@misc{oai:az.repo.nii.ac.jp:00003209,
 author = {坂本, 國昭 and Sakamoto, Kuniaki},
 month = {2013-02-19, 2014-08-18, 2013-02-19},
 note = {Inactivated rabies vaccines for humans previously used in Japan include Semple vaccine prepared from an emulsion of rabbit or goat brain infected with Nishigahara strain of fixed rabies virus inactivated with phenol, which was used from 1953-1973, and rabies vaccine inactivated by ultraviolet rays which was in use during the period 1960-1973.
 In 1973, Fuenzalida vaccine was developed. It was prepared from infected suckling mouse brain free of myelin, which was culpable for adverse reactions of rabies vaccine, by treating with phenol or ultraviolet rays. Known as "Inactivated rabies vaccine", it is presently listed among the minimum requirement of biological products.
 The existing rabies vaccine, however, has the potential of producing adverse reactions, since it is also made from nervous tissue and the manufacturing process involves no particular step of purification or elimination of brain substance except for low speed centrifugation.
 Therefore, prophylactic pre-exposure application of the vaccine is limited at present to special high risk situations (laboratory staffs, physicians, veterinarians, quarantine station personnel, dog catchers, etc.).
 No cases of rabies have been reported in Japan since 1957 because of enforcement of rabies prophylactic law in 1950 except for such patients as became infected abroad and suffered the disease after returning to Japan. In recent years the number of people who have been in hyperendemic areas (South East Asia, Middle and Near East, Africa, South America, etc.) abroad where occasions of rabies infection have been increasing, have increased.
 Under such circumstances, since 1974 I have made efforts to establish a dried inactivated tissue culture rabies vaccine for prophylactic use based on the techniques developed by A.Kondo who was a technical officer at the National Institute of Health. As a result, dried inactivated tissue culture rabies vaccine for human use derived from chick embryo cell culture (hereafter referred to as TC vaccine) was successfully developed as a commercial product, which satisfactorily met the current requirement for inactivated rabies vaccine (hereafter referred to as SMB vaccine) on efficacy and extremely reduced the adverse reactions.
 The details of studies are presented here along with the results obtained.

1. Basic studies on preparation of TC vaccine
 1) There was no difference in preparing CE cells suitable for large scale culture among the Dulbecco's method (pipetting at room temperature for 2 min), Bodian's method (trypsinization with stirrer at cold room for 60 min) and Youngner's method (trypsinization with stirrer at room temperature 2 times for 20 min each).
 2) HEP Flury strain of rabies virus grown in massive culture at 35 ℃ and PH7.8-8.0 had an infectious titer of 10^7.0 - 10^7.5 LD50/ml with an HA titer of 1 : 32-128 at 6 days after inoculation. On the basis of these results the 6th-day virus culture fluid was harvested.
 3) Complete inactivation of virus was achieved by adding of 0.02 v/v % BPL to the virus culture fluid at 37℃ for 45 min. For caution's sake, the same procedure was repeated.
 4) Filtration procedure could be used for avoidance of microbe contamination in vaccine production using G company's membrane filter without virus adsorption.
 5) The mean protein nitrogen content of the TC vaccines prepared by ultrafiltration and ultracentrifugation was 0.021mg/ml, or approximately 1/2 in comparisone to the SMB vaccine. The mean protein nitrogen content of the TC vaccines prepared by sucrose density gradient centrifugation was 0.007mg/ml, or approximately 1/3 in comparison to the TC vaccine prepared by ultrafiltration and ultracentrifugation. Since this method has a possibility of microbe contamination, the ultrafiltraton - ultracentrifugation method was rather used for concentration and purification of the virus. The variance of protein nitrogen contents was found among the TC vaccine lots, but it was not abnormal value as a criterion for external value.
 6) The potency of the TC vaccine was ranged between 10^4.64 -10^5.75 as a Habel index and between 1.0-13.2 as an Antigenic value, respectively. The TC vaccine fullfilled the current standard. There was no correlation between potency and protein nitrogen contents(r=-0.036).
 7) When the TC vaccine was stored in a thermostatic chamber for 36 months at 4 ℃, no changes were observed in its appearance, moisture content, hydrogen ion concentration and potency, and neither abnormal toxicity nor contamination was seen. When the TC vaccine was stored for 6 months at 37℃, there was a slight decrease in potency. However, it corresponded to the SMB vaccine potency test standard even after 36 months of storage. There were no changes regarding items (appearance, pH, abnormal toxity) other than potency. The stability of the TC vaccine to the external temperature was confirmed.
