was cultured in an MRS liquid medium at a set temperature of 37 ° C. for 24 hours, and the cells were collected by centrifugation at 7000 rpm for 10 minutes. The collected cells were washed with a PBS buffer, and then crushed with an ultrasonic crusher (output 4, 2 minutes × twice / Tomy Seiko Co., Ltd.). The disrupted solution was suspended in a sample buffer, heat denatured at 100 ° C. for 10 minutes, and subjected to SDS-polyacrylamide gel electrophoresis. After electrophoresis, it was transferred to a PVDF membrane (Millipore). The PVDF membrane was shaken with blocking buffer solution (PBS, 5% skim milk) for 1 hour to block, and then reacted with pg40 polyclonal antibody (received by Nihon University) at 37 ° C. for 1 hour as the primary antibody. . After completion of the reaction, the membrane was washed with a TBST buffer solution and reacted with biotin-labeled anti-rabbit IgG antibody (Vector) at 37 ° C. for 1 hour. After completion of the reaction, the membrane was washed, avidinized with Vectastein ABC Kit standard (Vector), and further developed with Peroxidase substrate kit DAB / Ni substrate (Vector) to confirm the expression of pgsA-pg40 fusion protein. The results are shown in FIG.
The pKT1 / pgsA-pg40 transformed strain obtained by the above steps 1 to 5 was used in the vaccine as it was (live bacteria) or as dead bacteria. The killing process of this strain is described in the following 6. It was shown to.

6). Death of the pKT1 / pgsA-pg40 transformed strain The pKT1 / pgsA-pg40 transformed strain obtained by the above steps 1 to 5 was killed by the method described in JP-A No. 2007-131610. That is, 0.1 to 1% by weight of a surfactant and 0.01 to 0.1% by weight of carbonate are added to the basic medium of lactic acid bacteria, and the pH of the culture solution is maintained at 6.0 to 7.0 during culture After culturing, the culture was killed by heat treatment at 80 to 120 ° C. for 5 to 30 minutes.

<Confirmation of ability to induce immune response of vaccine for transmucosal administration>
1. Materials and Methods 1) Mouse Mouse (BALB / C) 8 week old female was purchased from Sankyo Lab Service Co., Ltd.
2) Vaccine for transmucosal administration A freeze-dried preparation of a dead strain of the pKT1 / pgsA-pg40 transformed strain prepared in Example 1 above was used as a vaccine.
3) Immunization method To a BALB / C mouse, a phosphate buffer solution in which a vaccine for mucosal immunity administration (hereinafter referred to as BLS (PG40)) (1 mg / 20 μl) is dissolved is dropped into the nasal cavity of the mouse 10 μl per nose. Administration was performed 12 to 15 times, 3 times a week (3 consecutive days). Untransformed Lb. Casei L-49 strain (hereinafter referred to as “BLS”) was used, and 40-k-OMP (hereinafter referred to as “PG40”) was used for comparison, and the same was administered to the nasal cavity of mice.

4) Detection of specific antibody The antigen-specific IgA antibody titer in the saliva of mice immunized with BLS (PG40), and the antigen-specific IgA and IgG antibody titers in serum were measured using the Enzyme-linked immunosorbent assay (ELISA) method. It was measured. The antibody titer was determined by serially diluting the immune group (BLS (PG40) or PG40) and the control group (BLS) and comparing the absorbance of each well. did. The number of antigen-specific antibody-producing cells was measured using the Enzyme-linked Immunospot (ELISPOT) method. The number of spots in each well was calculated with a microscope (OLIMPUS SZH10).

5) Separation and adjustment of lymphocytes from salivary glands Salivary glands extracted from immunized mice were treated with RPMI 1640 containing collagenase (0.3 mg / ml) three times at 37 ° C. for 20 minutes, and the resulting cell suspension was used. Centrifuged at 400 × g for 8 minutes. Next, 100% Percoll solution (Pharmacia) was diluted with RPMI 1640 containing 2% NBS, adjusted to 50% Percoll solution, and the cells were suspended. In addition, after adjusting the 75% Percoll solution, the 75% Percoll solution was overlaid with the 50% Percoll solution, and centrifuged at 20 ° C. at 600 × g for 20 minutes. Thereafter, lymphocytes were separated from the layer between the 75% Percoll solution and the 50% Percoll solution, washed, trypan blue was added, and lymphocytes were measured with a hemocytometer. The obtained lymphocytes were suspended in RPMI 1640 containing 2% NBS and centrifuged at 400 × g for 8 minutes. After washing, the number of lymphocytes was measured with a hemocytometer.

