JP4183754B2 - Use of chemokine receptors to inhibit HIV-1 infection - Google Patents

Description

表１の説明：
Lussoら（12）に記載のとおりにPM1細胞を培養した。研究所労働者（LW）からフィコール／ハイパックで単離したPBMCを、抗CD8イムノマグネティックビーズ（DYNAL, Great Neck, NY）でのCD8⁺リンパ球の枯渇前の72時間、PHAで刺激した。文献記載（3）のとおりに、インターロイキン-2（100U/ml;Hofmann LaRoche, Nutley, NJ）を含有する培地内でCD4+リンパ球を維持した。NL4/3Δenv-ルシフェラーゼベクターとHIV-1 env発現ベクターとでコトランスフェクトされた293細胞からの上清（10〜50ngのHIV-1 p24）を標的細胞（1〜2×10⁵個）に感染させた（10,11）。β-ケモカイン（R & D Systems, Minneapolis）を、第１欄の括弧内に示されている最終濃度（ng/ml）で、ウイルスと同時に該標的細胞に加えた。従前の研究（2,3）に基づき、β-ケモカインの濃度範囲を選択した。２時間後、該細胞をPBSで２回洗浄し、β-ケモカインを含有する培地に再懸濁し、48〜96時間維持した。文献記載（10,11）のとおりに、細胞ライセート中のルシフェラーゼ活性を測定した。示されている値は、β-ケモカインを欠くウイルス対照培養（100％）に対して表されたルシフェラーゼ活性（cpm）/ng p24/mgタンパク質を意味し、二重または六重の測定の平均である。ndは、実施せず。R/Mα/Mβ、RANTES＋MIP-1α＋MIP-1β。
RANTESおよびMIP-βは、別々に加えた場合、強力な活性を示し、一方、他のβ-ケモカイン（MIP-1α、MCP-1、MCP-2およびMCP-3（参照文献13〜15））は、より弱い阻害物質であった（表1a）。MIP-1α、MIP-1βおよびRANTESの組合せは、TCLA株NL4/3およびHxB2による又は両種指向性マウス白血病ウイルス（MuLV-Ampho）シュードタイプによるPM1細胞の感染を阻害しなかった（表1a）。したがって、HIV-1エンベロープ糖タンパク質の表現型特性は、ウイルス侵入アッセイにおけるβ-ケモカインに対するそれらの感受性に影響を及ぼす。
env補足アッセイを用いて、２つの対照個体（LW4およびLW5）に由来するCD4⁺Ｔ細胞内へのHIV-1の侵入を評価した。MIP-1α、MIP-1βおよびRANTESは、LW4およびLW5のCD4⁺Ｔ細胞へのNSI初代株JR-FLによる感染を強く阻害し、LW細胞へのHxB2の感染を若干減少させたが（表1b）、このことは、種々のウイルス株による活性化CD4⁺Ｔ細胞上の受容体の利用において或る程度の重複が存在しうることを示唆している。また、感受性においてドナー間で有意な変動があるものの、正常PBLにおけるBaL env媒介性複製はMIP-1α、MIP-1βおよびRANTESにより阻害された（データは示していない）。
PM1細胞への感染の完全な阻害が、ADAまたはBaL env補足ウイルスの添加後５時間までのβ-ケモカインの連続的な存在を必要とすることを示すことによに２時間または24時間、該細胞をβ-ケモカインで前処理し、ついで、ウイルスの添加前に該細胞を洗浄した場合には、阻害効果は得られなかった。さらに、ウイルス添加の２時間後にβ-ケモカインを加えた場合には、ウイルスの侵入に対する最小限の影響が生じたにすぎなかった（図1a）。つぎに、感染の10時間後、PM1細胞内のHIV-1初期DNA逆転写物を検出するために、PCRに基づくアッセイを用いた。MIP-1βおよびRANTESの存在下では、NL4/3でなくADAの逆転写を検出することはできなかった（図1b）。したがって、β-ケモカインによる阻害は、HIV-1複製の初期段階（ウイルスの付着、融合および初期逆転写）の少なくとも１つにおいてそれが存在することを要する。
まず、β-ケモカインが、可溶性CD4に対するJR-FLまたはBRU gp120の結合、またはオリゴマーエンベロープ糖タンパク質を発現するHeLa-JR-FL細胞に対する四量体CD4-IgG2の結合を阻害するか否かを試験することにより、これらの作用部位を識別した（17）。いずれのβ-ケモカインも、いずれのアッセイにおいても阻害を示さなかったが、OKT4a CD4-Mabは、両方で強力な阻害性を示した（データは示していない）。したがって、エンベロープ糖タンパク質のコンホメーション変化がウイルスと細胞膜との融合を引き起こす場合には、β-ケモカインは、CD4結合後の段階を阻害する（18）。また、細胞-細胞膜融合は、gp120-CD4相互作用により誘導され、細胞膜内に取込まれた蛍光染料間の共鳴エネルギー移動（RET）により直接モニターすることができる（17）。RETアッセイにおいては、JR-FLまたはBRUのいずれかのエンベロープ糖タンパク質を発現するHeLa細胞（それぞれHeLa-JR-FLまたはHeLa-BRU）とのPM1細胞の膜融合をOKT4aは完全に阻害し、このことは、この過程の特異性を証明するものである（17）。RANTES、MIP-1β（および、より低度ではあるがMIP-1α）は、PM1細胞とHeLa-JR-FL細胞との膜融合を強力に阻害したが、PM1細胞とHeLa-BRU細胞との融合は、これらのβ-ケモカインに不感受性であった（表2a）。
表２：RETアッセイで測定した、HIV-1エンベロープ糖タンパク質媒介性膜融合

表２の説明：
CD4⁺標的細胞（マイトジェンにより活性化されたCD4⁺リンパ球またはPM1細胞）をオクタデシルローダミン（Molecular Probes, Eugene, OR）で標識し、HeLa-JR-FL細胞、HeLa BRU細胞（または対照HeLa細胞、示されていない）をオクタデシルフルオレセイン（Molecular Probes）で37℃で一晩標識した。等しい数の標識標的細胞とenv発現細胞とを96ウェルプレート内で混合し、β-ケモカイン（またはCD4 MAb OKT4a）を、第１欄の括弧内に示されている最終濃度（ng/ml）で加えた。細胞混合の４時間後にフルオレセインの発光値を測定した（17）。細胞融合が生じたら、合体した膜内に該染料を密接に結合させて、フルオレセインの450nmでの励起が、共鳴エネルギー転移（RET）およびローダミンによる590nmでの発光を引き起こすようにする。融合率（％）は、100×［（Exp RET−最小RET）／（最大RET−最小RET）］〔式中、最大RET＝HeLa-EnvとCD4⁺細胞とを混合した場合に得たRET（％）、Exp RET＝融合阻害化合物の存在下でHeLa EnvとCD4⁺細胞とを混合した場合に得たRET（％）、および最小RET＝HeLa細胞（HIV-1エンベロープ糖タンパク質を欠くもの）とCD4⁺細胞とを混合した場合に得たRET（％）〕に等しいと定義する。RET値（％）は、文献記載（17）の計算により定義し、それぞれは三重の測定の平均である。これらの値は、HeLa-JR-FL細胞およびHeLa-BRU細胞に関して、それぞれ、PM1細胞11.5％、10.5％；LW5 CD4⁺細胞、6.0％、10.5％；R/Mα/Mβ、RANTES＋MIP-1α＋MIP-1βであった。
LW5からの初代CD4⁺Ｔ細胞で、同様の結果を得た（表2b）（初代細胞における膜融合を阻害するためには、PM1細胞における膜融合を阻害するより高濃度のβ-ケモカインが必要であった）。したがって、β-ケモカインの作用は、PM1細胞系に限定されるものではない。RETアッセイは、β-ケモカインがenv媒介性膜融合を阻害することを裏付けている。
これらの結果の最も簡単な解釈は、あるβ-ケモカインのその受容体に対する結合が、直接的またはそれ以外の様態で、CD4⁺Ｔ細胞とHIV-1との融合を妨げる、というものである。HIV-1がCD4⁺Ｔ細胞内への侵入のために第２の受容体を必要とすることは、十年前から公知である（19-21）。TCLA株の場合には、この機能はフシン（fusin）により付与される（9）。MIP-1α、MIP-1βおよびRANTESのいくつかの受容体が同定されており（6,7）、β-ケモカインは、受容体の利用において相当な交差反応性を示す（4-8）。しかしながら、C-C CKR-1、および特にC-C CKR-5は、組織発現パターンとMIP-1α、MIP-1βおよびRANTESに対するそれらの結合能とに基づき、最も有望な候補体であると目されている（4,7,8,15,22）。C-C CKR-1、C-C CKR-5およびLESTRはそれぞれ、PM1細胞および初代マクロファージ内でmRNAレベルで発現される（データは示していない）。したがって、これら及び他のβ-ケモカイン受容体をPCR増幅し、クローニングし、発現させた。
HIV-1侵入のenv補足アッセイにおいては、HeLa-CD4（ヒト）、COS-CD4（サル）および3T3-CD4（マウス）細胞内でのC-C CKR-5の発現により、初代NSI株ADAおよびBaLは、それらの各々に容易に感染することが可能となった

表３の説明：
ケモカイン受容体遺伝子C-C CKR-1、C-C CKR-2a、C-C CKR-3、C-C CKR-4およびC-C CKR-5はイントロンを有しておらず（4-8,15,22）、７人の健康なドナーのPBMC由来のヒトゲノムDNAプール上で直接行なうPCRにより、それらを単離した。ATGおよび停止コドンと重複し、pcDNA3.1発現ベクター（Invitrogen Inc.）内への定方向（directional）クローニングのためのBamHIおよびXhoI制限部位を含有するオリゴヌクレオチドを使用した。NIHデータベースに由来する配列を用いてPM1細胞に由来するcDNA上で直接行なうPCRにより、LESTR（フシンまたはHUMSTRとしても公知である）（4,9,24）をクローニングした。ヒトゲノムDNAに直接由来するCKR遺伝子およびPM1 cDNA由来のLESTRの第１（5-1および3-1）および第２（5-2および3-2）ラウンドPCR増幅において用いる5’および3’プライマー対を、以下に挙げる。CKR-5の増幅には、単一のプライマーセットのみを使用した。

ヒトCD4発現細胞系HeLa-CD4（P42）、3T3-CD4（sc6）およびCOS-CD4（Z28T1）（23）に、リン酸カルシウム法により種々のpcDNA3.1-CKR構築物をトランスフェクトし、48時間後、β-ケモカイン（それぞれ400ng/mlのRANTES、MIP-1αおよびMIP-1β）の存在下または不存在下で種々のレポーターウイルス（200ngのHIV-1 p24/10⁶細胞）を感染させた。48時間後、細胞ライセート内のルシフェラーゼ活性を測定した（10,11）。C-C CKR-5以外のC-C CKR遺伝子により媒介される感染は、阻害の定量が可能となるほと強くなかったため、β-ケモカインの遮断データは、C-C CKR-5だけについて示されている。PCRに基づくHIV-1侵入のアッセイにおいては、C-C CKR-1発現細胞内へのNL4/3およびADAの低レベルの侵入が、一貫して認められた（データは示していない）。
このアッセイでは、LESTRもC-C CKR-1、-2a、-3または-4も、C-C CKR-5を置換することができなかった。COS-CD4および3T3-CD4内でのLESTRの発現はHxB2の侵入を許容せず、トランスフェクトされていない（または対照プラスミドでトランスフェクトされた）HeLa-CD4細胞にはHxB2は容易に侵入した（表３）。合計３つのC-C CKR-5発現細胞系内へのBALおよびADAの侵入は、MIP-1α、MIP-1βおよびRANTESの組合せによりほぼ完全に阻害され、一方、LESTR発現細胞内へのHxB2の侵入は、β-ケモカインに不感受性であった（表３）。これらの結果は、C-C CKR-5が、初代NSI HIV-1株に対するβ-ケモカイン感受性第２受容体として機能することを示唆している。
C-C CKR-5の第２受容体の機能は、env媒介性膜融合のアッセイで確認した。ヒトCD4を恒久的に発現するCOSおよびHeLa細胞系内でC-C CKR-5を一過性に発現させた場合には、両細胞系は、JR-FLエンベロープ糖タンパク質を発現するHeLa細胞と強力に融合し、一方、対照プラスミドを使用した場合には、融合は生じなかった（データは示していない）。C-C CKR-5の代わりにLESTRを発現させた場合には、COS-CD4細胞またはHeLa-CD4細胞とHeLa-JR-FL細胞との融合は可能でなかったが、COS-CD4細胞とHeLa-BRU細胞との融合は可能になった（データは示していない）。