2. Safety and antigenicity of TC vaccine
 1) A comparative acute toxicity test of the TC vaccine was carried out using mice and rats together with the SMB vaccine as control. The TC vaccine, even when administered in the highest possible dose for mice and rats, proved not to be lethal, so its LD50/ml was indeterminable. The maximum dose administered was 120ml/kg intravenously, orally or subcutaneously in mice, and 95ml/kg intravenously and 120ml/kg orally or subcutaneously in rats. The vaccine given in these doses produced no reactions indicative of toxity. The SMB vaccine as control was administered in the highest possible dose when given by the oral or subcutaneous routes. No deaths occurred among the animals so treated, and hence the LD50 of the vaccine was also not determinable. The SMB vaccine, however, was lethal for some of the mice when administered intravenously in a dose of 20ml/kg or more, its LD50 being 30ml/kg in males and 29ml/kg in females. The vaccine also proved to be lethal for some of the rats when given by the same route in a dose of 16ml/kg or more, the LD50 being 23.3ml/kg in males and 21.5ml/kg in females. In this regard there was no significant difference between sexes in either species. These results strongly suggest that the TC vaccine can be safely used in humans, having no serious acute toxicity.
 2) Systemic anaphylaxis tests were carried out in order to check the allergenic property of the TC vaccine. Tests were negative for all guinea pigs given the TC vaccine. The guinea pigs given the SMB vaccine as control, all displayed negative results after a subcutaneous eliciting injection, but 3 of 4 guinea pigs demonstrated slight anaphylactic symptoms after an intravenous eliciting injection. These results revealed the TC vaccine to be less allergenic than the SMB vaccine.
 3) Eight monkeys were given 10 successive subcutaneous injections of 1ml of the TC vaccine at a site of breast. Three-month observation of local and systemic reactions revealed no abnormalities. At the same time, the neutralizing antibody in the blood was titrated by means of the mouse neutralization test. One (12.5%) of the 8 monkeys demonstrated a neutralizing antibody titer of 1:10 on the 5th day. On the 10th day, the seroconversion was noted in all the monkeys, and the average level among all the monkeys was a titer of 1:33. The level increased remarkably and continued with high level of titer, and a titer of 1:1,770 on day 35. Based on these results, it was concluded that the TC vaccine had high productivity of neutralizing antibody and safety in monkeys.
3. Safety and productivity of neutralizing antibody of TC vaccine in human
 One ml of the TC vaccine was injected into 35 adult volunteers (14 males and 21 females) twice at the interval of 2 weeks, 3 weeks or 4 weeks. To 11 of them the 3rd dose of 1ml was given 6 months after the 2nd injection, and 6 of them were given 1 year and 4 months after the 2nd injection, and a booster (1ml/dose) was given at proper interval respectively to observe local and systemic reactions. Changes of neutralizing antibody titer in the blood were also studied in these 35 subjects. Flare(9%:3/35), swelling(6%:2/35), pain(23%:8/35) and itchiness(6%:2/35) at the 1st injection, flare(31%:11/35), swelling(3%:1/35), pain(20%:7/35) and itchiness(17%:6/35) at the 2nd injection and flare(7%:1/15), swelling(13%:2/15), pain(7%:1/15) and itchiness(7%:1/15) at the 3rd injection were observed at the injection site, respectively. However, all reactions were disappeared within 24-72hrs. Headache in 3% (1/35) at the 2nd injection and in 7% (1/15) at the 3rd injection were observed, but these reactions were also disappeared within 24hrs. However, neither other local or systemic reactions nor an increase of reaction due to given booster were seen. Besides, 2,447 of Japan Overseas Cooperation volunteers receiving pre-exposure immunization revealed flare in 1.1%(27/2,447) and pain in 0.3%(8/2,447) at the injection site slightly, and only a slight fever(around 37℃) in 0.2%(6/2,447) was observed as a systemic reaction. Two weeks after the 2nd injection, the neutralizing antibody titer reached a peek (mean titer=1:27・seroconversion=90%) and thereafter it was maintained for 6 months. The neutralizing antibody titer rose rapidly after the 3rd injection, that is, the titer reached a peek (mean titer=1:125・seroconversion=100 %) 1 week after the 3rd injection, and thereafter it was maintained for 6 months. When 1ml of booster injection was carried out while the neutralizing antibody was disappeared , the antibody titer again elevated rapidly 1 week after the injection, and reached a peek (mean titer=1:98・seroconversion=100%) 2 weeks after the injection. Thus, the booster injection was determined to be effective enough. As for productivity of neutralizing antibody with different intervals between 1st and 2nd injections at the pre-exposure immunization, 4-week interval was superior than 2-week or 3-week interval. From this results, the 4-week interval was decided to be the best for pre-exposure immunization.
