JPH0662431B2 - Oral vaccine for periodontitis prevention - Google Patents

Description

TECHNICAL FIELD The present invention relates to a vaccine which is orally administered to prevent periodontitis, and more specifically, Bacteroides gingivalis and Actiomyces actiomyces. viscosus) and other whole cells of periodontitis-causing bacteria, and pili for preventing periodontitis, which is an oral vaccine for preventing periodontitis.

Prior art and its problems The main cause of periodontal disease is bacteria in plaque accumulated in periodontal pockets. Gram-positive bacteria usually account for the majority in healthy periodontal pockets, but as periodontal disease progresses, bacteria such as Bacteroides gingivalis and Actinomyces biscoudith increase. In fact, Bacteroides gingivalis and Actinomyces viscodis are particularly frequently isolated from lesions of patients with severe adult periodontal disease, and there are many cases in which the antibody in the patient serum against this bacterium is also increased. It has also been shown that inoculation of Bacteroides gingivalis into animals exacerbates periodontal inflammation.
These results indicate that Bacteroides gingivalis and Achnomyces biscoudis play important roles in the establishment of periodontal disease.

Bacteroides gingivalis and Actinomyces biscoudisus have cell surface substances such as pili on the cell surface, and they are said to adhere to the periodontal mucosa, proliferate and adversely affect the periodontium. There is. In order to prevent periodontitis due to such causes, it is effective to prevent these periodontitis-causing bacteria from establishing in the oral cavity or suppress proliferation.

Heretofore, as a method of preventing the colonization of such periodontitis-causing bacteria and controlling the growth, a method of administering a vaccine using pili of the periodontitis-causing bacteria or an extract thereof as an antigen has been proposed ( JP-A-59-128338). However, this method uses a vaccine that is directly injected into the living body, and there is a problem in safety from the viewpoint of toxicity in the body.

SUMMARY OF THE INVENTION In view of the above circumstances, the present inventors have conducted a diligent study on a method that is more safe and effectively prevents periodontitis, and as a result, teeth such as Bacteroides gingivalis, Actinomyces viscous, etc. Oral administration of a vaccine that uses the pilus or capsule of the periodontitis-causing bacteria as an antigen prevents the periodontitis-causing bacteria from colonizing and suppresses the growth, and effectively prevents periodontitis. It was possible to carry out the present invention, and the method was orally administered with an antigen derived from oral bacteria, and it was found that the safety is higher than that of internal administration (injection administration), and the present invention has been completed. It is a thing.

Therefore, the present invention provides an oral vaccine for the prevention of periodontitis, which is orally administered using the pili or capsule of periodontitis-causing bacteria as an antigen.

Hereinafter, the present invention will be described in more detail.

Constitution of the Invention The oral vaccine of the present invention uses, as described above, the causative bacteria of periodontitis, particularly preferably Bacteroides gingivalis and Actinomyces biscoudis pili or capsule.

Here, bacteria causing periodontitis such as Bacteroides gingivalis and Actinomyces biscoudis are found in For in Boston.
Strains distributed from syth Dental Center, strains isolated from local lesions of periodontitis and the like are used, but particularly preferred strains are Bacteroides gingivalis 381.
Actinomyces biscoudis NY-1.

As a vaccine for oral administration of the present invention, a pili antigen and a bacterial cell extract (capsule) antigen are used. These antigens can be obtained by dividing and separating from the bacterial cells according to a known method. Specifically, the pili antigens can be obtained by stirring the above-mentioned bacteria with glass beads in distilled water. After separating the pili from the bacterial cells by passing through a 25-injection needle, 8000 rpm,
The supernatant obtained by centrifugation for 15 minutes can be obtained by a method such as freeze-drying, and the bacterial cell extract (capsule) antigen can be obtained by adding 60% of the above bacteria in 0.01 M EDTA-phosphate buffer. After reacting at ℃ for 30 minutes, N
o. The capsule can be separated from the cells through a 25-injection needle, and the supernatant obtained by centrifugation at 8,000 rpm for 15 minutes can be further centrifuged at 40,000 rpm for 2 hours to collect the precipitate. It should be noted that these antigens are stored in a freezer (for example, -80 ° C) and, if necessary, thawed before use.

The vaccine of the present invention is used by orally administering the above-mentioned antigen, and as the administration method, in the case of whole cell antigen, 10 ⁴ to 10 ¹⁰ whole cells /
The bacterial solution prepared in 0.1 ml was orally administered continuously in an amount of 0.1 to 10 ml for 3 to 15 days continuously, and in the case of pili antigen and extract antigen, 0.01 to 10 mg / ml of these were prepared. 1-1
A method of orally administering 0 ml each continuously for 3 to 15 days can be adopted.

