JPH0456011B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a vaccine for preventing dental caries, and particularly to a vaccine for preventing dental caries that uses a part of the bacterial cells of a Streptococcus microorganism that has the ability to induce caries as an antigen. Dental caries (caries) is a disease of tooth enamel, dentin, and cementum that begins on the exposed tooth surfaces in the oral cavity and progressively destroys tooth tissue. The direct cause of tooth decay is bacteria in the oral cavity, and it is said that these can be roughly divided into the following three types based on the initial site of tooth decay and the causative bacteria (Shigeo Kitano,
Bulletin of the Japanese Dental Association, Volume 7, No. 1, Page 21, 1981
year) Type Age of occurrence Site A Pit and fissure caries 4-8
Baby molars, permanent teeth, tooth fissures B Smooth surface caries 11-18 Smooth surface C Root surface caries 50-60 Tooth neck, root cementum According to the above report by Mr. Kitano, the causative bacteria of tooth decay are A. Bacillus acidophilus, Lactobacillus casei, Streptococcus miutans, Streptococcus sanguis B Streptococcus miutans Streptococcus sanguis C Actinomyces viscouzis. It is also known that Streptococcus miutans and Streptococcus sanguis are involved in root caries. Therefore, P. miutans is the most important bacterium that causes dental caries. Bacteria belonging to the genus Streptococcus have fimbriae on the surface of their bacterial bodies, which allow them to adhere to the surface of the bacteria and, in the presence of sucrose, form sticky dental plaque made of insoluble dextran-like polysaccharides. (plaque) and produces lactic acid. This lactic acid is said to destroy teeth. Vaccines for preventing dental caries that use whole cells, part of the cells, or extracellular enzymes of Streptococcus bacteria that have the ability to induce dental caries as antigens are known.
For example, Japanese Patent Publication No. 49-62624 discloses a vaccine for preventing caries on smooth surfaces that uses the whole bacterial body of Streptococcus miutans, a part thereof (especially the cell wall), or an extracellular enzyme as an antigen. However, this publication states that the causes of smooth surface caries and other caries are essentially different. Further, the vaccines described in the Examples are whole cell vaccines in which all the cells are inactivated with formalin or phenol. However, there is no description that this known vaccine is effective against caries other than smooth surface caries. Next, Japanese Patent Publication No. 47-35121 discloses a vaccine for preventing dental caries that uses levan polysaccharide extracted from a levan-producing bacterium of the genus Streptococcus as an antigen, but the specifically disclosed antigen is Streptococcus strain SS2, which has the ability to produce levan, but not from Streptococcus miutans, which has the ability to produce dextran-like polysaccharides.
(ATCC27006) is a levan polysaccharide obtained from a culture. However, there is still room for improvement in these known vaccines for preventing dental caries that use bacterial cells or parts thereof as antigens. In other words, it is known that vaccines that use the whole body of Streptococcus bacteria cause side effects such as allergic reactions, and that vaccines that use the cell wall of Streptococcus miutans as an antigen cause cross-reactions with myocardial antigens. It is being In addition, vaccines using levan polysaccharide as an antigen are not effective against Streptococcus miutans, the most important of the cavities-causing bacteria. The present invention shows that by using pili on the surface of Streptococcus bacteria, which have the ability to induce dental caries, as a vaccine antigen, it is possible to not only significantly prevent or suppress the adhesion of these bacteria to the surface and fissures of the bacteria. This is based on the knowledge that it is possible to prevent the occurrence of side effects observed in vaccines that use cell walls as antigens. The object of the present invention is to use a part of the bacterial cells of a Streptococcus microorganism having the ability to induce dental caries as an antigen.
An object of the present invention is to provide an improved vaccine for preventing or suppressing dental caries (hereinafter referred to as prevention). The vaccine for preventing dental caries according to the present invention uses as an antigen a part of the bacterial cells of a Streptococcus bacterium capable of inducing dental caries, and is characterized in that the antigen is fimbriae on the surface of the bacterial cells or an extract thereof. There is. The vaccine according to the present invention can effectively prevent dental caries caused by Streptococcus bacteria that have the ability to induce caries, without fear of side effects. As mentioned above, among the microorganisms of the genus Streptococcus, Streptococcus mutans has a much stronger ability to induce dental caries than S. mutans and is the most important. The present invention will be explained using the case of miutans. P. miutans is a bacterium that was first reported by Clarke in 1924 and included in the British National Collection of Type Cultures.
