【発明の詳細な説明】[Detailed description of the invention]
本発明は精製柿タンニンの添加処理を行なつた
百日せきワクチンを過酸化水素処理することを特
徴とする百日せき改良ワクチンの製法に関するも
のである。
従来、百日せきの予防に対するワクチンの有効
性は認められているが、現行の死菌ワクチンには
かなり強い副作用があり、この副作用に関与する
毒性成分を除去あるいは不活化する改良ワクチン
を開発する必要性が強調され、その研究もさかん
に行なわれている。
本発明者等は、さきに市販用の百日せきワクチ
ンに精製柿タンニン（またはその精製によつて得
られる単一化合物である縮合タンニン）を添加す
ることによつて、ワクチン中に残存する副作用物
質の一部と考えられているリンパ球増多症促進因
子（LPF）あるいはヒスタミン増感作用因子
（HSF）が不活化あるいは減毒化され、しかも目
的とする免疫原性を損わないことを報告した。
〔特開昭53―59023号、特開昭53―109912号および
三共研究所年報30、104〜111（1978）〕
しかしながら、上記の精製柿タンニン（または
その精製単一化による縮合タンニン）で処理した
百日せきワクチンには淡褐色の着色が残存すると
云う問題があつた。本発明者等は、この課題を解
決すべく鋭意研究を進め、過酸化水素を脱色剤と
して選んで処理したところ、得られたワクチンは
白色微細な安定化菌体浮遊液となり、前記の精製
柿タンニンの添加によるワクチン中の副作用物質
の減毒と云う長所を維持し、しかも免疫原性を損
わないことを確認して、本発明を完成するに到つ
た。
本発明の方法を実施するにあたつて、精製柿タ
ンニンで添加処理された百日せきワクチンは、百
日せき死菌体の生理食塩水浮遊液と精製柿タンニ
ンあるいは更にその精製によつて得られた縮合タ
ンニンの水溶液とを常温で混合して遠心分離する
ことによつて、淡褐色の沈澱菌体として採取され
る。（特開昭53―59023号および特開昭53―109912
号参照）
このようにして得られた精製柿タンニン処理に
よる着色菌体を使用して、この菌体を生理食塩水
またはPH７付近のリン酸緩衝液中に浮遊した液と
過酸化水素水とを混合し、放置することによつ
て、本発明の脱色安定化をはかることができる。
使用する過酸化水素水の濃度は３乃至10％が好適
である。その処理温度は好ましくは０乃至10℃で
ある。その処理時間は菌の濃度および過酸化水素
水の濃度に関与するが、通常は１乃至数日間であ
り、好適には24乃至48時間である。
上記の処理の結果、脱色を確認した後、菌体浮
遊液を遠心沈澱して菌体を採取し、常法に従つて
生理食遠水で１乃至数回遠心洗浄して過酸化水素
を除去することによつて、目的の百日せき改良ワ
クチンを得ることができる。
ここで白色微細な安定化した浮遊液として得ら
れた百日せきワクチンは、その色調にいて市販ワ
クチンとの差が認められないものであり、免疫原
性を保持し、なおかつ上述した副作用物質である
LPFあるいはHSFの活性を示さず、しかも動物
の足蹠注射による刺戟性においては現行ワクチ
ン、精製柿タンニン処理ワクチンに比べて遥かに
低いと云う優れた性質を有することを確認するこ
とができた。
以下に実施例をあげて、本願発明の百日せき改
良ワクチンの製法並びに得られたワクチンの効力
（抗原性）および局所刺戟性に関する比較試験に
ついて更に詳しく説明する。
実施例 １
縮合タンニン処理百日せきワクチンの過酸化水
素処理
ボルデ・ジヤング培地に培養した百日せき菌東
浜Ｉ相菌の死菌ワクチンを作り、生理食塩水に浮
遊後、常温で10分0.0625％縮合ンニン水溶液と等
量混合し再び遠心により沈澱菌体を採取し、この
着色菌体の生理食塩水浮遊液を10％の過酸化水素
水と等量混合後、４℃に48時間放置、脱色を確認
後、遠心沈澱（10000g、10分）で菌体を採取
し、これを生理食塩水にて２回遠心洗浄を行ない
過酸化水素を除去し、目的の改良ワクチンを得
た。
この改良ワクチンは白色微細な安定化した浮遊
液であり、市販のワクチンと肉眼的に差が認めら
れないものであり、更に下記の色差試験法によつ
ても、無処理ワクチンとの色差は認められなかつ
た。
すなわち、無処理百日せきワクチンと縮合タン
ニン処理ワクチン並びに本願の過酸化水素処理ワ
クチンとの間のLab系による色差〔△Ｅ(Lab)〕
を、JISZ8730「色差表示方法」に従つて１cmの
液層で透過法により求めた結果を表１に示す。
なお、検体ワクチンは20ビリオン／mlの浮遊液
を使用し、色差計はスガ試験機(株)製AUD―CH―
１型を使用した。
The present invention relates to a method for producing an improved pertussis vaccine, which comprises treating a pertussis vaccine that has been treated with purified persimmon tannin with hydrogen peroxide. The effectiveness of vaccines in preventing pertussis has been recognized in the past, but the current killed vaccines have quite strong side effects, and we need to develop an improved vaccine that removes or inactivates the toxic components that are involved in these side effects. The need for this has been emphasized, and research on it is being actively conducted. The present inventors first added purified persimmon tannin (or condensed tannin, a single compound obtained by its purification) to a commercially available pertussis vaccine, thereby eliminating the side effects remaining in the vaccine. It has been reported that lymphocytosis-promoting factor (LPF) or histamine-sensitizing factor (HSF), which are thought to be part of the substance, is inactivated or attenuated and does not impair the desired immunogenicity. did.
