JP2004099603A - Dna転写ユニットの接種による免疫化 - Google Patents
Dna転写ユニットの接種による免疫化 Download PDFInfo
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- JP2004099603A JP2004099603A JP2003281600A JP2003281600A JP2004099603A JP 2004099603 A JP2004099603 A JP 2004099603A JP 2003281600 A JP2003281600 A JP 2003281600A JP 2003281600 A JP2003281600 A JP 2003281600A JP 2004099603 A JP2004099603 A JP 2004099603A
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Abstract
【解決手段】脊椎動物の治療、例えば、免疫化、避妊又は腫瘍治療に用いられる、プロモーター領域に有効に結合した、目的の治療剤をコードするDNAを含有するDNA転写ユニットを含む生産物。
【選択図】なし
Description
手順
インフルエンザウイルス血球凝集素7型(H7)遺伝子を発現する、複製能を有するトリ白血病ウイルス(avian leukosis virus)をコードするpP1/H7というDNA転写ユニット(図1)を既報[ハントら(Hunt et al.)、J. of Virology, 62(8):3014-3019 (1988)]に従い構築した。pP1/H7からXbaI断片を欠失させることによって、H7を発現するがトリウイルスベクターポリメラーゼとエンベロープタンパク質を欠損している複製欠損pP1/H7誘導体をコードするDNAユニットp188(図2)を構築した。トリ白血病ウイルスベクターをコードし、かつインフルエンザウイルス挿入断片を有しないDNAユニットpRCAS(図3)を既報[フーゲスら(Hughes et al.)、J. of Virology, 61:3004 (1987)]に従い構築した。DNAユニットは0.2mlあたり100μgの濃度で食塩水に希釈して、接種に使用した。
H7発現DNA転写ユニットは、pP1/H7またはp188を接種した各ニワトリを防御した(表1)。一方、対照DNAであるpRCASを接種すると、ニワトリを致死的ウイルスチャレンジから防御することができなかった。対照群のトリは、チャレンジ後2日目から疾患徴候を示しはじめた。3日目までに、6羽の対照のトリのうちの3羽が死亡し、5日目までにすべての対照のトリが死亡した。血球凝集素発現DNAを接種されたトリは全く疾患徴候を示さなかった。チャレンジ後1.5週目までに、これら2群いずれにも高レベルのHI抗体が現われた。
複製能を欠くH7発現DNAによる免疫化で惹起された防御の再現性を評価するために、p188およびpRCASのDNAだけを接種に用いて、実施例1に説明する実験を3回反復した。この反復実験の結果、H7発現p188DNAは致死的チャレンジに対する防御をもたらしうることが確認された(表2)。p188接種ニワトリのすべてが致死的チャレンジに耐えて生存した最初の実験とは対照的に、第2、第3、および第4の実験における免疫化では、部分的な防御しか得られず、ワクチン接種を受けたトリのうち28〜83%だけが生存した。さらに、ワクチン接種を受けたトリが疾患徴候を示さなかった第1の実験とは対照的に、反復実験に耐えて生存したトリは、ほとんどのものがチャレンジ後に一時的な疾患徴候を示した。最初の実験の場合と同様、対照DNAは防御をもたらさなかった。4つの実験をまとめると、p188ワクチン接種を受けた56羽のトリのうち28羽が生存したのに対し、55羽の対照DNA接種トリのうちの1羽だけしか生存しなかった。したがって、変動があるにせよ、十分な免疫が達成されたといえる。
手順
p188−H7をコードするDNAおよび対照DNAの、致死的インフルエンザウイルスチャレンジに対抗する防御をもたらす能力を再び調べた。本実験では、3種類の接種経路(すなわちip、iv及びsc)でワクチン接種と追加免疫を行なう1群、同様に3つの接種経路でワクチン接種するが追加免疫は行なわない1群、1つの接種経路だけでワクチン接種と追加免疫を行なう複数の小さな群、および抗インフルエンザウイルス薬である塩酸アマンタジン(amantadine-HCL)で処理する対照群を設けた。最後の群は、ワクチン接種ニワトリと非接種ニワトリにおける、チャレンジウイルスに対する抗体反応を比較することができるように設けたものであった。アマンタジン処理されたトリには、チャレンジ8時間後から飲料水に0.01%のアマンタジンを混入させたものを与え、チャレンジ後24時間目と48時間目に1.0mlの0.1%アマンタジンをip注射した。