また、RETアッセイにおいて、β-ケモカイン受容体の融合能を試験した。C-C CKR-1、-2a、-3または-4ではなくC-C CKR-5の発現が、HeLa-CD4細胞とHeLa-JR-FL細胞との強力な融合を可能にした。HeLa-JR-FL細胞とC-C CKR-5発現HeLa-CD4細胞との融合の度合は、HeLa-BRU細胞とHeLa-CD4細胞との融合の構成的レベルより大きかった（図２）。したがって、初代NSI HIV-1株に関するC-C CKR-5の融合付与機能は、２つの独立した融合アッセイで確認されている。
実験の考察
総合すると、前記の結果は、MIP-1α、MIP-1βおよびRANTESが、CD4結合に次ぐウイルス細胞融合反応を妨げることにより、HIV-1の感染を侵入段階で阻害することを裏付けている。また、C-C CKR-5が、CD4+Ｔ細胞内へのHIV-1の初代NSI株の侵入のための第２の受容体として役立ちうること、およびβ-ケモカインとC-C CKR-5との相互作用が、HIV-1融合反応を阻害することが示された。
前節の参照文献

第２の一連の実験
実験方法
試薬の入手源：組換えヒトβ-ケモカインはR & D Systems（Minneapolis）から購入し、¹²⁵I-MIP-1β（比活性2,200Ci nmol^-1）はDupont-NENから購入した。抗CD4モノクローナル抗体は、Q. Sattentau（16）から入手した。ただし、5A8はL. Burkly（Biogen）（27）およびL120（UK MRC AIDS Reagent Repository）（16）から入手した。可溶性CD4は、文献に記載されている（17）。gp120に対するモノクローナル抗体は、23A（gp120C末端、J. Robinsonから）、447-Dおよび697-D（参照文献28; Cellular Products Inc.）および83.1（参照文献29; Repligen）以外は、文献（19,20）に挙げられているドナーから得た。組換えgp41（IIIB）エクトドメインはViral Therapeutics Inc.から、V3ペプチドは、RepligenまたはUK MRC AIDS Reagent Repositoryから得た。組換えMN gp120（Genentech）、SF-2 gp120（Chiron）およびCM243 gp120はNIAID AIDS Reagents Repositoryから提供され、W61D gp120（SmithKline Beecham, Belgium）はUK MRC AIDS Reagent Repositoryから得た。
ジヒドロ葉酸レダクターゼ発現カセットを含有する、Progenics Pharmaceuticalsで開発されたベクターを使用して、組換え単量体JR-FLおよびLAI gp120（共に完全長であり、V3ループが欠失している）を発現させた。該遺伝子の発現は、サイトメガロウイルス最初期プロモーターの制御下に置く。Δ-V3 gp120の場合には、重複伸長によるスプライシング技術（splicing-by-overlap-extension technique）によりV3ループを切除し、この際、該ループを規定するシステインが保有され、ペプチド配列TGAGHにより架橋される（Spanned）ようにする。すべての構築物を配列決定して、クローニング操作中に突然変異が導入されていないことを確認した。安定にトランスフェクトされたチャイニーズハムスター卵巣細胞（DXB-11）内で、該タンパク質を発現させ、ヌクレオシドを含まない培地内で選択し、メトトレキセートを使用して増幅した（文献記載（17）の方法に従い行なった）。分泌したタンパク質を、イオン交換、ガランツス・ニバラス（Galanthus nivalus）レクチンアフィニティーおよびゲル濾過クロマトグラフィーを含む非変性法により、>95％均一になるまで精製した。精製されたタンパク質は、ナノモラーのアフィニティーでsCD4に結合した（18）。
SF162 gp160の発現の場合には、env遺伝子を含有する3.5kbのEcoRI-BamHI断片を、SV40に基づくベクターであるpSMから切り出し、β-アクチンに基づく発現ベクターであるpCAGGSのR1/Bg1lll部位内にサブクローニングした。SF170 gp160の発現の場合には、env遺伝子を含有する3.8kbの断片を、pBSKS⁺プラスミドから切り出し、T4 DNAポリメラーゼでの処理により平滑末端化し、pCAGGSのRV/Xho1部位内にサブクローニングした。該発現プラスミドを、リン酸カルシウム共沈法により293T細胞内にトランスフェクトした。３日後、培養上清内の可溶性gp120を集め、0.2μmのフィルターで濾過し、Amicon 1000メンブレン上で濃縮した。
JR-FLおよび94RW020エンベロープ糖タンパク質の可溶性オリゴマー形態（および92TH014からの単量体gp120）の調製を、以下のとおりに行なった。JR-FL env遺伝子は、I. Chen（UCLA）により提供され、92TH014および92RW020のenv遺伝子は、NIAID AIDS Reagent Repositoryから得た（30）。gp120およびgp41エクトドメイン（JR-FLおよび92RW020のもの）、92TH014のgp120のみをコードする可溶性発現プラスミドを、文献記載（30）のとおりに構築し、リン酸カルシウム法によりチャイニーズハムスター卵巣細胞内にトランスフェクトした。JR-FLおよび92TH014 gp120およびgp41部分間の切断部位を保有させ、タンパク質をオリゴマーとして分泌させた（J.A., J.M.B.およびJ.P.M.,未公開データ）。エンベロープ糖タンパク質を、固定化金属アフィニティークロマトグラフィーにより培養上清から部分精製した。残基457での単点突然変異によりCD40結合が不能になったgp120/gp41分子を含有する対照調製物93MW959（c）は、¹²⁵I-MIP-1βと競合しなかった。未精製培養上清内の単量体gp120またはオリゴマーgp120/gp41の濃度の推定を、該タンパク質を変性させ、ニトロセルロースメンブレン上にドットブロットし、連続エピトープに対するマウスモノクローナル抗体の混液（19）、ついで抗マウスIgG-HRP結合体およびECL化学発光系（Amersham）でgp120を検出することにより行なった。精製された単量体JR-FL gp120を濃度基準として使用した（17）。オリゴマーgp120/gp41複合体の濃度は、該調製物内の単量体gp120サブユニットの全濃度と定義した。酵素結合抗体免疫アッセイ（ELISA）により、gp120に対する高いアフィニティーのCD4結合を確認した（19）。
細胞および細胞系
PBMCを、フィコール・ハイパック遠心法により血液ドナーから単離し、フィトヘマグルチニン（5μg ml^-1）およびIL-2（100U ml^-1）（Roche）で２〜３日間刺激した。抗CD4イムノマグネティックビーズ（Dynal Inc）を使用する陽性選択により、活性化PBMCからCD4⁺Ｔ細胞を精製した。精製されたリンパ球を、¹²⁵I-MIP-1β結合アッセイで使用する前に、IL-2（200U ml^-1）を含有する培地内で2×10⁶/mlで少なくとも３日間培養した。CCR-5欠損対立遺伝子に関して該細胞をスクリーニングし（14）、野生型ドナーからの細胞だけを使用した（特に示されている場合を除く）。リン酸カルシウム法を用いてpcDNA3.1-ckr-5（参照文献１）を293細胞にトランスフェクトし、1μgml^-1ネオマイシン（G418;Sigma）を含有する培地内で耐性クローンを選択した。耐性細胞をサブクローニングし、¹²⁵I-MIP-1βを使用する結合アッセイにおいてCCR-5発現に関して試験した。
MIP-1β結合アッセイおよびgp120競合
精製されたCD4⁺Ｔ細胞を、氷冷結合緩衝液（１％BSA、25mM HEPES、0.05％アジ化ナトリウムを含有するRPMI1640培地）中で２回洗浄した。二重のサンプル（200μl中の2×10⁶個の細胞）を、0.1nM ₁₂₅I-MIP-1β（2,200Ci nmol^-1;0.25μCml^-1）と共に氷上で２時間インキュベートした。放射能標識リガンドを加える直前に、未標識リガンドまたはgp120（適宜、モノクローナル抗体と混合する）を細胞に加えた。抗CD4モノクローナル抗体を、gp120と同時に該細胞に加えた。ついで、これらの細胞を、油（80％シリコーンオイル，Aldrich; 20％鉱油，Sigma）による遠心分離（60秒、14,000g）により未結合リガンドから分離し、細胞ペレット内の放射能をガンマカウンターにより測定した。100倍過剰の未標βを加えることにより、¹²⁵I-MIP-1βの特異的結合を推定した。各実験は、異なるドナーからの細胞を使用して少なくとも２回繰返した。293-CCR-5細胞での実験では、該細胞を1mM EDTAで解離させ、ついで結合緩衝液で２回洗浄した。サンプル（5×10⁵個の細胞）を0.5nM ¹²⁵I-MIP-1βと共にインキュベートし、ついで前記のとおりに加工した。¹²⁵I-MIP-1βの濃度を0.1nMまで減少させた場合には、特異的結合は検出されなかった。
概括
β-ケモカイン受容体CCR-5は、非シンシチウム誘導（non-syncytium-inducing（NSI））表現型のHIV-1株とCD4⁺Ｔ細胞との融合のための必須の補因子である（1-5）。ヒト免疫不全ウイルス（HIV）-1の初期結合部位は、CD4分子であり、その相互作用は、ウイルス表面糖タンパク質gp120により媒介される（6,7）。HIV-1の侵入中のCCR-5機能のメカニズムは明らかにされていないが、本発明者らは、そのβ-ケモカインリガンドが、HIV-1と該細胞との融合を妨げることを既に示している（1）。したがって、本発明者らは、CCR-5が、gp120とCD4との相互作用と同時に又はその後でHIV-1の第２の結合部位として作用するか否かを検討した。本発明者らは、CCR-5リガンドであるマクロファージ炎症性タンパク質（MIP）-IPが活性化CD4⁺Ｔ細胞またはCCR-5陽性CD4細胞上のその受容体に結合するのをgp120が阻害することに基づく競合アッセイを用いた。本発明者らは、CD4結合が、gp120-CCR-5相互作用に絶対に必要というわけではないが、その効率を著しく増大させると結論した。V3ループとCD4で誘導されたエピトープとを含むgp120上のいくつかの部位に対する中和モノクローナル抗体は、gp-120-CD4結合に影響を及ぼすことなく、gp120とCCR-5との相互作用を阻害した。HIV-1の受容体（CD4およびCCR-5）の一方または両方に対するHIV-1結合の阻害は、ウイルスの中和の重要なメカニズムかもしれない。
MIP-1βは、CCR-5に対する最も特異的なリガンドである（8-10）。なぜなら、MIP-1αおよびRANTESは、リンパ球系細胞上のβ-ケモカイン受容体ファミリーの他のメンバーにも高いアフィニティーで結合するからである（8-11）。したがって、本発明者らは、競合アッセイにおいてMIP-1βをCCR-5リガンドとして使用した。この受容体ファミリーの他のメンバーと同様に（12）、CCR-5は、マイトジェン応答遺伝子である。静止期にある精製されたCD4⁺Ｔ細胞内でのその発現は、通常、最小であるが、フィトヘマグルチニンおよびインターロイキン（IL）-2による該細胞の活性化の３日後、本発明者らは、CCR-5メッセンジャーRNAと¹²⁵I標識MIP-1β結合との著しい増加を観察した（データは示していない）。特異性対照として、本発明者らは、欠損CCR-5対立遺伝子に関してホモ接合の個体からのCD4⁺Ｔ細胞を使用した（13,14）。そのような３個体からの細胞に対する特異的（すなわち、非放射性MIP-1βと競合した）¹²⁵I-MIP-1β（0.1nM）結合の量は、2×10⁶個の細胞当たり92±12c.p.m.（平均±s.d.）であった。これに対して、21の対照個体からの細胞に対する平均結合は、2×10⁶個の細胞当たり1,044±1,073（範囲、222〜4,846c.p.m.）であった。したがって、活性化CD4⁺Ｔ細胞内との¹²⁵I-MIP-1βの反応性のほとんどは、CCR-5に対する結合に由来する。
組換え単量体gp120を¹²⁵I-MIP-1βと共に活性化CD4⁺Ｔ細胞に加えた場合には、本発明者らは、NSI株JR-FL［これは、侵入のためにCCR-5を使用した（1）］からのgp120が、MIP-1β結合を強力に阻害することを見出した（図3a：表４）。

表４の説明：
