JP2657787B2 - Preparation of a caries prevention vaccine - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a vaccine for preventing tooth decay, which comprises a part of the microorganisms of the genus Streptococcus having the ability to induce tooth decay as an antigen.

[0002]

BACKGROUND OF THE INVENTION Dental caries is a disease of the tooth enamel, dentin and cementum that begins on the surface of the tooth exposed in the oral cavity and progressively destroys the tissue of the tooth. It is said that the direct cause of tooth decay is bacteria in the oral cavity, which can be roughly classified into the following three types based on the initial site of the tooth decay and the causative bacteria (Kitano Shigeo, Bulletin of the Japanese Society of Dental Medicine) , Seventh
Vol. 1, No. 21, p. 1981). Type Multiple age Site A A pit fissure caries 4-8 Primary teeth, permanent teeth, fissures of teeth B Smooth caries 11-18 Smooth faces C Root caries 50-60 Neck, root cementum

According to Kitano's report, the causative bacteria of caries are A lactobacillus acidophilus, Lactobacillus casei, Streptococcus mutans, Streptococcus sanguis, B Streptococcus mutans, Streptococcus sangis, and C. actinomyces. Viscousis. It is also known that Streptococcus mutans and Sangis are involved in root caries. Therefore, mutans is the most important causative fungus of dental caries.

[0004] Streptococcus bacteria have fimbriae on the surface of the cells, which adhere to the surface of the tooth and, in the presence of sucrose, for example, sticky plaque (for example, of an insoluble dextran-like polysaccharide). Plaque) to form lactic acid. This lactic acid is said to destroy teeth. A vaccine for preventing tooth decay using an antigen of whole cells, a part of the cells, or an extracellular enzyme of a Streptococcus bacterium capable of inducing dental caries is known.

[0005] For example, Japanese Patent Publication No. 49-626
No. 24 discloses a vaccine for preventing smooth caries using whole Streptococcus mutans cells, a part thereof (particularly, cell wall) or an extracellular enzyme as an antigen. He says that the causes of eclipse and other caries are essentially different. The vaccine described in the Examples is a whole cell vaccine in which all cells are inactivated with formalin or phenol. However, there is no description that this known vaccine is effective for caries other than smooth caries.

Next, Japanese Patent Publication No. 47-35121
Discloses a vaccine for preventing tooth decay using levan polysaccharides extracted from levan-producing bacteria of the genus Streptococcus as antigens, and the specifically disclosed antigens are Streptococcus miu having dextran-like polysaccharide-producing ability. A levan polysaccharide obtained from a culture of Streptococcus strain SS2 (ATCC 27006) having levan-producing ability, not from tans.

[0007] However, vaccines for preventing dental caries using these known cells or a part thereof as antigens still have room for improvement. That is, it is known that vaccines using whole cells of Streptococcus bacteria cause side effects such as allergic reactions, and that vaccines using the cell wall of Streptococcus mutans as an antigen cause cross-reactivity with myocardial tissue. . In addition, a vaccine using levan polysaccharide as an antigen has no effect on Streptococcus mutans, which is the most important of the cariogenic bacteria.

According to the present invention, the use of fimbria on the surface of a Streptococcus bacterium capable of inducing caries or an extract thereof as a vaccine antigen significantly prevents the adhesion of these bacteria to the surface and fissures of the bacteria. It is based on the finding that not only can it be suppressed, but it can also prevent the occurrence of side effects observed in known vaccines using cell walls as antigens. Fimbriae is a fibrous structure with a length of 0.5 to several μm protruding from the bacterial cell surface, protrudes from the protoplasm, and is tubular (Iwanami Biological Dictionary, 2nd edition, 5th printing, 1
981, 693 ).

[0009]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an improved vaccine for preventing dental caries, which comprises a part of the cells of a microorganism of the genus Streptococcus having the ability to induce dental caries.

[0010]

The vaccine for preventing tooth decay according to the present invention is characterized by using, as an antigen, fimbriae on the surface of cells of Streptococcus bacterium capable of inducing dental caries or an extract thereof. INDUSTRIAL APPLICABILITY The vaccine according to the present invention can effectively prevent tooth decay due to Streptococcus bacterium capable of inducing caries, without fear of side effects.

