JP4958773B2 - C型肝炎ウイルス感染の診断、予防および治療方法 - Google Patents
C型肝炎ウイルス感染の診断、予防および治療方法 Download PDFInfo
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Description
(I-1) 白血球を対象として、当該白血球に存在するC型肝炎ウイルス蛋白(HCV蛋白)を検出する工程を有する、C型肝炎ウイルス(HCV)感染の診断方法。
(I-2) 白血球が単核球である、(I-1)記載の診断方法。
(I-3) HCV蛋白の検出を当該蛋白に対する抗体を用いて行う、(I-1)または(I-2)記載の診断方法。
(I-4) HCV蛋白の検出をHCVのコア蛋白またはNS4蛋白に対するモノクローナル抗体を用いて行う、(I-1)または(I-2)記載の診断方法。
(II-1) HCV蛋白に対する抗体を有効成分とする、HCV感染予防剤。
(II-2) 抗体が、HCVのコア蛋白またはNS4蛋白に特異的に反応する抗体である(II-1)に記載の予防剤。
(II-3) 抗体が、HCV蛋白に対するモノクローナル抗体である(II-1)または(II-2)に記載の予防剤。
(II-4) 白血球へのHCV感染を阻止するための製剤である(II-1)乃至(II-3)のいずれかに記載の予防剤。
(II-5) 上記白血球が単核球である、(II-4)記載の予防剤。
(II-6) 被験者にHCV蛋白に対する抗体を有効量投与することからなる、HCV感染の予防方法。
(II-7) 抗体が、HCVのコア蛋白またはNS4蛋白に特異的に反応する抗体である(II-6)に記載の予防方法。
(II-8) 抗体が、HCV蛋白に対するモノクローナル抗体である(II-6)または(II-7)に記載の予防方法。
(II-9) HCV蛋白に対する抗体の、HCV感染予防剤の調製のための使用。
(III-1) HCV感染者の血液の中からHCVに感染した白血球を除去または低減する工程を有する、HCV感染症の治療または改善方法。
(III-2) 下記(1)〜(3)の工程を有する、(III-1)記載の治療または改善方法:
(1)HCV感染症患者の体内から血液を取り出す工程、
(2)取り出した血液から、HCVに感染した白血球を除去する工程、および
(3)工程(2)により白血球が除去または低減した血液を、上記HCV感染症患者の体内に戻す工程。
(III-3) 白血球が単核球である、(III-1)または(III-2)記載の治療または改善方法。
(IV-1) 下記(A)〜(D)の工程を有する候補抗HCV薬のスクリーニング方法:
(A)被験物質の存在下で、HCVとHCV感染していない単核球とを接触させる工程、
(B)工程(A)で得られた単核球のHCV蛋白量を測定する工程、
(C)工程(B)で測定したHCV蛋白量と、被験物質非存在下でHCVと接触させた対照単核球中のHCV蛋白の量(対照HCV蛋白量)を比較する工程、および
(D)工程(B)で得られたHCV蛋白量が対照HCV蛋白量より低い場合に、当該被験物質を候補抗HCV薬として選択する工程。
(a)被験物質を、HCVに感染した単核球を接触させる工程、
(b)工程(a)で得られた単核球のHCV蛋白の量(HCV蛋白量)を測定する工程、および
(c)工程(b)で測定したHCV蛋白量が、被験物質を接触させる前の単核球中のHCV蛋白の量、または被験物質を接触させない対照のC型肝炎ウイルス感染単核球中のHCV蛋白の量よりも低い場合に、当該被験物質を候補抗HCV薬として選択する工程。
本発明のHCV感染の診断方法は、被験者の白血球を対象として、当該白血球内に存在するHCV蛋白を検出することを特徴とする。
実施例3に示すように、HCV感染症患者の血清をHCVに感染していない健常者の白血球(単核球)に接触させると当該白血球にHCVが感染するが、この系にHCV蛋白に対する抗体(抗HCV抗体)を存在させておくと、上記白血球へのHCV感染を阻止することができる。また、実施例5(2)に示すように、公知の抗HCV薬であるインターフェロン-α(IFN-α)にも同様に、かかる白血球へのHCV感染阻止作用が認められた。さらに、実施例4に示すように、HCVに感染した白血球(単核球)を公知の抗HCV薬であるインターフェロン-α(IFN-α)とともに培養すると当該白血球中のHCV抗原量が低下する。
