JP4011881B2 - HIV drug resistance test method - Google Patents

Description

【００４０】
３．表現型（phenotype）と遺伝子型（genotype）の比較
このＭＡＧＩＣ−５Ａを用いて、得られた臨床分離株１２９検体と実験室株２検体を用いて、各薬剤の耐性検査を行った。さらに臨床分離株は、ＡＺＴ４６検体、ｄ４Ｔ４７検体、３ＴＣ４８検体、ＲＴＶ４１検体、ＳＱＶ４１検体、およびＮＦＶ４３検体について、遺伝型と解析結果と比較した。また無治療での各薬剤に対するＩＣ５０の値をＴａｂｌｅ．２に示した。無治療であるにもかかわらず、ＡＺＴ、ＲＴＶ、ＳＱＶ、ＮＦＶについては、ＮＬ４３２と比べ４倍以上耐性に傾いていることが認められた。
【００４１】
【表２】

【００４２】
以下、それぞれの薬剤に対する耐性検査結果を記載する。
ＡＺＴ（図１を参照）：ＮＬ４３２を用いて得られた値を１倍とし、無治療群でのＡＺＴに対するphenotypeでの耐性の度合いは、０．１〜５倍（平均１．２倍）であった。それに比べgenotypeでの耐性変異がアミノ酸配列番号４１，２１５位の単独もしくは、両者の組合わさった変異が認められた場合、２．６倍〜１０６倍（平均７０倍）と耐性に傾くことがわかった。また以前から報告があるように、４１，２１５，１８４位が変異すると耐性ではなく、０．５３〜１４倍（平均６．７倍）と感受性を取り戻していることが確認された。そしてこの３つの組み合わせに、６７，２１９位の耐性変異が加算された場合には、２．６〜１０６倍（１１２倍）となり、再び高度耐性を獲得していることがわかった。
【００４３】
３ＴＣ（図２を参照）：無治療群での３ＴＣに対するphenotypeでの耐性の度合いは、０．１２〜３．８６（平均１．４倍）であった。それに比べgenotypeで１８４位の耐性変異が認められた場合、すべての検体において１２０倍以上の高度耐性を獲得していた。また１８４位がwild typeであっても他の逆転写酵素阻害剤や３ＴＣの治療歴がある場合は０．１２〜３１倍（平均９．５倍）と低い耐性が認められた。
【００４４】
ｄ４Ｔ（図３を参照）：無治療群でのｄ４Ｔに対するphenotypeでの耐性の度合いは、０．４〜３．２倍（平均１倍）であった。ｄ４Ｔでは耐性に関連するアミノ酸置換は不明な部分が多く、現在知られている７５位の変異との相関を調べてみたが、当センターにおいて７５位に対する変異を獲得したウイルスは見つかっているものの、耐性変異と報告されているアミノ酸置換ではなかった。しかし３ＴＣと同様，７５位がwild typeであっても他の逆転写酵素阻害剤や３ＴＣの治療歴がある場合は０．０４〜８．１８倍（平均２．３倍）と低い耐性が認められた。
【００４５】
ＲＴＶ（図４を参照）：無治療群１５名、またＲＴＶの治療歴があるがgenotypeでの耐性変異が全く認められない６名に対するphenotypeでの耐性変異の度合いは、０．３３〜２．５３倍（平均０．８倍）であった。primary mutationである８２位がwild typeであり、secondary mutationが１つ（３６か７１位）の変異があれば０．３３〜１．３３倍であるが、２つ以上の変異が伴うと１２〜１０６倍（平均７２倍）と中等度耐性から高度耐性を獲得していた。そしてprimary mutationである８２位の耐性変異が伴うと、secondary mutationの数にかかわらず、すべて１００倍前後（平均１０５倍）の高度耐性を獲得していた。
【００４６】
ＳＱＶ（図５を参照）：無治療群１６名、またＳＱＶの治療歴があるがgenotypeでの耐性変異が全く認められない１２名に対するphenotypeでの耐性変異の度合いは、０．１〜１０倍（平均１．９倍）であった。またsecondary mutationが１つ（１０か８２位）の変異であれば、０．３４〜３．２倍であるが、primary mutationである９０位に耐性変異が認められると６．６〜１００倍（平均６０．７倍）と中等度耐性から高度耐性を獲得していた。
【００４７】
ＮＦＶ（図６を参照）：無治療群４名、またＮＦＶの治療歴があるがgenotypeでの耐性変異が全く認められない２名に対するphenotypeでの耐性変異の度合いは、０．３３〜１０倍であった。そして無治療であるにもかかわらず、secondary mutationである３６、６３、７７位の単独変異もしくは６３，７７位の組合わさった変異については、ウイルスのpolymorphismであると考えられた。そのため無治療群ではsecondary mutationが１つであれば、０．３〜４．６倍（平均１．２倍）、２つ同時に認められれば０．３〜１．５倍（平均１．３倍）とどちらも感受性を示しているのに対して、治療群については１つであれば、０．６〜１０３倍（平均２１．３倍）、２つ同時に認められれば１０．６〜３３３倍（平均１１５倍）と中等度耐性から高度耐性を獲得していた。またprimary mutationである３０位に耐性変異が認められると５３〜３３３（平均１６５倍）と高度耐性を獲得していた。
【００４８】
ＭＡＧＩＣ−５ＡとＭＡＧＩＣ−５／ＳＥＡＰ細胞を用いた薬剤耐性試験の相関：上記で基本的にＭＡＧＩＣ−５Ａを用いたBlue Cell Countで計測した検体を同時に、ＭＡＧＩＣ−５ＡとＭＡＧＩＣ−５／ＳＥＡＰ細胞を用いた感染価測定系で平行してその薬剤耐性を検討した。ＡＺＴ，ＮＶＰおよびＮＦＶについてそれぞれ２６検体を、横軸にＳＥＡＰ、縦軸にBlue Cell Countで得られた各薬剤のＩＣ５０を示す濃度をプロットすると、高濃度域から低濃度域にわたる広い濃度域で高い相関を示した。またこの相関は、実験室株ＨＩＶ−１ ＮＬ４３２に対する相対的な耐性度としてプロットしても同様に、高い相関を示した（図７及び図８を参照）。ＭＡＧＩＣ−５Ａ細胞を用いたBlue Cell CountとＭＡＧＩＣ−５／ＳＥＡＰ細胞を用いたChemiluminescence測定は、途中の培養過程に係わる労力はほとんど変わらないが、最終段階の測定の際、Blue Cell CountはComputer Softによる画像処理によりその労力は軽減するもの、マイクロプレート１枚を読み切るには１時間弱の時間がかかるのに対して、マイクロプレート対応のLuminometerを用いれば数十秒で測定が終了するのは、多検体処理が必要な際は、かかる労力は大きくことなる。検出感度の点では、例えば９６穴マイクロプレートの穴に接種された検体中に感染性ＨＩＶ−１が十数個の時でも、ＭＡＧＩＣ−５Ａ細胞では固定して直接顕微鏡下で判定できる利点があるが、ＭＡＧＩＣ−５／ＳＥＡＰでもＨＩＶ−１非感染のバックグラウンドの数倍の有意な化学発光値が得られ、なおかつ培養を継続して翌日にはより明確な計測値が得られる利点もある。しかしながら、薬剤耐性試験に関しては、培養の初めに充分な感染価（１００〜３００ｂｃｃ／ｗｅｌｌ）のウイルスを接種するので実際には問題にはならない。またデータには示さないが、ｐ−ニトロフェノールホスフェートを基質とした比色反応でも化学発光での測定系と比べて感度の点では劣るものの、充分実用に耐える成績が得られた。
【００４９】
次に、新たに許可された抗ＨＩＶ薬であるアバカビア（ＡＢＣ）、ネビラピン（ＮＶＰ）およびアンプレナビア（ＡＰＶ）について、標準ウイルス株としてＨＩＶ―１ＮＬ４３２を用いて薬剤耐性試験の再現性を検討した。その結果、表３に示すように良好な再現性が得られた。以下、それぞれの薬剤に対する耐性試験結果を記載する。
【００５０】
【表３】

【００５１】
アバカビア（ＡＢＣ）（図９を参照）：逆転写酵素阻害剤であるアバカビア（ＡＢＣ）は、６５，７４，１１５，１８４の耐性変異が知られている。無治療者では平均して１６．２倍と耐性に傾いた。さらに１８４に７４の耐性変異が加わると２６．１倍と耐性度が上昇した。
【００５２】
ネビラピン（ＮＶＰ）（図１０を参照）：非拡散系逆転写酵素阻害剤であるネビラピン（ＮＶＰ）は１０３，１０６，１０８、１８１，１９０の耐性変異が知られている。これらはすべてＮＶＰに対するprimary mutationである。無治療者が１．０５倍であり、逆転写酵素阻害剤を使用したことがある患者においても１．５１倍と感受性の範囲であった。つまり逆転写酵素阻害剤とネビラピンでは交差耐性は認められなかった。また１８１の変異の２７．６倍を除いて、そのほか全ての（１０６、１０３、１９０）耐性度が１００倍以上と値が振り切れており、高度耐性を獲得していた。また、非拡散系逆転写酵素阻害剤のほとんどの耐性変異は交差耐性として認められているが、唯一ＮＶＰの１０６の耐性変異はＥＦＶ（エファビレンツ）に対して交差耐性が認められていない。今回測定した１０６の耐性変異が認められた検体は、ＮＶＰに対して１３１倍以上と高度耐性であるが、ＥＦＶに対して５．７倍と耐性を獲得していないことがわかった。
【００５３】
アンプレナビア（ＡＰＶ）（図１１を参照）：無治療者では１．４７倍であり、また無治療でありながらポリモフィズムとして１０に変異を獲得している検体でも１．０４倍と感受性の範囲であった。しかし同じ１０に変異が認められ、治療を行っているものについては１９．６倍と耐性度が上昇していることがわかった。さらにアンプレナビアのsecondary mutationである１０，４６，８４がそろうと、平均して２３２倍と耐性度が上昇していた。プロテアーゼ阻害剤であるアンプレナビアは、primary mutationである５０が他のプロテアーゼ阻害剤に対する耐性変異ではないため交差耐性の少ない薬剤として知られていたが、しかし、secondary mutationだけでも複数存在すれば他のプロテアーゼ同様耐性に傾くことがわかった。また以前にＰａｒｋｉｎらによると過去にＩＤＬ、ＮＦＶを使用した患者の２０％で見られるＮ８８Ｓの変異は、ＡＰＶに対しても感受性が戻ると報告（Journal of Viorology, 2000, 4414-4419）していた。今回のアッセイにおいても４６の耐性変異が認められる群では２４．２倍耐性であるのに対し、４６にＮ８８Ｓが認められていると５．５倍と感受性が戻り、これらの患者は過去にＮＦＶによる治療を受けていたことがわかった。
