JP2016198107A - 万能ウイルス様粒子(vlp)インフルエンザワクチン - Google Patents
万能ウイルス様粒子(vlp)インフルエンザワクチン Download PDFInfo
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- JP2016198107A JP2016198107A JP2016131988A JP2016131988A JP2016198107A JP 2016198107 A JP2016198107 A JP 2016198107A JP 2016131988 A JP2016131988 A JP 2016131988A JP 2016131988 A JP2016131988 A JP 2016131988A JP 2016198107 A JP2016198107 A JP 2016198107A
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Abstract
【解決手段】切断、再設計、または改変された1つまたは複数のHA分子をその表面に提示するインフルエンザウイルス様粒子(VLP)。少なくとも1つの基質タンパク質;および修飾インフルエンザHAポリペプチドを含み、前記修飾は、前記HAポリペプチドの膜貫通および細胞質尾部ドメインの外側に1つまたは複数のアミノ酸残基の欠失を含むウイルス様粒子(VLP)。
【選択図】図1
Description
本出願は、その開示内容を本明細書に援用する、どちらも2010年2月18日に出願された米国仮特許出願第61/305,768号明細書および米国仮特許出願第61/305,759号明細書の優先権を主張する。
該当なし。
本明細書で使用する場合、「サブウイルス粒子」「ウイルス様粒子」または「VLP」という用語は、複製しないウイルス殻をいう。VLPは一般に、以下に限定されるものではないが、カプシドタンパク質、コートタンパク質、殻タンパク質、表面タンパク質および/もしくはエンベロープタンパク質と呼ばれるタンパク質などの1つまたは複数のウイルスタンパク質、またはこれらのタンパク質に由来する粒子形成ポリペプチドからなる。VLPは、適切な発現系でタンパク質を組換え発現させると、自然に形成され得る。特定のVLPを産生する方法は、当該技術分野において公知であり、以下でより詳細に考察する。ウイルスタンパク質の組換え発現後のVLPの存在については、電子顕微鏡、生物物理学的特徴付けおよび同種のものなどにより当該技術分野において公知の従来の技術を用いて検出することができる。たとえば、Baker et al.,Biophys.J.(1991)60:1445−1456;Hagensee et al.,J.Virol.(1994)68:4503−4505を参照されたい。たとえば、VLPは、密度勾配遠心分離により単離し、および/または特徴的な密度バンディング(たとえば、実施例)により特定することができる。あるいは、対象のVLP調製物のガラス化した水性サンプルを用いて低温電子顕微鏡観察を行い、適切な撮影条件下で画像を記録してもよい。VLP精製の別の方法として、親和性法、イオン交換法、サイズ排除法および逆相法などのクロマトグラフィー技術があるが、これに限定されるものではない。
本明細書に記載するのは、インフルエンザ感染からのヒトの保護、および/またはヒトの処置に使用することができるインフルエンザVLPである。特に、本明細書に記載するのは、ウイルスのイムノドミナント領域および/または超可変領域(たとえば、抗原部分)の一部を形成または構成するアミノ酸残基およびペプチド配列の欠失により様々な異なるインフルエンザ(flu)ウイルスに対して強い免疫学的防御応答を誘導するVLPインフルエンザワクチンである。これにより、本来免疫学的に隠れたエピトープがドミナントになり、高い免疫抗原性を示すようになる。
インフルエンザタンパク質をコードする配列が真核細胞に発現すると、そのタンパク質は、非感染性ウイルス様粒子(VLP)に自己集合することが明らかにされている。Latham & Galarza(2001)J.Virol.75(13):6154−6165;Galarza et al.(2005)Viral.Immunol.18(1):244−51;ならびに米国特許出願公開第2008/0233150号明細書;米国特許出願公開第2008/0031895号明細書および米国特許出願公開第2009/0022762号明細書を参照されたい。
上述のように、本明細書に記載のVLPは、修飾(たとえば、切断、欠失、ハイブリッド等)インフルエンザ抗原ポリペプチドを含む。
本明細書に記載するように産生されるVLPは、標準的な組換え技術を用いて調製すると都合がよい。VLP形成タンパク質をコードするポリヌクレオチドを宿主細胞に導入し、細胞にタンパク質を発現させると、VLPにアセンブルする。
VLPに取り込まれるように、所望のポリペプチドをコードする配列を含むコンストラクトを合成したら、コンストラクトを任意の好適な発現用ベクターまたはレプリコンにクローニングしてもよい。多くのクローニングベクターが当業者に公知であり、当業者であれば、発現に関する本明細書の教示内容および当該技術分野において公知の情報に照らして任意の特定の宿主細胞型に適切なベクターおよび制御エレメントを容易に選択することができる。一般に、Ausubel et al,上掲またはSambrook et al,上掲を参照されたい。
上述のように、真核宿主細胞に発現したインフルエンザタンパク質は、非感染性ウイルス様粒子(VLP)に自己集合することが明らかになっている。これを踏まえ、次に本明細書に記載の配列および/またはベクターを使用して適切な宿主細胞を形質転換する。本明細書に記載のVLPを形成するタンパク質をコードするコンストラクトは、様々な異なる細胞型、以下に限定されるものではないが、昆虫細胞、真菌(酵母)細胞および哺乳動物細胞を用いてインフルエンザVLPを産生するための効率的な手段となる。