2. Results 1) Specific antibody production ability BLS (PG40) was administered three times a week in order to determine whether the antigen-specific immune response was effectively induced in the mucosal system by nasal immunization with BLS (PG40) ( (3 consecutive days), nasally administered a total of 12 to 15 times, and after 3 weeks, 4 weeks and 5 weeks after the first administration, the antigen-specific IgA antibody titer in saliva was determined by ELISA. The results of measurement using FIG. It was shown to.
Figure 4a. As can be seen, no increase in antibody titer was observed in the saliva of the group immunized with PG40. However, a significant antigen-specific antibody titer was detected in the saliva of the group immunized with BLS (PG40).
The antigen-specific antibody titer of the group immunized with BLS (PG40) shows an immunopotentiating effect comparable to that of a 40-k-OMP vaccine using cholera toxin as an adjuvant (see reference, FIG. 1A). ), It was confirmed that the BLS (PG40) of the present invention maintained the same level of immunopotentiating effect as a conventional vaccine using cholera toxin as an adjuvant.
Moreover, the measurement result of the antigen-specific antibody titer in the serum of the mouse immunized intranasally is shown in FIG. As can be seen from FIG. 5, both the group immunized with PG40 and the group immunized with BLS (PG40) showed an increase in antigen-specific IgG and IgA antibody titers in the serum. Note that FIG. FIG. 5 shows the average value and standard deviation when the test was performed 3 times in total using 4 mice per group.
Reference: JP 2003-286191 A

2) Measurement of the number of antigen-specific antibody-producing cells Whether the IgA antibody in saliva obtained by nasal immunization with BLS (PG40) is derived from salivary gland-derived antibody-producing cells, or contamination from serum In order to clarify whether or not BLS (PG40) is administered nasally 3 times a week (3 consecutive days), a total of 12 to 15 times, antigen-specific salivary glands 5 weeks after the first administration The results of measuring the number of antibody-producing cells by the ELISPOT method are shown in FIG. It was shown to. BLS was used as a control and PG40 was used as a comparison.
Figure 4b. As can be seen, a significant number of antigen-specific IgA antibody-producing cells were detected in the salivary glands of the group immunized with BLS (PG40). 4b. Shows the average value and standard deviation when the test was performed 3 times in total using 4 mice per group.
The number of antigen-specific antibody-producing cells in the group immunized with BLS (PG40) is similar to the number of antigen-specific antibody-producing cells in the salivary gland when a 40-k-OMP vaccine using cholera toxin as an adjuvant is used. As a result (see reference, FIG. 2A), it was confirmed that the BLS (PG40) of the present invention maintained the same level of immunopotentiating effect as a conventional vaccine using cholera toxin as an adjuvant.

<P. Confirmation of inhibitory effect of gingivalis on hemagglutination activity>
1. Materials and Methods 1) Collection of vesicles gingivalis 381 was cultured, and the culture supernatant containing the vesicles was collected and concentrated with an ultrafiltration system (Millipore Co.). After concentration, the mixture was dialyzed against 50 mM Tris-HCl containing 0.5 mM dithiothreitol, and vesicles were collected by centrifugal precipitation.
2) Antibody Purification IgG antibody (hereinafter referred to as BLS + PG40) was purified from mouse serum using HiTrap (registered trademark) protein G HP column (Amersham Biosciences).

3) Hemagglutination test A well of vesicle suspension (0.5 μg / 50 μl) pre-cultured with BLS + PG40 at each concentration (0, 50, 100, 150 μg / ml) at 37 ° C. for 1 hour in a well of a 96-well microtiter plate and 1 % Mouse erythrocyte suspension (50 μl) was mixed and cultured at 37 ° C. for 1 hour. The presence or absence of erythrocyte aggregation in the well after culture was confirmed.