【００５４】
（考察）
現在、薬剤耐性変異ウイルスを検出するためには、（１）薬剤作用領域の塩基配列の変異を調べる遺伝子型（genotype法）と（２）直接ＨＩＶを薬剤存在下で培養し、増殖を抑えることのできる濃度（ＩＣ５０、ＩＣ９０）を求めて評価する表現型（phenotype法）、そして（３）組換え（recombinant）ＤＮＡ技術を利用し、患者由来の感染性recombinant ＨＩＶを作り、phenotype法を行うrecombinant virus assayの３つが挙げられる。このうち短時間で、操作性にも優れ、比較的容易に結果が得ることができるgenotype法が主流となっているが、その反面、報告されていない耐性変異の出現や複数の変異蓄積した場合での総合的な判断は難しいとされている。それに比べ、耐性度を総合的に判断することができるphenotype法での検出方法は、現在急速に改善されつつあるとはいえ、いまだに時間や費用、労力が必要であると考えられ、一般的には行われていない。またrecombinant virus assayについても、患者由来のウイルスの薬剤作用領域をＨＩＶベクタープラスミドに組み込む技術が非常に熟練を要し、得られた組換えウイルスが増殖しないことも多く、さらにｐｏ１遺伝子などの薬剤作用領域以外の領域がＨＩＶ−１粒子形成に関与していることが知られており、得られた組換えウイルスが実際の患者体内のウイルスの性状を反映しているか判明していないことからphenotype法と同様、一般的にはおこなわれていない。
【００５５】
そこで今回本発明者らは、ＭＡＧＩＣ−５ＡとＭＡＧＩＣ−５／ＳＥＡＰ細胞株によるphenotype法を用いて、これらの問題を解決し、臨床分離株から直接、感受性試験を迅速に行うことを目的とし、検査を行った。従来でのＰＢＭＣを用いたphenotype法では、結果が得られるまでに数ヶ月を要し、また判定するのにｐ２４を測定するという点でコストがかかるが、この細胞を用いることによりウイルス分離から薬剤感受性試験までわずか２週間ほどで結果が得ることができ、細胞の染色もしくは培養上澄の酵素活性測定により感染の有無を迅速に判定することができた。
【００５６】
本実施例の結果から、無治療の患者ではコントロールであるＮＬ４３２に比べ、耐性の度合いが平均して１倍前後であったが、中には１０倍という検体が認められた。これらのウイルスは、genotype法でも耐性変異が認められていないにもかかわらず、phenotype法では低度耐性を獲得していることが判明し、今後臨床において治療開始時の薬剤選択の判断に参考となるデータであることがわかった。さらにgenotype法とphenotype法を比較したところ、アミノ酸配列番号７５位の耐性変異を検出出来なかったｄ４Ｔを除く薬剤については、良好な相関が得られた。しかし３ＴＣ、ｄ４Ｔについては、wild typeであっても、逆転写酵素阻害剤の治療歴があるものでは、低度耐性が認められた。
【００５７】
またＡＺＴについても４１，１８４，２１５位のアミノ酸変異が同時に認められた場合は、感受性が復帰していることも、この細胞を用いて確認された。しかしこのようなアミノ酸変異によって起こる感受性の変化が、酵素と基質との反応、相互作用という点からどのように起こっているかについては、現段階の技術と知識では明らかにされていない。さらにプロテアーゼ阻害剤では、primary mutationが認められると１００倍前後の高度耐性を示し、genotype法とphenotype法で得られた結果が一致したことが認められた。ＨＩＶ−１ウイルスは薬剤投与下に適応するためsecondary mutationを獲得することが知られている。他のプロテアーゼ阻害剤で治療歴のある患者では、ＮＦＶに対する反応が５０％程度しかないということが報告されたが、今回のこのようなケースにおいてＮＦＶに対するprimary mutationが認められなくてもsecondary mutationが見つかった。このsecondary mutationは、無治療の患者では感受性に示しているものの、プロテアーゼ阻害剤に治療歴がある患者については、耐性を示しているということが、phenotype法により示唆された。これらの成績からＭＡＧＩＣ−５Ａを用いたphenotype法は、genotype法で判断できなかった耐性の度合いを明確にし、今後の多剤併用療法で使用する薬剤選択の判断の一つとして有用であると考えられた。
【００５８】
薬剤耐性試験におけるＭＡＧＩＣ−５ＡとＭＡＧＩＣ−５／ＳＥＡＰ細胞を用いた成績の比較から、両者間では高い相関が得られた。このことはＭＡＧＩＣ−５／ＳＥＡＰ細胞がＭＡＧＩＣ−５Ａ細胞を親株としてさらにＨＩＶ−１感染によりＳＥＡＰを細胞外に分泌するように改変したことからも予測された。しかもReporterとしてはＳＥＡＰを用いた系の方がβ−ガラクトシダーゼに比べ、化学発光試薬を加えたあとの安定性及び操作の迅速簡便性においても格段に優れていた。検出感度の点についてもＳＥＡＰの方が優れていたため、接種するウイルス量が少なくても測定が可能であった。またＭＡＧＩＣ−５／ＳＥＡＰを用いたＡｓｓａｙ系は、細胞を播き、薬剤と同時にウイルスを接種し、培養上澄を採取して酵素基質液を添加して測定機にかけるのみで実測値が得られ、現在用いられているｐ２４ ＥＬＩＳＡあるいはＲＴ Ａｓｓａｙとくらべ操作手順は格段に簡略化されており、Work Stationによるロボット化への変換も可能であると考えられる。ＭＡＧＩＣ−５Ａ細胞株による患者検体からのウイルス分離とＭＡＧＩＣ−５／ＳＥＡＰによる薬剤耐性Phenotyping Assayにより概略２週間程の短期間で薬剤耐性が判明することは、早期の耐性ウイルスの検出と、感受性薬剤の選定に有用であり、今後新たに承認される薬剤についても測定を行い、Conbination Therapyとして様々な薬剤の組み合わせに対応しえるHigh Throughputな薬剤耐性試験系の構築になくてはならない測定系となることが期待される。
【００５９】
また、新たに許可された抗ＨＩＶ薬であるＡＢＣ、ＮＶＰおよびＡＰＶについても本試験法により、効率的なおかつ鋭敏に耐性ウイルスを検出しえることが判明した。このことは今後、さらに開発され臨床応用が期待されている様々な抗ＨＩＶ薬についても本試験が対応しえる柔軟性をもつことを意味する。
【００６０】
【発明の効果】
本発明により、ＨＩＶ−１感染価測定細胞株を用いた迅速簡便な薬剤耐性試験方法、特に多検体を迅速に処理することができる薬剤耐性試験方法を提供することが可能になった。
【図面の簡単な説明】
【図１】図１は、表現型でのＡＺＴ耐性と遺伝型との相関関係を示す図である。
【図２】図２は、表現型での３ＴＣ耐性と遺伝型との相関関係を示す図である。
【図３】図３は、表現型でのｄ４Ｔ耐性と遺伝型との相関関係を示す図である。
【図４】図４は、表現型でのＲＴＶ耐性と遺伝型との相関関係を示す図である。
【図５】図５は、表現型でのＳＱＶ耐性と遺伝型との相関関係を示す図である。
【図６】図６は、表現型でのＮＦＶ耐性と遺伝型との相関関係を示す図である。
【図７】図７は、ＭＡＧＩＣ−５／ＳＥＡＰ細胞を用いた化学発光アッセイとＭＡＧＩＣ−５Ａ細胞を用いたBlue Cell Countとにおける、抗ＨＩＶ−１剤濃度に対する相関関係を示す図である。
【図８】図８は、ＭＡＧＩＣ−５／ＳＥＡＰ細胞を用いた化学発光アッセイとＭＡＧＩＣ−５Ａ細胞を用いたBlue Cell Countとにおける、抗ＨＩＶ−１剤濃度に対する相関関係を示す図である。
【図９】図９は、表現型でのＡＢＣ耐性と遺伝子型との相関関係を示す図である。
【図１０】図１０は、表現型でのＮＶＰ耐性と遺伝子型との相関関係を示す図である。
【図１１】図１１は、表現型でのＡＰＶ耐性と遺伝子型との相関関係を示す図である。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method for testing drug resistance of HIV. More specifically, the present invention relates to a method for testing HIV drug resistance by detecting a reporter protein secreted into a culture supernatant by a target cell infected with HIV-1 in the presence of a test drug.
[0002]
[Prior art]
Human Immunodeficiency Virus type 1 (HIV-1) is a retrovirus belonging to Lentivirus, and is a pathogen of acquired immune deficiency syndrome AIDS. Already more than ten years after its discovery, initially, the immune system of the infected host was inevitable destroyed, and most of the infected people were recognized as infectious diseases resulting in immunodeficiency and death. Recently, however, various inhibitors of reverse transcriptase essential for HIV-1 Life Cycle and proteases involved in cleavage of mature protein necessary for virus particle formation have been developed, and combinations thereof are powerful anti-HIV-1 agents. (So-called HAART therapy (Highly Active AntiRetrovirus Therapy)) makes it possible to keep the amount of virus in the infected body below the detection limit by a highly sensitive PCR method. Infectious diseases have reached the point where they are considered chronic infections.