本明細書に記載するように産生されるVLPを使用して被検体に投与すると、免疫応答を惹起することができる。上記で論じたように、VLPは、様々な抗原(たとえば、1つまたは複数の株または分離株由来の1つまたは複数の修飾インフルエンザ抗原)を含んでもよい。精製されたVLPは、通常ワクチン組成物の形態で脊椎動物被検体に投与してもよい。また、混合ワクチンを使用してもよく、こうしたワクチンは、たとえば、インフルエンザもしくは他の生物に由来する他のサブユニットタンパク質および/またはこうした抗原をコードする遺伝子送達ワクチンを含む。
VLP、およびそうしたVLPを含む組成物は、たとえば、非経口注射(たとえば皮下、腹腔内、静脈内、筋肉内または間質の組織腔)、または直腸投与、経口投与(たとえば錠剤、噴霧剤)、経膣投与、局所投与、経皮投与(たとえば国際公開第99/27961号パンフレットを参照)または経皮投与(たとえば国際公開第02/074244号パンフレットおよび国際公開第02/064162号パンフレット)、経鼻投与(たとえば国際公開第03/028760号パンフレット)、点眼投与、点耳投与、経肺投与または他の粘膜投与など任意の送達方法で被検体に投与してもよい。同一または異なる経路で反復投与を行ってもよい。好ましい実施形態では、投与を経鼻で行う。
様々な修飾HA分子を以下の通り設計した。
改変HA 1TAのヌクレオチド配列(配列番号1):
ATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGTGATCAGATTTGCATTGGTTACAGGATGGAGTTCTTCTGGACAATTTTAAAGCCGAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAACAATTATGAAAAGTGAATTGGAATATGGTAACGGAAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGCATGCCATTCCACAATATACACCCTCTCACCATTGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCGACTGGGCTCAGAAATAGCCCTCAAAGAGAGAGAAGAAGAAAAAAGAGAGGATTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAACAACTTAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCATAAATGTGATAATGAATGTATGGAAAGTGTAAGAAATGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCGAGACTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATAGGAATTTACCAAATACTGTCAATTTATTCTACAGTGGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCCTTATGGATGTGCTCCAATGGGTCGTTACAATGCAGAATTTGCATTTAA
改変HA−1TAのアミノ酸配列(配列番号2):
MEKIVLLFAIVSLVKSDQICIGYRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNGNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI。
改変HA−2TAのヌクレオチド配列(配列番号3):
ATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGTGATCAGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGAAAAGAAACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTAGGATGGAGTTCTTCTGGACAATTTTAAAGCCGAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAACAATTATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGCATGCCATTCCACAATATACACCCTCTCACCATTGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCGACTGGGCTCAGAAATAGCCCTCAAAGAGAGAGAAGAAGAAAAAAGAGAGGATTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAACAACTTAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCATAAATGTGATAATGAATGTATGGAAAGTGTAAGAAATGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCGAGACTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATAGGAATTTACCAAATACTGTCAATTTATTCTACAGTGGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCCTTATGGATGTGCTCCAATGGGTCGTTACAATGCAGAATTTGCATTTAA
改変HA−2TAのアミノ酸配列(配列番号4):
MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI
改変HA−3TAのヌクレオチド配列(配列番号5):
ATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGTGATCAGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGAAAAGAAACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTGACATAGGACCAGGAAAGGTAGGATACGGACCAGGAATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGCATGCCATTCCACAATATACACCCTCTCACCATTGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCGACTGGGCTCAGAAATAGCCCTCAAAGAGAGAGAAGAAGAAAAAAGAGAGGATTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGACGGAAGGGAATTTAACAACGGAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCATAAATGTGATAATGAATGTATGGAAAGTGTAAGAAATGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCGAGACTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATAGGAATTTACCAAATACTGTCAATTTATTCTACAGTGGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCCTTATGGATGTGCTCCAATGGGTCGTTACAATGCAGAATTTGCATTTAA
改変HA−3TAのアミノ酸配列(配列番号6):
MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPDIGPGKVGYGPGMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEADGREFNNGERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI
改変HA−4TAのヌクレオチド配列(配列番号7):
ATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGTGGATTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAACAACTTAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCATAAATGTGATAATGAATGTATGGAAAGTGTAAGAAATGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCGAGACTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATAGGAATTTACCAAATACTGTCAATTTATTCTACAGTGGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCCTTATGGATGTGCTCCAATGGGTCGTTACAATGCAGAATTTGCATTTAA
改変HA−4TAのアミノ酸配列(配列番号8):
MEKIVLLFAIVSLVKSGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI
改変HA−5TAのヌクレオチド配列(配列番号9):
ATGGAGAAAATAGTGCTTCTTTTTGCAATAGTCAGTCTTGTTAAAAGTGATCAGATTTGCATTGGTTACCATGCAAACAACTCGACAGAGCAGGTTGACACAATAATGGAAAAGAACGTTACTGTTACACATGCCCAAGACATACTGGAAAAGAAACACAACGGGAAGCTCTGCGATCTAGATGGAGTGAAGCCTCCACAGAGAGAAAGAAGAAGAAAGAAGAGACTAATTTTGAGAGATTGTAGCGTAGCTGGATGGCTCCTCGGAAACCCAATGTGTGACGAATTCATCAATGTGCCGGAATGGTCTTACATAGTGGAGAAGGCCAATCCAGTCAATGACCTCTGTTACCCAGGGGATTTCAATGACTATGAAGAATTGAAACACCTATTGAGCAGAATAAACCATTTTGAGAAAATTCAGATCATCCCCAAAAGTTCTTGGTCCAGTCATGAAGCCTCATTAGGGGTGAGCTCAGCATGTCCATACCAGGGAAAGTCCTCCTTTTTCAGAAATGTGGTATGGCTTATCAAAAAGAACAGTACATACCCAACAATAAAGAGGAGCTACAATAATACCAACCAAGAAGATCTTTTGGTACTGTGGGGGATTCACCATCCTAATGATGCGGCAGAGCAGACAAAGCTCTATCAAAACCCAACCACCTATATTTCCGTTGGGACATCAACACTAAACCAGAGATTGGTACCAAGAATAGCTACTAGATCCAAAGTAAACGGGCAAAGTGGAAGGATGGAGTTCTTCTGGACAATTTTAAAGCCGAATGATGCAATCAACTTCGAGAGTAATGGAAATTTCATTGCTCCAGAATATGCATACAAAATTGTCAAGAAAGGGGACTCAACAATTCCACAGAGAGAAAGAAGAAGAAAGAAGAGAATGAAAAGTGAATTGGAATATGGTAACTGCAACACCAAGTGTCAAACTCCAATGGGGGCGATAAACTCTAGCATGCCATTCCACAATATACACCCTCTCACCATTGGGGAATGCCCCAAATATGTGAAATCAAACAGATTAGTCCTTGCGACTGGGCTCAGAAATAGCCCTCAAAGAGAGAGAAGAAGAAAGAAGAGAGGATTATTTGGAGCTATAGCAGGTTTTATAGAGGGAGGATGGCAGGGAATGGTAGATGGTTGGTATGGGTACCACCATAGCAATGAGCAGGGGAGTGGGTACGCTGCAGACAAAGAATCCACTCAAAAGGCAATAGATGGAGTCACCAATAAGGTCAACTCGATCATTGACAAAATGAACACTCAGTTTGAGGCCGTTGGAAGGGAATTTAACAACTTAGAAAGGAGAATAGAGAATTTAAACAAGAAGATGGAAGACGGGTTCCTAGATGTCTGGACTTATAATGCTGAACTTCTGGTTCTCATGGAAAATGAGAGAACTCTAGACTTTCATGACTCAAATGTCAAGAACCTTTACGACAAGGTCCGACTACAGCTTAGGGATAATGCAAAGGAGCTGGGTAACGGTTGTTTCGAGTTCTATCATAAATGTGATAATGAATGTATGGAAAGTGTAAGAAATGGAACGTATGACTACCCGCAGTATTCAGAAGAAGCGAGACTAAAAAGAGAGGAAATAAGTGGAGTAAAATTGGAATCAATAGGAATTTACCAAATACTGTCAATTTATTCTACAGTGGCGAGTTCCCTAGCACTGGCAATCATGGTAGCTGGTCTATCCTTATGGATGTGCTCCAATGGGTCGTTACAATGCAGAATTTGCATTTAA
改変HA−5TAのアミノ酸配列(配列番号10):
MEKIVLLFAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKKHNGKLCDLDGVKPPQRERRRKKRLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPVNDLCYPGDFNDYEELKHLLSRINHFEKIQIIPKSSWSSHEASLGVSSACPYQGKSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPRIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIPQRERRRKKRMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGIYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI
この例では、VLPの産生に必要なコンストラクトの作製について記載する。作製されるコンストラクトの一般的な構造を図6に示す。遺伝子発現の高度で持続可能なレベルだけでなく、安定にトランスフェクトされた細胞の産生速度をも高める特有の調節配列を含む哺乳動物プラスミド(ベクター、図6、パネルI)に目的の遺伝子をサブクローニングする。VLPを産生する別の方法は、バキュロウイルス−昆虫細胞発現系の利用である。作製されるコンストラクトの全体的な構造を図6のパネルIIに示す。
実施例2に記載されているような所望のベクターの作製後、ベクターを利用して哺乳動物細胞(CHO、Vero、MDCK、WI−38またはMRC5)でVLPを産生した。
トランスフェクションのため、5mlのCHO−S−SFM II培地(CHO細胞)、または5%ウシ胎仔血清(FBS)(Invitrogen,Carlsbad,CA)を補充した10mlのDMEM(Vero、MDCK、MRC5、WI−38)を用いて哺乳動物細胞(CHO、Vero、MDCK、MRC5またはWI−38)を1.5×106〜2.5×106細胞/mlの密度で適切な培養容器(CHO細胞の場合、25cm2フラスコ、またはVero、MDCK、MRC5またはWI−38細胞の場合75cm2フラスコ)に蒔いて調製した。トランスフェクション手順の開始の24時間前に接着細胞(Vero、MDCK、MRC5、WI−38)を蒔いた。
エレクトロポレーションあるいは化学的トランスフェクションにより未切断環状プラスミドの組み合わせを哺乳動物細胞に導入してVLP産生の評価を行った。この例では、M1/M2ベクターをNA/HA(改変、図1)と組み合わせて、上述の2つのトランスフェクション方法のどちらかにより哺乳動物細胞(CHO、MDCK、MRC5またはWI−38)に送達した。
直線化したプラスミドDNAを哺乳動物細胞にトランスフェクトすると、プラスミドが宿主細胞のゲノムに組み込まれる。実施例1に記載したコンストラクトのいずれかの直鎖形態を送達すると、プラスミドが組み込まれ、その後ベクターDNAが有する遺伝子が発現する。この戦略により、目的の遺伝子産物を構成的に発現する安定にトランスフェクトされた細胞株の産生が可能になる。
次いで目的の遺伝子をコードする直線化したプラスミドをトランスフェクトした哺乳動物細胞を抗生物質処理に付し、トランスフェクトされたプラスミドが宿主細胞ゲノムに組み込まれた細胞を選別した。トランスフェクトされたプラスミド は、ハイグロマイシン、ピューロマイシンまたはネオマイシンのいずれかに耐性を付与する抗生物質耐性カセットを含む。
上記のように得た細胞を組織培養フラスコから回収し、500×gで5分間連続遠心分離により3回洗浄し、続いてリン酸塩緩衝生理食塩水(PBS)に再懸濁した。3回目の洗浄の後、細胞をカウントし、それぞれ5×106細胞の3つのアリコートに分けた。細胞表面のM2発現でソートすることになっていた第1のアリコートは、PBSで1:500に希釈した抗M2モノクローナル抗体(Abcam Inc,Cambridge,MA)と室温で1時間インキュベートした。第2のアリコートは、PBSで1:200に希釈したインフルエンザAヌクレオプロテインに対するモノクローナル抗体(Meridian Life Sciences,Saco,ME)と室温で1時間インキュベートした。このアリコートは、フローサイトメトリー実験のアイソタイプコントロールとした。第3のアリコートは、本実験の未染色の対照とした。
