JPS61140527A - Oral vaccin for preventing pheridontosis - Google Patents

Abstract

PURPOSE:The titled vaccin for oral administration that contains, as an antigen, cell bodies of periodontosis-causative bacterium or their cillia or extract. CONSTITUTION:The objective vaccin for preventing periodontosis is obtained by using, as an antigen, the whole cell bodies of periodontosis causative bacteria, preferably bacteriodes gingivalis and Actimonyces viscosus or their cillia or their extract. When it is given orally, the fixation of the bacteria causing periodontosis is prevented to inhibit their proliferation. This prevention is higher in safety than parenteral administration. The dose of the somatic antigen is 0.1-10ml of the vaccin containing 10<4>-10<10>/ml of the whole cell bodies and is given continuously for 3-15 days. Fimbrila antigen and extract antigen are given, respectively, 0.1-10ml of 0.01-10mg/ml concentratin for 3-15 days.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a vaccine that is orally administered and used for the prevention of periodontitis, and more specifically, it relates to a vaccine that is administered orally and is used for the prevention of periodontitis.
s) and Actinomyces viscouzis (Action
The present invention relates to an oral vaccine for the prevention of periodontitis that uses the whole population of periodontitis-causing bacteria such as Cys yces viscosus, its fimbriae, or an extract thereof as an antigen.

The main cause of periodontal disease is plaque in the dental plaque that accumulates in periodontal pockets. In healthy periodontal pockets, Durham-positive bacteria usually make up the majority, but as periodontal disease progresses, bacteria such as Bacteroides gingivalis and Actinomyces viscouzis increase.

In fact, Bacteroides gingivalis and Actinomyces viscoudis are particularly frequently isolated from the lesions of adult patients with severe periodontal disease, and there are many cases in which the level of antibodies against these bacteria is increased in the patient's serum.

Furthermore, it has been shown that inoculating animals with Bacteroides gingivalis exacerbates periodontal inflammation. These results indicate that Bacteroides gingivalis and Achnomyces viscolagis play an important role in the establishment of periodontal disease.

Bacteroides gingivalis and Actinomyces viskouzis have fimbriae-like bacterial surface substances on their bacterial surfaces, which are said to cause them to adhere to the periodontal mucosa, multiply, and have a negative impact on periodontal health. There is. In order to prevent periodontitis caused by such causes, it is effective to prevent these periodontitis-causing bacteria from colonizing the oral cavity or to suppress their proliferation.

Conventionally, as a method to prevent colonization and suppress the growth of periodontitis-causing bacteria, a method has been proposed in which a vaccine containing the fimbriae of periodontitis-causing bacteria or an extract thereof as an antigen is administered ( JP-A-59-128338). However, this method uses a vaccine that is directly injected into a living body, which poses safety problems due to toxicity within the body.

In view of the above circumstances, the present inventors have conducted intensive studies on a method that is safer and can effectively prevent periodontitis. When administered orally, a vaccine containing whole cells of periodontitis-causing bacteria or their fimbriae or extracts thereof as an antigen is highly effective in preventing colonization and suppressing proliferation of periodontitis-causing bacteria; It was found that periodontitis can be effectively prevented, and that this method, which orally administers antigens derived from intraoral su@, is safer than internal administration (injection). However, the present invention has been completed.

Accordingly, the present invention provides an orally administered light vaccine for preventing periodontitis, which uses the whole body of periodontitis-causing bacteria, their fimbriae, or their extracts as antigens.

The present invention will be explained in more detail below.

, 01L As mentioned above, the light vaccine of the present invention uses as an antigen the whole cells of periodontitis-causing bacteria, particularly preferably Bacteroides gingivalis and Actinomyces viscouzis, their fimbriae, or their extracts. be.

Here, periodontitis-causing bacteria such as Bacteroides gingivalis and Actinomyces viscouzis are found in the Boston F.
Bacterial strains distributed from Orsyth [) Dental Center, strains isolated from the focal area of periodontitis, etc. are used, but a particularly preferred bacterial cell is Bacteroides gingivalis 381. It is Actinomyces viscouzis NY-1.

The vaccine for oral administration of the present invention is prepared as an inactivated vaccine obtained by inactivating these bacteria or a bacterial component vaccine obtained by extracting these bacteria. In this case, the inactivated vaccine is prepared by culturing the above-mentioned bacteria in a todhepitprose medium supplemented with, for example, hemin and menadione, washing the grown bacteria, and then
% formalin solution overnight and washing thoroughly, whole bacterial antigens are used. In addition, as a one-component vaccine, fimbriae antigens and national extract (capsule) antigens are used. These antigens can be obtained by dividing and separating Kokutai according to known methods. Specifically, fimbrial antigens can be obtained by stirring the above $1 bacteria in hot water with glass peas, No. After separating the fimbriae from the bacterial cells through a 25-syringe needle, the supernatant obtained by centrifugation at 8,000 rpm for 15 minutes can be obtained by freeze-drying. No. After reacting the above sii in O, OIM-EDTA-phosphate buffer at 60°C for 30 minutes.

The capsule was separated from the national body by passing it through a 25-syringe needle, and 8000 r
pm, 'The supernatant obtained by centrifugation for 15 minutes was further
It can be obtained by a method such as centrifuging at 0000 rpm for 2 hours and collecting the precipitate. Note that these antigens are stored in a freezer (for example, at -80°C) and used after thawing, if necessary.

The vaccine of the present invention is used by orally administering the above-mentioned antigen, and in the case of a national antigen, the total bacterial cells are 104 to 10 cells/-.
0.1 to 101 of the bacterial solution prepared in A! In the case of fimbriae antigens and extract antigens, these were prepared at 0.01 to 10+g/lf and administered lightly for 3 to 15 days.
~10ν! A method of continuous oral administration for 3 to 15 days at a time may be adopted.