As mentioned above, among the Streptococcus genus microorganisms having this kind of caries-inducing ability, Streptococcus mutans is far more potent than Sangis and is most important. The present invention will be described in the case of Mututance.

[0012] Meutance was in 1924 Clark.
e) the bacteria reported by
NCTC1 for Collection of Type Culture
No. 0449, and ATCC 25175, 273 with the American Type Culture Collection.
51, 27352, 27607, 27947. The serotypes of these deposited strains are not described, but appear to be c-type or g-type.

In the Barges Manual of Deterministic Bacteriology, p. 502, 1974, it is stated that mutans is similar to S. salivarius.
According to other reports, Meutance, Sangis (S. sangu
is), Salivarius is considered to be practically difficult to distinguish because of its similarity to caries induction. Now, however, it differs from other oral bacteria of the genus Streptococcus in that it has the ability to decompose mannitol and sorbit.Mutance degrades sucrose to produce a water-insoluble, sticky dextran-like polysaccharide. Sangis produces water-soluble dextran-like polysaccharides from sucrose, but its adhesion is very low, and Sangis degrades arginine to produce ammonia, and Salivarius produces fructans from sucrose In that respect, mutans are distinguished from other Streptococcus oral bacteria.

In this specification, for example, a report by Mr. Toshio Morioka, "Streptococcus mutans" (Journal of the Japanese Society of Dental Medicine, Vol. 6, No. 12, pages 21-24, 19)
(1980) with reference to the biochemical and serological properties of the strain. S. mutans is classified from type a to type g according to the serological specificity of the polysaccharide contained in its cell wall. Types a and b are present in the oral cavity of rats and the like, whereas types c-g are It is known to be present in the human oral cavity.

The present invention is based on the fact that the immunological specificity of the antigens contained in the pili of these bacteria is not significantly different from the viewpoint of dental caries prevention. It is based on the surprising finding that fimbria obtained from Meutans can be used. In fact, there are mutans ranging from c-type to g-type in the human oral cavity, but 93% or more of them have c-type (Makoto Sato, Journal of Oral Health,
Vol. 28, No. 2, pp. 100-123, July 1978
Therefore, this finding is important from a practical point of view.

[0016] As a raw material of the vaccine of the present invention, fimbriae of an oral microorganism of the genus Streptococcus capable of inducing tooth decay or an extract thereof can be used, and pili of a mutant strain derived from these strains or an extract thereof can be used. Things can also be used. In particular, the use of the Streptococcus mutans mutant K-Dp (Microtechnical Research Laboratories No. 317) as a raw material provides excellent results as described below. This strain is obtained by mutagenizing c-type mutans isolated from the human oral cavity by the present inventors, and has remarkably stronger caries-inducing ability than the parent strain. K-Dp
The mycological properties of the strain are as follows.

(I) Morphology Form and size Streptococcus 1 μ × 1-2 μ, Gram stain positive (II) Growth status in each medium (37 ° C., 24 hours, anaerobic culture) (1) TYC agar plate medium ( (pH about 7.2) (Stoppelaar
JD et al. Archs.Oral.Biol., 12.1199-1201.196
7) Creates a heterogeneous settled colony covered by a large quantity of qualitatively hard insoluble dextran-like polysaccharides. (2) Dot Hewitt broth agar plate (pH
(Approximately 7.8) (Baltimore Biological Research Laboratories, USA; hereinafter abbreviated as BBL) Creates a small, flat, slightly opaque, glossy, somewhat viscous settlement. (3) Tryptic case soy broth (pH about 7.
3) (manufactured by BBL, USA) Grow from the lower part of the medium. (4) Dot Hewitt broth (pH about 7.8)
It grows from the lower part of the medium (manufactured by BBL, USA).

(III) Physiological properties A large amount of water-insoluble dextran-like polysaccharide is produced. (1) Wire plaque forming ability +++ (2) Cell aggregation by polysaccharide + (3) Pigment formation. -(IV) Growth range pH5-8 Temperature 10-39 ° C

(V) Glycolysis Glucose, galactose, maltose, sucrose, lactose, sorbitol, mannitol, inulin, melibiose, cellobiose + raffinose, salicin-esculin + ~-

(VII) Various properties Esculin hydrolysis + Arginine hydrolysis ± Hemolytic (sheep blood) γ

The K-Dp strain was obtained by the present inventor from c-type mutans isolated from the human oral cavity. Human-type Mutant strains other than c-type and mutants thereof can be used for the purpose of the present invention, although they have different sugar-degrading properties. A mutant strain suitable as a source of the vaccine according to the present invention can be obtained, for example, by the following method.