Deposited as NCTC 10449 and also in the American Type Culture Collection at ATCC.
Deposited as 25175, 27351, 27352, 27607, 27947. Although the serotypes of these deposited strains have not been described, they are believed to be type C or type G. Burgess Manual of Determinative Bacteriology, p. 502, 1974.
miyutans has been described as similar to Streptococcus salivarius (S. salivarius). According to other reports, the fungi-inducing effects of S. miutans, S. sanguis, and S. salivarius are so similar that it is difficult to distinguish them in practice. However, it is now known that S. miutans differs from other oral bacteria of the genus Streptococcus in that it has the ability to degrade mannite and sorbitol, and that S. miutans breaks down sucrose to produce a water-insoluble sticky dextran-like polysaccharide. Although S. sangis produces a water-soluble dextran-like polysaccharide from sucrose, its adhesiveness is very low. S. sanguis breaks down arginine to produce ammonia, and S. salivarius produces fructans from sucrose. In this, S. miutans is distinguished from other oral bacteria of the genus Streptococcus in the oral cavity. In this specification, for example, Mr. Toshio Morioka's report "Streptococcus miutans" (Japan Dental Association Bulletin, Vol. 6, No. 12, pp. 21-24, 1980)
The B. miutans strain was identified from the biochemical and serological properties of the strain, with reference to 2010). Bacillus miutans is classified into types A to G depending on the serum specificity of the polysaccharide contained in its cell wall. Types A and B exist in the oral cavity of rats, etc., but types C and G are found in the oral cavity of rats. It is known to exist in the human oral cavity. The present invention proposes that the immunological specificity of the antigens contained in the fimbriae of these bacteria shows no significant difference from the perspective of preventing dental caries. This research is based on the surprising finding that pili can be utilized. In fact, there are types C to G in the human oral cavity, and more than 93% of them are type C (Makoto Sato, Journal of the Oral Hygiene Society, Vol. 28, No. 2,
100-123, July 1978), this finding is important from a practical point of view. As raw materials for the vaccine of the present invention, pili of oral microorganisms of the genus Streptococcus or extracts thereof having the ability to induce caries can be used, but pili of mutant strains derived from these strains or extracts thereof may be used. You can also use In particular, when Streptococcus miutans mutant strain K-Dp (Feikoken Joyori No. 317) is used as a raw material, excellent results as described below can be obtained. The K-Dp strain was obtained by the present inventor through mutation treatment of type C miutans isolated from the human oral cavity, and has significantly stronger caries-inducing ability than the parent strain. The mycological properties of this strain are as follows. () Morphology Bacterial shape and size Streptococcus 1μX1-2μ Gram staining Positive () Growth status in each medium (37℃, 24 hours, anaerobic culture) TTY agar plate medium (PH approximately 7.2) (Stoppelaar, JD, et al .Archs.Oral.
Biol., 12, 1199-1201, 1967) It forms a fixed, heterogeneous colony covered with a large amount of qualitatively hard and insoluble dextran-like polysaccharide. Dot Human Broth Agar Plate Medium (PH7.8) (manufactured by Baltimore Biological Laboratories, USA, hereinafter referred to as BBL) A perfectly round, flat, slightly opaque, glossy medium.
Creates small, slightly viscous settlements. Tryptocase soy broth (PH7.3) (manufactured by BBL, USA) Proliferates from the bottom of the medium. Dot Huyt Broth (PH7.8) (manufactured by BBL, USA) Proliferates from the bottom of the medium. () Physiological properties Produces large amounts of water-insoluble dextran-like polysaccharides. Wire plaque forming ability +++ Bacterial aggregation by polysaccharides + Pigment production - () Growth range PH5-8 Temperature 10-39℃ () Glycolysis glucose, galactose, maltose, sucrose, lactose, sorbitol, mannitrate, inulin, melibiose , cellobiose + raffinose, salicin - esculin + ~ - () Properties Aesculin hydrolysis + arginine hydrolysis ± hemolytic (sheep blood) γ The K-Dp strain is a type C miutans that the present inventor isolated from the human oral cavity. It was derived from. Field strains of human type C. miutans other than type c and their mutant strains differ in sugar decomposition ability, etc.