[0003]
However, when multidrug administration is discontinued, HIV-1 rapidly proliferates, and due to the high mutation rate of reverse transcriptase, which is a characteristic of this virus, a virus resistant to the administered drug easily appears and is not It has been reported that the selection of a prepared drug causes a virus resistant to a plurality of drugs based on the same mechanism of action, resulting in difficulty in subsequent treatment.
[0004]
In the current situation, where curative treatment is unlikely to be available in the near future, anti-HIV-1 therapy strategies can quickly detect the emergence of resistant viruses, combine the most effective drugs with individual patient viruses, It is thought that the center of gravity shifts in keeping the amount of virus in a low steady state. Genotyping and Phenotyping drug resistance tests are examples of criteria for effective drug selection in HIV treatment methods diversified using a wide variety of drugs, including newly developed anti-HIV-1 drugs.
[0005]
In Genotyping, amplification of the HIV-1 pol gene region by Primer Design and PCR method and analysis of its base sequence have already been made into a kit, and have been technically established and spread. However, it is often pointed out that there is a discrepancy between the drug resistance mutation due to the amino acid mutation of the gene encoding the reverse transcriptase or protease of the virus genome in the sample and the results regarding the biological drug resistance of the patient virus.
[0006]
On the other hand, although Phenotyping reflects actual virus resistance, it is pointed out that the method using peripheral blood mononuclear cells (PBMC) currently used as a standard method still has many problems to be solved in the standardization. Has been. For example, resistance tests using human PBMC often take several months to obtain results, and it is difficult to compare results obtained due to variations in HIV susceptibility among PBMC donor lots. However, it is not suitable for processing many specimens because it requires labor, and the cost required for resistance testing is increased due to measurement of virus growth by HIV-1 gag protein p24 ELISA. The current situation is not done. However, since the Phenotyping method reflects the biological resistance of the virus, it provides indispensable information as a criterion for drug selection.
[0007]
In order to overcome these problems, the present inventors have reported a drug resistance test using a rapid and simple HIV-1 infectivity titer measuring cell line MAGIC-5A (Reiko Hachiya, Saori Aizawa, Mari Tanaka, Yukiko Takahashi). , Yoshihiro Hirabayashi, Setsuko Ida, Masashi Tsuji, Shinichi Oka, Examination of anti-HIV drug resistance test using CCR5-expressing HeLa / CD4-LTR-βGal cells (MAGIC5 clone 1-10) December 1999 Japan Society for AIDS Research Tokyo, Hachiya, A., Aizawa-Matsuoka, S., Tanaka, M., Takahashi, Y., Ida, S., Gatanaga, H., Hirabayashi, Y., Kojima, A., Tatsumi, M. And Oka , S .: Rapid and simple phenotypic assay for drug susceptibility of human immunodeficiency virus type 1 by using CCR5-expressing HeLa / CD4 cell clone 1-10 (MAGIC-5) Antimicrob. Agents Chemother. 45: 495-501, 2001.) . This cell line MAGIC-5A was transformed to stably express the HIV-1 receptor CD4 and co-receptors CXCR4 and CCR5 in the human cervical cancer cell line HeLa, and the Tat protein produced by the infected HIV-1 Is an indicator cell designed to drive β-Galactosidase with added SV40 nuclear translocation signal downstream of the incorporated LTR and to measure the infectious titer of HIV-1 in a sample specimen quickly and easily by X-Gal staining. is there. It has been reported that by using this cell line, rapid virus isolation from patient serum and subsequent detection of resistant strains against anti-reverse transcription and anti-protease drugs can be detected within 2 weeks.