The field strain of Moutans collected from human plaque was subjected to mutagenesis treatment with nitrogen mustard by a conventional method, and a strain having a high ability to produce insoluble dextran-like polysaccharide was selected and separated. If desired, the mutagenesis treatment is repeated, and those with a high production rate of insoluble dextran-like polysaccharide are collected and purely cultured. If desired, a known mutagenesis treatment other than nitrogen mustard, such as ultraviolet irradiation or nitrosoguanidine, may be used.

The number and time of the mutation treatment required to obtain the mutant strain according to the present invention vary depending on various conditions. For the K-Dp strain, for example, a TYC agar plate medium (pH
(7.8; 24 hours at 37 ° C.), and the occurrence of reverse mutation was observed when artificial mutation treatment such as nitrosoguanidine, nitrogen mustard, and ultraviolet irradiation was repeated. Not only can not be
No significant changes were observed in the above-mentioned mycological properties, nor in the penetration or adhesion into the fissures, which are considered to be closely related to caries. The K-Dp strain was stable when subcultured using various media for 6 years or more.

The microorganisms described in the examples are K-Dp strain and Streptococcus mutans attenuated strain K-III.
(Microtechnical Laboratory No. 316). The K-Dp strain has much stronger dextran-like polysaccharide-producing ability, caries-inducing ability and lactate hydrogenase activity than the C. mutans field strain capable of inducing caries, and the K-III strain has a dextran-like polysaccharide. Loss of productivity (-), loss of caries induction (-) and loss of lactate hydrogenase activity (-) or meaningless (-
Or ±).

The K-Dp strain and the K-III strain were prepared by artificial mutagenesis such as nitrosoguanidine, nitrogen mustard, ultraviolet irradiation, or the like.
It was confirmed by a caries score for molars after long-term oral administration to rats and hamsters that no reverse mutation was observed even after passage of 0 or more passages.

The method for culturing the mutant strain according to the present invention is the same as the known method for culturing Streptococcus microorganisms.
That is, the culture may be under aerobic or anaerobic conditions,
Practically, the latter is preferable. The medium may be a natural medium or a synthetic medium, but a liquid medium is suitable for mass production. The pH of the medium is 5.6-8.0, for example 7.0-7.2,
The culture temperature is 23-39 ° C, for example, 37 ° C. Acid production is rapid and the pH of the medium is usually about 4.2 hours within 48 hours.
And the bacteria do not grow thereafter.

For culturing the strain, various media suitable for culturing Streptococcus microorganisms can be used. The composition of the media used in the examples is as follows. Medium (A) Polypeptone 1.7% Polypeptone S 0.3% Yeast extract 0.5% Dipotassium phosphate 0.25% Sodium chloride 0.5% Glucose 0.25% (pH 7.0-7.8)

Next, the present invention provides a method for culturing a microorganism of the genus Streptococcus capable of inducing caries in a medium, and suspending the cultured cells in a substantially neutral hypertonic buffer to obtain a desired fimbrial antigen. A method for producing a vaccine for preventing dental caries, comprising extracting and selectively recovering the antigen from the extract.

After completion of the culture, the culture solution is usually about 100 million cells / ml.
Contains live bacteria. For example, centrifugation (8000
The cells are separated from the culture solution by a conventional method such as rpm). For example, 0.1-1M acetic acid / sodium acetate buffered 1M saline (pH 6.0-7.8) or 0.01-M
0.75M phosphate buffered 1M saline (pH 6.8-7.
0) (The salt concentration of the phosphate buffered 1M saline solution is about 6.8% unless otherwise specified) Other suitable hypertonic buffers, such as Triton X100 (Rome and Haas, USA) Suspended in a solution to which a nonionic surfactant is added, and subjected to ultrasonic treatment (for example, 10-20 kHz, 5-2).
0 min) to selectively extract the pilus antigen. The obtained extract is used alone or in combination, for example, by a known column fractionation method, isoelectric focusing method, cold solvent fractionation precipitation method, membrane concentration method, ammonium sulfate salting out method or sucrose density gradient ultracentrifugation method. For purification by fractionation.