[JP-A-53-59023, JP-A-53-109912, and Sankyo Research Institute Annual Report 30, 104-111 (1978)] However, when treated with the above purified persimmon tannin (or condensed tannin by its purification and unification) There was a problem with the pertussis vaccine that left a light brown coloration. The present inventors conducted intensive research to solve this problem, and when treated with hydrogen peroxide as a decolorizing agent, the obtained vaccine became a white fine stabilized bacterial cell suspension, and the purified persimmon The present invention was completed after confirming that the addition of tannin maintains the advantage of reducing the toxicity of side effects in vaccines and does not impair immunogenicity. In carrying out the method of the present invention, a pertussis vaccine supplemented with purified persimmon tannin is prepared by combining a suspension of killed pertussis bacteria in physiological saline and purified persimmon tannin or by further purifying the same. By mixing the resulting aqueous solution of condensed tannin at room temperature and centrifuging the mixture, a light brown precipitated bacterial body is collected. (JP-A-53-59023 and JP-A-53-109912
(Refer to No.) Using the thus obtained colored microbial cells treated with purified persimmon tannin, the microbial cells were suspended in physiological saline or a phosphate buffer solution with a pH of around 7, and hydrogen peroxide solution was added to the solution. By mixing and standing, the decolorization of the present invention can be stabilized.
The concentration of the hydrogen peroxide solution used is preferably 3 to 10%. The treatment temperature is preferably 0 to 10°C. The treatment time depends on the concentration of bacteria and the concentration of hydrogen peroxide solution, but is usually one to several days, preferably 24 to 48 hours. After confirming decolorization as a result of the above treatment, the bacterial suspension is centrifuged to collect the bacterial cells, and the hydrogen peroxide is removed by centrifugal washing once or several times with physiological saline using conventional methods. By doing so, the desired improved pertussis vaccine can be obtained. The pertussis vaccine obtained here as a white, fine, stabilized suspension shows no difference in color from commercially available vaccines, retains immunogenicity, and is free from the above-mentioned side effects. be
It was confirmed that the vaccine does not exhibit LPF or HSF activity, and has excellent properties such that the irritation caused by injection into the footpad of animals is far lower than that of the current vaccine and the purified persimmon tannin-treated vaccine. Examples are given below to explain in more detail the method for producing the improved pertussis vaccine of the present invention and comparative tests regarding the efficacy (antigenicity) and local irritation of the obtained vaccine. Example 1 Hydrogen peroxide treatment of condensed tannin-treated pertussis vaccine A killed vaccine of B. pertussis Higashihama I phase bacteria cultured in Bordet-Jeyang medium was prepared, suspended in physiological saline, and incubated at 0.0625% at room temperature for 10 minutes. Mix an equal amount with the condensed nin aqueous solution and centrifuge again to collect the precipitated bacterial cells. Mix the colored bacterial suspension in physiological saline with an equal amount of 10% hydrogen peroxide solution and