[0008]
In addition, since this cell line constitutes the basic technique of measuring the infectious titer of HIV-1, it may be applicable to many HIV-1 research areas. So far, it has been applied to drug-resistant Phenotype Assay and anti-HIV drug screening. Although it is quicker, simpler and more economical than the commonly used HIV-1 gag protein p24 measurement by ELISA or reverse transcriptase activity measurement, the HIV infectivity titer measurement by this cell line is a microscope. Since it is based on the count of cell nuclei stained with X-gal below, it was not suitable for rapid multi-sample processing. In the future, since development of anti-HIV-1 drugs has progressed and many drugs are expected to be applied clinically, a quicker and simpler measurement system has been demanded.
[0009]
[Problems to be solved by the invention]
The present invention has been made to solve the above-described problems of the prior art. That is, this invention made it the subject which should be solved to provide the quick and easy drug-resistance test method using the HIV-1 infectivity titer measuring cell line. In particular, an object of the present invention is to provide a drug resistance test method capable of rapidly processing multiple specimens.
[0010]
[Means for Solving the Problems]
As a result of diligent studies to solve the above problems, the present inventors have developed an indicator cell MAGIC-5A with an LTR for the purpose of further rapidly and easily evolving a Phenotyping drug resistance test using MAGIC-5A. We succeeded in establishing a high throughput measurement system by further incorporating an expression unit incorporating SEAP (secreted alkaline phosphatase) downstream, and detecting alkaline phosphatase secreted into the culture supernatant by HIV infection by chemiluminescence. . Furthermore, it has been found that drug resistance tests using these cells can evaluate the drug resistance of HIV against various anti-HIV drugs. The present invention has been completed based on these findings.
[0011]
  That is,The present invention relates to an animal cell transformed so as to stably express the HIV-1 receptor CD4 and co-receptors CXCR4 and CCR5 in human HeLa cells, as a secreted reporter protein downstream of the HIV-1 LTR sequence. Human HeLa cell-derived animal cell line FERM P-18078 capable of expressing a secreted reporter protein by HIV-1 infection by introducing an expression vector having a gene encoding secreted alkaline phosphatase SEAP Using the established animal cell line, the animal cell line is brought into contact with a sample containing HIV-1 in the presence of the test drug, and the culture supernatant is obtained by HIV-1 infection. By detecting the reporter protein secreted by Quickly, in which it succeeded in establishing a method of testing drug resistance of HIV that can be tested with high sensitivity.
[0012]
Preferably, the secretory reporter protein is a protein that can be detected by one or more measurement systems of colorimetric reaction, fluorescence, or chemiluminescence. Particularly preferably, the secreted reporter protein is alkaline phosphatase, luciferase or peroxidase.
[0013]
  BookAnimal cells used in the invention are cells obtained by transforming an expression vector having a gene encoding a secreted reporter protein downstream of the HIV-1 LTR sequence.. BookAnimal cells used in the invention are cells derived from mammalian animal cells,Human HeLa cellsIt is a derived cell. Used in the present inventionMovementThe animal cell is an animal cell having a deposit number of FERM P-18078stockIt is.
[0014]
Preferably, the test drug is an anti-HIV drug containing a reverse transcriptase inhibitor or a protease inhibitor, and a combination of two or more drugs can be used as the test drug.
Preferably, the reporter protein secreted in the culture supernatant is detected by one or more measurement systems of colorimetric reaction, fluorescence emission or chemiluminescence.
[0015]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the present invention will be described in detail.
In the method for testing drug resistance of the present invention, an animal cell capable of expressing a secreted reporter protein by HIV-1 infection is contacted with a sample containing HIV-1 in the presence of a test drug, and cultured by HIV-1 infection. It is characterized by detecting a reporter protein secreted into the supernatant. First, an animal cell capable of expressing a secreted reporter protein by HIV-1 infection used in the present invention will be described.
[0016]
  Animal cells used in the present inventionAs animal cells for establishing strainsIs not particularly limited as long as it is a cell capable of being infected by HIV-1.But,Usually, HIV-1 virus receptor CD4 is required for HIV-1 infection, and therefore it is preferable that the animal cells used in the present invention have HIV-1 virus receptor CD4. In addition, as described above in this specification, in addition to CD4 molecule, which is a major viral receptor on the cell side, HIV cell-directed HIV-1 induces CXCR4, macrophage-directed in the process of HIV-1 entry into target cells. Since HIV-1 uses CCR5 as a co-receptor, it is preferable that the animal cells used in the present invention also have these co-receptors. When it is intended to analyze a broad range of HIV-1 infectivity, it is preferable to use animal cells expressing both CXCR4 and CCR5.
[0017]
These HIV-1 virus receptor CD4 and HIV-1 virus co-receptor may be used as they are when they are originally retained and expressed by the starting animal cell. If the starting cell does not express them, a recombinant expression vector in which a gene encoding the above receptor or co-receptor is incorporated into a suitable expression vector is introduced into the starting animal cell, so that the receptor or The co-receptor can be expressed in the animal cell.
[0018]
The type of starting cell that can be used to establish animal cells used in the present invention is not particularly limited, but is preferably a mammal (eg, a primate such as a human or a monkey, or a rodent such as a mouse, rat or hamster). And more specifically, human-derived HeLa cells or human-derived HOS cells.
[0019]
As a technique for selecting cells expressing the receptor or co-receptor of interest, an antibiotic resistance gene can be expressed together with the receptor or co-receptor in a recombinant expression vector containing the receptor or co-receptor. And cells that express the target receptor or co-receptor can be selected by culturing in a medium containing the antibiotic. A limiting dilution method can be used as a method for cloning a target cell as a single cell derived from one cell.
[0020]
For example, when a HeLa cell constitutively expressing CXCR4 is used as a starting cell, a mouse retroviral vector carrying an antibiotic resistance gene (for example, a neomycin (Neo) resistance gene) is transformed with the HeLa cell. CD4 is expressed by conversion, and if desired, the LTR-βGal expression unit is transformed together with a hygromycin resistance gene to select growing cells. Further, an expression vector for imparting CD4 expression and Puromycin resistance to the transformant for enhancing CD4 expression, and an expression vector for conferring CCR5 expression and Blasticidin resistance to the transformant are transformed into the above cells, and by the limiting dilution method. Clone the cells of interest. The target cells cloned by this operation are resistant to a total of four selective drugs, G418, Hygromycin, Puromycin and Blasticidin.
[0021]
The animal cell used in the present invention is characterized in that it can express a secreted reporter protein by HIV-1 infection. The animal cells used in the present invention express a secreted reporter protein by HIV-1 infection, and the expressed secreted reporter protein is secreted outside the cell and released into the culture medium. By adding a sample containing HIV-1 to a culture system containing animal cells used in the present invention, collecting the culture solution, and detecting or analyzing the reporter protein secreted in the culture solution by an appropriate method, The infectious titer of HIV-1 in the sample can be measured.