組換えタンパク質を、0.1nM ¹²⁵I-MIP-1βの存在下で力価測定し、活性化CD4⁺Ｔ細胞に加えた。各gp120濃度における¹²⁵I-MIP-1β結合の阻害率（％）が示されており、それらは、２〜４個の独立した実験の平均±s.d.である。値の記載がないものは、その濃度でgp120分子を試験しなかったことを示す（いくつかの分子は、>1μg ml^-1で利用できなかった）。
^*オリゴマー性gp120/gp41複合体
半最大阻害は、gp120-CD4相互作用に関する会合定数と同様の値である（7）0.1〜1.0μg ml^-1（0.8〜8nM）のgp120で生じた。これに対して、Ｔ細胞系順化（T-cell-line adapted（TCLA））SI株LAIからのgp120は無効であった（図3a;表４）。このウイルスは、CCR-5でなくフシン（CXCR-4）を侵入のために用いる（1-5）。JR-FLの突然変異体と、V3ループ（Δ-V3 gp120）を欠くがCD4に高いアフィニティーで結合するLAI gp120とは、MIP-1β結合を遮断しなかった（図3a）。しかしながら、以下の株のV3ループからのペプチド（特に示されていない限り15残基）も不活性であった：JR-FL（32残基）、RA、VS、Case-B（それぞれNSI）；HxB2、MN、SF-2（それぞれTCLA）（60μml^-1 gp120のおよそのモル当量、1μg ml^-1でペプチドを加えた）。gp41のエクトドメインと非共有結合しているJR-FL gp120のオリゴマー複合体は、MIP-1β結合の有効な阻害物質であったが、gp41エクトドメインだけを含む組換え分子はそうではなかった（表４）（後者の分子は、天然構造に折り畳まれないことがある（15））。
遺伝子亜型Ａ、Ｂ、ＣおよびＥからのHIV-1株は、侵入のためにCCR-5を利用し（3）、本発明者らは、MIP-1βが、亜型Ａ〜Ｅからのほとんどの初代NSI HIV-1株の複製を阻害することを見出した。CCR-5とのHIV-1の反応性のこのような広さは、¹²⁵I-MIP-1β競合アッセイに及ぶものである。以下のNSI初代株からの組換えgp120は競合性であり、MIP-1βの半最大阻害は約0.1〜0.5μg ml^-1の濃度で生じた：JR-FL（亜型Ｂ）、SF162（Ｂ）、W61D（Ｂ）、92TH014（Ｂ）、SF170（Ａ）、92RW020（Ａ）およびCM243（Ｅ）（表４）。これに対して、TCLA亜型Ｂ株LAI、MNおよびSF-2からのgp120との競合は認められなかった（表４）（ただ、それらのそれぞれは、CD4と高いアフィニティーで結合する（示されていない））。したがって、CCR-5との相互作用の判定において、gp120分子が由来するウイルスの表現型は、ウイルス遺伝子型より重要である。
本発明者らは、gp120-CD4相互作用のアンタゴニストを使用することにより、NSI gp120とMIP-1βとの競合におけるCD4の役割を評価した。CD4のドメイン１と反応してgp120結合を阻害するモノクローナル抗体Q4120およびL77（16）、ならびにgp120と反応してCD4結合を阻害する可溶性CD4（sCD4）（17）は共に、CCR-5に対するMIP-1β結合のJR-FL gp120による阻害を逆転させた（図3b；表５）。

表５の説明：
活性化CD4⁺Ｔ細胞に対する¹²⁵I-MIP-1β結合のJR-FL gp120（2μg ml^-1）による阻害を、sCD4（50μg ml^-1）またはCD4に対するモノクローナル抗体（50μg ml^-1）またはgp120に対する抗体（20μg ml^-1）の存在下または不存在下で試験した。各抗体の存在下のgp120の競合効果の平均逆転率（％）（±s.d; n=２〜４個の独立した実験）を示す。gp120の存在下かつ抗体の不存在下で記録された特異的¹²⁵I-MIP-1β結合（c.p.m.）のレベルを０％と設定した。gp120および抗体の両方の不存在下で記録されたレベルを100％と設定した。負の逆転率（％）は、¹²⁵I-MIP-1β結合に対するgp120の競合効果が、該抗体の存在下で増加したことを示す。また、文献（19,20）で定義されているgp120上の抗体エピトープのおおよその位置も記載されている。抗体の起源についての言及は文献（19,20）に記載されているか、「方法の部」に記載されている。
^*HIV-1_JR-FLを中和しうる抗gp120モノクローナル抗体（またはsCD4）
⁺他のHIV-1株（初代またはTCLA）に対する中和活性を有するモノクローナル抗体
CD4の他のドメインに対するモノクローナル抗体［これは、gp120-CD4結合を遮断しない（16）］は無効であり（表５）、gp120の不存在下では、sCD4（50μg ml^-1）は、MIP-1βの阻害を引き起こさなかった（データは示していない）。したがって、gp120がCCR-5と効率的に相互作用しMIP-1β結合を遮断するのに、細胞表面CD4との相互作用は重要である。CD4が絶対要件であるか否かを確認するために、本発明者らは、安定なヒトCD4^- CCR-5⁺293細胞系を作製した。これらの細胞は、¹²⁵I-MIP-1β（5×10⁵個の細胞当たり2,500c.p.m.までの特異的結合）に結合し、一方、トランスフェクトされていない293細胞は結合しない（<50c.p.m.の特異的結合）。CD4^- CCR-5⁺293細胞に対する¹²⁵I MIP-1βの結合は、JR-FL gp120により単発的に阻害されたが、これは、試験した最高gp120濃度のみで認められた（50〜100μg ml^-1）。これらの細胞上で認められた最強の競合は、50μg ml^-1のJR-FL gP120によるMIP-1β結合の73％阻害であったが（比較しうる阻害が、他の２つの実験で認められた）、競合が何ら検出されないことが多く、本発明者らは、より低いgp120濃度でのMIP-1β結合の阻害を全く観察しなかった。該CD4^- CCR-5⁺細胞に過剰のsCD4を加えても、JR-FL gp120の阻害効果は減少も増加もしなかった（データは示していない）。
JR-FL gp120とCCR-5との相互作用は、CD4^-細胞上ではCD4⁺細胞上より少なくとも100倍高いgp120濃度を要する。これは、該細胞表面上のCD4に対するgp120の結合がgp120-CCR-5相互作用の確率を増加させることによるものである、と本発明者らは示唆する。すなわち、gp120-CD4-CCR-5三重複合体が形成されるか、またはgp120とCD4、ついでCCR-5との逐次的な相互作用が生じるかのいずれかである。１つの可能性は、gp120とCD4との間の高いアフィニティーの会合が、gp120とCCR-5とのより低いアフィニティーの相互作用の確率を増加させること（近接効果）である。これは、sCD4が細胞表面CD4（少なくとも、JR-FL gp120を有するもの）を置換しないという知見と一致する。あるいは、CD4に対する結合は、gp120上のCCR-5結合部位を（より良好に）露出させるために必要かもしれない。これは、単量体gp120上で接近可能なオリゴマー性エンベロープ糖タンパク質の領域のいくつか（V3ループを含む）がCD4結合前に最適に露出されないビリオンの場合には特に重要である（18）。
gp120-CD4複合体が活性化CD4⁺Ｔ細胞上のCCR-5と如何にして相互作用するかに関する更なる知見を得るために、本発明者らは、JR-FL gp120に結合可能であることが各々確認されているHIV-1を中和する及び中和しない抗gp120モノクローナル抗体（19,20）のパネルを用いた。MIP-1β結合部位上のgp120の競合効果の逆転に関して、該抗体を試験した（表５）。sCD4の場合と同様に、CD4ガインディング（ginding）部位と重複するコンホメーションエピトープ（15eおよびIgG1b12）または線状エピトープ（G3-508）に対する抗体は（20）、JR-FL gp120がMIP-1βと競合するのを妨げた。しかしながら、CD4に対する単量体gp120の結合に影響を及ぼさないいくつかの抗体も（20）、gp120-CCR-5相互作用を阻害した（表５）。これらは、V3ループに対して３個（447-D、10bおよび83.1）、C3-V4領域内の保存エピトープに対して１個（2G12）、保存されたCD4誘導性エピトープに対して２個（48dおよび17b）含んでいた。A32以外のこれらのすべては、本発明者らがgp120中和面と定義した位置（20）にマッピングされる。JR-FL gp120がMIP-1β結合を遮断するのを妨げなかった他の８個のモノクローナル抗体は（表２）、本発明者らがgp120非中和面と定義した位置（20）にクラスター化する。それらのエピトープは、単量体gp120に接近しうるが、オリゴマーの場合には、それらは他のgp120サブユニットまたはgp41分子により塞がれる（19,20）。無効なこれらの抗体は、A32のものと重複するエピトープに対する2/11cを含む。この理由により、また、A32はHIV-1株を強力には中和しないため、gp120-CCR-5相互作用に対するA32の部分阻害作用の意義は明らかでない。また、V2ループに対する２つのモノクローナル抗体（697-DおよびSC259）も無効であった。V2ループ構造はgp120中和面（20）上にモデル化されるが、これらの２つ抗体は、HIV-1_JR-FLを中和する能力を有していない。また、モノクローナル抗体2G12、17b、447-D、48d、IgG1b12、G3-508および697-Dを、オリゴマー性JR-FL gp120/gp41タンパク質に対して試験し、697-D以外のすべてが、このタンパク質とCCR-5との相互作用を阻害した（示されていない）。
したがって、gp120の中和面に対するこれらの抗体のほとんどは、CD4に対するgp120の結合を妨げるか、CCR-5第２受容体との後続の相互作用を妨げた。初代ウイルスは中和に抵抗し、組換えタンパク質での研究は中和効力を不正確に予測しうるにすぎないため（18）、gp120のこの面に対するすべての抗体が、HIV-1_JR-FLを実際に中和するわけではない。しかしながら、本発明者らの知見は、HIV-1が如何にして抗体に中和されるかに関する理解を示唆しているかもしれない。該ウイルスの第１または第２受容体相互作用の遮断が特に重要であるかもしれない。
Δ-V3 JR-FL gp120がMIP-1β結合を遮断する能力を有していないこと（図3a）についての最も簡単な解釈は、CCR-5結合部位がV3ループ内に含有されている、というものである。これは、V3ループがHIV-1表現型および指向性の重要な決定因子を含有するという多数の観察（18,21）と一致し、第２受容体の利用に影響を及ぼしうる（3）。しかしながら、本発明者らは、CCR-5結合部位がV3ループに限定されないと考えている。CCR-5と相互作用する亜型Ａ、ＢおよびＥのgp120のV3配列は、相当に可変性である（表４）。さらに、いくつかのサル免疫不全ウイルス（SIV）株もまた、第２受容体としてヒトCCR-5を使用しうるが（Z.W. ChenおよびP. Marx,私信）、HIV-1およびSIVのV3領域は、一次配列の相同性をほとんど有していない。これらすべての配列のそれぞれが、同じ保存された細胞タンパク質に対する結合部位を形成することは、TCLA HIV-1株からの同様のV3配列がそのようにできない場合に可能なのであろうか（表４）。リガンドとケモカイン受容体との相互作用の双状部位モデル（twin-site models）（8）は、比較的保存されたV3ループ部が、gp120上のCCR-5に対する複数点（multi-point）結合部位の一成分となりうる可能性を依然として残している。しかしながら、本発明者らは、CCR-5結合部位が、HIV-1遺伝子亜型間だけでなく霊長類免疫不全ウイルス間で強く保存されているgp120領域を含んでいるにちがいないと示唆する。これがCXCR-4とのHIV-1相互作用にも当てはまるか否かは、依然として明らかでない。
V3ループの構造は、gp120上のCCR-5に対する複雑な結合部位の性質に影響を及ぼすかもしれない。モノクローナル抗体48dおよび17bのCD4誘導性エピトープと重複している（必ずしも同一というわけではない）gp120の領域は、そのような部位の優れた候補である。これらの抗体は、V1、V2およびV3ループの塩基の周囲におそらく位置するコンホメーション的に感受性の同様の構造を認識する（22,23）。HxBc2 gp120およびJR-Fl gp120の両方からのV3ループの欠失は、48dおよび17bエピトープを破壊し（22,23）（未公開データ）、これは、Δ-V3 JR-FL gp120がCCR-5と相互作用する能力を有していないことと関連している可能性があり（図5A）、HIV-1_LAIのV3およびC4領域内の単一のアミノ酸の変化も、これらのエピトープの構造に大きな影響を及ぼすかもしれない（24）。