leave at 4°C for 48 hours to decolorize. After confirming this, the bacterial cells were collected by centrifugal sedimentation (10,000 g, 10 minutes) and centrifugally washed twice with physiological saline to remove hydrogen peroxide, thereby obtaining the desired improved vaccine. This improved vaccine is a white, fine, stabilized suspension, and there is no visible difference between it and the commercially available vaccine, and there is no difference in color between it and the untreated vaccine using the color difference test method described below. I couldn't help it. That is, the color difference between the untreated pertussis vaccine, the condensed tannin-treated vaccine, and the hydrogen peroxide-treated vaccine of the present application due to the Lab system [△E (Lab) ]
Table 1 shows the results obtained using a transmission method using a 1 cm liquid layer according to JIS Z8730 "Color Difference Display Method". The sample vaccine used was a suspension of 20 billion/ml, and the color difference meter was AUD-CH- manufactured by Suga Test Instruments Co., Ltd.
Type 1 was used.
【表】
実施例 ２
効力（抗原性）に関する検討
0.0625％の縮合タンニン水溶液と百日せき死菌
液を等量混合し常温で10分放置後、生理食塩水に
て菌体を遠心洗浄したのち、過酸化水素で処理を
行なつた。菌液は20ビリオン／mlに作製し、この
２倍および10倍希釈菌液をマウスの腹腔内に0.5
ml１回注射し10日後に200LD50（2.5×104／
mouse）の百日せき菌東浜相菌を１群12匹のマ
ウス脳内に攻撃し、14日間にわたり生死を観察し
た。
結果は本ワクチンの２倍希釈液では12匹中９匹
生残し、10倍希釈液では５匹生残の成績を得て、
縮合タンニン処理ワクチン並びに無処理ワクチン
と同程度の致死防禦効果が認められた。
実施例 ３
LPF作用に関する検討
検体は以下に示すようにして調製した。
検体１＝0.0625％の縮合タンニン水溶液と百日
せき菌液を等量混合し常温で10分放置後、生理食
塩水にて菌体を遠心洗浄。
検体２＝検体１を実施例１と同様に過酸化水素
で処理。
検体３＝無処理百日せき菌液。
菌液は20ビリオン／mlのものを使用した。検体
をマウスの腹腔内に0.5mlずつ注射し72時間後に
マウスの静脈より採血を行ない、白血球数を算出
した。成績は表２に示す。[Table] Example 2 Study on efficacy (antigenicity) Equal amounts of 0.0625% condensed tannin aqueous solution and killed pertussis solution were mixed, left at room temperature for 10 minutes, and the bacterial cells were centrifugally washed with physiological saline. , treated with hydrogen peroxide. The bacterial solution was prepared at 20 virions/ml, and the 2-fold and 10-fold diluted bacterial solution was intraperitoneally injected into mice at 0.5 virions/ml.
ml once and 10 days later 200LD 50 (2.5×10 4 /
A group of 12 mice were challenged with Bordetella pertussis Higashihama bacteria (mouse) into the brains of 12 mice, and their survival and death were observed for 14 days. As a result, 9 out of 12 mice survived with the 2-fold dilution of this vaccine, and 5 survived with the 10-fold dilution.
The same level of lethal protection effect as the condensed tannin-treated vaccine and the untreated vaccine was observed. Example 3 Study on LPF action Specimens were prepared as shown below. Specimen 1 = Mix equal amounts of 0.0625% condensed tannin aqueous solution and Bordetella pertussis solution, leave at room temperature for 10 minutes, and centrifugally wash the bacterial cells with physiological saline. Specimen 2 = Specimen 1 treated with hydrogen peroxide in the same manner as in Example 1. Specimen 3 = untreated pertussis liquid. The bacterial solution used was 20 billion/ml. 0.5 ml of the sample was injected intraperitoneally into mice, and 72 hours later, blood was collected from the veins of the mice and the number of white blood cells was calculated. The results are shown in Table 2.