It can be utilized for the purpose of the present invention. A mutant strain suitable as a raw material for the vaccine according to the present invention can be obtained, for example, by the following method. M. miutans field strains collected from human plaques are subjected to mutation induction treatment with nitrogen mustard using a conventional method, and those with a high ability to produce insoluble dextran-like polysaccharides are selected and isolated from the strains obtained. Then, by repeating the mutation induction treatment, those with a high ability to produce insoluble dextran-like polysaccharides are collected and pure cultured. If desired, known mutation induction treatments other than nitrogen mustard, such as ultraviolet irradiation, nitrosoguanidine, etc., may be used. The number and time of mutation treatment required to obtain the mutant strain of the present invention differs depending on various conditions, but for K-Dp strain, for example, TYC agar plate medium (PH7.8, 37°C, 24 hours ), or when artificial mutation induction such as nitrosoguanidine, nitrogen mustard, or ultraviolet irradiation was repeated, not only was no reverse mutation observed, but the aforementioned bacteria No significant changes were observed in the chemical properties or in the ability to penetrate into fissures or adhere to the fissures, which are considered to be closely related to dental caries. The K-Dp strain remained stable even when it was subcultured using various media for over 6 years. The microorganisms described in the examples include the K-Dp strain and the attenuated Streptococcus miutans strain K-
It is a stock (Feikoken Joyori No. 316). The K-Dp strain has much stronger dextran-like polysaccharide-producing ability, caries-inducing ability, and lactate dehydrogenase activity than the C. miutans field strain, which has the ability to induce caries, and the K- strain has a much stronger ability to produce dextran-like polysaccharides, caries-inducing ability, and lactate dehydrogenase activity. It is characterized by a lack of activity (-), a lack of caries-inducing ability (-), and a lack of or no significance (- or ±) of lactate dehydrogenase activity. The K-Dp strain and the K- strain do not exhibit reverse mutation even when induced by artificial mutagenesis such as nitrosoguanidine, nitrogen mustard, or ultraviolet irradiation, or by subculturing for more than 100 generations on various media. ,
This has been confirmed by caries scores on molars of rats and hamsters after long-term oral administration. The method for culturing the mutant strain according to the present invention is similar to the known culture method for microorganisms of the genus Streptococcus. That is, culture may be carried out under aerobic or anaerobic conditions, but the latter is preferred from a practical standpoint. 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, e.g. 7.0-7.2, and the culture temperature is 23-39℃, e.g. 37
It is ℃. Acid production is rapid, and the pH of the medium usually drops to about 4.2 within 48 hours, after which the bacteria do not grow. Although various media suitable for culturing Streptococcus microorganisms can be used to culture the bacterial strain, the composition of the culture medium 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% (PH7.0-7.8) Next, the present invention focuses on the Streptococcus sp. To provide a method for producing a fimbrial antigen for preventing dental caries, which comprises a step of suspending microbial cells in a substantially neutral hypertonic buffer solution and recovering the desired fimbrial antigen by treating with ultrasound. . The culture solution after culturing usually contains about 100 million viable bacteria/ml.
The bacterial cells are separated from the culture medium by a conventional method such as p, m), and the bacterial cells are injected into, for example, a 0.1-1M acetic acid/sodium acetate buffer (PH6.0-7.8), 0.01-
0.75 phosphate buffered 1M saline (PH6.8-7.0) (salt concentration in phosphate buffered 1M saline is approximately 6.8% unless otherwise specified), or other suitable buffer solution, or triton and Haas Co., Ltd.) to which a nonionic surfactant has been added, and then subjected to ultrasonic treatment (for example, 10 to 20 kHz,
5 to 20 minutes) to separate and extract the fimbriae. The obtained extract is subjected to 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 alone or in combination. Then fractionate and purify. The purified material was diluted with 1/75M phosphate buffered saline (PH approximately 6.2-6.5) to a protein content of 0.05 to 0.5 mg/ml, and aluminum hydroxide gel was used as an adjuvant to adjust the final aluminum dose. is 0.2
mg/ml to adsorb the antigen, and as a preservative, for example, thimerosal 0.01% (w/v).