 Of seventeen persons suspected of exposing to rabies in 8 countries, four were exposed to rabid dogs, 8 to suspect dogs, 2 to suspect monkeys and 3 to unknown dogs. They were given post-exposure treatment with the TC vaccine. None revealed any sign of rabies infection. Thus, the effectiveness of post-exposure treatment with the TC vaccine was proved.

 The TC vaccine newly developed with a chick embryo cell culture had less protein content (0.021mg/ml), no preservative (formalin or thimerosal), no serious acute toxicity in mice and rats, less allergenic property in guinea pigs, excellent induction of neutralizing antibodies in monkeys and humans and no severe local and systemic reactions in humans compared with the SMB vaccine derived from mouse cerebral tissue. These results strongly suggests that the TC vaccine can be used safely and effectively for prophylaxis to rabies infection in human. Besides, it is considered that the TC vaccine is sufficiently stable to withstand abnormal external temperature because of its freeze-dried form; therefore, it can be used even in tropical areas where rabies is likely to occur.
 The WHO recommends about the dosage and administration of rabies vaccine, that is, as the first 2 doses 1ml each should be given subcutaneously at interval of 1 month, followed by the 3rd dose of 1ml 6 months after the 2nd injection for pre-exposure prophylaxis, and 6 doses of 1ml each subcutaneously on days 0, 3, 7, 14, 30 and 90 for post-exposure treatment. It was concluded that the TC vaccine satisfactorily can meet the dosage and administration of the rabies vaccine recommended by the WHO judging from the results of potency test and the neutralizing antibody response.
 Here, a new type rabies vaccine (Tissue culture) which had no severe side effects and had high effectiveness for rabies prophylaxis was developed in the long history of the rabies vaccine development which had begun at Pasteur's vaccine derived from dried attenuated rabbit spinal cord at 1885., わが国における人体用不活化狂犬病ワクチンは，1953～1973年は狂犬病固定毒西ケ原株感染ウサギまたはヤギ脳乳剤に，石炭酸を加えて不活化したSemple型ワクチンが用いられ，また，1960～1973年は紫外線で不活化したSemple型ワクチンも併用されてきた。