また本発明は、C型肝炎ウイルス感染症の治療または改善方法を提供する。
(1)C型肝炎ウイルス感染症患者の体内から血液を取り出す工程、
(2)取り出した血液から白血球を除去する工程、および
(3)(2)の工程で浄化された血液を、上記C型肝炎ウイルス感染症患者の体内に戻す工程。
(IV-1) 候補HCV感染予防薬のスクリーニング方法
前述するように、実施例3および実施例5(2)の結果から、生体のHCV感染には、白血球、特に単核球へのHCVの感染が関与しており、当該白血球に対するHCV感染を阻止することにより、生体のHCV感染を防御(予防)することができる。すなわち、白血球に対するHCV感染を阻止する作用を有する物質は、抗HCV薬、特にHCV感染予防薬として有用である。
(A)被験物質の存在下で、C型肝炎ウイルスとHCVに感染していない単核球とを接触させる工程、
(B)工程(A)で得られた単核球のC型肝炎ウイルス蛋白の量(HCV蛋白量)を測定する工程、
(C)工程(B)で測定したHCV蛋白量と、被験物質非存在下でC型肝炎ウイルスと接触させた対照の単核球中のHCV蛋白の量(対照HCV蛋白量)を比較する工程、および
(D)工程(B)で得られたHCV蛋白量が対照HCV蛋白量より低い場合に、当該被験物質を候補抗C型肝炎ウイルス薬として選択する工程。
工程(B)において単核球中のHCV蛋白量の測定は、HCV蛋白に対する抗体を用いた免疫測定法に従って行うことができる。その詳細は(I)で説明した通りであり、好ましくはサンドイッチ法によるELISA法を用いることができる。
また実施例4において、HCV治療薬として公知のIFN-αに、白血球、特に単核球中のHCV抗原量を低減させる作用があることを示した。このことは、白血球、特に単核球中のHCV抗原量を低減させる作用がある物質は、抗HCV薬、特にHCV治療薬として有用であることを示すものである。
(a)被験物質をC型肝炎ウイルスに感染した単核球と接触させる工程、
(b)工程(a)で得られた単核球のC型肝炎ウイルス蛋白(HCV蛋白)の量を測定する工程、
(c)工程(b)で測定したHCV蛋白量が、被験物質と接触させる前の単核球のHCV蛋白量より低下している場合、または被験物質を接触させない対照のC型肝炎ウイルス感染単核球中のHCV蛋白の量よりも低い場合に、当該被験物質を候補抗C型肝炎ウイルス薬として選択する工程。
(1)HCV感染症患者から入手した肝臓、心臓、腎臓、大動脈、リンパ節及び骨髄組織の各ホルマリン固定標本(一片5μmの切片)を、キシレンを用いて脱パラフィン処理した後、アルコールで脱水し、次いで過酸化水素で処理して内因性のベルオキシダーゼを不活性化した。なお、ここでHCV感染患者としては、血中の抗HCV抗体が陽性である患者を対象とした。
(1)HCV 感染症患者及びHCV非感染の健常者よりそれぞれ末梢血を採取し、フィコールパック液(アマシャム社、U.S.A.)を用いた比重遠心法により、該血液から白血球のうち単核球を分離した。これをスライドガラス上に塗布し、乾燥後、エタノールで固定した。この単核球を検体として、実施例1と同様にして免疫染色を行った。具体的には、HCV 感染症患者及びHCV非感染者の単核球に、コア抗体またはNS4抗体をそれぞれ0.2mL(20μg/mL)加えて、4℃で一夜インキュベートし、次いでマウスIgG用ベクタスタインABCキットを用いて免疫染色してDAB発色させた。
HCV 感染症患者の血清(HCV定量値:225KIU/mL)をリン酸緩衝液にて100倍希釈したもの10μLに、(1)コア抗体(実施例1に記載のもの)10μg/mL、(2)NS4抗体(実施例1に記載のもの)60μg/mL、及び(3)対照としてマウスIgG(Dako社製)10μg/mLを、それぞれ10μL添加混合し、4℃で一夜インキュベートした。