The purified material was dissolved in 1/75 M phosphate buffered saline (pH of about 6.2 to 6.5) and the protein N content was adjusted to 0.
The dilution was adjusted to be 0.05-0.5 mg / ml, and an aluminum hydroxide gel was added as an adjuvant so that the final aluminum concentration was 0.2 mg / ml, and the antigen was adsorbed.
As a preservative, for example, thimerosal 0.01% (w /
When v) is added, the vaccine for preventing dental caries according to the present invention is obtained. Although the culture method and the method for producing a vaccine have been described above for the mutant K-Dp strain, the above description is also applicable to the case where a mutans other than c-type and a Streptococcus microorganism having a caries-inducing ability other than mutans are used. .

The use of the vaccine of the present invention is as follows. When used, the dosage is, for example, 0.2 to 1.0 ml for a human being injected subcutaneously or intramuscularly, preferably into the oral mucosa. When this vaccine is administered to humans or animals, it is recognized that immune antibodies are formed, especially in saliva. This antibody is mainly IgA and has the property of inhibiting the invasion and colonization of the fungal field-causing field strain on the surface or in the fissure, but does not generate agglutinin, and the growth of the corresponding field strain itself And does not prevent glucan production. However, even if these field strains proliferate, their plaque-forming ability on the tooth surface is suppressed, and as a result, they can be easily removed from the oral cavity by ordinary methods such as tooth brushing, and therefore, the prevention and control of tooth decay can be prevented. The goal can be achieved.

The field strain used in the examples is of type c, but can be used in combination with other strains. Cultivation in the following Examples, Test Examples and Reference Examples was performed under a mixed gas atmosphere of nitrogen gas (90%), carbon dioxide gas (5%) and hydrogen gas (5%) or a gas pack system (BBL, USA) unless otherwise specified. Anaerobic cultivation using the method described above.

[0033]

Example 1 Preparation of a Streptococcus mutans mutant K-Dp strain (Microtechnical Laboratories No. 317). After identifying the field strain isolated from human plaque as a Streptococcus mutans c-type bacterium from various biochemical and serological properties, the strain was identified as dot Hewitt broth (pH 7.8, manufactured by BBL, USA) ) The cells were cultured at 37 ° C for 24 hours using 20 ml, and the cells were centrifuged at 4 ° C from the culture solution (8000 rpm / 20 minutes) to obtain 1/7.
3M with 5M phosphate saline (pH 6.8, 100ml each)
Washing was performed by centrifugation (the same conditions as before). Next, the viable cell count was reduced to about 10 in 1/75 M phosphate buffered saline (pH 6.8, 10 ml) containing 0.1 % of nitrogen mustard.
The suspension was suspended at a concentration of 10,000 / ml and kept at 37 ° C. until about 90% of the viable cell count was killed.

This was centrifuged at 4 ° C. (under the same conditions as before), and the isolated cells were cultured in 20 ml of dot Hewitt broth under the same conditions as described above. One platinum loop of the culture was placed on a TYC agar plate (Stoppelaar plate). JD et al.) (PH about 7.
After anaerobic culture at 37 ° C for 48 hours at 2,15 ml),
The culture was allowed to stand at room temperature for 24 hours, and colonies with a high production rate of insoluble dextran-like polysaccharide were selected and separated. If desired, the same operation was repeated to collect colonies with a high production rate of the insoluble dextran-like polysaccharide and purely cultured them. It was confirmed by oral administration to hamsters that the thus obtained strain had extremely high caries induction ability.

The mutant strain may be subcultured on a TYC agar plate medium (pH about 7.2; 37 ° C., 24 hours) for 100 or more passages, or may be a known mutation such as nitrogen mustard, nitrosoguanidine, or ultraviolet irradiation. Even after the induction treatment, no deterioration of various properties or appearance of other mutant strains was observed. This bacterium was founded on January 22, 1987 by
No. 17 has been deposited internationally.

