JP2011140522A - 多価髄膜炎菌多糖−タンパク質複合ワクチン - Google Patents
多価髄膜炎菌多糖−タンパク質複合ワクチン Download PDFInfo
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Abstract
【解決手段】本ワクチンは、単回投与ワクチンとして処方された4つの別個の多糖−タンパク質複合体から成る。髄膜炎菌血清群A、C、W−135、およびY由来の精製莢膜多糖を化学的に活性化させ、さらに共有化学結合によって担体タンパク質に選択的に結合させて、子供ならびに大人において様々な髄膜炎菌株に対して永続的な免疫を誘導できる多糖−タンパク質複合体を形成する。
【選択図】なし
Description
髄膜炎菌血清群A、C、W−135、およびY群の湿式凍結種細胞を解凍し、液体ワトソン・シャープ(Watson Scherp)培地を用いて解凍および回収し、ミューラー・ヒントン寒天培地を含むブレークボトル(Blake bottle)に播いた。ブレークボトルを35℃から37℃でCO2雰囲気中において15時間から19時間インキュベートした。インキュベーション時間に続いて、ブレークボトルから増殖細胞を取り除き、ワトソン・シャープ培地を含む4Lフラスコに添加した。プラットフォームシェーカーにおいて、フラスコを35℃から37℃で3時間から7時間インキュベートした。4Lフラスコの内容物を、ワトソン・シャープ培地含有発酵容器に移した。栄養補助物質の供給および消泡剤の添加によって溶解酸素含量およびpHを制御しながら、発酵容器を35℃から37℃で7時間から12時間インキュベートした。インキュベーション時間後に、発酵容器の内容物を500Lタンクに移し、セタブロン(Cetavlon)を添加し、さらに1時間混合した。セタブロン処理増殖細胞を、約15,000から17,000xgで、約30から70リットル/時間の流速で遠心した。第2のセタブロン沈殿で上清から粗多糖を沈殿させた。セタブロンを上清に添加し、室温で1時間以上混合した。材料を1℃から5℃で8時間から12時間保管した。約45,000から50,000xgで、300から400ml/分の流速で遠心して、沈殿多糖を採取した。集めたペーストをさらに加工するまで−60℃以下で保管した。
不活化ペーストを解凍し、ブレンダーに移した。ペーストを0.9M塩化カルシウムと混合して、均一な懸濁液を得た。懸濁液を約10,000xgで15分間遠心した。上清をリントフリーのパッドを通して、第1抽出物として静かに容器に注いだ。第2容量の0.9M塩化カルシウムをペーストに添加し、さらに混合して均一懸濁液を得た。懸濁液を上記のように遠心して、その上清を第1抽出から得た上清と混ぜ合わせた。全体で4回の抽出を実施し、上清をプールした。10−30kDA MWCO渦巻状限外濾過ユニットを用いて限外濾過することによって、プールした抽出物を濃縮した。
調製に用いられる材料として、髄膜炎菌血清群A、C、W−135、およびY由来の精製莢膜多糖粉末(実施例1に基づいて調製)、滅菌50mM酢酸ナトリウムバッファー(pH6.0)、滅菌1N塩酸、滅菌1N水酸化ナトリウム、30%過酸化水素、および滅菌生理食塩水(0.85%塩化ナトリウム)が挙げられる。
この調製で用いられる材料として、髄膜炎菌血清群A、C、W−135、およびYの過酸化水素解重合莢膜多糖(実施例2に基づいて調製)、アジピン酸ジヒドラジド、血清群Aのみに関して1−エチル−3−(3−ジメチルアミノプロピル)カルボジイミド(EDAC)、シアノボロ水素化ナトリウム (sodium cyanoborohydride)、滅菌1N塩酸、滅菌1N水酸化ナトリウム、滅菌塩化ナトリウム、および滅菌生理食塩水(0.85%塩化ナトリウム)が挙げられる。
粗ジフテリアトキソイドタンパク質の調製