CCR-5結合部位についての本発明者らの理解を深めるためには、さらなる研究が必要であろう。β-ケモカインが末梢血単核細胞（PMBC）内でNSI初代分離株の複製を阻害する効率は、亜型ではなく株に左右され（未公開データ）、このことは、おそらく、gp120とCCR-5上のβ-ケモカイン結合部位との重複の程度がgp120間で異なることを示唆している。もしそうだとしたら、gp120上のCCR-5結合部位は、CD4結合部位より柔軟かもしれない。最後に、HIV-1および（特に）HIV-2およびSIVのいくつかの株では、sCD4はCD4⁺細胞上のJR-FL gp120とCCR-5との相互作用を阻害したが、sCD4は、第２受容体相互作用の効率を増強して、CD4またはCD4⁺細胞内へのこれらの霊長類免疫不全ウイルスの侵入を容易にしている可能性がある（25,26）。
第２の一連の実験の参照文献

第３の一連の実験
概括
C-Cケモカイン受容体CCR5は、CD4⁺細胞の原形質膜とのＭ指向性HIV-1株の効率的な融合に必要であり（1-5）、ウイルス表面糖タンパク質gp120と直接的に相互作用する（6,7）。受容体キメラ研究は有用な情報を提供しているが（8-10）、HIV-1の侵入のために機能するCCR5のドメイン（gp120相互作用の部位を含む）は明確には同定されていない。負に荷電した残基Asp-2（D2）、Asp-11（D11）およびGlu-18（E18）の置換だけが、単独で又は一緒になって、ADAおよびJR/FL M指向性株およびDH123二重指向性（dual-tropic）株へのCCR5媒介性HIV-1侵入を損なうか破綻することを示すために、本発明者らはここで、CCR5の部位特異的アラニンスキャニング突然変異誘発を用いる。また、これらの突然変異は、env媒介性膜融合およびgp120-CCR5相互作用を損なう。これらの３つの残基のうち、D11だけが、HIV-1侵入のCC-ケモカイン媒介性阻害に必要であるが、それは、他の細胞外CCR5残基にも左右される。したがって、gp120およびCC-ケモカイン結合部位（CCR5上）は部分的に重複するにすぎず、前者は、Ｎ末端CCR5ドメイン内の負に荷電した残基を必要とする。
結果
gp120結合およびHIV-1侵入に関与するCCR5の領域を同定するために、本発明者らは、荷電残基がCC-ケモカインとその受容体との相互作用に予め関与していることに基づいて、Ｎ末端（Nt）および３つの細胞外ループ（ECL１〜３）内の負（Ｄ、Ｅ）または正（Ｋ、Ｒ、Ｈ）に荷電した残基のアラニンスキャニング突然変異誘発を行なった（11 12）。また、本発明者らは、ヒトCCR5とそのマウスホモログ（これは、HIV-1侵入には非機能的である）（9,10）との間で異なるいずれかの残基を、該相違が荷電変化を伴うたびに改変した。全部で、15個の一重突然変異体、４個の二重突然変異体および１個の三重突然変異体を、これらの研究で試験した（図４）。
野生型および突然変異CCR5タンパク質（検出を容易にするためにＣ末端でHA標識されている）を、U87MG-CD4およびSCL-1-CD4細胞内で一過性に発現させ、HIV-1エンベロープ糖タンパク質により媒介される侵入をそれらが補助する能力を、ルシフェラーゼで読み取るenv補足アッセイを用いて測定した（図５）（1,2）。これらの非リンパ球系ヒト細胞系を選択したのは、それらが、CCR5、CCR3およびCXCR4共受容体を欠き、トランスフェクトされた共受容体の不存在下でHIV-1による感染に抵抗するからである（2,4,13,14）（少数の例外的なHIV-1株は、未知の経路でU87MG-CD4細胞に侵入するが（15）、本発明者らはそれらを使用しなかった）。両方の細胞系で、ほぼ同じ結果を得た。CXCR4ではなくCCR5を使用する２つのＭ指向性ウイルス（ADAおよびJR-FL）（1-5）、およびCCR5およびCXCR4の両方を同等に十分に利用する１つの二重指向性ウイルス（DH123）（15）を用いて、突然変異体CCR5タンパク質がHIV-1の侵入を補助しうるか否かを試験した。トランスフェクトされた各CCR5突然変異体の発現レベルを、ウエスタンブロット法により評価し、共受容体の効率の測定の際に考慮した。
15個の一重突然変異のうち３個だけが、CCR5の共受容体の機能に対する有意な阻害効果を有していた（図５）。これらは、D2A、D11AおよびE18Aであり、すべて、CCR5のNtドメイン内に位置していた（図４）。E18A置換だけが、CCR5の機能を15〜20倍減少させるのに十分であった。二重突然変異体D2A/D11Aは、いずれの一重突然変異体よりも低活性であり、三重突然変異体（D2A/D11A/E18A）はほぼ完全に不活性であった（野生型と比べて>50倍の侵入の減少）（図5A）。残りの置換はいずれも、このアッセイにおいてはCCR5の機能に有意な影響を及ぼさなかった（図5B、Ｃ）。両方のＭ指向性エンベロープ糖タンパク質で同様の結果を得た。二重指向性DH123エンベロープで認められた唯一の相違は、D11A置換に対する感受性の有意な増加であった（図5A）。このように、CCR5 Nt内の負に荷電した残基は、この分子の共受容体の機能に大きな影響を及ぼす。
HIV-1 env機能に関する独立したアッセイにおいてD2A、D11AおよびE18A置換の効果を研究するために、本発明者らは、JR-FL env遺伝子を安定に発現するHeLa細胞と、野生型または突然変異体CCR5で一過性にトランスフェクトしたHeLa-CD4細胞とを混合する膜融合アッセイを用いた（図６）（1,18）。それらの２つの細胞型を種々の蛍光プローブで標識し、それらの２つの染料が同一膜内に存在する場合にだけ生じる共鳴エネルギー転移（RET）（1,18）により融合をモニターする。本発明者らは、D2A、D11AおよびE18A一重突然変異体ならびに二重および三重突然変異体を、RETアッセイにおいて野生型CCR5と比較して試験した。各突然変異体は、ウイルス侵入アッセイで観察されたのと同じ表現型をこの融合アッセイにおいて有していた（図5A、６）。E18Aならびに二重および三重突然変異体は、env媒介性膜融合を補助することが完全に不可能であった。しかしながら、本発明者らが、ワクチン-T7ポリメラーゼ（vFT7-pol）系を用いて共受容体の発現を約100倍増強した場合には（1,4.5,13）、該CCR5突然変異体のそれぞれは、野生型タンパク質より低効率ではあったものの、膜融合を補助することができた（図６）。本発明者らは、CCRのCC-ケモカインリガンドが膜融合を阻害する能力をCCR過剰発現が破綻させることを既に認めており、このことは、CCR5発現が高すぎる場合にはいくつかの表現型変化が生じない可能性があることを示唆している（1）。しかしながら、vac-T7pol系での結果は、三重突然変異体（D2A/D11A/E18A）でさえ、共受容体として完全に不活性というわけではなく、非常に強く損なわれているにすぎないことを示している。
つぎに、本発明者らは、HIV-1の侵入を補助するCCR5突然変異体が、CCR5のCC-ケモカインリガンド（MIP-1α、MIP-1βおよびRANTES）の阻害効果に対して感受性であるか否かを試験した（表１）（19-21）。

表６：CC-ケモカインによる共受容体機能の阻害
U87MG-CD4細胞に、野生型または突然変異体CCR5を一過性にリポフェクションし、ついでNlluc/JR-FLを感染させた（2μg/mlのMIP-1α、MIP-1βおよびRANTESの存在下または不存在下で行なった）。72時間後、ルシフェラーゼ活性を測定した（1,2）。各突然変異体に対するCC-ケモカインによる相対阻害率（％）を、［1−（ケモカイン存在下のルシフェラーゼc.p.s.／ケモカイン不存在下のルシフェラーゼc.p.s.）］／［1−（ケモカイン存在下の野生型ルシフェラーゼc.p.s.／ケモカイン不存在下の野生型ルシフェラーゼc.p.s.）］×100％と定義する。各値は、それぞれ四重に行なった３つの独立した実験の平均値である。相対阻害率（％）が野生型CCR5で認められるものの<50％である突然変異共受容体には、陰影が付されている。
（D2A、D11AおよびE18A突然変異体はHIV-1侵入に関して損なわれているが、それらは、阻害に対する感受性を測定するに十分な程度の侵入を補助したことに注意されたい。しかしながら、これは、Nt二重および三重突然変異体には当てはまらなかった）。U87MG-CD4細胞では、他の非リンパ球様細胞の場合と同様に（1,2,22）、CC-ケモカインは、HIV-1感染を完全には阻止せず、効果を得るためには高濃度を要する。したがって、本発明者らは、突然変異体および野生型CCR5受容体上でCC-ケモカインにより得られる阻害の程度を比較した（個々のリガンドに応じて40〜60％、それぞれの効力はRANTES>MIP-1β>MIP-1α）。以下の突然変異体は、CC-ケモカインの１以上の作用に対して比較的に不感受性であった:D11A、K22A、R31A（Nt）、H181A、Y184A、K171A/E172A、K191A/N192A（ECL2）、R274A/D276A（ECL3）。これらのうち、D11Aだけが、HIV-1侵入および侵入のCC-ケモカイン阻害の両方に関して損なわれた。したがって、ある位置（ほとんどは、NtおよびECL2内）でのアミノ酸置換は、CCR5のHIV-1共受容体機能に影響を及ぼさないが、この過程のCC-ケモカイン媒介性阻害には影響を及ぼす（表６）。これらの置換がCC-ケモカインの作用に影響を与える様態は、明らかでない。しかしながら、最も簡単な解釈は、CCR5上のCC-ケモカイン結合部位とHIV-1相互反応部位とが同一ではなく、ECL2およびECL3内の或る置換がCC-ケモカイン結合部位にだけ影響を及ぼす、というものである。
Nt置換が如何にしてCCR5のHIV-1共受容体機能に影響を及ぼすのかを理解するために、本発明者らは、それらがgp120結合に影響を及ぼすか否かを確認した。本発明者らは、CCR5に対する標識化gp120の結合を直接測定することができなかった。なぜなら、一過性にトランスフェクトされた細胞上でのCCR5発現のレベルが低すぎて、試験したいくつかの結合アッセイのいずれにおいても、再現可能なシグナルを得ることができなかったからである。したがって、本発明者らは、フィコエリトリン（PE）標識化CCR5特異的MAb（2D7-PE）（23-25）に対する結合をgp120（JR-FL）（7）が阻害する能力を測定する競合アッセイを用いた。このMAbに対するエピトープはECL2内に位置し、本発明者らは、それが、CD4およびCCR5 Nt突然変異体でコトランスフェクトされたHeLa細胞に効率的に結合しうることを見出した。
gp120結合の検出を可能にする程度にまでCCR5を過剰発現するマウスL1.2細胞系（25）上のCCR5に対する（125）I標識gp120の結合を2D7が阻害することが、独立した研究で示されている（6,25）。ここでは、本発明者らは、野生型CCR5に対する2D7-PEの結合が、gp120を予め加えることにより強力（70％）に阻害されたことを示す。このことは、gp120および2D7と該受容体との相互作用が、互いに排他的であることを示している（図７）。しかしながら、gp120は、D2A、D11AおよびE18A突然変異体に対する2D7-PEの結合を部分的（40％）に阻害したにすぎず、二重および三重Nt突然変異体に対する2D7-PE結合の遮断に関してはほとんど無効であった（それぞれ25％および15％の阻害）（図７）。注目すべきは、HIV-1侵入に関して最も損なわれている突然変異体（図５）が、同時に、2D7-PE結合がgp120阻害に対して最も低感受性であるものでもあったことである（図７）。この結果についての最も確かな解釈は、gp120が、ECL2に対する2D7-PEの結合を立体的に妨害するように野生型CCR5分子に結合するが、Nt突然変異体には不完全にしか結合しない、というものである。それより低い可能性は、gp120がNt突然変異体に効率的に結合するものの、その結合が、ECL2に対する2D7-PE結合を阻害する可能性を低下させる異常な配向で生じる、というものである。後者の場合には、CCR5内のドメイン間相互作用の幾何構造は、CCR5共受容体機能を損なうNt置換により改変されている。