The oral vaccine for preventing periodontitis of the present invention uses whole cells of periodontitis-causing bacteria, their fimbriae, or extracts as antigens, so that oral administration improves the immune system of the living body, especially in the oral cavity. stimulates the local immune system in the IoA, Ia
Antibodies such as M provide specific protection against periodontitis-causing bacteria.

Therefore, according to the present invention, vaccination can be effectively used to prevent periodontitis, and in particular, vaccination at young ages confers long-term immunity, so it is effective in preventing adult periodontitis. be.

Furthermore, since the vaccine of the present invention is administered orally, it is safer than injection administration.

Examples are shown below.

[Example 1] Bacteroides gingivalis strain 381 was cultured for 2 days in Todohepitprose supplemented with hemin and menadione, and then the bacterial cells were collected by centrifugation at 8,000 rpm for 15 minutes. After washing with No. 4 phosphate buffer, the mixture was treated with 0.5% formalin overnight and used as a national antigen to obtain an inactivated vaccine.

[Example 2] Bacteroides was cultured for 2 days in the same manner as in Example 1.
After collecting bacteria from Gingivalis and washing, they were gently stirred with glass beads in hot water for 2 days, and then used with a No. 25 injection needle (
0.5×25wns) three times to separate pili from the bacterial cells. Next, the bacterial cells and pili in the upper groove were separated by centrifugation at 8,000 rpm for 15 minutes, and the supernatant was dialyzed with hot water and freeze-dried to obtain a pili antigen (11! hair component vaccine). The yield is bacterial cell moisture I! It was 0.0042% based on the amount.

[Example 3] Bacteroides was cultured for 2 days in the same manner as in Example 1.
After collecting and washing the gingivalis, use a phosphate buffer (0.05M) containing 0.01M-EDTA.

After reacting at 60°C for 30 minutes at pH 7,4),
The capsule was separated from the bacterial cells by passing it through a No. 25 injection needle three times. Next, the supernatant from which bacterial cells were removed by centrifugation at 8,000 rpm for 15 minutes was ultracentrifuged 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 bacterial cells.

The vaccine of Example 1 has a total of 106 71! ~
Orally administered as a bacterial solution prepared at 1012 cells/xl,
In addition, the vaccine of Example 2.3 has 0.01 m/
It is used by orally administering the product prepared at lf to 1011 g/if. The dosage for each adult is 0.1 to 101 per day! It is administered orally continuously for 3 to 15 days.

Oral vaccines for Actinomyces viscouzis and fimbriae components are also prepared and administered in the same manner as in the above example.

Next, experimental examples will be shown to specifically explain the effects of the present invention.

[Experimental Example 1] Using each antigen derived from Bacteroides gingivalis, rats with periodontitis frequently occurring in the mandibular anterior gum region (ODU rats)
immunized.

The formalin-inactivated whole cells of Bacteroides gingivalis (inactivated vaccine) obtained by the method of Example 1 were
-i~10 (5x109~5x1010CFLI) 17) The rODU was orally administered into the uterine cavity once/day for 24 consecutive times as a li solution. In addition, Bacteroides obtained in Examples 2 and 3
gingivalis pili antigen (fimbrial component vaccine) and capsular antigen (extract vaccine) are 0.1 mg to 1.0 m/
It was orally administered as a suspension of lf in the same manner as above. For each antigen administration group, the 1aA antibody titer in the saliva was measured by ELISA four times after the end of administration, and 6 to 8 individuals/group with high antibody titers were subjected to a colonization inhibition experiment.

In the colonization prevention experiment, live bacterial solution of Bacteroides gingivalis (OD O.'t0.1) was mixed with Q, 1fff (5X1011
CFU) was administered to the anterior gingival region of the mandible, and the colonization rate was examined on days 1, 3, and 6 after infection. As a control group, a group to which no vaccine was administered was used and infected.

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

Bacteroides colonization rate=g゛"<l The effectiveness is recognized.

[Experimental Example 2] Using each antigen derived from Actinomyces viscouzis,
1 star rats were immunized. Formalin-inactivated whole cells of Actinomyces viscodis (inactivated vaccine) were prepared on an OD evening. -1~10 (5X109~5X10
CFU) was orally administered to rats as a bacterial suspension once per day for 21 consecutive times. In addition, the fimbrial antigen (fimbrial component vaccine) of Actinomyces viscouzis is O,
"It was orally administered as a suspension of 1m to 1.0119/fir in the same manner as above. After the end of administration, I
The antibody titer of oA was measured by ELISA, and individuals with high antibody titers were subjected to a colonization inhibition experiment.

The colonization inhibition experiment was performed using the native liquid of Actinomyces viscouzis. L: rO, 1) to 0.1tf (5x1
06CFLI) was administered to the molar region gingiva of the upper and lower jaws, and the colonization rate was examined 1.3 weeks after infection. In addition,
As a control group, a group to which no vaccine was administered was used and infected.

The results are shown in Table 2. Here, the colonization rate of Actinomyces biscouzis was calculated from the following formula.

Actinomyces colonization rate - 1 (11 Z people - x 100 ± SD% total anaerobic bacteria count Table 2) From the results in Table 2, it is clear that the experimental group was more colonized than the control group. The oral administration of Actinomyces viscodis vaccine is effective.

Claims (1)

[Scope of Claims] 1. An oral vaccine for preventing periodontitis, which uses whole cells of periodontitis-causing bacteria, their fimbriae, or an extract thereof as an antigen and is orally administered. 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 viscouzis.