[0036]

Example 2 Production of vaccine 500 ml of the above-mentioned medium (A) was sterilized as a seed medium, and the K-Dp strain obtained in Example 1 was inoculated into the medium, and
After culturing for 24 hours at 100 ° C., 100 ml of the medium was added to the medium (A)
The medium was transferred to 15000 ml of the same main medium as in Example 2 and cultured under the same conditions. After the culture, the culture was centrifuged at 4 ° C. (8000
r. p. m. / 20 minutes) to separate the cells, and then add 1M acetic acid
The suspension was suspended in a sodium acetate buffer (pH 6.8-7.0) and sonicated (10 kHz) on ice for 10 minutes. To this was added a saturated ammonium sulfate solution so as to be 60% saturated, and the mixture was allowed to stand at a temperature of 4 ° C. for 48 hours. The obtained precipitate was collected by centrifugation (8000 rpm / 20 minutes) at 4 ° C., and then 0.1 M acetic acid / sodium acetate buffer solution (pH
6.8-7.0) Suspend thickly in about 20 ml, and use cellophane dialysis tube at 4 ° C. for 48 hours, 3000 ml
Dialysis was performed in the above buffer solution. The inner dialysate was centrifuged at 4 ° C. (2000 rpm / 20 minutes) to remove bacterial cells and other impurities, and about 60 ml of the supernatant was recovered. The protein N content of this supernatant was about 2 mg / ml.

The supernatant is transferred to a cellophane dialysis tube and reduced to 1/10 volume using Ficoll 100 (copolymer of sucrose and epichlorhydrin, manufactured by Pharmacia Fine Chemical Co., Sweden). Concentrated.
The obtained concentrate is placed on 30 ml of a 3-30 sucrose density gradient solution in a cellulose tube, and ultracentrifuged (35,000 rpm) at 4 ° C. for 4 hours using a SW # 25.1 rotor (manufactured by Beckman, USA). After that, fractionation of 1 ml at a time using Density, Gradent, Fractionator, Isco 200 (made by Isco, USA) gave a specific gravity of 1.31-1.
35, an effective antigen was recovered near the fraction with a sucrose density of 10-13%. The protein N content is about 0.25-0.5mg / ml
Met.

The obtained material was diluted with 1/75 M phosphate buffered saline (pH 6.2-6.5) to contain 0.05 mg of protein N.
/ ml and dilute aluminum hydroxide gel as adjuvant to a final aluminum concentration of 0.2 mg / m2.
l and adsorbed, and 0.01% (v / w) of thimerosal was added as a preservative.

Using the vaccine obtained in Example 2 and the reference vaccine obtained in Reference Example 2, a notification was issued by the Ministry of Health and Welfare,
Staining tests, bacterial culture tests, and acute abnormal toxicity tests were conducted according to the Biologics Standards General Tests, A, and Test Methods, but no abnormalities were found. The reference vaccine showed strong acute abnormal toxicity. Using the present vaccine and the reference vaccine, the following caries prevention effect test was performed. The test animals used were golden hamsters, and the number of animals was 10 per group unless otherwise specified.

[0040]

[Test Example 1] (1) A caries suppression test was performed using a 21-day-old hamster. In order to examine the immune effects of the vaccines described in the examples, the mutans mutant strain KD having strong caries induction potential was used.
Culture medium (viable cell count 100)
Each of the test animals was continuously administered 0.1 ml / day into the buccal pouch of the test animal for 5 days from the 21st day of age, and at the same time, was raised only with 10-30 g / day of caries-inducing diet (Diet 2000, manufactured by Funabashi Farm). The vaccine and the reference vaccine were subcutaneously injected into the cheek pouch at 0.2 ml each once a day at 21 days and 27 days of age. Animals bred for 60 days were anesthetized with pentobarbital, and 0.75 pilocarpine hydrochloride solution (0.1 ml per 100 g body weight) was intraperitoneally injected to collect saliva. Thereafter, the whole blood was collected and sacrificed, the jaw was removed, and this was treated in an autoclave at 120 ° C. for 1-2 minutes to remove soft tissue, and the remainder was thoroughly washed with water and dried to obtain a sample. The control group was treated as the test group, but did not receive the vaccine.