本実験の結果、複製能を欠くH7発現DNA(p188)は致死的ウイルスチャレンジに対する防御をもたらすことが確認された(表3)。p188で免疫化したトリは、チャレンジ後に一時的な疾患徴候を示した。先の実験の場合と同様、対照DNAは防御をもたらさなかった。5羽のアマンタジン処理対照トリはいずれも発病した。これらのうちの4羽はチャレンジに耐えて生存し、免疫化されたニワトリおよび免疫化されていないニワトリにおける、抗インフルエンザウイルス反応の経時変化と特異性を比較するのに使用することができる血清が提供された(以下の実施例5参照)。
手順
異なる免疫化経路の有効性を評価するために、試験群のトリの数を増やして、第3の実験を開始した。本実験では、12羽のヒヨコにiv、ip、およびsc経路で100μgのp188を接種し、8羽にはivだけ、別の8羽にはipだけで接種した。対照として、12羽のヒヨコにpRCASを接種し、別の12羽には接種しなかった。いずれの免疫化の場合も、最初の接種後4週目に追加免疫を行なった。追加免疫には、ワクチン接種の場合と同じDNA量と接種部位を使用した。追加免疫後1〜2週目に対照動物および免疫化動物に対し、ck/vic/85によるチャレンジを行なった。1〜2週以内に完全に100%の致死率が得られるよう、高いチャレンジ投与量を用いた。
ここで得られた結果でも、p188による防御が証明された(表4)。12羽のp188免疫化トリのうち8羽が生存したが、対照pRCASニワトリは12羽すべてが死亡した。未処理対照群の12羽も生存したものはなかった。ivだけでp188接種を受けた8羽のニワトリのうちの6羽は生存したが、ipだけで接種を受けた8羽のニワトリはいずれも生存しなかった。
手順
ワクチン接種ニワトリと非接種ニワトリにおける、チャレンジウイルスに対する抗体反応の比較を可能とするために、実施例2(表2)の実験2に、抗インフルエンザA型ウイルス薬である塩酸アマンタジンにより救われた非ワクチン接種群を加えた(表2)[ウエブスターら(Webster, R.G., et al.)、J. Virol. 55:173-176 (1985) ]。5羽のアマンタジン処理されたトリはいずれも発病した。これらのうちの4羽が生存し、免疫化ニワトリと非免疫化ニワトリにおける抗体反応の比較に使うことができる血清が得られた(表6)。
ワクチン接種とアマンタジンによって救われたトリにおける抗体反応の分析で、p188接種はH7に対する抗体反応を誘導することが証明された(表6)。実験1の場合と同様(表1)、DNAワクチン接種および追加免疫は、低いH7抗体力価しか誘導しなかったが、チャレンジ1週間以内に、DNA免疫化群は高いHI力価とH7中和活性を示した。これらの力価は、翌週には例えあるにしても殆ど上昇しなかった。さらに、ワクチン接種されたトリにおけるチャレンジ後抗体のほとんどがH7を標的とするものであった。この特異性は、H7ウイルス(免疫血球凝集素タイプ)およびH5ウイルス(トリに投与していない血球凝集素タイプ)のELISA抗体力価を比較することで示された。上記チャレンジ後血清は、H5ウイルスの場合よりH7の場合の方が20倍高いELISA抗体価を示した(表6)。一方、アマンタジンに救われた群では、チャレンジ後2週目までは抗体は現われなかった。この反応のほとんどはH7特異的ではなかった。このことは、H5インフルエンザウイルスとH7インフルエンザウイルスのいずれの場合もELISA抗体価が同等である、アマンタジンによって救われたトリから採取したチャレンジ後血清によって証明された(表6)。
手順
レトロウイルスDNAを欠くDNA転写ユニットは本明細書で説明する方法によってニワトリとマウスのいずれにおいても防御免疫反応を作り出す目的に効果的に使うことができることを証明するために本実験を実施した。本実験でニワトリとマウスのワクチン接種に使用したベクターを図4A〜4Cに示した。図4Aは、サイトメガロウイルス(CMV)即時型初期プロモーター(immediate early promoter)の転写制御下にインフルエンザウイルスH7サブタイプ血球凝集素を発現する能力を有するプラスミドであるpCMV−H7の概略図である。図4Bは、CMV即時型初期プロモーターの転写制御下にインフルエンザウイルスH1サブタイプ血球凝集素を発現する能力を有するプラスミドであるpCMV−H1の概略図である。このものが、マウス実験で使用したDNA転写ユニットである。図4Cは、インフルエンザ抗原発現能力を有しない対照プラスミドであるpCMVを示す。これらのプラスミドは、ブライアン−クレン博士(Dr. Brian Cullen, Duke University, Durham, North Carolina )のpBC12/CMVベクターの誘導体である。
ワクチン接種用非レトロウイルス由来ベクター(pCMV−H7)を用いる5つのニワトリ試験において(図4A)、約60%のニワトリが防御免疫を獲得した。1つのマウス試験では、6匹のワクチン接種マウスのうちの6匹全部、および6匹の対照マウスのうちの1匹だけが生存した。したがって、非レトロウイルスDNA発現ベクター(ウイルス抗原をコードするDNA転写ユニットを含有)を用いて動物をワクチン接種することによってかなりの防御が達成された。例えば表5参照。
手順