[0022]
Although the detection method of the reporter protein secreted in the culture solution is not particularly limited, for example, a method of detecting by a colorimetric reaction, fluorescence emission, chemiluminescence, or the like can be mentioned. The type of secretory reporter protein is not particularly limited, but it is preferable that various enzyme-active substrates are easily available, have high measurement sensitivity, and can simplify the measurement technique, such as alkaline phosphatase, luciferase, or peroxidase. Of these, alkaline phosphatase is particularly preferable.
[0023]
As one of preferable methods for producing an animal cell that expresses a secreted reporter protein by HIV-1 infection according to the present invention, an expression vector having a gene encoding a secreted reporter protein downstream of the HIV-1 LTR sequence is used. A method for transforming animal cells can be mentioned. The expression vector used in the above method preferably contains a drug resistance gene for selecting a desired cell.
[0024]
For example, a secreted reporter protein is expressed against cells that are resistant to a total of four selective drugs, G418, Hygromycin, Puromycin, and Blasticidin as described hereinabove, and that express CXCR4, CD4, and CCR5. Animal cells used in the present invention can be prepared by transforming an expression vector having the gene to be encoded. In this case, the drug resistance gene that can be incorporated into an expression vector having a gene encoding a secreted reporter protein may be other than the above four drug resistance genes, and examples thereof include a Zeocin resistance gene.
[0025]
More specifically, an expression vector having a gene encoding a secreted reporter protein is transfected into a cell having HIV-1 virus receptor CD4 and HIV-1 virus co-receptor and selected with a drug to obtain a surviving colony be able to. Cloned cells that have been grown on these are plated twice on a microplate, one infected with HIV-1 and the other as a non-infected control. Two days after infection, the culture supernatant is collected and treated at 65 ° C. for 15 minutes to inactivate endogenous alkaline phosphatase and HIV. SEAP secreted into the culture supernatant can be measured and selected, for example, by a colorimetric reaction using p-Nitrophenol phosphate as a substrate. The target cells can be established by selecting clones that have low SEAP activity when HIV is not infected and exhibit high SEAP activity due to HIV infection.
An example of a cell that can be established by the above-described method is a cell having a deposit number of FERMP-18078.
[0026]
The animal cell culture method and culture conditions used in the present invention can be appropriately selected according to the cell type and properties. For example, in the case of a cell line derived from HeLa cells, 5% CO at 37 ° C. in Dulbecco's Modified Minimum Essential Medium (High glucose; Gibco) containing antibiotics (for example, Kanamicin etc.) and supplemented with 5% FCS.₂Can be cultured.
[0027]
In the method for testing drug resistance of the present invention, an animal cell as described above is contacted with a sample containing HIV-1 in the presence of a test drug, and a reporter protein secreted into the culture supernatant by HIV-1 infection is detected. It is characterized by that.
The test drug is preferably an anti-HIV drug, and more specifically, an anti-HIV drug containing a reverse transcriptase inhibitor or a protease inhibitor is preferable. As reverse transcriptase inhibitors, AZT / ZDV (zidovudine: 3'-Azido-3'-deoxythymidine), d4T (sanilvudine: 2 ', 3'-didehydro-3'-deoxy-thymidine), 3TC (lamivudine: 2 '-deoxy-3'-thiacytidine), COM (ZDV / 3TC mixture), ddI (didanosine: 2', 3'-dideoxyinosine), ddIEC (didanosine: 2 ', 3'-dideoxyinosine), ddC (zalcitabine: 2 ', 3'-dideoxycytidine), ABC (abacavir), NVP (nevirapine), EFV (efavirenz), DLV (delavirdine), and the like, and protease inhibitors include RTV (ritonavir), SQV (saquinavir), NFV ( nelfinavir), IDV (indinavir), APV (amprenavir), LPV (ritonavir) and the like.
Moreover, a combination of two or more kinds of drugs can be used as the test drug.
[0028]
To carry out the method of the present invention, first, animal cells capable of expressing a secreted reporter protein by HIV-1 infection are inoculated on a microplate, and a sample containing HIV-1 is diluted stepwise with a medium. Add to each hole. Furthermore, an appropriate dilution series of anti-HIV drug is added, and appropriate culture conditions (for example, 5% CO at a temperature of 37 ° C.₂), And the supernatant of each well is collected. It is then heated for 15 minutes at 65 ° C. for inactivation of endogenous reporter protein and HIV. Using this supernatant, the reporter protein can be assayed according to a conventional method. When the reporter protein is alkaline phosphatase, for example, a colorimetric reaction using p-nitrophenol phosphate as a substrate, fluorescence emission using MUP as a substrate, and chemiluminescence measurement system using CSPD as a substrate can be used. . The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to the examples.
[0029]
【Example】
Example 1: Establishment of animal cells capable of expressing secreted reporter protein by HIV-1 infection
All HeLa cell-derived cell lines contain 50 μg / ml Kanamicin as an antibiotic and Dulbecco's Modified Minimum Essential Medium (High glucose; Gibco) supplemented with 5% FCS at 37 ° C. with 5% CO2.₂In culture. The CCR5-expressing MAGI cell line MAGIC-5A already established by the present inventor expresses CD4 by a mouse retroviral vector in which a Neo resistance gene is placed on the original parent strain HeLa cell, and the LTR-βGal expression unit together with the Hymymycin resistance gene Expression vector pEFBOSpacCD4 (confers CD4 expression and Puromycin resistance to Transformant) and expression for CCR5 expression on MAGI cell line, which is a cell line selected by FACS (Fluorescent Activated Cell Sorter) A cell line transformed with the vector pEFBOSbsr (confers CCR5 expression and Blasticidin resistance to Transformant) and established by limiting dilution. It is resistant to a total of four selective drugs: G418, Hygromycin, Puromycin and Blasticidin. It was.
[0030]
Construction of an expression vector that incorporates secreted alkaline phosphatase (SEAP) as a reporter gene from pSEAP2-Basic (Clontech) and Transcriptional Blocker downstream of the LTR derived from HIV-1 subtype B, and further carries a Zeocin resistance gene as a new selection gene did. After transfection of this expression vector into the MAGIC-5A cell line, 165 colonies were obtained by selective culture with Zeocin. These expanded clonal cells were double seeded in 96-well microplates, one infected with HIV-1 and the other as a non-infected control. Two days after infection, 100 μl of culture supernatant was collected, treated for 15 minutes at 65 ° C. for inactivation of endogenous alkaline phosphatase and HIV, and stored at −20 ° C. until measurement. Secreted SEAP in the sample was measured and selected by a colorimetric reaction using p-nitrophenol phosphate added with L-Homoarginine as an endogenous alkaline phosphatase inhibitor (Berger J. et al., Secreted placental alkaline phosphatase). : a powerull new quantitative indicator of gene expression in eukaryotic cells. Gene 66: 1-10, 1988).
[0031]
A clone having a low SEAP activity in non-HIV infection and a high SEAP activity by HIV infection was selected and subjected to serial dilution twice in the absence of a selective agent. That is, a total of 162 surviving colonies were obtained by selective culture in the presence of 200 μg / ml Zeocin. However, most surviving colonies secreted high SEAP with or without HIV infection. Clone MAGIC that showed stable HIV-1 susceptibility by two limiting dilutions from a group of cells that could detect only about Backgroud in HIV non-infected from two few colonies and secreted a large amount of SEAP by HIV infection. -5 / SEAP 3-1-1 was established.
MAGIC-5 / SEAP 3-1-1 is FERM P-18078 as of October 16, 2000, Institute of Biotechnology, Institute of Industrial Technology, 1-3 1-3 Higashi, Tsukuba City, Ibaraki, Japan ( Postal code 305-8856).