　1973年以降は，副反応(注射後麻痺)の原因物質とされている，脳由来のミエリンを含まない感染哺乳マウス脳を，石炭酸あるいは紫外線で不活化したFuenzalida型のワクチンが開発され，不活化狂犬病ワクチンとして生物学的製剤基準で規定されて，1980年まで使用されてきた。
　しかし，このワクチンも脳組織を原材料としており，低速遠心で脳物質を除去するほかは，脳物質を特に除去することなく製品化しているので，依然として副反応の可能性が残されている。それ故，ウイルス暴露前に予防的に使用するのは，狂犬病ウイルス感染の危険度の高いヒト(研究室，検査室の職員，医師，獣医師，検疫所職員，野犬捕獲員など)の一部にかぎられ，大量に使用されている諸外国では副反応を認めている。
　わが国では，1950年(昭和25年)に狂犬病予防法が施行され，イヌへの予防が功を奏し1957年以来発生は見られていないが，最近，海外で狂犬病罹患動物の咬傷をうけて帰国する人も見られるようになり，また，特に東南アジア，中近東，アフリカおよび南アメリカなどの狂犬病常在地に向け出国するヒトも増加し，罹患する機会が多くなってきている。
　このような背景のもとに，予防的に使用可能で副反応の少ない組織培養ワクチンの開発を考え，国立予防衛生研究所・近藤技官の技術指導のもとに，1974年以来実験を重ねた結果，有効性については従来の哺乳マウス脳由来の不活化狂犬病ワクチン(SMBワクチン)基準を十分に満足し，副作用の危険性を遥かに低減するニワトリ胚由来の乾燥組織培養不活化狂犬病ワクチン(TCワクチン)の実用化に成功し，次のような成績を得た。

1. ワクチン調製法に関する基礎的研究
　1)大量培養に適するCE細胞調製法の検討を行った結果，従来のピペッティングによる細胞分散法と，マグネチック・スターラーを用いた室温20分間，2回および4℃60分間の細胞分散法との間に差は認められず，いずれの細胞分散法を用いても細胞調製が可能であることがわかった。
　2)HEP Flury狂犬病ウイルス株を，ニワトリ胚細胞に接種し35℃で培養した場合，維持培地のpHを7.8～8.0に維持することで，単層細胞の維持も良く，ウイルスは接種後6日に最高値(10^7.0 ～10^7.5 LD50/ml，HA価32～128倍)を示した。この成績より，ウイルス採取時期をウイルス接種後6日とした。
　3)狂犬病ウイルスの不活化は，ベータープロピオラクトンを0.02v/v%になるように添加し，37℃の温浴中に45分間おくことで完全に不活化されたが，ワクチンの安全性を考慮し，さらに1回，同一操作を繰り返し実施した。
　4)ウイルス吸着作用のない，G社製メンブランフィルターを用いることにより，濾過によるウイルスのロスを防止することが可能となり，ワクチン調製工程中にウイルス浮遊液の除菌濾過工程を導入することができた。
　5)限外濾過-超遠心法により調製したTCワクチンの平均蛋白窒素量は，0.021mg/mlであり従来のSMBワクチンの約1/20であった。また，蔗糖密度勾配遠心法により調製したワクチンの平均蛋白窒素量は0.007mg/mlであり，限外濾過-超遠心法で調製したワクチンの1/3量を示したが，無菌性に問題があることから，ウイルス浮遊液の濃縮・精製には限外濾過-超遠心法を採用した。ワクチンのロット間に蛋白窒素含量のバラツキを認めたが，棄却検定の結果，異常値は認められなかった。
　6)TCワクチンの力価は，Habel指数で10^4.64 ～10^5.75，Antigenic value(A.V.)で1.0～13.2を示し，いずれも基準に適合した。ワクチンの力価と蛋白窒素含量とに相関は認められなかった(相関係数r=-0.036)。
　7)TCワクチンを4℃の恒温室に保存した場合，36ケ月間全ての項目(外観，含湿度，水素イオン濃度，無菌性，異常毒性および力価)において変化は認められなかった。37℃の恒温室に保存した場合，6ケ月より若干の力価の低下が認められたが，36ケ月でもなお従来のSMBワクチンの力価試験の基準に適合する成績であった。力価試験以外の全ての項目(外観，水素イオン濃度，異常毒性)において変化は認められず，TCワクチンの外部温度に対する安定性が確認された。
2. ワクチンの安全性と抗原性
　1)TCワクチンをマウスおよびラットに投与可能な最大量(マウス静脈内，経口投与および皮下投与で120ml/kg，ラットの静脈内投与で95ml/kg，経口および皮下投与で120ml/kg)を投与したが，致死動物は見られず，したがってLD50値を算出することはできなかった。また，いずれの投与量においても毒性を示すような所見は全く認められなかった。対照として用いたSMBワクチンでは，経口および皮下投与の場合，投与可能な最大量を投与したが致死動物は見られず，LD50値を算出することはできなかったが，静脈内投与ではマウスの場合20ml/kgより致死動物が見られ，LD50値は雄で30ml/kg，雌で29ml/kgであり，ラットの場合は16ml/kgより致死動物が見られ，LD50値は雄で23.3ml/kg，雌で21.5/kgであり，TCワクチンの安全性が示唆された。また，いずれの動物においても性差は認められなかった。