マウス適応(mouse adapted) A/PR/8/34 H1N1インフルエンザウイルスによる致死的チャレンジに対抗する免疫化をマウスに行なうために、pCMV−H1(図4Bに示す)というDNA転写ユニットを使用し、好成績を得た。この転写ユニットは、CMV即時型初期プロモーターの転写支配下にインフルエンザH1型血球凝集素をコードする。本構築物中で使用したH1インフルエンザウイルス血球凝集素遺伝子については、既報[ウインタースら(Winters et al.)、Nature 292;72 (1981) ]に詳細な説明がある。
この一連の実験で得られた生存率データ、体重減データ、および初期血清データから、多くの接種経路が防御免疫をもたらしうることがわかる。また、これらのデータは、鼻腔内接種(DNA点鼻剤をメトファン麻酔マウスに投与)は、致死的ウイルスチャレンジに対する防御免疫をもたらしうることを証明するものである。したがって、本明細書で説明する方法は粘膜免疫刺激手段(means of stimulating mucosal immunity) を提供する(表7および図5)。最後に、これらのデータは、一部の接種経路は他の経路より防御免疫反応を作り出す効果が高いことを証明するものである(表8)。
pCMV−H7ワクチン接種ニワトリにおける血清反応を分析する実験を実施例4と同様にして行なった。pCMV−H7免疫化は抗体反応を誘導し、H7に対する高い抗体力価がチャレンジ後に現われた(表9)。
Claims (12)
- 脊椎動物の治療、例えば、免疫化、避妊又は腫瘍治療に用いられる、プロモーター領域に有効に結合した、目的の治療剤をコードするDNAを含有するDNA転写ユニットを含む生産物。
- 目的の抗原に対する体液性免疫応答、細胞性免疫応答又はその両方を誘導することにより、脊椎動物の免疫化に用いる薬剤を製造するための、プロモーター領域に有効に結合した、目的の抗原をコードするDNAを含有するDNA転写ユニットの使用。
- プロモーター領域に有効に結合した、目的の抗原をコードするDNAを含有するDNA転写ユニットを脊椎動物へ投与し、それにより目的の抗原に対する体液性免疫応答、細胞性免疫応答又はその両方が誘導されることからなる脊椎動物の免疫化の方法。
- 目的の抗原が、感染源(infectious agent)に対して防御免疫応答を誘導する能力を有するものである請求項2記載の使用又は請求項3記載の方法。
- 薬剤が生理的に許容される担体を含有し、並びに粘膜内、鼻腔内、静脈内、筋肉内、腹腔内、皮内及び皮下から選ばれる経路によって投与できるものである請求項2又は4記載の使用。
- 生理的に許容される担体中にあるDNA転写ユニットが、鼻腔内、静脈内、筋肉内、腹腔内、皮内及び皮下から選ばれる投与経路を通して脊椎動物に投与されるものである請求項3又は4記載の方法。
- 生理的に許容される担体中にあるDNA転写ユニットと脊椎動物の粘膜表面とが接触することにより、DNA転写ユニットを脊椎動物に投与する請求項3又は4記載の方法。
- プロモーター領域に有効に結合した、目的の抗原をコードするDNAを含有する、生理的に許容される担体中にあるDNA転写ユニットを脊椎動物の(例えば鼻の)粘膜表面に投与し、それにより目的の抗原に対して体液性若しくは細胞性免疫応答、又はその両方が誘導され、それにより脊椎動物が感染源により引き起こされる疾患から防御されることからなる、感染源に対する脊椎動物の免疫化の方法。
- DNA転写ユニットが非レトロウイルス由来のものである上記請求項いずれか記載の生産物、使用又は方法。
- 抗原がウイルス性のものである請求項2〜9いずれか記載の使用又は方法。
- ウイルスがインフルエンザウイルス、例えば、ウイルス血球凝集素である請求項10記載の使用又は方法。
- 脊椎動物が哺乳動物、例えば、ヒトである上記請求項いずれか記載の生産物、使用又は方法。
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US20030026782A1 (en) * | 1995-02-07 | 2003-02-06 | Arthur M. Krieg | Immunomodulatory oligonucleotides |
US6207646B1 (en) * | 1994-07-15 | 2001-03-27 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
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US6127116A (en) | 1995-08-29 | 2000-10-03 | Washington University | Functional DNA clone for hepatitis C virus (HCV) and uses thereof |
US6270795B1 (en) | 1995-11-09 | 2001-08-07 | Microbiological Research Authority | Method of making microencapsulated DNA for vaccination and gene therapy |
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