[0032]
Example 2: Drug resistance test
(Method)
1. Target person and virus isolation
From December 1997 to January 1999, 245 specimens of 137 patients who visited the National Medical Center AIDS Treatment Research and Development Center were examined. Blood was collected from a patient who gave consent and the plasma was isolated. MAGIC5A suspended in Dulbecco's MEM medium supplemented with 5% FCS the day before isolation (Masashi Tsuji, Asato Kojima: Establishment and application of CCR5-expressing HeLa / CD4-LTR-beta Gal cells suitable for HIV-1 infectivity measurement The 10th Annual Meeting of the Japan Society for AIDS Research, Kumamoto, December 1997. Tsumi, M. and Kojima, A .: Establishment of HeLa / CD4-LTR = bGal expressing CCR5 suitable for infectivity assay of T- and M-tropic HIV-1 The XIIth International Congress of AIDS, Geneve, Switzerland, June, 1998) was seeded at 20,000 cells / well in a 48-well microplate, and the cells were allowed to adhere overnight. 1 ml of plasma derived from the patient on the day of infection was centrifuged at 15,000 rpm for 40 minutes in a microcentrifuge to precipitate the virus, 900 μl of supernatant was removed, and 400 μl of Infection Medium (20 μg / ml DEAE-Dexran 5% FCS-D′MEM) was added. In addition, the suspension was inoculated into MAGIC-5A cells after suspension. 37 ℃ 5% CO₂After culturing in an incubator for 2 hours, the culture medium was replaced with 5% FCS-D'MEM medium, and then the observation culture was continued until virus separation was positive. The isolated virus was stored at −80 ° C. until use.
[0033]
1. Drug resistance test
Infectious titer measurement:
The day before infection, inoculate and culture MAGIC-5A cells in a 96-well microplate at 10,000 cells / well. On the day of infection, the virus was thawed and serially diluted 1- to 1000-fold with Infection Medium, and 100 μl was inoculated into each well. Then, the cells were cultured for 2 hours. After washing, a fresh culture solution was added and further cultured. After 48 hours, the culture solution was removed, washed with PBS, fixed solution (1% formaldehyde, 0.2% glutaraldehyde, in PBS) was added at 100 μl / well, fixed at room temperature for 5 minutes, washed again with PBS, and stained. Liquid (4 mM potassium ferrocyanate, 4 mM potassium ferricyanate, 16 mM MgCl₂, 400 μg / ml X-gal in PBS) was added at 100 μl / well, incubated at 37 ° C. for 2 hours, and the number of cells in which the cell nucleus was stained blue was counted under a microscope to obtain the HIV-1 infectivity titer in the sample specimen. In the subsequent drug resistance test, the specimen was diluted and used so as to be 100 to 300 bcc (Blue cell count) in each hole of the 96-well microplate.
[0034]
Reverse transcriptase inhibitors:
On the day before infection, MAGIC-5A or MAGIC-5 / SEAP cells were seeded at 10,000 cells / well in a 96-well microplate and cultured to allow the cells to adhere beforehand. On the day of infection, 100 to 300 bcc of virus was diluted with Infection Medium, and 100 μl was added. Further, 100 μl each of antiviral drugs prepared in a 10-fold dilution series was added, and the mixture was incubated at 37 ° C., 5% CO 2.₂The cells were cultured in an incubator. After 48 hours, MAGIC-5A cells were stained as described above, and the number of Blue cells was counted, while MAGIC-5 / SEAP cells (accession number FERM P-18078) were collected from the culture supernatant, The alkaline phosphatase activity was measured with a Luminometer using Reporter Assay Kit-SEAP- (SAK-101; Toyobo Co. Japan), and at a wavelength of 405 nm in a colorimetric reaction using p-nitrophenol phosphate as a substrate. did. The value of wells to which no drug was added was defined as 100%, and the concentration at which 50% growth inhibition was obtained was defined as IC50.
[0035]
Protease inhibitors:
Virus at a concentration of 100 to 300 bcc was diluted with Infection Medium, and 100 μl was added to each MAGIC-5A cell previously seeded in a 96-well microplate. Further, 100 μl each of antiviral drugs prepared in a 10-fold dilution series was added, and the mixture was incubated at 37 ° C., 5% CO 2.₂The cells were cultured in an incubator. 72 hours later, 100 μl of the supernatant and 100 μl of Infection Medium were added to a 96-well plate of MAGIC-5A and MAGIC-5 / SEAP cells prepared the day before and cultured. After 48 hours, in the same manner as in the reverse transcriptase inhibitor drug section, the value of a well containing no inhibitor was taken as 100%, and the concentration at which 50% growth inhibition was obtained was taken as IC50.
For all resistance tests, the infectious molecular clone HIV-1 NL432 strain was assayed in parallel as a control for inhibitor-susceptible virus, and the resistance of clinical isolates was determined as the resistance to the IC50 value of this strain. Expressed.
[0036]
Resistance test with Genotype:
RNA was extracted from 100 ml of patient plasma using a high-purity RNA isolation kit (Roche), and the po1 region of HIV-1 was amplified using a One Step RNA PCR Kit (Perkin Elmer). The PCR reaction product was concentrated using Wizad ™ PCR Prep DNA Purification System (Promega), electrophoresed, and then the gel site containing the desired fragment was excised and purified using SUPREC-01 (Takara Co.). . The base sequence of each sample thus obtained was determined using an Auto sequencer (ABI model 310). The amino acid sequence was estimated from the base sequence and examined for resistance.
[0037]
(result)
1. Correlation between MAGIC-5A and viral load
Using MAGIC-5A, the virus was isolated from 129 samples out of 210 samples. Further, when the amount of virus in the patient plasma and the virus separation rate were examined, the separation rate was 77% when the virus amount was 4th power or more and 8% when it was less than 4th power. From this, it was recognized that virus separation was possible using this method if the amount of virus was 4th power or more.
[0038]
2. Reproducibility in NL432
AZT (3'-Azido-3'deoxythymidine), d4T (2 ', 3'-didehydro-3'-deoxy-thymidine), and 3TC (2'-deoxy-3'-thiacytidine) as reverse transcriptase inhibitors In addition, when reproducibility (triplicate × 5 times) of drug resistance test with NL432 as HIV-1 Wild strain sensitive virus was investigated for RTV (ritonavir), SQV (saquinavir) and NFV (nelfinavir) as protease inhibitors, Good reproducibility was shown as shown in Table 1.
[0039]
[Table 1]

[0040]
3. Comparison of phenotype and genotype
Using this MAGIC-5A, 129 samples of clinical isolates and 2 samples of laboratory strains obtained were used to test the resistance of each drug. Furthermore, the clinical isolates of AZT46, d4T47, 3TC48, RTV41, SQV41, and NFV43 were compared with the genotype and analysis results. In addition, the IC50 value for each drug without treatment is shown in Table. It was shown in 2. Despite no treatment, AZT, RTV, SQV, and NFV were found to be more than 4 times more resistant than NL432.
[0041]
[Table 2]

[0042]
Hereinafter, resistance test results for each drug are described.
AZT (see FIG. 1): The value obtained using NL432 is multiplied by 1 and the phenotype resistance to AZT in the untreated group is 0.1 to 5 times (average 1.2 times) there were. On the other hand, the resistance mutation in genotype is found to be 2.6-106 times (average 70 times) and tends to resistance when a single mutation at amino acid sequence Nos. 41 and 215 or a combination of both is observed. It was. In addition, as previously reported, it was confirmed that when positions 41, 215, and 184 were mutated, they were not resistant, but recovered sensitivity by 0.53 to 14 times (average 6.7 times). And when resistance mutation of the 67,219 position was added to these three combinations, it became 2.6-106 times (112 times), and it turned out that high tolerance is acquired again.