安定にトランスフェクトされた細胞集団からクローン細胞株を取得するため、エンドポイントクローニングを実施した。細胞を回収し、血球計数器を用いてカウントした。次いで最終濃度が10細胞/mlになるように適切な量の培養基で細胞を希釈した。この細胞調製物を穏やかに撹拌して細胞が均一に分布するようにしてから、100μl/ウェルで滅菌96ウェルプレートに蒔いた。次いでプレートを5%CO2の湿潤雰囲気、37℃でインキュベートし、クローンの細胞増殖について定期的にモニターした。活発に増殖している細胞を含むウェルを一定期間にわたり特定した。これらのウェルのクローン細胞が約70〜80%コンフルエントになったとき、6ウェルプレート、25cmおよび75cmフラスコに連続継代してスケールアップした。
トランスフェクトされた哺乳動物細胞の細胞ペレットおよび上清についてタンパク質発現の解析を行った。トランスフェクトされた(Tranfected)細胞を回収し、1×PBSで洗浄し、RIPA緩衝液[トリス−HCl(pH7.4)50mM、塩化ナトリウム150mM、EDTA1mM、1%トリトンX−100、1%NP−40、0.1%硫酸ドデシルナトリウム]に再懸濁し、次いで凍結融解サイクルにより破壊し、ピペッティングし、最後にその後使用するまで−20℃で保存した。各サンプルの総タンパク質濃度をBradford法により推定した。簡単に説明すると、10μlのサンプルを、室温に予め温めておいた1.0mlの1×Bradford色素試薬(Bio−Rad Inc.,Hercules,CA)に加えた。次いでこの反応混合物を激しく振盪して均一な溶液を得た。分光光度計を使用して595nmの波長で各サンプルの吸光度を測定した。Bradfordアッセイを用いて既知濃度のウシ血清アルブミンの595nmの吸光度を測定してプロットした標準曲線を用いて各サンプルのタンパク質濃度を判定した。
M1およびM2インフルエンザ遺伝子を有する直線化したDNAベクターを、MDCK細胞およびCHO細胞にエレクトロポレーションによりトランスフェクトした。ハイグロマイシンによる選択後、細胞を抗体の組み合わせ(最初に一次抗体として、細胞表面に発現したM2タンパク質と反応した抗M2マウスモノクローナル、続いて二次抗体としてフルオレッセインコンジュゲート抗マウス抗体)で標識してから、蛍光活性化セルソーター(FACS)を用いて単細胞クローンを単離した。
M1/M2を産生する細胞における改変HA/NAタンパク質の発現
NAを含むまたは含まない改変HA分子(図6パネルI−HA/NA)を有するDNAベクターを、M1/M2DNAベクター(図1−パネルI−M1/M2)と一緒に、あるいは、M1/M2を発現している細胞にトランスフェクトすると、これらのタンパク質が発現し、タンパク質は、細胞ライセートだけでなく、細胞上清および濃縮精製された上清にも存在した。
ネガティブ染色電子顕微鏡観察によるインフルエンザVLPの調査
トランスフェクト細胞の上清を200,000×g、4℃で1.5時間超遠心に付した。超遠心後のペレットをPBSに再懸濁し、再懸濁したペレットの20μlを電子顕微鏡観察用に確保した。この20μlのサンプルを4%パラホルムアルデヒドで固定してから、固定材料の5μlを200メッシュのカーボンコートしたグリッド(EMS,Hatfield,PA)に載せ、グリッドを5分間カバーし、次いで水で3回洗浄した。この後、コートしたグリッドに2%酢酸ウラニルを、グリッドを酢酸ウラニルで過剰染色しないように素早く5滴滴下した。グリッドから過剰な溶液をブロットし、次いで風乾してからこれをJOEL JEM 100CX透過型電子顕微鏡に導入した。サンプルは、60,000×〜100,000×の倍率で観察した。
Claims (1)
- 少なくとも1つの基質タンパク質;および
修飾インフルエンザHAポリペプチドを含み、前記修飾は、前記HAポリペプチドの膜貫通および細胞質尾部ドメインの外側に1つまたは複数のアミノ酸残基の欠失を含むことを特徴とする
ウイルス様粒子(VLP)。
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EP2610345B1 (en) | 2007-11-27 | 2015-08-19 | Medicago Inc. | Recombinant influenza virus-like particles (VLPS) produced in transgenic plants expressing hemagglutinin |
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EP3089755A1 (en) | 2014-01-03 | 2016-11-09 | Fundacion Biofisica Bizkaia | VLPs, METHODS FOR THEIR OBTENTION AND APPLICATIONS THEREOF |
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US9352031B2 (en) | 2016-05-31 |
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US20110212128A1 (en) | 2011-09-01 |
JP6524555B2 (ja) | 2019-06-05 |
US11529410B2 (en) | 2022-12-20 |
CN102858368B (zh) | 2017-04-12 |
WO2011102900A1 (en) | 2011-08-25 |
CA2789945A1 (en) | 2011-08-25 |
EP2536428A4 (en) | 2014-04-09 |
US20190091323A1 (en) | 2019-03-28 |
US20160303223A1 (en) | 2016-10-20 |
EP2536428A1 (en) | 2012-12-26 |
CN102858368A (zh) | 2013-01-02 |
US10695418B2 (en) | 2020-06-30 |
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