本研究において本発明者らは、共受容体機能のCC-ケモカイン阻害を必然的に妨げることなくCCR5の共受容体機能に影響を及ぼすアミノ末端ドメイン内の負に荷電した３個の残基での点置換を同定した。同じ置換は、おそらくgp120-CCR5相互作用のアフィニティーを減少させることにより、gp120がCCR5と正しく相互作用する能力に影響を及ぼす。これは、共受容体欠損表現型を説明するのに十分であるかもしれない。CCR5内のNt置換により生じるgp120に対するアフィニティーの喪失は、突然変異共受容体の過剰発現により部分的に補われうる（図６）。これはおそらく、低いアフィニティーの共受容体の数の増加が、膜融合を開始させるエンベロープ糖タンパク質内のコンホメーション変化に適合するのに十分な程度に迅速に、首尾良いgp120-CCR5相互作用を引き起こしうるからであろう（26-28）。
したがって、CCR5上のgp120結合部位はNt内の残基に左右され、実際には、別個のgp120結合ドメインがNtに限局されている可能性がある。キメラ受容体または欠失突然変異体を用いる従前の研究は、共受容体機能に対するCCR5およびCXCR4 Ntの重要性を示している（8-10,29）。また、該キメラ研究は、CCR5とHIV-1との相互作用の部位が、比較的広く、ある程度柔軟であると示唆している（8-10）。この可能性を無視すべきではないが、受容体キメラの細胞外ループ内の改変がCCR5 Ntの配向（したがって、それがgp120と正しく相互作用する能力）に間接的に影響を及ぼしている可能性もある。gp120結合部位とは対照的に、CCR5上のCC-ケモカイン結合部位は、Nt内と細胞外ループ内との両方の残基に左右される（ECLが、独占的ではないものの顕著である）。したがって、gp120とCC-ケモカイン結合部位との間には或る程度の重複があるが（CCR5に対するCC-ケモカイン結合のgp120阻害により示されるとおり）（6,7）、それらは同一ではなく、シグナル伝達および共受容体活性がCCR5の分離可能な機能であることを示す研究と一致した結論となる（10,30,31）。
HIV-1の標的細胞内への侵入のためにHIV-1がCCR5を如何に使用するかを明らかにし、そしておそらくこの過程の阻害剤を開発するためには、gp120およびCC-ケモカインに対するCCR5上（およびCCR5に対するこれらの分子上）の相互活性部位についてのより詳細な理解が必要となろう。また、CCR5 Nt内で本発明者らが同定した負に荷電した残基が、gp120内、V3ループ内および／またはその他の部位内の正に荷電したアミノ酸と直接的または間接的に相互作用するか否かを確認することが重要となろう。
方法
リポフェクションおよびレポーター遺伝子アッセイ
突然変異cDNAをpcDNA3.1（Stratagene）発現ベクター内にサブクローニングした。U87MG-CD4およびSCL-1-CD4細胞を、リポフェクチン（5μg/ml）およびpSVlacZ（5μg/ml）、または突然変異体DNA（4μg/ml）＋pSVlacZ（1μg/ml）（OPTI-MEM（Gibco BRL）内）と共に、37℃で５時間インキュベートした。24時間後、200〜500ng/ml p24を含有するNLluc/Env上清を、該細胞に37℃で２時間感染させた。HIV-1の侵入のCC-ケモカインによる阻止のために、2μg/mlのMIP-1α、MIP-1βまたはRANTES（R & D System）を、HIV-1（50〜100ng/ml p24）と同時に加え、該培養内に12時間維持した。感染の72時間後、細胞サンプルを100μlの溶解緩衝液（Promega）で処理し、ルシフェラーゼ（luc）およびβ-ガラクトシダーゼ活性（OD₄₂₀）を測定した（1,2）。標準化ルシフェラーゼ活性を、（luc c.p.s／ng/ml p24／（OD₄₂₀÷対照OD₄₂₀）／（突然変異体CCR5バンドに関するr.f.e.÷野生型CCR5バンドに関するr.f.e.））と定義する（以下を参照されたい）。Luc c.p.s.値は、野生型CCR5に関しては5×10⁵〜2×10⁶の範囲であった。
全細胞抽出物におけるCCR5発現の免疫ブロット分析
この研究で使用するすべてのCCR5分子は、検出を容易にするために、Ｃ末端伸長として９残基の赤血球凝集素（HA）-タグを有していた。60mm組織培養プレートからのリポフェクション化U87MG-CD4細胞を、１％ドデシルマルトシドナトリウム、10mM Tris-HCl（pH6.8）、50mM NaCl、1mM CaCl₂、0.1mM PMSF、5μg/mlロイペプチン、10μg/mlアプロチニン、0.7μg/mlペプスタチンおよび10mM EDTAに再懸濁した。該懸濁液を４℃で30分間インキュベートし、上清画分を集めた。全タンパク質濃度を、Bio-Rad DC Protein Assayを用いて測定した。ついでタンパク質（15μg）を、予め煮沸することなく、SDS-ポリアクリルアミドゲル上で分画した。タンパク質をImmobilon-Pメンブレン（Millipore）にトランスファーし、CCR5に関してウサギ抗HAタグ抗体（1:500希釈;Berkeley Antibody Company）およびAP標識ヤギ抗ウサギIgG（1:10⁴希釈;Amersham）でプローブし、ついで化学蛍光基質（Vistra ECF, Amersham）と共にインキュベートした。450nmで励起された免疫反応性バンボの相対蛍光発光（r.f.e.）を、レーザーベーススキャナー（laser-based scanner）（Molecular Dynamics Storm 860）上で検出した。全細胞抽出物と原形質膜抽出物とで同一の発現パターンが得られた。CCR5突然変異体の発現レベルは、野生型タンパク質の20％〜100％で変化した。野生型CCR5の発現レベルとHIV-1の侵入効率との関係は、10倍の範囲にわたり直線的であることを確認した（データは示していない）。
CCR5結合に関するgp120と2D7MAbとの競合
リポフェクチン（10μg/ml）およびpCDM8 CD4発現ベクター（3.75μg/ml）＋野生型または突然変異型CCR5プラスミド（1.25μg/ml）と共に、HeLa細胞（2×10⁶個）をOPTI-MEM内で５時間インキュベートした。ついで、CCR5発現を増強するために、該細胞に2×10⁷p.f.u.のvFT7を12時間感染させ、その細胞を2mM EDTA/PBSで解離させ、結合緩衝液（DPBS内の１％BSA、0.05％アジド）で１回洗浄した。細胞（1×10⁶個）を、10μg/mlのgp120（JR-FL）（7）の存在下または不存在下、37℃で１時間インキュベートした後、PE標識2D7 MAb（23,24）（20ng/ml）を４℃で30分間加えた。該細胞を結合緩衝液で１回、PBSで１回洗浄し、１％ホルムアルデヒド／PBSに再懸濁し、FACSにより分析して、平均蛍光強度（m.f.i.）を測定した。Leu3Aでの染色によりCD4の発現をモニターした。該発現は、サンプル間で±10％以下で変動した。CCR5突然変異体の発現レベルは、野生型CCR5の発現レベルの20％〜100％の範囲であった
第三の一連の実験の参照文献

第四の実験
CCR5 ⁺ CD4 ^- 細胞へのHIV-1 _JR-FL gp120の直接的結合
CCR5⁺CD4^-細胞へのHIV-1_JR-FLgp120の直接的結合を実証した。この場合には、gp120をsCD4又は他のCD4ベースの分子とプレインキュベートすることが必要であるが、おそらく、これによってケモカイン受容体結合部位を露出せしめるgp120の立体構造の変化がもたらされるためであると思われる。図８は、ヒトCCR5を有するマウスL1.2細胞へのsCD4/gp120複合体の直接的結合を測定するためのフローサイトメトリーの使用を表している。結合のバックグラウンドレベルは、ビオチン化されたタンパク質のみ、又は実験室に適合させたHIV-1LAI株を用いるならばHIV-1_JR-FLgp120の代わりの何れかによって観察した（図には示されていない）。
該アッセイは、薬物スクリーニングの目的のために、sCD4/gp120複合体のCCR5⁺/CD4^-細胞への結合が、蛍光プレートリーダーを用いて測定される96穴マイクロプレートフォーマットに適合させた。一つの方法は、以下のとおりである。
1）L1.2-CCR5⁺細胞を播種する（約500,000／ウェル）
2）室温で１時間阻害剤を添加する
3）洗浄し、ビオチン化されたsCD4（2.5μg/mL）及びビオチン化されたHIV-1_JR-FLgp120（5μg/mL）を加え、次に室温で２時間インキュベートする
4）洗浄し、ストレプトアビジン−フィコエリトリン（100ng/nL）とともにインキュベートする
5）洗浄し、530nmでの励起、及び590nmの測定蛍光で蛍光プレートリーダーを用いて、結合したgp120/sCD4の量を測定する
該方法を用いて、CCケモカインによるgp120/sCD4へのCCR5の阻害（図９）及びHIV-1感染をブロックするCCR5に対する抗体（図には示されていない）が実証された。
CCR5の細胞外ドメインによるHIV-1エンベロープを介した膜融合の阻害
クオリティー・コントロールド・バイオケミカルズ（Quality Controlled Biochemicals;Hopkinton, MA）によってヒトCCR5の４つの細胞外ドメインに相当する合成ペプチドを作成し、上述した共鳴エネルギー移動（RET）アッセイを用いて、HIV-1のLAI又はJR-FL株のエンベロープ糖タンパク質によって介される膜融合を阻害する能力をテストした。ECL2ペプチドを用いると、JR-FLエンベロープによって介される融合の特異的な阻害がみられたが、他のペプチドではみられなかった。ECL2は、100μg/mLで97％、33μg/mLで65％、及び11μg/mLで15％、HeLa-env_JR-FL細胞とPM1細胞との融合を阻害した（二つのアッセイの平均）。ECL2はHeLa-env_LAIとPMI細胞との融合又はHeLa-env_LAIとHeLa-CD4細胞との融合を阻害しなかった。これらの結果は、おそらくHIV-1JR-FL gp120とCCR5との相互作用をブロックすることによって、CCR5 ECL2が融合を特異的に阻害することを強く示唆している。テストした他のペプチドは、有意なレベルの特異的な融合の阻害を与えなかった。
バイサイクラム、JM3100によるHIV-1エンベロープを介した膜融合の阻害
J.ムーア博士（Dr. J. Moore; Aaron Diamond AIDS Research center, NY）から入手したバイサイクラムが、上述した共鳴エネルギー移動（RET）アッセイを用いて、HIV-1のLAI又はJR-FL株のエンベロープ糖タンパク質によって介される膜融合を阻害する能力をテストした。図10に示されているように、該分子は、HIV-1_LAI由来のgp120/gp41によって介される融合を特異的且つ強力に阻害するが、HIV-1_JR-FL株由来のgp120/gp41によって介される融合は阻害しない。これらのデータは、HIV-1_LAIgp120とCXCR4との相互作用をブロックすることによって、該分子がHIVの融合を特異的に阻害することを示唆している。
配列表
（１）一般情報：
（i）出願人：アラウェイ，グラハム Ｐ
ドラジク，タティヤナ
リトウィン，バージニア Ａ
マドン，ポール Ｊ
ムーア，ジョン Ｐ
（ii）発明の名称：ＨＩＶ−１感染を阻害するためのケモカイン受容体の使用
（iii）配列の数：３０
（iv）通信宛先：
（Ａ）名宛人：クーパー・アンド・ダンハム ＬＬＰ
（Ｂ）通り：１１８５ アヴェニュー・オブ・ジ・アメリカズ
（Ｃ）市：ニューヨーク
（Ｄ）州：ニューヨーク
（Ｅ）国：アメリカ合衆国
（Ｆ）郵便番号：１００３６
（ｖ）コンピュータ読取り可能な形態：
（Ａ）媒体タイプ：フロッピーディスク
（Ｂ）コンピュータ：ＩＢＭ・ＰＣコンパチブル
（Ｃ）オペレーティングシステム：ＰＣ−ＤＯＳ／ＭＳ−ＤＯＳ
（Ｄ）ソフトウェア：Patent Inリリース#1.0、バージョン#1.30
（vi）現在の出願データ：
（Ａ）出願番号：
（Ｂ）出願日：
（Ｃ）分類：
（viii）アトニー／エージェント情報：
（Ａ）氏名：ホワイト，ジョン Ｐ．
（Ｂ）登録番号：２８，６７８
（Ｃ）参照／ドケット番号：５１３２０
（ix）電信情報：
（Ａ）電話：（２１２）２７８−０４００
（Ｂ）テレファックス：（２１２）３９１−０５２５
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This application claims priority from US Provisional Application Publication No. 60 / 019,941, filed June 14, 1996, the contents of which are incorporated herein by reference.