[0043]
3TC (see FIG. 2): The degree of resistance with phenotype against 3TC in the untreated group was 0.12 to 3.86 (average 1.4 times). In contrast, when a resistance mutation at position 184 was observed in the genotype, all specimens had acquired a high tolerance of 120 times or more. In addition, even when the position 184 was wild type, a low tolerance of 0.12 to 31 times (average 9.5 times) was observed when there was a history of treatment with other reverse transcriptase inhibitors or 3TC.
[0044]
d4T (see FIG. 3): The degree of tolerance with phenotype against d4T in the untreated group was 0.4 to 3.2 times (average 1 time). In d4T, there are many unknown amino acid substitutions related to resistance, and we investigated the correlation with the currently known mutation at position 75, but although a virus that acquired a mutation at position 75 was found at this center, It was not an amino acid substitution reported as a resistance mutation. However, as with 3TC, even when position 75 is a wild type, it has a low tolerance of 0.04 to 8.18 times (average 2.3 times) when other reverse transcriptase inhibitors or 3TC have been treated. It was.
[0045]
RTV (see FIG. 4): The degree of resistance mutation in phenotype for 15 patients in the untreated group and 6 persons who have a history of RTV treatment but no genotype resistance mutation is 0.33-2. It was 53 times (average 0.8 times). The 82nd primary mutation is wild type, and if there is one secondary mutation (position 36 or 71), it is 0.33 to 1.33 times, but if there are two or more mutations, 12 ~ High tolerance was obtained from moderate tolerance of 106 times (average 72 times). When the 82nd resistance mutation, which is the primary mutation, accompanies the primary mutation, all have acquired high resistance of about 100 times (average 105 times) regardless of the number of secondary mutations.
[0046]
SQV (see FIG. 5): The degree of resistance mutation in phenotype is 0.1 to 10 times for 16 untreated group and 12 who have a history of SQV treatment but no genotype resistance mutation is observed. (Average 1.9 times). If the secondary mutation is one mutation (position 10 or 82), it is 0.34 to 3.2 times, but if a resistance mutation is found at position 90, which is the primary mutation, 6.6 to 100 times ( The average resistance was 60.7 times) and high tolerance was obtained from moderate tolerance.
[0047]
NFV (see FIG. 6): The degree of resistance mutation in phenotype is 0.33 to 10 times for 4 untreated groups and 2 who have a history of NFV treatment but no genotype resistance mutations Met. In spite of no treatment, the single mutations at positions 36, 63, and 77, or mutations combined at positions 63 and 77, which are secondary mutations, were considered to be polymorphism of the virus. Therefore, in the untreated group, if there is one secondary mutation, it is 0.3 to 4.6 times (average 1.2 times), and if two are recognized simultaneously, 0.3 to 1.5 times (average 1.3 times) ) And both show sensitivity, but in the case of one treatment group, 0.6 to 103 times (21.3 times on average), and 10.2 to 333 times if two are recognized simultaneously (Average 115 times) and high tolerance from moderate tolerance. Moreover, when a resistance mutation was recognized in the 30th position which is a primary mutation, 53-333 (an average of 165 times) and high tolerance were acquired.
[0048]
Correlation of drug resistance test using MAGIC-5A and MAGIC-5 / SEAP cells: MAGIC-5A and MAGIC-5 / SEAP cells were analyzed at the same time using the Blue Cell Count basically using MAGIC-5A. The drug resistance was examined in parallel with an infectivity titration measurement system using Plotting 26 samples each for AZT, NVP and NFV, the horizontal axis indicating SEAP, and the vertical axis indicating the IC50 of each drug obtained by Blue Cell Count, the concentration is high in a wide concentration range from the high concentration range to the low concentration range Correlation was shown. This correlation was also high when plotted as relative resistance to the laboratory strain HIV-1 NL432 (see FIGS. 7 and 8). The Blue Cell Count using MAGIC-5A cells and the Chemiluminescence measurement using MAGIC-5 / SEAP cells have almost no change in the labor involved in the culturing process. However, it takes less than an hour to read a single microplate, but using a Luminometer for microplates, the measurement can be completed in tens of seconds. When multi-sample processing is required, this labor is significant. In terms of detection sensitivity, for example, even when there are dozens of infectious HIV-1 in a sample inoculated in a hole of a 96-well microplate, there is an advantage that MAGIC-5A cells can be fixed and determined directly under a microscope. However, even with MAGIC-5 / SEAP, a significant chemiluminescence value several times the background of HIV-1 non-infection can be obtained, and there is also an advantage that a clearer measurement value can be obtained on the next day by continuing the culture. However, the drug resistance test is not actually a problem because a sufficient infectious titer (100 to 300 bcc / well) is inoculated at the beginning of the culture. Although not shown in the data, a colorimetric reaction using p-nitrophenol phosphate as a substrate was sufficiently inferior to the chemiluminescence measurement system in terms of sensitivity, but was able to withstand practical use.
[0049]
Next, the reproducibility of the drug resistance test was investigated for newly approved anti-HIV drugs Abacavia (ABC), Nevirapine (NVP) and Amprenavia (APV) using HIV-1NL432 as a standard virus strain. As a result, good reproducibility was obtained as shown in Table 3. Hereinafter, the resistance test results for each drug are described.
[0050]
[Table 3]

[0051]
Abacavia (ABC) (see FIG. 9): Ababavia (ABC), a reverse transcriptase inhibitor, is known to have 65, 74, 115, and 184 resistance mutations. Non-treated subjects were on average 16.2 times more resistant. Further, when 74 resistance mutations were added to 184, the resistance increased by 26.1 times.
[0052]
Nevirapine (NVP) (see FIG. 10): Nevirapine (NVP), a non-diffusion reverse transcriptase inhibitor, has known resistance mutations 103, 106, 108, 181, 190. These are all primary mutations for NVP. The number of untreated patients was 1.05 times, and even in patients who had used reverse transcriptase inhibitors, the sensitivity range was 1.51 times. In other words, no cross-resistance was observed between the reverse transcriptase inhibitor and nevirapine. Except for 27.6 times the mutation of 181, all the other (106, 103, 190) resistance levels were 100 times or more, and high tolerance was obtained. In addition, most resistance mutations of non-diffusion type reverse transcriptase inhibitors are recognized as cross resistance, but the only resistance mutation of NVP 106 is not cross resistance to EFV (efavirenz). It was found that the specimens with 106 resistance mutations measured this time were highly resistant to NVP 131 times or more but not 5.7 times to EFV.