By adding , the vaccine for preventing dental caries according to the present invention can be obtained. The above describes the culture method and vaccine production method for the mutant strain K-Dp, but the above method also applies when using Streptococcus microorganisms other than type C and other microorganisms of the genus Streptococcus that have the ability to induce dental caries. be understood. The use of the vaccine of the present invention is as follows. The dosage for use is, for example, 0.2 to 1.0 ml per human injection subcutaneously or intramuscularly, preferably into the oral mucosa. When this vaccine is administered to humans or animals, it is observed that immune antibodies are produced, especially in saliva. This antibody is mainly IgA
It has the property of inhibiting the invasion and colonization of the surface and fissures of field strains that have the ability to induce caries.
It does not generate agglutinin, nor does it inhibit the growth itself or glucan production ability of the corresponding field strain. However, even if these field strains proliferate, their ability to form plaque on tooth surfaces is suppressed, and as a result, they can be easily removed from the oral cavity by conventional methods such as tooth brushing. The purpose of suppression can be achieved. The field strain used in the examples is type c, but
It can also be used in combination with other types of strains. Unless otherwise specified, culturing in the following examples, test examples, and reference examples was carried out under a mixed gas atmosphere of nitrogen gas (90%), carbon dioxide gas (5%), and hydrogen gas (5%) or in a gas pack system (USA).
The culture was carried out anaerobically using BBL (manufactured by BBL). Example 1 Streptococcus miutans mutant strain K-
Creation of Dp strain (Feikoken Joyori No. 317). After fixing the field strain isolated from human dental plaque to be Streptococcus miutans type C based on biochemical and serological properties, it was infused into Dots Human Broth (PH7.8, BBL in the United States).
Cultured at 37℃ for 24 hours using 20ml of 20ml (manufactured by Sekisui Chemical Co., Ltd.), and centrifuged the bacterial cells from the culture solution at 4℃ (8000r.pm/20min).
Separate with 1/75M phosphate saline (PH6.8, each
1/75M phosphate buffered saline containing 0.1% nitrogen mustard (PH 6.8, 10 ml) after washing by centrifugation three times (same conditions as before)
Suspend the bacteria so that the number of viable bacteria is approximately 1 million/ml,
The cells were kept at 37°C until more than 90% of the viable bacteria were killed.
This was centrifuged at 4°C (same conditions as before), the isolated bacterial cells were cultured in 20 ml of Dot Huyt Broth in the same manner as above, and one platinum loop of the culture was transferred to TYC agar plate medium [Stoppelaar et al. .，Archs.Oral
Biol., 12, 1190-1201 (1967)] (PH approx. 7.2, 15ml)
After incubation anaerobically for 48 h at 37°C, the culture
The colonies were allowed to stand at room temperature for 24 hours, and colonies with a high ability to produce insoluble dextran-like polysaccharides were selected and isolated.
If desired, the same operation was repeated to obtain a pure culture of colonies with a high ability to produce insoluble dextran-like polysaccharide. It was confirmed by oral administration to hamsters that the bacterial strain thus obtained had an extremely high ability to induce dental caries. This mutant strain was grown on TYC agar plate medium ((PH approx. 7.2, 37
Even if the strain is subcultured for more than 100 generations at 24 hours (°C, 24 hours) or subjected to known mutagenesis treatments such as nitrogen mustard, nitrosoguanidine, or ultraviolet irradiation, no deterioration of various properties or the appearance of other mutant strains was observed. I couldn't help it. This bacterium was internationally deposited on January 22, 1957 as FIKEN Article No. 317. Example 2 Production of vaccine Sterilize 500 ml of the above medium (A) as a seed medium,
The K-Dp strain obtained in Example 1 was planted in a medium and cultured at 37°C for 24 hours, and then 100 ml of it was transferred to 15,000 ml of the same main medium as medium (A) and cultured under the same conditions. After culturing, the culture solution was centrifuged at 4°C (8000 rpm/20 minutes) to separate the bacterial cells.