The invention described in this application was made with the assistance of Grants Al35522, Al36057, Al36082, and Al38573 from the National Institutes of Health, US Department of Health and Human Services. Accordingly, the United States government has certain rights in the invention.
Throughout this application, various references are cited in Arabic numerals in parentheses. In order to more fully describe the current state of the art to which this invention pertains, the entire disclosure content of these documents is incorporated herein by reference. A complete literature list of these references is listed at the end of each experiment.
Background of the Invention
Replication of primary non-syncytium-inducing (NSI) HIV-1 isolates in CD4 + T cells is inhibited by the C-Cβ chemokines MIP-1α, MIP-1β and RANTES However, T cell line adapted (TCLA) primary or syncytium inducing (SI) primary strains are insensitive (2, 3). β-chemokines are small (8 kDa) closely related proteins that are active on lymphoid and monocyte cells (4-8). These receptors are members of the 7-transmembrane, G protein-coupled superfamily, one of which (LESTR orphan receptor) has been identified as the second receptor of the TCLA HIV strain and is now fusine ( fusin) (9). Fusin is not known to be a β chemokine receptor (7-9).
Summary of the Invention
The present invention relates to CD4 of HIV-1.⁺A polypeptide having a sequence corresponding to a partial sequence of a chemokine receptor capable of inhibiting fusion to a cell and thereby inhibiting infection of the cell is provided. In certain embodiments, the chemokine receptor is C-C CKR-5. CCKR-5 is also called CCR5. In another embodiment, the polypeptide comprises an amino acid having the sequence of the extracellular domain of C-at least one C-C CKR-5.
In one preferred embodiment, the portion of the chemokine receptor comprises the amino acid sequence MDYQVSSPIYDINYYTSEPCQKINVKQIAAR (SEQ ID NO: 5). In another preferred embodiment, said portion comprises the amino acid sequence HYAAAQWDFGNTMCQ (SEQ ID NO: 6). In yet another preferred embodiment, said portion comprises the amino acid sequence RSQKEGLHYTCSSHFPYSQYQFWKNFQTLKIV (SEQ ID NO: 7). In another preferred embodiment, said portion comprises the amino acid sequence QEFFGLNNCSSSNRLDQ (SEQ ID NO: 8). A portion of the chemokine receptor C-C CKR-5 may comprise one or more of the above sequences. The polypeptide may contain a portion of the above sequence that can still inhibit HIV-1 infection. The minimum number of amino acids sufficient to inhibit HIV infection can be determined by the RET or infection assay shown below.
The present invention also provides a pharmaceutical composition comprising an effective amount of one or more of the above polypeptides and a pharmaceutically acceptable carrier.
The present invention also provides a polypeptide having a sequence corresponding to a partial sequence of HIV-1 glycoprotein that can specifically bind to the chemokine receptor C-C CKR-5.
The present invention relates to an effective amount of one or more polypeptides having a sequence corresponding to a partial sequence of HIV-1 glycoprotein capable of specifically binding to the chemokine receptor CC CKR-5 and a pharmaceutically acceptable carrier. A pharmaceutical composition is provided.
The present invention relates to CD4⁺Provided are antibodies or portions of antibodies that can bind to chemokine receptors on cells and can inhibit HIV-1 infection of cells.
The present invention relates to CD4⁺Also provided is a pharmaceutical composition comprising an effective amount of an antibody capable of binding to a chemokine receptor on a cell and inhibiting HIV-1 infection of the cell and a pharmaceutically acceptable carrier.
The present invention provides a method of treating a patient infected with HIV-1, comprising administering to the patient the polypeptide, antibody and pharmaceutical composition described above.
The present invention provides a method for reducing the rate at which a subject is infected with HIV-1, comprising administering the pharmaceutical composition to a subject.
The present invention relates to CD4⁺CD4 and a non-chemokine agent that can bind to the chemokine receptor C-C CKR-5 in an amount and under conditions that inhibit the fusion of HIV-1 to the cell⁺HIV-1 CD4 comprising contacting cells⁺Methods of inhibiting fusion to a cell are provided.
The present invention relates to CD4⁺CD4 and a non-chemokine agent that can bind to the chemokine receptor C-C CKR-5 in an amount and under conditions that inhibit the fusion of HIV-1 to the cell⁺CD4 comprising inhibiting cell HIV-1 infection by contacting the cell⁺Methods are provided for inhibiting cells from being infected with HIV-1.
The present invention provides non-chemokine agents capable of inhibiting binding to the chemokine receptor C-C CKR-5 and HIV-1 infection.
The present invention relates to CD4 other than chemokine receptors so that binding of a non-chemokine agent to a chemokine receptor does not prevent the ligand from binding to another receptor.⁺Provided are molecules capable of inhibiting binding to the chemokine receptor C-C CKR-5 and HIV-1 infection comprising a non-chemokine agent linked to a ligand capable of binding to a cell surface receptor of a cell.
The present invention relates to CD4 of HIV-1.⁺Provided is a pharmaceutical composition comprising a substantial amount of the above-described molecule effective to inhibit fusion to cells and a pharmaceutically acceptable carrier.
The present invention is capable of binding to the chemokine receptor C-C CKR-5 comprising a non-chemokine agent coupled to a compound capable of increasing the in vivo half-life of the non-chemokine agent, and CD4 of HIV-1⁺Molecules that can inhibit fusion to cells are also provided.
The present invention further includes CD4⁺Binds to the chemokine receptor CC CKR-5 with a non-chemokine agent coupled to a compound that can increase the in vivo half-life of a non-chemokine agent effective to inhibit HIV-1 fusion to cells There is provided a pharmaceutical composition comprising a substantial amount of a molecule capable of inhibiting HIV-1 infection and a pharmaceutically acceptable carrier.
The present invention provides a method of reducing the rate of HIV-1 infection in a subject comprising administering the pharmaceutical composition to the subject.
The present invention provides a method of treating HIV-1 infection in a patient comprising administering to the patient the pharmaceutical composition described above.
The present invention provides that a non-chemokine agent is CD4⁺, C-C CKR-5⁺A method for determining whether HIV-1 fusion to a cell can be inhibited, comprising:
(A) HIV-1 CD4⁺, C-C CKR-5⁺CD4 on cells expressing HIV-1 envelope glycoprotein on its surface in the absence of drugs known to inhibit cell fusion⁺, C-C CKR-5⁺Under conditions that allow the cells to fuse, cells labeled with a second dye that expresses the appropriate HIV-1 envelope glycoprotein on its surface in the presence of excess drug, CD4 labeled with⁺, C-C CKR-5⁺Contacting the cells, and the first and second dyes are selected to be capable of resonance energy transfer between the dyes;
(B) contacting the product of step (a) under conditions that cause resonance energy transfer if fusion occurs;
(C) determine if resonance energy transfer is present and the absence or decrease of transfer indicates that the drug is CD4⁺And C-C CKR-5⁺Being an indicator of the ability to inhibit the fusion of HIV-1 to cells;
A method comprising:
The present invention also provides a non-human transgenic animal comprising an isolated DNA molecule encoding the chemokine receptor C-C CKR-5. In certain embodiments, the non-human transgenic animal further comprises an isolated DNA molecule encoding a portion of the CD4 molecule sufficient to be able to bind HIV-1 envelope glycoprotein.
The present invention further provides a non-human transgenic animal comprising an isolated DNA molecule encoding the chemokine receptor C-C CKR-5 and an isolated DNA molecule encoding fusin. In certain embodiments, the non-human transgenic animal further comprises an isolated DNA molecule that encodes a full-length CD4 molecule or a portion of a CD4 molecule sufficient to bind an HIV-1 envelope glycoprotein. .
The invention also provides a transformed cell comprising an isolated nucleic acid molecule encoding the chemokine receptor C-C CKR-5.
Finally, the present invention provides an agent capable of inhibiting HIV-1 infection and binding to a chemokine receptor without substantially affecting the ability of the chemokine receptor to bind to chemokines. .
Description of drawings
Figure 1: Specificity, time course, and stage (1A) of inhibition of HIV-1 replication by β chemokines, along with RANTES + MIP-1α + MIP-1β (R / Mα / Mβ; 100 ng / mL each) PM1 cells ( 1 × 10⁶) For 24 hours (-24 h) or 2 hours (-2 h) and then washed twice with phosphate buffered saline (PBS). After adding HIV-1 (complemented with BaL env) virus (50 ng p24; see footnote in Table 1) for 2 hours, before measuring luciferase activity in the cell eluate as previously described (10,11) The cells were washed and incubated for 48 hours. Virus and R / Mα / Mβ were added to the cells simultaneously, and at the indicated time points (1h, 3h), the cells were washed twice in PBS and resuspended in culture until 48 hours before luciferase assay. Incubated for hours. Zero time is a positive control with no added beta chemokine. + 2h indicates that the cells and virus are mixed for 2 hours before washing twice in PBS, R / Mα / Mβ is added and the culture is continued for an additional 48 hours before the luciferase assay.
(1B) CEM cells (NL4 / 3; in the presence of 500 ng / mL RANTES (lanes 1 and 5) or MIP-1β (lanes 2 and 6) or in the absence of β-chemokines (lanes 4 and 8); Lanes 1-4) or HIV-1 (500 pgp24) grown in macrophages (ADA; lanes 5-8) are PM1 cells (1 × 10⁶). Lanes 3 and 7 are negative controls (no virus). All virus strains used in the PCR assay were treated with DNAse for 30 minutes at 37 ° C and tested for DNA contamination prior to use. After 2 hours, the cells were washed and after an additional 8 hours in the medium containing the same chemokine, the cells were washed and resuspended in the medium containing the same chemokine for an additional 8 hours. Next, DNA was extracted from the infected cells using a DNA / RNA isolation kit (US Biochemicals). The first round of nested PCR uses primers: U3 +, 5'-CAAGGCTACTTCCCTGATTGGCAGAACTACACACCAGG-3 '(SEQ ID NO: 1) preGag, 5'-AGCAAGCCGAGTCCTGCGTCGAGAG-3' (SEQ ID NO: 2), and the second round of nested PCR uses primers : LTR-test, 5′-GGGACTTTCCGCTGGGGACTTTC3 ′ (SEQ ID NO: 3) LRC2,5′-CCTGTTCGGGCGCCACTGCTAGAGATTTTCCAC3 ′ (SEQ ID NO: 4) in a Perkin Elmer 2400 cycler with the following amplification cycle (94 ° C. for 5 minutes, 94 ° C. 30 Seconds, 55 ° C. for 30 seconds, 72 ° C. for 30 seconds, 72 ° C. for 7 minutes and 35 cycles). M represents a 1 kb DNA ladder, and 1,10,100,1000 represents the number of reference plasmid (pAD8) copies. The assay can detect 100 copies of reverse transcript.