凍結乾燥種細胞を再生させて16から18時間インキュベートした。培養液の一部を、増殖培地を含有する0.5リットルフラスコに移し、回転式振とう培養器において、34.5から36.5℃で7から9時間培養フラスコをインキュベートした。増殖培地を含有する4リットルフラスコに培養フラスコから一部を移し、回転式振とう培養器において、34.5から36.5℃で14から22時間培養フラスコをインキュベートした。4リットルフラスコからの培養液を用いて、増殖培地含有発酵槽に播種した。発酵槽を34.5から36.5℃で70から144時間インキュベートした。発酵槽の内容物を、デプスフィルターを通して回収容器に濾過した。0.2%濃度になるように、回収物に37%ホルムアルデヒド溶液を添加した。pHを7.4から7.6に調整した。0.2μmフィルターカートリッジを通して滅菌20リットルボトルに回収物を濾過した。34.5から36.5℃で7日間ボトルをインキュベートした。0.4%濃度になるように、各20リットルボトルに37%ホルムアルデヒド溶液を添加した。混合物のpHを7.4から7.6に調整した。振とう撹拌器において、34.5から36.5℃で7日間ボトルをインキュベートした。0.5%濃度になるように、各20リットルボトルに37%ホルムアルデヒド溶液を添加した。混合物のpHを7.4から7.6に調整した。34.5から36.5℃で8週間ボトルをインキュベートした。無毒化に関して粗トキソイドを試験した。試験期間の間、1から5℃でボトルを保管した。
粗トキソイドを室温まで暖め、20リットルボトルの内容物を合わせて精製タンクに入れた。トキソイドのpHを7.2から7.4に調整し、活性炭を粗トキソイドに添加して2分間混合した。活性炭−トキソイド混合物を1時間放置し、その後デプスフィルターカートリッジを通して第2精製タンクに濾過した。70%飽和になるように固形硫酸アンモニウムを濾液に添加した。pHを6.8から7.2に調整し、溶液を16時間放置した。沈殿したタンパク質を濾過によって回収し、70%飽和硫酸アンモニウム溶液(pH7.0)で洗浄した。沈殿物を滅菌蒸留水中に溶解させ、タンパク質溶液をステンレス回収容器に濾過した。pHを6.8から7.2に調整し、40%飽和になるように硫酸アンモニウムを添加した。溶液のpHを7.0から7.2に調整し、溶液を16時間放置した。濾過によって沈殿物を除去し、捨てた。60%飽和になるように濾液に硫酸アンモニウムを添加し、さらにpHを7.0から7.2に調整した。混合物を16時間放置し、沈殿したタンパク質を濾過によって回収した。沈殿物を滅菌蒸留水に溶解させ、溶けていないタンパク質を濾過によって除去し、さらに0.85%生理食塩水に対して限外濾過した。
15g/リットルまでタンパク質溶液を濃縮し、3000MWCO再生セルロースカートリッジを用いて、10容量の0.85%生理食塩水に対して限外濾過した。0.2μmメンブランを通して濾過することによって、濃縮タンパク質を滅菌した。複合体に加工するまで、タンパク質溶液を1から5℃で保管した。
この調製に用いられる材料として、髄膜炎菌血清群A、C、W−135、およびYのアジピン酸誘導体化多糖(実施例3に基づいて調製)、滅菌ジフテリアトキソイドタンパク質(実施例4に基づいて調製)、EDAC、硫酸アンモニウム、滅菌1N塩酸、滅菌1N水酸化ナトリウム、および滅菌生理食塩水(0.85%)が挙げられる。
ならびに50%飽和硫酸アンモニウム溶液(血清群C)、続いて20容量の生理食塩水(0.85%)に対して、複合体形成反応混合物を限外濾過した。限外濾過した複合体を、最初に1.2μmおよび0.45マイクロmフィルターを備えたフィルターカプセルを通して濾過し、続いて0.22μmフィルターを備えた第2のフィルターカプセルを通して濾過した。