(2) In order to compare the occurrence of dental caries between the test group and the control group, the average caries score of the caries formed on all molars of the animals of each group was determined by a conventional method [Keyes, PH (J. Dent. Res. . twenty three,
439-444, 1944)].

(3) In order to examine the relationship between the antibodies retained in the test group and the control group, antibodies in serum and saliva collected from animals were compared and evaluated by a quantitative agglutination reaction using a microtiter plate and an adhesion inhibition test.

As antigens for the agglutination reaction, tryptocase soy broth (0.5% yeast extract to each of the mutant mutans K-Dp and human (cg type) mutans described in Example 1) was used. pH 7.8, 15 ml, US B
(Manufactured by BL Co., Ltd.) for 24 hours at 37 ° C., and the culture was centrifuged (8000 rpm / 20 minutes). The resulting cells were diluted with 1/75 M phosphate buffer containing 0.2 M glutaraldehyde. The cells were suspended in saline (pH 7.0, 100 ml) to collect the cells, and the cells were suspended in 1/75 M phosphate buffered saline (pH 7.0).
(0, 300 ml) so that OD550nm = 0.50, and used as an antigen. The antigen (0.02 each) was added to each of the double-diluted solutions (0.025 ml each) of the collected saliva and serum.
5 ml), reacted at 37 ° C. for 4 hours, allowed to stand at 5 ° C. overnight, and visually observed.

The adhesion inhibition test was performed as follows. TYC liquid medium (Stoppelaar JD et al.
Archs. Oral. Biol., 12. 1199-1201. 1967) (pH 7.
2) was added 10-fold, sterile-filtered through a membrane filter (Millipore, USA, 0.45 μm), and then diluted 2-fold with the same medium.

Separately, K-Dp strain (Microtechnical Lab. No. 317)
And human (cg) mutans bacteria separately from Dot Hewitt broth (BBL, USA, pH 7.
8, 10 ml each) at 37 ° C. for 24 hours. One platinum loop of each culture was inoculated into each of the above dilutions (3 ml each) and cultured at 37 ° C. for 24 hours. Each culture was removed from the culture test tube, and the test tube was washed with water, and then stained with a methylene blue staining solution for adhesion to the tube wall, and visually determined. A similar test was performed using the reference vaccine. The results are shown in Tables 1 and 2 below.

[0046]

[Table 1]

[0047]

[Table 2]

When the caries development of all molars obtained from the immunized group and the control group was compared, a significant difference was found in the average caries score of the carious teeth formed on all molars of the animals of each group. Generation was strongly suppressed. In addition, when the antibodies in the serum and saliva of the immunized group were examined, no agglutinin was found in both, but a high adhesion-inhibiting ability was found in saliva. No significant difference in caries score was found between the reference vaccine immunized group and the control group. Agglutinin was found in the serum of the reference vaccine group, but no anti-adherent antibody was found.

[0049]

[Test Example 2] Combination test of caries preventive vaccine and live caries preventive vaccine:
The dental caries prevention vaccine (hereinafter referred to as the present vaccine) is a vaccine described in Examples. A live vaccine for preventing dental caries (hereinafter referred to as a live vaccine) is a mutans having the activity of suppressing the growth of a field-mutant field strain disclosed in “Live vaccine for preventing dental caries” filed by the applicant on the same day as the present application. The present invention relates to a live vaccine for preventing dental caries, which is characterized in that the method for producing the vaccine is described in Reference Example 1 below.

According to this test example, when a live vaccine is administered simultaneously with or before the administration of the present vaccine, the occurrence of human caries can be significantly prevented or suppressed. The first to third groups of hamsters were test groups, and the fourth group was a control group. From the age of 21 days after birth of each group, the above K-
0.2 ml for each culture containing about 10 million / ml of viable bacteria of Dp strain
Was administered to the cheek pouch for 5 consecutive days, and raised only with a caries-inducing diet (Diet 2000, manufactured by Funabashi Farm) at a daily dose of 10 to 30 g to induce dental caries. Next, a live vaccine containing about 10 million / ml live bacteria was used. The test period was 60 days.