Fig. 2: Membrane fusion of cells transiently expressing CC CKR-5 via HIV-1 env Membrane fusion mediated by β-chemokine receptors expressed in HeLa cells is as follows: Demonstrated. Cells were transfected with control plasmid pcDNA3.1 or plasmid pcDNA3.1-CKR construct using Lipofectin (Gibco BRL). The pcDNA3.1 plasmid carries the T7 polymerase promoter, and transient expression of the β chemokine receptor is 1 × 10 4 encoding T7 polymerase (vFT7.3) 4 hours after lipofection.⁷Enhanced by infecting cells with pfu vaccinia (9). The cells are then cultured overnight in R18-containing media and tested by RET assay for their ability to fuse with HeLa-JR-FL cells (black columns) or HeLa-BRU cells (shaded columns). did. The% RET for control HeLa cells was between 3-4% regardless of the transfected plasmid.
Figure 3: CD4-dependent competition between gp120 and MIP1β for binding to CCR-5
(3A) CD4 activated JR-FL gp120 (black square), LAIgp120 (black triangle), JR-FL-ΔV3gp120 (white square) or LAI-ΔV3gp120 (white triangle)⁺Added to T cells and specific¹²⁵The extent of I-MIP-1β binding was determined. The data shown is the average of three independent experiments and each experiment was performed twice.
(3B) CD4 activated in the presence of monoclonal antibody Q4120 (black circle) or sCD4 (black square)⁺JR-FL gp120 (2 μg ml)^-1)as well as¹²⁵I-MIP-1β (0.1 nM) was added at the indicated concentration. Specific¹²⁵The degree of I-MIP-1β binding was determined and the percent inhibition of the gp120 competition effect was calculated for each antibody concentration (no inhibition is 0% inhibition). The experiment shown is one of the two performed, each with similar results.
Figure 4: Mutation into four predicted extracellular domains of CCR
The amino acid sequence of human CCR5 amino terminus (Nt) and three extracellular loops (ECL1-3) are shown (19,20 in the third experiment). Below the main sequence, the polarity (+ or-) of the charged residue is shown, along with the identification of residues that differ from mouse CCR5. Negative or positively charged human CCR residues (white squares), positively charged side chains (black squares), and residues that differ from mouse CCR5 (white circles) are all PCR or site-specific mutations Modified to alanine by introduction. The accuracy was confirmed by sequencing both strands of the entire CCR coding region. In some cases, double mutants K171A / E172A, K191A / N192A, and R274A / D276A were created to preserve the net total charge of those domains. Nt double and triple mutants, D2A / D11A and D2A / D11A / E18A were based on initial results with single residue mutants.
Figure 5: HIV-1 co-receptor function of CCR5 mutant
We tested the effect of substitutions in selected mouse residues different from human sequences on HIV-1 entry: (A) negatively charged residues; (B) positively charged residues; (C) human sequences. U87MG-CD4 cells are transiently lipofected with CCR5 mutants, followed by NLluc / ADA (dark slashed bars), NLluc / JR-FL (light slashed bars), or NLluc / DH123 (white bars) luciferase Infected with the expression chimeric virus (1,2 of the third experiment). Luciferase activity (luc c.p.s.) was measured 72 hours after infection (1,2) and normalized for lipofection efficiency and receptor expression level. The co-receptor activity of each variant shown on the X-axis is expressed as a percentage of wild-type co-receptor activity (100%), and is the mean ± sd of three independent experiments, each experiment being 4 I went twice. (^*) Indicates that the amino acid is also different in mouse CCR5. Similar results (not shown in the figure) were obtained with SCL-1-CD4 cells.
Figure 6: Membrane fusion activity of CCR5 Nt mutant
HeLa-CD4 cells were lipofected with the indicated Nt mutant (or pcDNA3.1 negative control plasmid) and tested 48 hours later for their ability to fuse with the HeLa cells expressing the JR-FL env gene (black). Bar) (1.18 of the third experiment). To increase co-receptor expression, the vFT7pol system was used (shaded bars) (1,4,5,13 in the third experiment). The extent of cell-cell fusion was determined using the RET assay (1.18 of the third experiment). The% RET values shown are the mean ± sd of three independent experiments performed twice for each experiment.
Figure 7: Competition between gp120 and CCR5 Mab 2D7 for CCR5 binding
Before adding 2ng / mL PE-labeled 2D7MAb (23,24 in the third experiment) and before FACS analysis to determine mean fluorescence intensity (mfi), CD4 and wild type CCR5 or mutant HeLa cells co-transfected with either CCR5 and infected with vFT7pol to increase receptor expression were preincubated with or without 10 μg / mL gp120 (JR-FL) (7). Inhibition of 2D7-PE binding is expressed as [1- (m.f.i. With gp120 added / m.f.i. Without gp120)] × 100%, and is the mean ± s.d. Of three independent experiments.
Figure 8: (B) CCR5^-And (B) CCR5⁺Flow cytometric analysis of sCD4-gp120 complex binding to L1.2 cells, mouse pre-B lymphoma lineage
sCD4 and biotinylated HIV-1_JR-FLCells were incubated for 15 minutes with an equimolar (˜100 nM) mixture of gp120, then stained with streptavidin-phycoerythrin conjugate, fixed with 2% formaldehyde and analyzed by FACS. The number of cells was plotted on the Y axis.
Figure 9: Butylated L1.2 CCR5 of HIV-1JR-FL gp120 complexed with sCD4⁺Inhibition of cell binding
Inhibitors were CC chemokines MIP-1β or RANTES at the concentrations indicated on the X-axis.
Figure 10: Inhibition of cell fusion through the HIV-1 envelope by bicyclam JM3100
Inhibition was measured with the indicated cell combinations using the RET assay.
Detailed Description of the Invention
The present invention provides a polypeptide having a sequence corresponding to a partial sequence of a chemokine receptor that can inhibit the fusion of HIV-1 to CD4 + cells and thereby inhibit infection of cells. In certain embodiments, the chemokine receptor is C-C CKR-5 (CCR5). In another embodiment, the fragment comprises the extracellular domain of at least one chemokine receptor C-C CKR-5. In a further embodiment, the extracellular domain is the second extracellular loop of CCR5.
In another embodiment, the chemokine receptor is CCR3 or CKR-2b (31,32).
Some sequences of chemokine receptors include the original amino acids or modified amino acids obtained from the receptors, derivatives and analogs thereof. Such a sequence must retain the ability to inhibit HIV-1 infection. The sequence of fusin is also included.
In one preferred embodiment, the portion of the chemokine receptor comprises the amino acid sequence MDYQVSSPIYDINYYTSEPCQKINVKQIAAR (SEQ ID NO: 5). In another preferred embodiment, said portion comprises the amino acid sequence HYAAAQWDFGNTMCQ (SEQ ID NO: 6). In yet another preferred embodiment, said portion comprises the amino acid sequence RSQKEGLHYTCSSHFPYSQYQFWKNFQTLKIV (SEQ ID NO: 7). In another preferred embodiment, said portion comprises the amino acid sequence QEFFGLNNCSSSNRLDQ (SEQ ID NO: 8). A portion of the chemokine receptor C-C CKR-5 may comprise one or more of the above sequences. The polypeptide may contain a portion of the above sequence that can still inhibit HIV-1 infection. The minimum number of amino acids sufficient to inhibit HIV-1 infection can be determined by the RET or infection assay shown below.
The polypeptide described above can be a fusion molecule in which a fragment is linked to another molecule. In certain embodiments, the molecule is a CD4-based molecule. CD4-based molecules are known in the art and are described in Allaway et al. (1996), Patent Cooperation Treaty Application PCT / US95 / 08805, Publication No. WO96 / 02575, see contents thereof. Incorporated into the present application as a document. In another embodiment, the polypeptide contains multiple units of one or more portions of a chemokine receptor. In certain preferred embodiments, the polypeptide contains a sequence corresponding to multiple units of one or more extracellular domains of the chemokine receptor C-C CKR-5.
The present invention also provides a pharmaceutical composition comprising an effective amount of the above polypeptide and a pharmaceutically acceptable carrier.
As used herein, “pharmaceutically acceptable carrier” refers to PBS solutions, water, and emulsions (such as oil / water or water / oil emulsions), and various types of wetting agents. Includes any standard pharmaceutical carrier.
The invention also provides a polypeptide having a sequence corresponding to a partial sequence of HIV-1 envelope glycoprotein that can specifically bind to chemoreceptor C-C CKR-5. Such sequences can be identified by routine experimentation. For example, overlapping synthetic peptides corresponding to fragments of gp120 or gp41 express cells that express envelope glycoproteins of clinical isolates of HIV-1 and cells that express CD4 and CC CKR-5 The ability to inhibit fusion with can be tested in a RET assay. Peptides that inhibit fusion in the assay were also screened in the RET assay for the ability to inhibit fusion-mediated by laboratory-adapted HIV-1 strain envelope glycoproteins and inhibited in the latter assay Discard the peptide showing sex. Alternatively, the peptides may be tested for the ability to compete with chemokine binding to cells expressing C-C CKR-5.
The present invention provides a pharmaceutical composition comprising an effective amount of a polypeptide comprising a fragment of HIV-1 glycoprotein capable of specifically binding to the chemokine receptor CC CKR-5 and a pharmaceutically acceptable carrier. To do.
The present invention relates to CD4⁺Provided are antibodies or portions of antibodies that can bind to chemokine receptors on cells and can inhibit HIV-1 infection of cells.
The present invention also provides a pharmaceutical composition comprising an effective amount of an antibody capable of binding to a chemokine receptor and inhibiting HIV-1 infection and a pharmaceutically acceptable carrier.
The present invention provides a method of treating a patient infected with HIV-1 comprising administering to the patient the pharmaceutical composition described above.
The present invention provides a method for reducing the rate at which a subject is infected with HIV-1, comprising administering the pharmaceutical composition to a subject.
The present invention relates to CD4⁺CD4 and a non-chemokine agent that can bind to the chemokine receptor C-C CKR-5 in an amount and under conditions that inhibit the fusion of HIV-1 to the cell⁺CD4 comprising contacting the cells⁺Methods of inhibiting HIV-1 fusion to cells are provided.
The present invention relates to CD4⁺CD4 and a non-chemokine agent that can bind to the chemokine receptor C-C CKR-5 in an amount and under conditions that inhibit the fusion of HIV-1 to the cell⁺CD4 comprising inhibiting cell HIV-1 infection by contacting the cell⁺Methods of inhibiting infection of cells with HIV-1 are provided.
The non-chemokine agent of the present invention binds to a chemokine receptor and binds to CD4.⁺It can inhibit the fusion of HIV-1 to cells. Non-chemokine agents include (but are not limited to) chemokine fragments and chemokine derivatives and analogs, but not natural chemokines.
In certain embodiments, the non-chemokine agent is an oligopeptide. In another embodiment, the non-chemokine agent is a polypeptide. In yet another embodiment, the non-chemokine agent is a non-peptide agent.
The present invention relates to binding to the chemokine receptor C-C CKR-5 and CD4⁺Provided are non-chemokine agents capable of inhibiting HIV-1 fusion to cells.
The present invention relates to CD4 other than chemokine receptors so that binding of a non-chemokine agent to a chemokine receptor does not prevent the ligand from binding to another receptor.⁺Binding to the chemokine receptor C-C CKR-5 and CD4 with a non-chemokine agent linked to a ligand capable of binding to the cell surface receptor of the cell⁺Molecules capable of inhibiting HIV-1 fusion to cells are provided. In certain embodiments, the cell surface receptor is CD4. In another embodiment, the ligand comprises an antibody or a portion of an antibody.