1M acetic acid/sodium acetate buffer (PH6.8-7.0)
It was suspended in 150 ml and subjected to ultrasonication (10 kHz) for 10 minutes under ice cooling. A saturated ammonium sulfate solution was added to this to make it 60% saturated, and the mixture was allowed to stand at a temperature of 4° C. for 48 hours. The resulting precipitate was collected by centrifugation at 4°C (8000rpm/20min), and then mixed with 0.1M acetic acid.
Suspend in approximately 20 ml of sodium acetate buffer (PH6.8-7.0) and use a cellophane dialysis tube to remove
Dialysis was carried out in over 3000 ml of the above buffer for 48 hours at °C. Centrifuge the dialysis fluid at 4℃ (2000r.pm/20
6 minutes) to remove bacterial cells and other impurities, and then remove the supernatant for approximately 60 minutes.
ml was collected. The protein N value of this supernatant is approximately 2 mg/ml.
It was hot. The supernatant was transferred to a cellophane dialysis tube and concentrated to 1/10 volume using Fecol 100 (a copolymer of sucrose and epichlorohydrin, manufactured by Pharmacia Huain Chemical, Sweden). The obtained concentrate is cellulose
Place it on 30ml of 3-30% sucrose density gradient solution in the tube, and add it to SW#25.1 rotor (manufactured by Beckman, USA).
Ultracentrifugation for 4 hours at 4°C (35000r.pm) using
After that, 1
When fractionated by ml, specific gravity is 1.31~1.35, sucrose density is 10~
Effective antigen was recovered in the vicinity of the 13% fraction. The amount of protein N was about 0.25-0.5 mg/ml. The obtained material was diluted with 1/75M phosphate buffered saline (PH6.2-6.5) so that the protein N content was 0.05 mg/ml, and aluminum hydroxide gel was used as an adjuvant to give a final aluminum concentration of 0.2. mg/ml
0.01% (v/w) of thimerosal was added as a preservative. In addition, using the vaccine obtained in Example 2 and the reference vaccine obtained in Reference Example 2, staining tests, bacterial culture tests, and acute abnormal toxicity were conducted in accordance with the Ministry of Health and Welfare notification, Biological Products Standard General Test, and Test Method A. Tests were conducted, but no abnormality was found. The reference vaccine showed strong acute abnormal toxicity. Using this vaccine and a reference vaccine, the following caries prevention effect test was conducted. The test animals used were goldens and hamsters, and the number of animals was 10 per group unless otherwise specified. Test Example 1 (1) A caries inhibition test was conducted using 21-day-old hamsters. In order to investigate the immune effect of the vaccine described in the example, a culture solution (1 million live bacteria/ml) of the K-Dp strain (Keiken Joyori No. 317), which has strong caries-inducing ability described in the example, was used at 0.1 each. ml/day was continuously administered into the buccal pouch of test animals for 5 days from 21 days of age, and at the same time, they were fed only 10 to 30 g/day of caries-inducing feed (Diet 2000, manufactured by Funabashi Farm). This vaccine and the reference vaccine were injected subcutaneously into the cheek pouch at 0.2 ml each once a day at 21 and 27 days of age. Animals kept for 60 days were anesthetized with pentabarbital, and 100 g of 0.75% pilocarpine hydrochloride solution was applied to them.
0.1 ml per patient was injected intraperitoneally, and saliva was collected.
Thereafter, all the blood was collected and sacrificed, the jaws were removed, and the jaws were treated in an autoclave at 120°C for 1 to 2 minutes to remove soft tissues, and the remaining parts were thoroughly washed with water and dried to prepare specimens. The control group was treated similarly to the test group, but did not receive the vaccine. (2) In order to compare the occurrence of cavities between the test group and the control group, the average caries score of all molars of the animals in each group was calculated using the conventional method [Keyes, PH (J.Dent.
Res. 23, 439-444, 1944)]. (3) In order to investigate the relationship between the antibodies possessed by the test group and the control group, antibodies in the serum and saliva collected from the animals were comparatively evaluated by quantitative agglutination reaction using a microtiter plate and adhesion inhibition test. As antigens for the agglutination reaction, S. miutans K-Dp bacteria and human type (c-g type) described in Example 1 were used.