2. Results The results of the aggregation test are shown in FIG. As can be seen from FIG. 6, red blood cells were aggregated when BLS + PG40 was not added or 50 μl was added, but when 100 or 150 μl of BLS + PG40 was added, red blood cell aggregation did not occur. Thus, BLS + PG40 is It has been shown to inhibit the hemagglutination activity of gingivalis.

  From the above, the vaccine of the present invention showed the effects of increasing the serum IgG and IgA antibody titers and increasing the antigen-specific IgA antibody titer in saliva by transmucosal administration. And the induced antigen-specific antibody is P.I. Since it significantly suppresses the hemagglutination activity of gingivalis, it can be said to be an effective vaccine for the prevention or treatment of periodontal disease.

  The vaccine obtained by the present invention has high safety to the human body and is useful as a vaccine for the prevention or treatment of periodontal disease.

a. FIG. 3 shows shuttle vector pLCE3 (Example 1). b. It is the figure which showed expression vector pKT1 (Example 1). FIG. 1 shows shuttle vector pKT1 / pgsA-pg40 (Example 1). (Example 1) which confirmed the expression of pgsA-pg40 fusion protein. a. It is the figure which showed the antigen specific antibody production ability (Example 2). b. It is the figure which showed the antigen specific antibody producing cell number (Example 2). It is the figure which showed the antigen specific antibody production ability (Example 2). P. It is the figure which showed the inhibitory effect with respect to hemagglutination activity of gingivalis (Example 3).

Claims (12)

Periodontal disease, which is a vaccine for transmucosal administration for the prevention or treatment of periodontal disease including lactic acid bacteria expressing an antigen, wherein the antigen is an antigen that can elicit an immune response against the outer membrane protein of Porphyromonas gingivalis Vaccine for transmucosal administration for the prevention or treatment of cancer. The vaccine according to claim 1, wherein the antigen is the protein according to any one of 1) to 3) below.
1) Porphyromonas gingivalis outer membrane protein with a molecular weight of 40-kDa (40-k-OMP)
2) a protein comprising the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing 3) consisting of an amino acid sequence having 90% or more identity with the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing A protein that is capable of inducing an immune response to an outer membrane protein of Porphyromonas gingivalis The vaccine according to claim 1 or 2, wherein the lactic acid bacterium is Lactobacillus casei. The vaccine according to any one of claims 1 to 3, wherein the lactic acid bacterium expressing the antigen is NITE P-418. The vaccine according to any one of claims 1 to 4, wherein the lactic acid bacterium expressing the antigen is dead or live. The vaccine according to any one of claims 1 to 5 for preventing or treating periodontal disease, which inhibits hemagglutination activity of Porphyromonas gingivalis. The vaccine according to any one of claims 1 to 6, which is for intranasal administration. The prevention or treatment method of periodontal disease using the vaccine in any one of Claims 1-7. A shuttle vector comprising a base sequence encoding the protein according to any one of 1) to 3) below.
1) Porphyromonas gingivalis outer membrane protein with a molecular weight of 40-kDa (40-k-OMP)
2) a protein comprising the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing 3) consisting of an amino acid sequence having 90% or more identity with the 22-344th amino acid sequence shown in SEQ ID NO: 1 in the sequence listing A protein that is capable of inducing an immune response to an outer membrane protein of Porphyromonas gingivalis The shuttle vector according to claim 9, further comprising a surface expression vector containing any one or more of genes pgsB, pgsC and pgsA constituting the poly-γ-glutamate synthetase complex. A shuttle vector pKT1 / pgsA-pg40 comprising a base sequence encoding an outer membrane protein (40-k-OMP) having a molecular weight of 40-kDa of Porphyromonas gingivalis. The shuttle vector pKT1 / pgsA-pg40 according to claim 11, which comprises the base sequence set forth in SEQ ID NO: 2 in the sequence listing.