Group 1 (live vaccine alone) From the eruption of molars, only 0.2 ml of each live vaccine was administered once a day for 10 consecutive days. Group 2 (combination of live vaccine and the present vaccine)
0.2 ml each of the vaccine was subcutaneously injected into the buccal pouch on the day and the 27th day. Group 3 (this vaccine alone) 0.2 ml each of this vaccine alone at 14 days and 21 days after birth
Was injected subcutaneously into the buccal pouch. Group 4 (untreated) No vaccine was given.

According to Table 3 showing the results, the caries suppressing effects of the first group (live vaccine alone) and the third group (this vaccine alone) are recognized. The low caries score of the second group (combination) indicates an excellent effect of the combination. No antibody was produced in blood or saliva by administration of the live vaccine. In the second and third groups to which this vaccine was administered, no agglutinin was produced, but an antibody having a high antiadhesion ability was produced in saliva.

[0053]

[Table 3] *: 9 animals per group

[0054]

[Reference Example 1] Production of a live vaccine for preventing tooth decay 500 ml of the above-mentioned medium A was sterilized and used as a seed medium, and cultured at 37 ° C for 24 hours at 37 ° C for attenuated Streptococcus mutans attenuated strain K-III. After that, 100 ml thereof was transferred to 15,000 ml of the same main medium, and cultured under the same conditions. After completion of the culture, the culture was centrifuged at 4 ° C. (8000 rpm for 20 minutes each) to separate the cells,
This was added to 1/75 M phosphate buffered saline (300 ml, pH
7.0) and centrifuged at 4 ° C (8000 rpm).
/ 20 minutes each), and washed with 1 / 75M
Diluted with phosphate buffered saline so that the number of viable bacteria is about 100 million / ml.
(W / v) and lactose 5% (w / v) were added, dispensed into vials and freeze-dried. When used, the number of viable bacteria is about 10
The solution was diluted with 1/75 M phosphate buffered saline (pH 7.0) so as to be 100,000 cells / ml.

[0055]

Reference Example 2 A vaccine was prepared using all the cells according to the method described in Example 1 of Japanese Patent Laid-Open Publication No. Sho 49-62624. Streptococcus mutans (NCTC1
0449) was cultured at 37 ° C. for 24 hours using Brain Heart Infusion Broth (manufactured by Difco, USA, pH 7.8, 10 ml). Transfer the culture solution to Rubin (30
0 ml) was spread on the surface of Brain Heart Infusion Agar Medium (manufactured by Difco, USA, pH 7.8, 100 ml) and cultured at 37 ° C for 24 hours. The grown cells were scraped off and suspended in sterile saline (0.85% w / v, NaCl). This was added to sterile saline (0.68% w /
v, 100 ml each) by centrifugation three times (8000 r.p.
m./20 minutes each), and washed the washed cells with a saline solution containing 0.6 formalin (0.85% w / v, 200 ml each).
ml) and left overnight at room temperature before adding saline (0.6 ml).
8% w / v, 100 ml each)
rpm / 20 minutes each).

Next, this was added to a saline solution (0.68% w / v, 1
0 ml). The suspension (0.1 ml) was diluted with a thioglycolic acid medium (BBL, USA, pH 7.2, 10 ml).
After culturing at 7 ° C. for 10 hours, a sterility test was performed. The remaining suspension was homogenized by sonication (20 kHz, 10 minutes), and saline containing thimerosal (0.01%) (0.68% w /
In v), the mixture was diluted to a final cell count of about 2 × 10 ⁸ ml.
Table 1 shows the test results. 0.5 instead of formalin
In addition to using% phenol, the results of the second reference vaccine obtained in a similar manner were even worse.

[0057]

As described above, the present invention provides a method for producing an improved vaccine for preventing dental caries, which comprises a part of the cells of a microorganism of the genus Streptococcus having the ability to induce dental caries. ADVANTAGE OF THE INVENTION The vaccine by this invention can prevent the caries by Streptococcus microbe which has the caries induction | guidance | derivation ability effectively without fear of side effects.

Claims (3)

(57) [Claims] Claims: 1. A step of culturing a Streptococcus genus microorganism capable of inducing caries in a medium, and suspending the cultured microorganism in a substantially neutral hypertonic buffer, thereby extracting a pilus antigen. How to make a preventive vaccine. 2. The method according to claim 1, wherein the microorganism is Streptococcus mutans. 3. The method according to claim 1, wherein a hypertonic buffer containing salt is used.