JP6166178B2 - 表面タンパク質の単純化したグリコシル化によるウイルス粒子の生産方法 - Google Patents
表面タンパク質の単純化したグリコシル化によるウイルス粒子の生産方法 Download PDFInfo
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Description
本出願は、2010年11月4日出願の米国仮特許出願第61/410,257号、表題「METHODS FOR PRODUCING MONO−GLYCOSYLATED INFLUENZA VIRUS HEMAGGLUTININ」の優先権を主張するものであり、その内容は引用により全体において本明細書に組み込まれる。
本発明は、ウイルスに関するものであり、特に、単純化したグリカンによるウイルス粒子の生産に関する。特に、本発明は、単純化したグリコシル化によるインフルウイルス粒子の生産に関する。具体的に、本発明は、モノグリコシル化した(mono−glycosylated)インフルエンザウイルス粒子の生産方法に関する。
本発明は、モノグリコシル化したインフルエンザウイルスの模範的な調製に関して、本明細書で開示される。同じ方法は、レトロウイルス(ヒト免疫不全ウィルス(HIV)など)、及びフラビウイルス科(デングウイルス、西ナイル熱ウイルス、C型肝炎ウイルス(HCV)など)を含む、単純化したグリカン構造により他のウイルスを調製するために使用され得ることが、熟慮される。
グリコシド加水分解酵素(グリコシダーゼ又はグリコシル加水分解酵素とも呼ばれる)は、より小さな糖を放出するためにグリコシド結合の加水分解を触媒する。それらは、抗細菌の防衛戦略(例えば、リゾチーム)における、病因機構(例えば、ウイルスのノイラミニダーゼ)における、及び標準の細胞の機能(例えば、N結合糖タンパク質の生合成に関与するマンノシダーゼをトリミングする)における、セルロース及びヘミセルロースなどのバイオマスの分解を含む性質における役割を有する一般的な酵素である。グリコシルトランスフェラーゼと共に、グリコシダーゼは、グリコシド結合の合成及び破壊のための主要な触媒機構を形成する。
高マンノース糖タンパク質を有するタンパク質を生産するため、インフルエンザウイルスを持つ細胞は、糖タンパク質処理を阻害するためにKif(及び/又はDMJ)に曝露される。モノグリコシル化したインフルエンザウイルスを生産するため、ウイルスは、キフネンシンなどのマンノシダーゼ阻害剤と共に、病原体の無い発育鶏卵の尿膜腔に注入される。
本明細書で開示されたモノグリコシル化したインフルエンザウイルス抗原は、インフルエンザウイルス感染に対して免疫化するために使用され得る。抗原が由来する具体的なウイルスは、保護を提供する具体的なウイルスと同じであるか、又はそれとは相違する。なぜなら、異なる分離株間での交差防御は、インフルエンザウイルスで、特に同じウイルスのサブタイプ内で起きることが既知であるためである。
本発明の範囲を限定する意図なしに、本発明の実施形態に従って、例示的な器具、装置、方法及びそれらに関連する結果を以下に提供する。読者の便宜のために、実施例において表題又は副題が使用され得るが、それらは決して、本発明の範囲を限定するものではないことに注意されたい。さらに、本明細書においては特定の理論が提示され、開示されている;しかしながら、決してそれらは、正しかろうと間違っていようと、いかなる特定の理論又は動作のスキームを考慮することなく、本発明に従って本発明を実施する限りにおいて、本発明の範囲を限定するべきではない。
インフルエンザAウイルス、NIBRG−14(H5N1)(Nicolson C, et al.(2005)Vaccine 23(22):2943−2952)を、孵卵後10日目の特定病原体除去(SFP)発育鶏卵(10−days−old specific pathogen free(SFP)embryonated chicken eggs)の尿膜腔に注入し(20μlのPBS中の1000 TCID50(50%の組織培養感染量))、通常の十分にグリコシル化したウイルス(WHO(2005)WHO manual on animal influenza diagnosis and surveillance.(World Health Organization,Geneva))を生産した。
ウイルス溶液を、銅グリッド(Electron Microscopy Science)の、コロジオン−炭素で覆われた表面に置いた。余分な液体を濾紙を用いて除去し、2%のタングステン酸メチルアミン(Ted Pella,Inc.)を加え、30秒間染色した。余分な液体を濾紙を用いて除去し、H2Oで30秒間洗浄した。その後、グリッドを4時間風乾し、ウイルスの粒子を透過型電子顕微鏡(Hitachi H−7000)によって撮影した。十分にグリコシル化した及びモノグリコシル化したインフルエンザNIBRG−14ウイルスは、大きさと形態共に類似している(図2)。
ウイルスを変性緩衝液(5%のSDS及び10%のβ−メルカプトエタノール)と混合し、10分間煮沸し、その後そのサンプルを10%のNP−40に加え、25℃で一晩にわたりPNGase F(New England Biolabs)で処置した。そのサンプルをSDS−PAGEサンプル緩衝液と混合し、ゲルに充填する前に10分間、95℃まで加熱した。そのゲルをSYPRO RUBY(登録商標)(Invitrogen)で染色し、VERSA DOC(登録商標)イメージングシステム(Bio−rad)で分析した。SDS−PAGE分析の前のPNGase Fの処置は、十分にグリコシル化したウイルスサンプル中のNPタンパク質からHA1を分離することである。これにより、ウイルス中の血球凝集素の定量化のため、SDS−PAGE上の十分にグリコシル化した及びモノグリコシル化したウイルスの両方からのHA1タンパク質の、直接的な強度の比較が可能となる(図3)。