この調製で用いられる材料として、髄膜炎菌血清群A、C、W−135、およびYの多糖−ジフテリアトキソイド複合体(実施例5に基づいて調製)、滅菌100mMリン酸ナトリウム緩衝生理食塩水(0.85%塩化ナトリウム)が挙げられる。
水酸化アルミニウム吸着複合体の調製
この調製で用いられる材料として、髄膜炎菌血清群A、C、W−135、およびYの多糖−ジフテリアトキソイド複合体(実施例5に基づいて調製)、滅菌生理食塩水(0.85%塩化ナトリウム)、および生理食塩水(0.85%)中の滅菌水酸化アルミニウムが挙げられる。
この調製で用いられる材料として、髄膜炎菌血清群A、C、W−135、およびY多糖−ジフテリアトキソイド複合体(実施例5に基づいて調製)、滅菌生理食塩水(0.85%塩化ナトリウム)、および生理食塩水(0.85%)中の滅菌リン酸アルミニウムが挙げられる。
臨床で評価する前に、実験小動物において免疫応答を誘導する能力に関して四価複合ワクチンを試験した。マウス、ラット、およびウサギを用いて、多糖ワクチンと比較した複合ワクチンの免疫原性を試験した。それら動物モデルは、それらの免疫応答パターンによって対応する多糖と複合ワクチンとを区別することができるため有用である。複合ワクチンは、それらモデルにおいてT−依存性様免疫応答を誘導するが、多糖ワクチンはT−非依存性様免疫応答を誘導する。
Claims (17)
- 2、3、または4つの別個のタンパク質−多糖複合体を含み、各複合体が、1以上の担体タンパク質に結合した2以上の髄膜炎菌血清群由来の莢膜多糖を含むことを特徴とする免疫組成物
- 前記莢膜多糖が、髄膜炎菌血清群A、C、W−135、およびY由来の莢膜多糖より成る群から選択されることを特徴とする請求項1記載の免疫組成物。
- 前記莢膜多糖が、髄膜炎菌血清群AおよびC由来の莢膜多糖であることを特徴とする請求項1記載の免疫組成物。
- 前記莢膜多糖が、髄膜炎菌血清群A、C,A−135、およびC由来の莢膜多糖であることを特徴とする請求項1記載の免疫組成物。
- 前記担体タンパク質がジフテリアトキソイドであることを特徴とする請求項1記載の免疫組成物。
- アジュバントをさらに含むことを特徴とする請求項1記載の免疫組成物。
- 前記アジュバントが水酸化アルミニウムであることを特徴とする請求項5記載の免疫組成物。
- 前記アジュバントがリン酸アルミニウムであることを特徴とする請求項5記載の免疫組成物。
- 髄膜炎菌の莢膜多糖に対する免疫応答を誘導するためにヒトまたは動物に投与されることを特徴とする請求項1記載の免疫組成物。
- 免疫学的効果量の2から4つの別個のタンパク質−多糖複合体から成り、各複合体が担体タンパク質に結合した異なる莢膜多糖を含み、各莢膜多糖が血清群A、C、W−135、およびY由来の莢膜多糖より成る群から選択されることを特徴とする多価髄膜炎菌ワクチン。
- 前記莢膜多糖が、髄膜炎菌血清群AおよびCから調製されることを特徴とする請求項10記載の多価髄膜炎菌ワクチン。
- 前記莢膜多糖が、髄膜炎菌血清群A、C,A−135、およびCから調製されることを特徴とする請求項10記載の多価髄膜炎菌ワクチン。
- 前記担体タンパク質がジフテリアトキソイドであることを特徴とする請求項10記載の多価髄膜炎菌ワクチン。
- アジュバントをさらに含むことを特徴とする請求項10記載の多価髄膜炎菌ワクチン。
- 前記アジュバントが水酸化アルミニウムであることを特徴とする請求項14記載の多価髄膜炎菌ワクチン。
- 前記アジュバントがリン酸アルミニウムであることを特徴とする請求項5記載の多価髄膜炎菌ワクチン。
- 髄膜炎菌に感染しやすいヒトまたは動物を防御するためにヒトまたは動物に投与されることを特徴とする請求項10記載の多価髄膜炎菌ワクチン。
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