[0053]
Amprenavia (APV) (see Fig. 11): 1.47 times for untreated individuals, and 1.04 times for specimens that have acquired no mutation as polymorphism while being untreated. It was. However, it was found that the same 10 mutations were observed, and those treated were 19.6 times more resistant. Furthermore, when 10, 46 and 84, which are amprenavian secondary mutations, are aligned, the tolerance level increased by an average of 232 times. Amprenavia, a protease inhibitor, was known as a drug with low cross resistance because 50, which is a primary mutation, is not a resistance mutation to other protease inhibitors. However, if there are multiple secondary mutations alone, other proteases are also present. It turned out to be inclined to resistance as well. Previously, Parkin et al. Reported that N88S mutations seen in 20% of patients who used IDL and NFV in the past returned to APV (Journal of Viorology, 2000, 4414-4419). It was. In this assay, 46 resistance mutations were found to be 24.2 times more resistant to the group, whereas N46S was found to be 5.5 times more sensitive if 46 was found, and these patients were previously NFV Was found to have been treated by
[0054]
(Discussion)
Currently, in order to detect drug-resistant mutant viruses, (1) genotype (genotype method) for examining mutations in the base sequence of the drug action region and (2) directly culturing HIV in the presence of the drug to suppress growth Phenotype (phenotype method) to evaluate and determine the concentration (IC50, IC90) that can be produced, and (3) recombinant using recombinant DNA technology to create infectious recombinant HIV from patients and perform phenotype method There are three virus assays. Of these, the genotype method, which has a short time, excellent operability, and results can be obtained relatively easily, is the mainstream, but on the other hand, the appearance of unreported resistant mutations and the accumulation of multiple mutations Comprehensive judgment is difficult. In comparison, the detection method using the phenotype method, which can comprehensively determine the degree of resistance, is considered to require time, cost, and labor, although it is currently improving rapidly. Is not done. In addition, for the recombinant virus assay, a technique for incorporating a drug action region of a virus derived from a patient into an HIV vector plasmid is very skillful, and the obtained recombinant virus often does not propagate, and further, a drug action such as a po1 gene. It is known that regions other than the region are involved in HIV-1 particle formation, and it is not known whether the obtained recombinant virus reflects the actual virus characteristics in the patient's body, so the phenotype method Like, it is not generally done.
[0055]
Therefore, the present inventors have aimed to solve these problems by using the phenotype method with MAGIC-5A and MAGIC-5 / SEAP cell lines, and to quickly conduct a sensitivity test directly from clinical isolates, Inspected. In the conventional phenotype method using PBMC, it takes several months until results are obtained, and it is costly in terms of measuring p24 to make a judgment. The results could be obtained in as little as two weeks until the susceptibility test, and the presence or absence of infection could be determined quickly by staining the cells or measuring the enzyme activity of the culture supernatant.
[0056]
From the results of this Example, the average degree of tolerance was about 1-fold in untreated patients compared to NL432 as a control, but 10-fold samples were observed. These viruses were found to have acquired low-level resistance by the phenotype method even though no resistance mutation was observed in the genotype method. It turned out that it was data. Further, when the genotype method and the phenotype method were compared, a good correlation was obtained for drugs excluding d4T for which no resistance mutation at amino acid sequence number 75 was detected. However, for 3TC and d4T, even if they were wild type, low tolerance was observed in cases where there was a history of treatment with a reverse transcriptase inhibitor.
[0057]
In addition, when amino acid mutations at positions 41, 184, and 215 were also observed for AZT, it was confirmed using these cells that sensitivity was restored. However, the technology and knowledge at this stage does not reveal how such sensitivity changes caused by amino acid mutations occur in terms of the reaction and interaction between the enzyme and the substrate. Furthermore, protease inhibitors showed high tolerance of about 100 times when primary mutation was observed, and it was confirmed that the results obtained by genotype method and phenotype method were consistent. The HIV-1 virus is known to acquire a secondary mutation because it adapts to drug administration. In patients with a history of treatment with other protease inhibitors, it was reported that the response to NFV was only about 50%. However, in this case, the secondary mutation was not observed even if the primary mutation for NFV was not observed. found. It was suggested by the phenotype method that this secondary mutation shows sensitivity in untreated patients but shows resistance in patients who have been treated with protease inhibitors. Based on these results, the phenotype method using MAGIC-5A clarifies the degree of resistance that could not be determined by the genotype method, and is considered to be useful as one of the decisions on drug selection to be used in future multi-drug combination therapy. It was.
[0058]
From the comparison of the results using MAGIC-5A and MAGIC-5 / SEAP cells in the drug resistance test, a high correlation was obtained between the two. This was also predicted from the fact that MAGIC-5 / SEAP cells were modified to secrete SEAP out of the cells by HIV-1 infection using MAGIC-5A cells as a parent strain. Moreover, as a reporter, the system using SEAP was much more excellent in stability after adding a chemiluminescent reagent and in the quick and convenient operation than β-galactosidase. Since SEAP was also superior in terms of detection sensitivity, measurement was possible even with a small amount of virus to be inoculated. In the assay system using MAGIC-5 / SEAP, the measured values can be obtained simply by seeding the cells, inoculating the virus simultaneously with the drug, collecting the culture supernatant, adding the enzyme substrate solution, and applying to the measuring machine. Compared with the currently used p24 ELISA or RT Assay, the operation procedure is greatly simplified, and it is considered that conversion to robotization by Work Station is also possible. Isolation of viruses from patient specimens using the MAGIC-5A cell line and drug resistance phenotyping assay using MAGIC-5 / SEAP reveals drug resistance in a short period of about 2 weeks. This is a measurement system that is indispensable for the construction of a high-throughput drug resistance test system that can measure combinations of newly approved drugs in the future and handle various combinations of drugs as combination therapy. It is expected.
[0059]
In addition, it was found that the newly approved anti-HIV drugs ABC, NVP and APV can detect resistant viruses efficiently and sensitively by this test method. This means that the study will be flexible enough to support various anti-HIV drugs that are further developed and expected to be clinically applied in the future.
[0060]
【The invention's effect】
According to the present invention, it is possible to provide a rapid and simple drug resistance test method using an HIV-1 infectivity titer measuring cell line, particularly a drug resistance test method capable of rapidly processing multiple specimens.
[Brief description of the drawings]
FIG. 1 is a diagram showing the correlation between AZT resistance and genotype in phenotype.
FIG. 2 is a diagram showing the correlation between phenotypic 3TC resistance and genotype.
FIG. 3 is a diagram showing a correlation between d4T resistance in phenotype and genotype.
FIG. 4 is a diagram showing a correlation between RTV resistance in phenotype and genotype.
FIG. 5 is a diagram showing a correlation between phenotype SQV resistance and genotype.
FIG. 6 shows the correlation between phenotype NFV resistance and genotype.
FIG. 7 is a diagram showing a correlation with anti-HIV-1 agent concentration in a chemiluminescence assay using MAGIC-5 / SEAP cells and a Blue Cell Count using MAGIC-5A cells.
FIG. 8 is a diagram showing a correlation with anti-HIV-1 agent concentration in a chemiluminescence assay using MAGIC-5 / SEAP cells and a Blue Cell Count using MAGIC-5A cells.
FIG. 9 shows the correlation between phenotype ABC resistance and genotype.
FIG. 10 is a diagram showing the correlation between phenotype NVP resistance and genotype.
FIG. 11 shows the correlation between phenotypic APV resistance and genotype.

Claims (5)

Human HeLa cells were transformed into animal cells so as to stably express the HIV-1 receptor CD4 and co-receptors CXCR4 and CCR5, and secreted alkaline phosphatase as a secreted reporter protein downstream of the HIV-1 LTR sequence. A human HeLa cell-derived animal cell line FERM P-18078 established by introducing an expression vector having a gene encoding SEAP and capable of expressing a secreted reporter protein by HIV-1 infection, A method for testing drug resistance of HIV, comprising contacting a sample containing HIV-1 in the presence of a test drug and detecting a reporter protein secreted into a culture supernatant by HIV-1 infection. 2. The drug resistance of HIV according to claim 1 , wherein the secretory alkaline phosphatase SEAP, which is a secreted reporter protein , is detected by one or more measurement systems of colorimetric reaction, fluorescence or chemiluminescence. test methods. The test drug for HIV drug resistance according to claim 1 or 2 , wherein the test drug is an anti-HIV drug containing a reverse transcriptase inhibitor or a protease inhibitor. Test method for drug resistance of HIV according to any one of claims 1 to 3 you characterized by using a combination of two or more agents as test drugs. Reporter protein colorimetric reaction secreted into the culture supernatant, of HIV according to any one of claims 1 to 4 you and detecting a fluorescent emission or chemiluminescence any one or more measurement system Test method for drug resistance .

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