EFFECTS OF THE INVENTION The oral vaccine for preventing periodontitis of the present invention uses the pilus or capsule of periodontitis-causing bacterium as an antigen, so that the oral administration of the vaccine results in the immunity of the living body, particularly the local immunity mechanism in the oral cavity. Is stimulated, and as a result, antibodies such as IgA and IgM specifically protect against periodontitis-causing bacteria. Therefore, according to the present invention, it is effectively used for the prevention of periodontitis, and since vaccination at a young age imparts long-term immunity, it is effective for preventing adult periodontitis and the like. is there.
In addition, the vaccine of the present invention is orally given, so that it is more safe than injection administration.

Examples will be shown below.

[Reference Example] After culturing Bacteroides gingivalis strain 381 strain in dot hebit broth containing hemin and menadione for 2 days, cells were collected by centrifugation at 8000 rpm for 15 minutes and
An inactivated vaccine was obtained by using a washing solution with a phosphate buffer of mM, pH 7.4 and treating it with 0.5% formalin overnight as whole cell antigen.

[Example 1] Bacteroides gingivalis, which had been cultured for 2 days in the same manner as in Reference Example, was collected and washed, and then gently stirred for 2 days together with glass beads in distilled water to prepare a No. 25 injection needle (0.5 x 25).
(mm) three times, and the pili were separated from the bacterial cells. Then 80
Cells and supernatant pili were separated by centrifugation at 00 rpm for 15 minutes, and the supernatant was dialyzed against distilled water and freeze-dried to obtain pili antigen (pili component vaccine). The yield was 0.0042% based on the wet weight of the cells.

[Example 2] Bacteroides gingivalis that had been cultured for 2 days in the same manner as in Reference Example was collected, washed, and then reacted with a phosphate buffer solution (0.05 M, pH 7.4) containing 0.01 M EDTA for 30 minutes at 60 ° C. No. The capsule was separated from the cells by passing it through a 25-needle 3 times. Then, the supernatant obtained by removing the bacterial cells by centrifugation at 8,000 rpm for 15 minutes was subjected to ultracentrifugation at 40,000 rpm for 2 hours, and the precipitate was used as a capsular antigen (extract vaccine). The yield was 0.09% based on the wet weight of the cells.

The vaccines of Examples 1 and 2 above contain 0.01 mg / ml-1
Used by oral administration of 0 mg / ml. The daily dose for adults is 0.
It is orally administered at 1 to 10 ml continuously for 3 to 15 days.

An oral vaccine of the pili component of Actinomyces biscoudisus is also prepared and administered in the same manner as in the above-mentioned example.

Next, the effects of the present invention will be specifically described with reference to experimental examples.

[Experimental Example 1] Rats (ODU rats) in which periodontitis is common in the anterior mandibular region of the lower jaw were immunized with each antigen derived from Bacteroides gingivalis.

OD of formalin inactivated whole cells of Bacteroides gingivalis (inactivated vaccine) obtained by the method of Reference Example
_As a bacterial solution of ₅₅₀ = 1 to 10 (5 × 10 ^{9 to} 5 × 10 ¹⁰ CFU), it was orally administered to the oral cavity of the ODU rat once per day for 24 consecutive times. In addition, the pili antigen (pili component vaccine) and capsular antigen (extract vaccine) of Bacteroides gingivalis obtained in Examples 1 and 2 were 0.1 mg to 1.0.
It was orally administered as a suspension of mg / ml in the same manner as above. For each antigen-administered group, the antibody titer of IgA in saliva was measured by ELISA 4 weeks after the end of administration, and 6 to 8 animals with high antibody titer /
Groups were subjected to colonization inhibition experiments.

The colonization prevention experiment is a live bacterial solution of Bacteroides gingivalis
(OD ₅₅₀ ≈0.1) was administered to the gingiva of the mandibular anterior gingiva by 0.1 ml (5 × 10 ⁶ CFU), and the colonization rate was examined at 1, 3 and 6 weeks after infection. In addition, a group to which no vaccine was given was used as a control group, and the group was infected.

Further, for comparison, the colonization rate when the above antigens were administered by injection was examined.

