JP2022500050A - アフリカ豚熱ウイルスワクチン - Google Patents
アフリカ豚熱ウイルスワクチン Download PDFInfo
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Abstract
Description
本明細書で使用される場合、「T細胞エピトープ」または「T細胞抗原」という用語は、抗原の細胞内プロセシング後に免疫系によって認識され得るエピトープを指す。プロセシング後、T細胞エピトープは少なくとも1つのMHC分子と結合し、抗原提示細胞の表面上にMHC−ペプチド複合体として発現する。MHCクラスI分子によって提示されるT細胞エピトープは、通常、長さが8〜11個のアミノ酸であるのに対し、MHCクラスII分子は、長さが約12〜25個のアミノ酸、好ましくは約13〜17個のアミノ酸のペプチドを提示し得る。例えば、タンパク質の両親媒性プロファイル、配列モチーフ、定量的マトリックス、人工ニューラルネットワーク、サポートベクターマシン、定量的構造活性相関、分子ドッキングシミュレーションに基づいて、タンパク質内の潜在的なT細胞エピトープを予測することができるソフトウェアプログラムが利用可能である[Desai and Kulkarni−Kale,2014.Methods Mol Biol 1184:333−64]。これらのプログラムには、IEDB Analysis Resource、ELISpot(PepScan,Lelystad,the Netherlands)、RANKPEP [Reche et al.,2004.Immunogenetics 56:405−19]、nHLAPred [Bhasin and Raghava,2007.J Biosci 32:31−42]、およびNetMHC[Lundegaard et al.,2008.Nucleic Acids Res 36:W509−12]が含まれる。
本発明は、アフリカ豚熱ウイルスのタンパク質由来のT細胞抗原を含むポリエピトープをコードする発現カセットを含む組換え核酸分子であって、T細胞抗原がプロテアソーム切断のためのシグナルを含むスペーサーによって、好ましくは1〜10個のアミノ酸残基のスペーサーによって分離されている、組換え核酸分子を提供する。
本発明はさらに、本発明によるアフリカ豚熱ウイルスのタンパク質由来のT細胞抗原を含むポリエピトープをコードする発現カセットを含む組換え核酸分子を含むウイルス粒子を提供する。
本発明はブタにおける免疫応答を刺激する方法を提供し、方法は本発明の組換え分子、および/または本発明のウイルス粒子を、免疫応答を誘導するのに有効な量でブタに投与することを含む。
方法:
既知のASF分離株の全ゲノム配列およびブタのゲノム配列に基づいて、エピトープ予測プログラムNetMHCpanを使用して、ウイルス株またはブタの品種に依存しないT細胞エピトープのリストを用意した(表1を参照)。上位19のT細胞エピトープを使用して、ポリエピトープ合成DNAワクチンを生成した。DNA配列は、プロテアソーム切断およびさらなるプロセシングのためのシグナルを含む3〜5アミノ酸のスペーサーによって分離された19個のノナペプチドエピトープをコードした(図1Aを参照)。エピトープ14は、正しいプロセシングを妨げる可能性があるため、使用しなかった。また、いわゆるPADREユニバーサルT細胞エピトープ配列がこの目的のために含まれていた(図1Bを参照)。プロテアソーム分解を促進するために、ユビキチンのためのヌクレオチド配列を合成遺伝子の5’末端に付加した。これにより、プロテアソームによるより効率的な分解と、宿主T細胞へのエピトープの提示の改善がもたらされる。最終に、CpGアジュバント配列を合成遺伝子の3’UTRに付加した。合成遺伝子のコドン最適化したものを、GenScript Corporationで化学的に合成し、NheIおよびNotI制限部位を使用してプラスミドpCVI(ClaI制限フラグメントを除去することによって得られるpCI−neo[Promega]の派生物)のCMVプロモーターの後ろにクローン化した。得られたプラスミドをpCVI−ASFDVAC2と名付けた。
非ワクチン接種対照群(群3)のチャレンジ感染は40%の生存率であった。ワクチン接種により、1群および2群では約83%の生存率であった(図2Aを参照)。2群のブタは、他の群のブタよりも総臨床スコアが有意に低かった(図2B)。この群のブタはまた、最初のワクチン接種後(p.v.)42日目(チャレンジから[p.c.]0日目)から実験の終了まで、ノナペプチドのカクテルに対して著しいIFN−γ分泌細胞応答を示した(図2Cを参照)。2群のブタは、p.v.49日目(p.c.7日目)からノナペプチドのカクテルに対して著しいIFN−γ分泌細胞応答を示した。ノナペプチドのカクテルに対するIFN−γ分泌細胞応答は、対照群では観察されなかった。すべての群は、p.v.56日目にウイルスに対して著しいIFN−γ分泌細胞応答を示した。(p.c.14日目)群間で血中のウイルスDNAレベルに有意差は見られなかった。ASF抗体ELISAのブロッキング率のレベルに有意差は観察されなかった。
方法:
ポリエピトープDNAワクチンの有望な結果に基づいて、合成ASFDVAC2遺伝子にMVA 13.5プロモーター配列を付加し、公開された手順(Cottingham,2012.Methods Mol Biol 890:37−57)に従ってBAC組換え工学法によってMVA(弱毒天然痘)ワクチンベクターのTK遺伝子に挿入した。得られたウイルスをMVA−VAC2と名付けた。MVA−VAC2の挿入部分は、5’末端から、TKの左隣接部、MVA 13.5Lプロモーター、コザック配列、合成ASFDVAC2遺伝子、mH5からの終止配列、およびTKの右隣接部を含む。