【表】
表２に示すごとく縮合タンニン処理後過酸化水
素処理ワクチン（検体２）注射群は最も白血球増
加が少く対照の正常マウス群と殆んど差を認めら
れない程度であつた。
実施例 ４
HSF作用に関する検討
１群８匹のマウスを使用し、実施例３と同一の
検体を腹腔内に0.5mlずつ注射し、対照群には生
理食塩水を注射し、注射４日後に腹腔内にヒスタ
ミン二塩酸塩を4.0mg／マウス注射を行ない生死
を観察した。その結果、縮合タンニン処理、縮合
タンニン処理後、過酸化水素処理および生理食塩
水注射対照群は何れも全例生残したが、無処理ワ
クチン注射群は８例中５例の死亡が認められた。
以上の成績より縮合タンニン処理、ならびに縮
合タンニン処理後過酸化水素処理ワクチンにはヒ
スタミン感受性増強作用は認められなかつた。
実施例 ５
刺戟性低下に関する検討
市販百日せきワクチン（千葉血清、Lot1.）を
用い、実施例１と同様な方法で処理した。菌数は
16×109／mlに調整しマウスの１側の足蹠内に
0.05ml注射し、１、２、３および７日後に両後肢
を距骨関節部分より切断し重量測定を行なつて、
腫脹率＝接種足重量／健足重量×100
（１群５匹の平均）
を求めた。成績は第図に示す。
第１図に示すごとく刺戟性は縮合タンニン処理
後、過酸化水素処理（検体２）が最も軽く、次い
で縮合タンニン処理（検体１）、無処理（検体
３）の順であつた。本成績に示すごとく過酸化水
素処理はワクチンの注射により起る局所の刺戟を
著しく軽減し得るものである。[Table] As shown in Table 2, the group injected with the condensed tannin-treated and hydrogen peroxide-treated vaccine (specimen 2) had the least increase in white blood cells, with almost no difference observed from the control normal mouse group. Example 4 Study on HSF effect Using 8 mice per group, 0.5 ml of the same sample as in Example 3 was injected intraperitoneally, and the control group was injected with physiological saline, and 4 days after injection, intraperitoneally injected the same sample as in Example 3. 4.0 mg/mouse of histamine dihydrochloride was injected into the mice, and their survival was observed. As a result, all cases in the condensed tannin treatment, post-condensation tannin treatment, hydrogen peroxide treatment, and physiological saline injection control groups survived, but in the untreated vaccine injection group, 5 out of 8 cases died. . From the above results, no histamine sensitization effect was observed in the condensed tannin-treated vaccine and in the condensed tannin-treated and hydrogen peroxide-treated vaccine. Example 5 Study on reduction in irritability A commercially available pertussis vaccine (Chiba Serum, Lot 1.) was used and treated in the same manner as in Example 1. The number of bacteria is
Adjust to 16×10 9 /ml and place it in the footpad on one side of the mouse.
Inject 0.05ml, and after 1, 2, 3, and 7 days, both hind limbs were cut off at the talar joint and their weight was measured. Swelling rate = weight of inoculated paw / weight of healthy paw x 100 (average of 5 animals in 1 group) I asked for The results are shown in the figure. As shown in FIG. 1, the irritation was the lowest after condensed tannin treatment, followed by hydrogen peroxide treatment (Sample 2), followed by condensed tannin treatment (Sample 1), and no treatment (Sample 3). As shown in the present results, hydrogen peroxide treatment can significantly reduce local irritation caused by vaccine injection.
【図面の簡単な説明】[Brief explanation of the drawing]
第１図は実施例５で得られたマウス後肢に対す
る縮合タンニン処理（検体１）、縮合タンニン処
理後過酸化水素処理（検体２）並びに無処理（検
体３）の各百日せきワクチンの局所刺戟性の成績
を示したものである。
Figure 1 shows the local stimulation of the pertussis vaccine on the hind limbs of mice obtained in Example 5, treated with condensed tannin (sample 1), treated with hydrogen peroxide after condensed tannin treatment (sample 2), and untreated (sample 3). It shows the sexual performance.