モノグリコシル化したウイルスのグリカンの組成物を、分析するために質量分析を使用した。ウイルス溶液を非還元のサンプル緩衝液と混合し、ゲルに充填する前に10分間、95℃まで加熱した。そのゲルをクマシーブルーで染色した。HA0バンドを、トリプシンによるゲル内でのトリプシン分解のために、小片に切り分けた。消化の後、サンプルをLC−MS−MSにより分析した(Wu Y,et al.(2010) Rapid Commun Mass Spectrom 24(7):965−972)(Agilent Technologies,Thermo Scientific)。インフルエンザNIBRG−14ウイルスの6つのN−グリコシル化部位が存在する。残基295(〜10% Man6(GlcNAc)2)及び残基489(〜10% Man7−9(GlcNAc)2)における高マンノース型グリカンの存在の低い割合を除いて、すべての他のグリカンは、付着される単一のGlcNAcサッカリド残基により、十分にグリコシル化した(図4)。
Claims (18)
- 免疫原性組成物の生産方法であって、該方法は:
a)マンノシダーゼ阻害剤の効果的な量を有する適切な宿主においてインフルエンザウイルス赤血球凝集素(HA)抗原を生産する工程であって、ここで、前記マンノシダーゼ阻害剤の濃度は、N−グリコシル化経路においてα−マンノシダーゼIを阻害するのに充分であり、前記インフルエンザウイルスHA抗原は、全粒子インフルエンザウイルスに含まれ、該全粒子インフルエンザウイルスは、特定病原体除去(SPF)発育鶏卵の宿主において増殖する工程;
b)工程aで生産されたインフルエンザウイルスHA抗原を回収する工程;
c)工程bで回収したインフルエンザウイルスHA抗原をエンドグリコシド(Endo H)と接触させる工程であって、ここで、Endo Hの濃度は、モノグリコシル化したインフルエンザウイルスHA抗原を生産するために高マンノース型グリカンを除去するのに充分である、工程;
d)工程cで生産されたモノグリコシル化したインフルエンザウイルスHA抗原を分離する工程;及び
e)工程dで分離して得られたモノグリコシル化したインフルエンザウイルスHA抗原と、少なくとも薬学的に許容可能な担体又はアジュバントとを含む免疫原性組成物を生産する工程
を含むことを特徴とする、方法。 - 前記インフルエンザウイルスHA抗原は、H5N1インフルエンザウイルスに由来することを特徴とする、請求項1に記載の方法。
- 前記H5N1インフルエンザウイルスは、NIBRG−14であることを特徴とする、請求項2に記載の方法。
- 前記マンノシダーゼ阻害剤はキフネンシン(Kif)であることを特徴とする、請求項1に記載の方法。
- 前記マンノシダーゼ阻害剤はデオキシマンノジリマイシン(DMJ)であることを特徴とする、請求項1に記載の方法。
- α−マンノシダーゼIの前記阻害は、Man9(GlcNAc)2を有する糖タンパク質をもたらすことを特徴とする、請求項1に記載の方法。
- 前記マンノシダーゼ阻害剤は、特定病原体除去(SPF)発育鶏卵の宿主の尿膜腔に加えられることを特徴とする、請求項1に記載の方法。
- 前記キフネンシンは、0.005乃至0.5mg/mlの濃度で加えられることを特徴とする、請求項4に記載の方法。
- 前記インフルエンザウイルスHA抗原の回収は:
インフルエンザウイルスを増殖するのに充分な時間、特定病原体除去(SPF)発育鶏卵の宿主においてインキュベートする工程;及びその後、
インフルエンザウイルスを含む尿膜液をSPF発育鶏卵から分離する工程
を含むことを特徴とする、請求項1に記載の方法。 - 回収された前記インフルエンザウイルスHA抗原は、0.1乃至100μg/mLのEndo Hにより処置されることを特徴とする、請求項9に記載の方法。
- インフルエンザウイルスHA抗原を含む前記インフルエンザウイルスは、密度勾配超遠心法を含む方法により分離されることを特徴とする、請求項9に記載の方法。
- 請求項1に記載の方法であって、該方法は更に、工程eの前に、限外濾過を含む方法によって、インフルエンザウイルスHA抗原を精製する工程を含むことを特徴とする、方法。
- 前記限外濾過は、0.2μmのフィルタの使用を含むことを特徴とする、請求項12に記載の方法。
- 請求項1に記載の方法であって、該方法は更に、電子顕微鏡検査によって、インフルエンザウイルスHA抗原を分析する工程を含むことを特徴とする、方法。
- 請求項1に記載の方法であって、該方法は更に、PNGaseによる処置を含む方法によって、インフルエンザウイルスHA抗原を定量化する工程を含むことを特徴とする、方法。
- 請求項1に記載の方法であって、該方法は更に、SDS−PAGE電気クロマトグラフィーを含む方法によって、モノグリコシル化したインフルエンザウイルスHA抗原を定量化する工程を含むことを特徴とする、方法。
- 請求項1に記載の方法であって、該方法は更に、質量分析方法を含む方法によって、インフルエンザウイルスHA抗原のグリカン組成物を分析する工程を含むことを特徴とする、方法。
- 前記インフルエンザウイルスは、インフルエンザウイルスHI、H3、及びH5から成る群から選択されることを特徴とする、請求項1乃至17のいずれかに記載の方法。
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