B. miutans was cultured at 37°C for 24 hours in tryptocase soy broth (manufactured by BBL, USA, pH 7.8, 15 ml) containing 0.5% yeast extract, and the culture solution was centrifuged for 20 minutes (8000 r.pm). The collected bacterial cells were added to 1/75M phosphate buffered saline (PH7.0, 100%
ml), treated at 37°C for 12 hours, centrifuged (8000 rpm/20 minutes) to collect the bacterial cells, and added to 300 ml of 1/75M phosphate buffered saline (PH
7.0) so that the OD550nm was 0.50 and used as an antigen. Antigen (0.025 ml each) was added to each diluted saliva and serum solution (0.025 ml each).
ml) and reacted at 37°C for 4 hours, then at 5°C.
After allowing it to stand overnight, it was observed with the naked eye. The adhesion inhibition test was conducted as follows. Serum or saliva was added to TYC agar plates (Stoppelaar et al., Archs. Oral Biol. 12, 1199).
-1201, 1967, PH7.2), and after sterile filtration with a membrane filter (Millipore, USA, 0.45μ), it was diluted 2 times with the same medium. Separately, K-Dp (Feikoken Jokyo No. 317) and human type (c-g type) Miutans were cultured separately in Dot Huyt broth (PH7.8, 10 ml each) at 37°C for 24 hours. One platinum loopful of each of the above culture solutions was inoculated into each of the above twofold dilutions (3 ml each) and cultured at 37°C for 24 hours. Remove each culture solution from the culture test tube,
After washing the test tube with water, the adhesion to the tube wall was stained with methylene blue staining solution and judged visually. A similar test was conducted using a reference vaccine. The results are shown in Tables 1 and 2 below.

【table】

[Table] In order to compare the occurrence of caries in all molars obtained from the immunized group and the control group, a significant difference was observed in the caries average score of all molars of animals in each group, and It strongly suppressed the occurrence of tooth decay. When antibodies in the blood and saliva of the immunized group were examined, no agglutinin was observed in either group, but a high level of adhesion-inhibiting antibodies was observed in the saliva. No significant difference in caries score was observed between the reference vaccine immunized group and the control group. Agglutinin was observed in the serum of the reference vaccine, but no anti-adhesion antibodies were observed. Test Example 2 Combination test of a cavity prevention vaccine and a cavity prevention live vaccine. Vaccine for preventing cavities (hereinafter referred to as this vaccine)
is the vaccine described in the example. The live vaccine for preventing dental caries (hereinafter referred to as "live vaccine") is disclosed in "Live vaccine for preventing dental caries", for which the applicant applied for a patent on the same day as this application, and is a strain of the same strain that has the activity of suppressing the growth of field strains of S. miutans. This is a live vaccine for preventing dental caries, which is characterized by containing a mutant strain of .The method for producing it is described in Reference Example 1 below. According to this test example, the occurrence of dental caries in humans can be significantly prevented or suppressed when a live vaccine is administered at the same time as or before the administration of this vaccine. Groups 1 to 3 of hamsters were the test group, and group 4 was the control group. From the age of 21 days after the birth of each group, 0.2 ml of each culture solution containing about 10 million viable bacteria/ml of the above-mentioned K-Dp strain was continuously administered into the cheek pouch for 5 days, and a caries-inducing food diet of 10 to 30 g per day was given. 2000 to induce caries. A live vaccine containing approximately 10 million live bacteria/ml was used. The test period was 60 days. Test Group 1 (Live vaccine alone) From the time of molar tooth eruption, 0.2 ml of each live vaccine was administered once a day for 10 consecutive days. Test group 2 (combination of live vaccine and this vaccine) In addition to administering the live vaccine as in test group 1, 0.2 ml each of this vaccine was administered at 21 and 27 days of age after birth.
was injected subcutaneously into the cheek pouch. Test group 3 (this vaccine alone) This vaccine alone was administered to 14 and 21 days old after birth.