実施例3と同様にHCV感染症患者から末梢単核球を分離し、これにHCV治療薬として知られているヒトインターフェロンα(ヒトIFN-α)1×105μg/mL (PeproTech EC社, London, UK.) を加えて5日間培養した。また比較実験としてヒトIFN-αを加えないで、HCV感染者の末梢単核球を同様にして5日間培養した。次いで、これらの各培養単核球を採取して、実施例1と同様にして一次抗体としてコア抗体を用いて免疫染色を行った。
(1)培養単球細胞株を用いたHCV感染系の確立
前単球系細胞株であるU937細胞株(ATCC CRL-1593.2:American Type Culture Collection, Manassas, VA, VSA)を、10%胎児牛血清を含むRPMI培地を用いて1×106個/mLに調整し、24穴プレートを用いて培養した。これに、HCV感染症患者の血清(HCV定量値:225KIU/mL)またはHCV非感染者の血清を加え、5%炭酸ガス環境下、37℃で2〜7日間培養を行った。次いで、各培養U937細胞株を採取して、実施例1と同様にしてコア抗体を用いて免疫染色を行った。
(1)と同様に、10%胎児牛血清を含むRPMI培地を用いて培養した前単球系細胞株(U937細胞株)に、HCV感染症患者の血清(HCV定量値:225KIU/mL)を加え、さらにこれに抗HCV薬として知られているヒトINF-α(1×105μg/mL)を加えて、5%炭酸ガス環境下、37℃で2〜7日間培養を行った。比較実験として、ヒトINF-αを加えない系についても同様に、5%炭酸ガス環境下、37℃で2〜7日間培養を行った。次いで、各培養U937細胞株を採取して、実施例1と同様にしてコア抗体を用いて免疫染色を行った。
あらかじめ無菌的に乾燥させた2g のG-1 ビーズ(日本抗体研究所、高崎市、日本)を5ml のシリンジに充填し、これにヘパリンを含有するHCV感染症患者の血液1.0ml を加え、これをインキュベーター(37℃)内に設置したローターRT-5(タイテック)にセットし、1時間反応(1分間に1回転)させた。
Claims (4)
- 下記(1)および(2)の工程を有する、C型肝炎ウイルス感染の有無の検出方法:
(1)被験者の単核球を、C型肝炎ウイルスのコア蛋白またはNS4蛋白に対するモノクローナル抗体と反応させ、単核球中のC型肝炎ウイルス蛋白の有無を検出する工程、
(2)単核球内にC型肝炎ウイルス蛋白が検出される場合に、当該被験者がC型肝炎ウイルスに感染していると決定する工程。 - 下記(A)〜(D)の工程を有する候補抗C型肝炎ウイルス薬のスクリーニング方法:
(A)被験物質の存在下で、C型肝炎ウイルスとC型肝炎ウイルスに感染していない単核球とを接触させる工程、
(B)工程(A)で得られた単核球のC型肝炎ウイルス蛋白の量(HCV蛋白量)を、C型肝炎ウイルスのコア蛋白またはNS4蛋白に対するモノクローナル抗体を用いて測定する工程、
(C)工程(B)で測定したHCV蛋白量と、被験物質非存在下でC型肝炎ウイルスと接触させた対照の単核球中のHCV蛋白の量(対照HCV蛋白量)を比較する工程、および
(D)工程(B)で得られたHCV蛋白量が対照HCV蛋白量より低い場合に、当該被験物質を候補抗C型肝炎ウイルス薬として選択する工程。 - 下記(a)〜(c)の工程を有する候補抗C型肝炎ウイルス薬のスクリーニング方法:
(a)被験物質をC型肝炎ウイルスに感染した単核球と接触させる工程、
(b)工程(a)で得られた単核球のC型肝炎ウイルス蛋白の量(HCV蛋白量)を、C型肝炎ウイルスのコア蛋白またはNS4蛋白に対するモノクローナル抗体を用いて測定する工程、および
(c)工程(b)で測定したHCV蛋白量が、被験物質と接触させる前の単核球のHCV蛋白量、または被験物質を接触させない対照のC型肝炎ウイルス感染単核球中のHCV蛋白の量よりも低下している場合に、当該被験物質を候補抗C型肝炎ウイルス薬として選択する工程。 - 単核球がU937細胞株である、請求項2又は3に記載のスクリーニング方法。
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