　2)TCワクチンの抗原性を検討するためのモルモットを用いた全身性アナフィラキシー試験では，TCワクチンについては全例陰性であった。対照として用いたSMBワクチンでは皮下惹起注射の場合，全例陰性であったが，静脈内注射の場合，4匹中3匹が軽度のアナフィラキシー症状を示し，TCワクチンのアレルギー原性の低さが認められた。
　3)TCワクチンを8頭のサルの胸部皮下に1mlずつ連続10回注射し，局所および全身反応について3ケ月間観察したが，注射局所および全身反応に全く異常は認められなかった。同時に血中中和抗体価をマウス中和試験法で測定した結果，初回注射後5日目で8頭中1頭(12.5%)が10倍の抗体価を示し，10日目では全例が抗体陽性となり平均抗体価は33倍であった。以後，高い抗体価の上昇持続が認められ，最終試験日の35日目には1,770倍に達し，TCワクチンのサルに対する安全性と良好な中和抗体産生能が確認された。
3. ヒトに対する安全性と中和抗体産生能
　当研究所成人男子14名，女子21名，計35名の志願者について，TCワクチン1mlを1回量として，2週間隔，3週間隔および4週間隔でそれぞれ2回注射し，6ケ月および1年4ケ月後に3回目を注射，さらに適当な間隔をおいて追加注射を行い，局所反応および全身反応を観察するとともに中和抗体の産生と持続について検討した。ヒトにおける安全性では，1回の注射で局所の発赤9%(3/35)，腫脹6%(2/35)，疼痛23%(8/35)，かゆみ6%(2/35)，2回目の注射では局所の発赤31%(11/35)，腫脹3%(1/35)，疼痛20%(7/35)，かゆみ17%(6/35)，3回目の注射では局所の発赤7%(1/15)，腫脹13%(2/15)，疼痛7%(1/15)，かゆみ7%(1/15)が認められたが，いずれの反応も一過性で注射後24～72時間には消失した。全身反応は2回目の注射で頭痛3%(1/35)，3回目の注射では7%(1/15)を認めたが，その程度は軽く注射後24時間には消失した。その他の局所反応，全身反応は認められなかった。さらに，国際協力事業団・海外青年協力隊員2,447名に行った暴露前免疫に関する調査では，局所の発赤，腫脹を認めたものが27名(1.1%)，注射部位の疼痛8名(0.3%)，全身症状として37℃台の発熱6名(0.2%)を認めた程度であった。中和抗体の産生と持続に関しては，抗体は2回目注射後2週で最高値(平均抗体価27倍・抗体陽性率90%)に達し，以後6ケ月間持続した。3回目注射後は抗体の上昇は早く，1週で最高値(平均抗体価125倍・抗体陽性率100%)に達し，以後6ケ月間持続した。さらに，抗体陰性期に1mlの追加注射を行ったところ，1週より抗体の産生を認め2週で最高値(平均抗体価98倍・抗体陽性率100%)に達し，明らかにブースター効果が認められた。初回注射間隔の違いによる抗体産生を比較したところ，4週間隔が一番高い値を示したことから，暴露前免疫における初回注射間隔を4週間とした。
　狂犬病ウイルス暴露後の治療効果について，海外で狂犬病罹患犬(4頭)あるいはその疑いのある動物(イヌ，サル)の咬傷を受けた17名に対して，TCワクチン注射を行った結果，全員が発症を免れ，本TCワクチンの暴露後治療効果が実証された。
　今回開発した乾燥組織培養不活化狂犬病ワクチンは，蛋白窒素含量が0.021mg/mlと少なく，一般のワクチンに含まれるような防腐剤(ホルマリン，チメロサールなど)を含んでおらず，脳組織を用いた従来のワクチンと比べて，マウスおよびラットを用いた毒性試験，モルモットを用いたアナフィラキシー試験，サルおよびヒトに対する接種試験で，その安全性と有効性が極めて高いことが確認された。本TCワクチンは凍結乾燥品であるため，4℃および37℃における経時安定性も良く，狂犬病発生の多い熱帯地方での使用も十分可能な成績が得られた。
　また，ワクチンの使用方法に関して，WHOでは狂犬病ウイルス暴露前のワクチン投与方法として，1ケ月間隔で2回，さらに6ケ月後1回の計3回，1mlを皮下注射すること，また，狂犬病ウイルス暴露後は，できるだけ速やかにワクチン注射を開始し，その第1回目を0日とし，以後3，7，14，30，90日の計6回，1mlを皮下注射するように推奨している。今回開発したTCワクチンは，力価およびヒトにおける中和抗体産生能試験の成績から，WHOの推奨するワクチン投与方法にも十分対応できるものと判断された。
　1885年，Pasteurのウサギ脊髄乾燥減毒ワクチンに始まる狂犬病ワクチン開発の長い歴史の中で，副反応が少なく，狂犬病発症阻止効果の高い，新しいタイプのワクチン(組織培養ワクチン)を開発することができた。},
 title = {人体用乾燥組織培養不活化狂犬病ワクチンに関する研究},
 year = {}
}