0.2 ml was injected subcutaneously into the cheek pouch area. Test group 4 (untreated) No vaccine was given. According to Table 3 showing the results, the effect of inhibiting caries induction was observed in Group 1 (live vaccine alone) and Group 3 (this vaccine alone). The low caries score of the second group (combination) indicates the excellent effect of the combination. Administration of the live vaccine did not produce antibodies in the blood or saliva. In the second and third groups administered with this vaccine, no agglutinin was produced, but antibodies with high adhesion-blocking ability were produced in saliva.

[Table] *: Reference example of 9 animals per group 1 Production of live vaccine for caries prevention Sterilize 500 ml of the above medium (A) and use it as a seed medium. No. 316) was cultured at 37℃ for 24 hours, then 100 ml of the culture was added to 15,000 ml of the same main medium.
and cultured under the same conditions. After culturing, the culture solution was centrifuged at 4°C (8000 rpm/20 minutes) to separate the bacterial cells, which were then added to 1/75M phosphate buffered saline.
After suspending in 300 ml (PH7.0) and washing the bacterial cells by centrifugation (8000 rpm/20 minutes) at 4°C,
The number of viable bacteria contained in 1/75M phosphate buffered saline is approximately 1.
Dilute to 100 million cells/ml, add 0.2% (w/v) gelatin and 5% (w/v) lactose as protective agents.
was added, dispensed into vials, and lyophilized. Before use, it was diluted with 1/75M phosphate buffered saline (PH7.0) so that the number of viable bacteria contained was approximately 10 million cells/ml. Reference Example 2 A vaccine using whole bacterial cells was produced according to the method described in Example 1 of Patent Publication No. 49-62624. Streptococcus miutans (NCTC10449) in Brain Heart Infusion Broth (manufactured by Difco, USA, PH7.8,
10ml) and cultured at 37°C for 24 hours. Transfer the culture solution to Brain Heart Infusion Agar medium (manufactured by Difco, USA) in Rubin (300 ml).
PH7.8, 100ml) and cultured at 37°C for 24 hours. The grown bacterial cells were scraped off and suspended in sterile saline (0.85% w/v, Nacl). This suspension was heated to 4°C in sterile saline solution (0.68% W/V, 100ml each).
The mixture was centrifuged three times (8000r.pm for 20 minutes each). The thus washed bacterial cells were suspended in saline containing 0.6% formalin (0.85% w/v, 200 ml each),
After leaving it at room temperature overnight, the bacterial cells were dissolved in saline solution (0.68%
centrifugation (8000 r.p. w/v, 100 ml each).
m./20 minutes). Next, add this to saline solution (0.68
%w/v, 10ml). Suspension liquid (0.1ml)
thioglycolic acid medium (manufactured by BBL, USA, PH7.2,
After culturing at 37°C for 10 hours in 10ml), sterility was tested. Sonicate the remaining suspension (20KHz, 10 minutes)
The mixture was homogenized and diluted with saline containing 0.01% thimerosal (0.68% w/v) until the final bacterial cell count was approximately 2×10 ⁸ ml. The test results are shown in Table 1. In addition to using 0.5% phenol instead of formalin,
The results of a second reference vaccine obtained in the same manner were poor.

Claims (1)

[Scope of Claims] 1. A vaccine for preventing dental caries that uses as an antigen a part of the bacterial cells of a microorganism of the genus Streptococcus that has the ability to induce dental caries, characterized in that the antigen is fimbriae on the surface of the bacterial cells or an extract thereof. vaccine. 2. The vaccine according to claim 1, wherein the microorganism is Streptococcus miutans. 3. The vaccine according to claim 2, wherein the serotype of Streptococcus miutans is type c-g. 4. The vaccine according to claim 2, wherein the microorganism is Streptococcus miutans mutant strain K-Dp (Feikoken Jokyo No. 317). 5. A method for producing a vaccine for preventing dental caries, which comprises the step of collecting fimbrial antigens from the bacterial cells of a Streptococcus microorganism capable of inducing dental caries by suspending them in a substantially neutral hypertonic buffer. . 6. The manufacturing method according to claim 5, which uses a hypertonic buffer containing salt. 7. The manufacturing method according to claim 5, wherein the bacterial cells are treated with ultrasound to collect fimbriae antigens.