非ワクチン接種動物(3群)のチャレンジ感染は、0%の生存率をもたらした(図3Aを参照)。4つのMVA組換え体すべての組み合わせでワクチン接種した(2群)10匹のブタのうち9匹が生存し、わずか数日間、軽度の病的状態のみを示した。MVA−VAC2のみをワクチン接種した1群の動物では、4匹のブタが生存した。この1群の動物は、2群の動物と比較して、病的状態をより多くかつより長い期間にわたり示した(図3Bを参照)。これらの結果は、T細胞ノナペプチドエピトープまたはASFV抗原全体のカクテルに対するIFN−γ分泌細胞応答で測定された細胞性免疫(CMI)とよく対応する(図3C)。
この試験では、ASFV T細胞ならびにB細胞エピトープを発現するMVAベクターウイルスからなる混合ワクチンを使用することにより、ASFVワクチンの開発に有望な結果が得られた。混合ワクチンは、チャレンジ後の死亡および臨床疾病に対する保護をもたらした。
Claims (16)
- アフリカ豚熱ウイルスのタンパク質由来のT細胞抗原を含むポリエピトープをコードする発現カセットを含む、組換え核酸分子、好ましくは組換えDNA分子であって、前記T細胞抗原が、プロテアソーム切断のためのシグナルを含むスペーサーによって、好ましくは、1〜10個のアミノ酸残基のスペーサーによって分離されている、組換え核酸分子。
- 前記コードされたポリエピトープが、T細胞抗原として2〜50個のペプチドを含む、請求項1に記載の組換え分子。
- 前記コードされたポリエピトープが、T細胞抗原として2〜50個のノナペプチド、好ましくは表1に示されるようにノナペプチド1〜13個および15〜20個を含み、約1〜5個のアミノ酸残基のスペーサー、好ましくは表2に示されるようにスペーサー配列1〜11によって分離されている、請求項1または2に記載の組換え分子。
- ユニバーサルT細胞エピトープをさらにコードする、請求項1〜3のいずれか一項に記載の組換え分子。
- ユビキチンのためのヌクレオチド配列を、好ましくは前記ポリエピトープの5’末端にさらに含み、前記組換え分子が、好ましくは図1Bに示される前記ヌクレオチド配列を含む、請求項1〜4のいずれか一項に記載の組換え分子。
- 請求項1〜5のいずれか一項に記載の組換え分子を含む、ウイルス粒子。
- マーカータンパク質をさらに含む、請求項6に記載のウイルス粒子。
- ブタにおける免疫応答を刺激する方法であって、請求項1〜5のいずれか一項に記載の組換え分子、および/または請求項6もしくは7に記載のウイルス粒子を、好ましくはアフリカ豚熱ウイルスのタンパク質p30、p54、p72、EP402R、A104Rおよび/またはB602Lから選択されるアフリカ豚熱ウイルスのB細胞抗原を含むウイルス粒子と組み合わせて、免疫応答を誘導するのに有効な量で前記ブタに投与することを含む、方法。
- 前記組換え分子および/または前記ウイルス粒子が、非経口的に投与される、請求項8に記載の方法。
- 前記組換え分子および/または前記ウイルス粒子が、2〜4回、好ましくは約2週間の間隔で投与され、前記組換え分子および/または前記ウイルス粒子の前記投与の少なくとも1つが、好ましくはアフリカ豚熱ウイルスのタンパク質p30、p54、p72、EP402R、A104Rおよび/もしくはB602Lから選択されるアフリカ豚熱ウイルスのB細胞抗原を含むウイルス粒子の投与と組み合わされる、請求項8または請求項9に記載の方法。
- 前記組換え分子および/または前記ウイルス粒子の前記投与の少なくとも1つが、アフリカ豚熱ウイルスのタンパク質由来の合成T細胞抗原の投与と組み合わされる、請求項8〜10のいずれか一項に記載の方法。
- ブタにおけるアフリカ豚熱ウイルスの感染および/もしくは伝播を予防または改善するための方法であって、請求項1〜5のいずれか一項に記載の組換え分子、および/または請求項6もしくは7に記載のウイルス粒子を少なくとも1匹のブタに投与することを含み、前記組換え分子および/または前記ウイルス粒子の前記投与が、好ましくはアフリカ豚熱ウイルスのタンパク質p30、p54、p72、EP402R、A104Rおよび/またはB602Lから選択されるアフリカ豚熱ウイルスのB細胞抗原を含むウイルス粒子の投与と組み合わされる、方法。
- 請求項1〜5のいずれか一項に記載の組換え分子を含むウイルス粒子と、好ましくはアフリカ豚熱ウイルスのタンパク質p30、p54、p72、EP402R、A104Rおよび/またはB602Lから選択されるアフリカ豚熱ウイルスのB細胞抗原を含む1つ以上のウイルス粒子とを含む、ウイルス粒子のセット。
- 請求項1〜5のいずれか一項に記載の組換え分子を含むウイルス粒子と、好ましくはアフリカ豚熱ウイルスのタンパク質p30、p54、p72、EP402R、A104Rおよび/またはB602Lから選択されるアフリカ豚熱ウイルスのB細胞抗原を含む1つ以上のウイルス粒子とを含む、パーツキット。
- アフリカ豚熱ウイルスによる続発性感染症からブタを保護する方法で使用するための、請求項13に記載のウイルス粒子のセット。
- アフリカ豚熱ウイルスによる続発性感染症からブタを保護する方法で使用するための、請求項14に記載のパーツキット。
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