The method of injection administration is as follows. After mixing the above-mentioned antigen for oral vaccine with an equal amount of Freund's complete adjuvant or Freund's complete adjuvant to form an emulsion, divide into 6 subcutaneous sites on the back of the rat. Was injected (primary immunization). 7 days after the first immunization,
14 days later, the skin was injected subcutaneously in the back and immunized three times in total. The colonization inhibition experiment was performed in the same manner as in the above oral immunization.

The results are shown in Table 1. Here, the fixing rate of Bacteroides gingivalis was calculated by the following formula.

From the results shown in Table 1, the colonization rate of the experimental group is clearly lower than that of the control group, and the effectiveness of the oral administration of Bacteroides gingivalis vaccine is confirmed.

Also, in this case, oral administration has a lower colonization rate and higher efficacy than injection administration.

[Experimental Example 2] W using each of the antigens derived from Actinomyces biscoudius
Immunized istar rats. All formalin-inactivated whole cells (inactivated vaccine) of Actinomyces viscosus are OD
Rats were orally administered 21 times continuously at a rate of once per day as a bacterial suspension of ₅₅₀ = 1 to 10 (5 x 10 ^{9 to} 5 x 10 ¹⁰ CFU). The pili antigen of Actinomyces biscodis (pili component vaccine) was orally administered in the same manner as above in the form of a suspension of 0.1 mg to 1.0 mg / ml. Four weeks after the completion of the administration, the antibody titer of IgA in saliva was measured by the ELISA method, and individuals with a high antibody titer were subjected to the colonization inhibition experiment.

The colonization prevention experiment is a live bacterium solution of Actinomyces biscoudius
(OD ₅₅₀ ≈0.1) was administered in 0.1 ml (5 × 10 ⁶ CFU) to the gingiva of the upper and lower jaws, and the colonization rate was examined 1 and 3 weeks after infection. In addition, a group to which no vaccine was administered was used as a control group, and the group was infected.

For comparison, the retention rate when injection was administered was examined. The method for injection and administration is as follows. After mixing the above-mentioned antigen for oral vaccine with an equal amount of Freund's complete adjuvant or Freund's incopret adjuvant to form an emulsion, the mixture was injected into 6 subcutaneous sites on the back of the rat. Initial immunization). Seven days after the first immunization, 14 days later, the animals were immunized three times by reinjection under the dorsal skin. The colonization inhibition experiment was performed in the same manner as in the above oral immunization.

The results are shown in Table 2. Here, the fixing rate of Actinomyces biscoudisus was calculated by the following formula.

From the results shown in Table 2, the colonization rate of the experimental group was clearly lower than that of the control group, and the effectiveness of the oral administration of Actinomyces viscous vaccine was confirmed.

Also, in this case, oral immunization (oral administration) was shown to be much more effective than injection administration.

[Experimental Example 3] The antibody titer of IgA in saliva against Bacteroides gingivalis was examined by the following method, and oral immunization and injection (subcutaneous)
The efficacy when immunized was examined. The results are shown in Table 3.

The rats in each group of Experimental Example 1 were anesthetized with Nembutal 4 weeks after the final immunization, and 0.3% pilocarpine hydrochloride solution was intraperitoneally injected at 0.1 ml per 100 g of body weight to collect saliva. The antibody titer of IgA in saliva against Bacteroides gingivalis was measured by ELISA.

From the results in Table 3, it is confirmed that the oral immunization method has a higher IgA antibody titer in saliva, which is more effective than the injection immunization.

[Experimental Example 4] The following method was used to compare the safety between oral immunization and injection (subcutaneous) immunization. The results are shown in Table 4.

The individual body weight of each group of rats in Experimental Example 1 was measured over time. Based on the body weight immediately before the first immunization, increase / decrease in individual body weight one week after the start of immunization was obtained by the following formula, and the average value was calculated for each group.

Increase / decrease in body weight = (body weight one week after the first immunization)-(body weight immediately before the first immunization) As is clear from the results in Table 4, it is suggested that the injected immunization group causes a significant weight loss and thus has a strong harmful effect. Further, among the oral immunization groups, the pili immunization group and the capsular immunization group are highly safe, whereas the whole cell immunization group is inferior in safety.

Claims (3)

[Claims] 1. An oral vaccine for preventing periodontitis, which is orally administered using pili or capsule of periodontitis-causing bacterium as an antigen. 2. The vaccine according to claim 1, wherein the periodontitis-causing bacterium is Bacteroides gingivalis. 3. The vaccine according to claim 1, wherein the periodontitis-causing bacterium is Actinomyces viscosus.