The present invention relates to CD4⁺Provided is a pharmaceutical composition comprising a substantial amount of the above molecule effective to inhibit fusion of HIV-1 to a cell and a pharmaceutically acceptable carrier.
The present invention is capable of binding to the chemokine receptor C-C CKR-5 comprising a non-chemokine agent coupled to a compound capable of increasing the in vivo half-life of the non-chemokine agent, and CD4 of HIV-1⁺Molecules that can inhibit fusion to cells are also provided. In certain embodiments, the compound is polyethylene glycol.
The present invention further includes CD4⁺Binds to the chemokine receptor CC CKR-5 with a non-chemokine agent coupled to a compound that can increase the in vivo half-life of a non-chemokine agent effective to inhibit HIV-1 fusion to cells CD4 can⁺A pharmaceutical composition is provided comprising a substantial amount of a molecule capable of inhibiting fusion of HIV-1 to a cell and a pharmaceutically acceptable carrier.
The present invention provides a method for reducing the rate at which a subject is infected with HIV-1, comprising administering the pharmaceutical composition to a subject.
The present invention provides a method of treating HIV-1 infection in a patient comprising administering to the patient the pharmaceutical composition described above.
The present invention provides that a non-chemokine agent is CD4⁺, C-C CKR-5⁺A method for determining whether HIV-1 fusion to a cell can be inhibited, comprising:
(A) HIV-1 CD4⁺, C-C CKR-5⁺CD4 on cells expressing HIV-1 envelope glycoprotein on its surface in the absence of drugs known to inhibit cell fusion⁺, C-C CKR-5⁺Under conditions that allow the cells to fuse, cells labeled with a second dye that expresses the appropriate HIV-1 envelope glycoprotein on its surface in the presence of excess drug, CD4 labeled with⁺, C-C CKR-5⁺Contacting the cells, and the first and second dyes are selected to be capable of resonance energy transfer between the dyes;
(B) contacting the product of step (a) under conditions that cause resonance energy transfer if fusion occurs;
(C) determine if resonance energy transfer is present and the absence or decrease of transfer indicates that the drug is CD4⁺And C-C CKR-5⁺Being an indicator of the ability to inhibit the fusion of HIV-1 to cells;
A method comprising: In certain embodiments, the agent is an oligopeptide. In another embodiment, the agent is a polypeptide. In yet another embodiment, the drug is a non-peptidic drug. In a further embodiment, the CD4⁺The cell is a PM1 cell. In another embodiment, the cell expressing HIV-1 envelope glycoprotein is HIV-1_JR-FLIt is a HeLa cell expressing gp120 / gp41.
The present invention also provides a non-human transgenic animal comprising an isolated DNA molecule encoding the chemokine receptor C-C CKR-5. In certain embodiments, the non-human transgenic animal further comprises an isolated DNA molecule encoding a portion of the CD4 molecule sufficient to be able to bind HIV-1 envelope glycoprotein.
The present invention further provides a non-human transgenic animal comprising an isolated DNA molecule encoding the chemokine receptor C-C CKR-5 and an isolated DNA molecule encoding fusin. In certain embodiments, the non-human transgenic animal further comprises an isolated DNA molecule encoding a portion of the CD4 molecule sufficient to bind the HIV-1 envelope glycoprotein.
One means that can be used to create transgenic animals, for example using mice, is as follows. Female mice are mated and the resulting fertilized egg is removed from the fallopian tube. The eggs are stored in a suitable medium such as M2 medium (Hogan B. et al., Manipulating the Mouse Embryo, A Laboratory Manual, Cold Spring Harbor Laboratory (1986)). DNA or cDNA encoding C-C CKR-5 chemokine receptor or CD4 is purified from the vector by methods well known in the art. An inducible promoter may be fused to the coding region of DNA to provide experimental means for regulating transgene expression. Alternatively or in addition, a tissue-specific regulatory element may be fused with the coding region to allow tissue-specific expression of the transgene. In a suitably buffered solution, place the DNA in a microinjection needle (which can be made from a capillary tube using a pipette aspirator) and inject the egg into a concave slide. The needle is inserted into the pronucleus of the egg and the solution of DNA is injected. The injected egg is then transplanted into the fallopian tube of a pseudopregnant mouse (a mouse that has been stimulated with the appropriate hormones to maintain pregnancy but is not actually pregnant), which migrates to the uterus. And then grow up to give birth. As mentioned above, microinjection is not the only method for inserting DNA into egg cells and is used here for illustration only.
The present invention provides a transformed cell comprising an isolated nucleic acid molecule encoding the chemokine receptor C-C CKR-5.
The present invention also provides an agent that can inhibit HIV-1 infection and substantially bind to a chemokine receptor without substantially affecting the ability of the chemokine receptor to bind to chemokine.
As used herein, “without substantially affecting” means that after binding of a drug to a chemokine receptor, the chemokine receptor must still be able to bind to the chemokine. Under certain conditions, after the drug has bound to the chemokine receptor, a higher concentration of chemokine is required to achieve the degree of binding seen when the chemokine receptor is not bound to the drug. In a preferred embodiment of the agent, the chemokine concentration required to achieve the same binding is doubled. In another embodiment, the chemokine concentration is 10 times.
In a preferred embodiment of the invention, the chemokine receptor is CCR5. In another embodiment, the chemokine receptor is CXCR4, CCR3, or CCR-2b.
The agent can be an oligopeptide, a non-peptidic agent, or a polypeptide. Alternatively, the agent can be an antibody or part of an antibody.
The present invention further includes CD4⁺Provided is a pharmaceutical composition comprising a substantial amount of the above agent effective to inhibit fusion of HIV-1 to cells and a pharmaceutically acceptable carrier.
The present invention relates to CD4⁺A method for inhibiting HIV-1 infection of a cell, which can inhibit HIV-1 infection and binds to a chemokine receptor without substantially affecting the ability of the chemokine receptor to bind to the chemokine. CD4 like this to get drugs⁺A method comprising contacting cells is provided.
The present invention relates to the ability of the chemokine receptor to bind to a chemokine that is capable of binding to the chemokine receptor CCR5, the chemokine receptor being linked to a compound that can increase the in vivo half-life of a non-chemokine agent. Without substantially affecting the CD4⁺Also provided are molecules that can inhibit the fusion of HIV-1 to cells. In certain embodiments, the compound is polyethylene glycol.
The present invention provides a pharmaceutical composition comprising a substantial amount of the above-described molecule effective to inhibit HIV-1 infection and a pharmaceutically acceptable carrier.
The present invention provides a method for reducing the rate at which a subject is infected with HIV-1, comprising administering the pharmaceutical composition to a subject.
The present invention provides a method of treating HIV-1 infection in a patient comprising administering to the patient the pharmaceutical composition described above.
The present invention is a method for determining whether an agent can inhibit HIV-1 infection, comprising:
(A) immobilizing a chemokine receptor on a solid phase matrix:
(B) contacting the drug with an immobilized chemokine receptor under conditions that allow the drug to bind to the chemokine receptor;
(C) removing unbound drug;
(D) contacting gp120 with the immobilized chemokine receptor obtained in step (c) in the presence of CD4 under the condition that gp120 / CD4 complex and chemokine receptor can bind in the absence of the drug And letting
(E) measuring the amount of bound gp120 / CD4 complex; and
(F) comparing the amount determined in the absence of the agent with the amount determined in step (d), and a decrease in the amount is an indication that the agent can inhibit HIV-1 infection;
A method comprising the steps of:
The present invention is a method for determining whether an agent can inhibit HIV-1 infection, comprising:
(A) immobilizing a chemokine receptor on a solid phase matrix:
(B) contacting the drug with an immobilized chemokine receptor;
(C) contacting gp120 with the mixture of step (b) in the presence of CD4 under conditions that allow the gp120 / CD4 complex and chemokine receptor to bind in the absence of the agent;
(D) measuring the amount of bound gp120 / CD4 complex; and
(E) comparing the amount determined in the absence of the agent with the amount determined in step (d), and that the decrease in amount is an indication that the agent can inhibit HIV-1 infection;
There is also provided a method comprising the steps of:
The present invention is a method for determining whether an agent can inhibit HIV-1 infection, comprising:
(A) Immobilizing the gp120 / CD4 complex on a solid phase matrix:
(B) contacting the drug with an immobilized gp120 / CD4 complex under conditions that allow the drug to bind to the gp120 / CD4 complex;
(C) removing unbound drug;
(D) contacting the chemokine receptor and the immobilized gp120 / CD4 complex obtained in step (c) under the condition that the gp120 / CD4 complex and the chemokine receptor can bind in the absence of the drug. When;
(E) measuring the amount of bound chemokine receptor; and
(F) comparing the amount determined in the absence of the agent with the amount determined in step (e), and a decrease in the amount is an indication that the agent can inhibit HIV-1 infection;
There is also provided a method comprising the steps of:
The present invention is a method for determining whether an agent can inhibit HIV-1 infection, comprising:
(A) Immobilizing gp120 / CD4 on a solid phase matrix:
(B) contacting the drug with an immobilized gp120 / CD4 complex;
(C) contacting the chemokine receptor with the mixture of step (b) under conditions that allow the gp120 / CD4 complex and the chemokine receptor to bind in the absence of the drug;
(D) measuring the amount of bound chemokine receptor; and
(E) comparing the amount determined in the absence of the agent with the amount determined in step (d), and that the decrease in amount is an indication that the agent can inhibit HIV-1 infection;
There is also provided a method comprising the steps of:
As used in these assays, CD4 includes a CD4 fragment or polypeptide that incorporates the CD4 gp120 binding site capable of binding soluble CD4, gp120, and allows gp120 to bind to the appropriate chemokine receptor. It is.
As used in these assays, gp120 is gp120 obtained from a suitable strain of HIV-1. For example, macrophage-directed clinical isolate HIV-1_JR-FLGp120 binds to the chemokine receptor CCR5, whereas the lab-adapted T-directed strain HIV-1_LAIBinds to the chemokine receptor CXCR4.
In a preferred embodiment of the above method, CD4 is soluble CD4. Chemokine receptors that can be used in the above assay include CCR5, CXCR4, CCR3 and CCR-2b.
In certain embodiments, the chemokine receptor is expressed on the cell. In certain preferred embodiments, the cells are L1.2 cells. In another embodiment, the chemokine receptor is purified and reconstituted into liposomes. Such chemokine receptors embedded in the lipid bilayer of the liposome retain the gp120 binding activity of the receptor.
In the above assay, gp120, CD4, or both may be labeled with a detectable marker. In the present invention, a marker containing an enzyme such as a radioisotope or horseradish peroxidase may be used.
In certain embodiments, the gp120 or CD4 or chemokine receptor is labeled with biotin. In a further embodiment, the biotinylated gp120, or CD4, or chemokine receptor is
(I) incubating with streptavidin-phycoerythrin;
(Ii) washing the incubated mixture obtained in step (i); and
(Iii) Measure the amount of bound gp120 using a plate reader with excitation at 530 nm and measurement fluorescence at 590 nm
Detected by.
The present invention also provides a conventionally unknown drug that has been shown to be capable of inhibiting HIV-1 infection by the above method.
The present invention also provides a pharmaceutical composition comprising an agent that has been shown to be capable of inhibiting HIV-1 infection by the above-described method and a pharmaceutically acceptable carrier. In certain embodiments, the agent is an oligopeptide. In another embodiment, the agent is a polypeptide. In yet another embodiment, the drug is a non-peptidic drug.
The present invention is capable of binding to the chemokine receptor CCR5, comprising the above-described agent conjugated to a compound capable of increasing the in vivo half-life of a non-chemokine agent, and CD4 of HIV-1⁺Molecules that can inhibit fusion to cells are also provided. In certain embodiments, the compound is polyethylene glycol. The present invention also provides a pharmaceutical composition comprising a substantial amount of the above-described molecule effective to inhibit HIV-1 infection and a pharmaceutically acceptable carrier.
The present invention provides a method of reducing the rate of HIV-1 infection in a subject comprising administering the pharmaceutical composition to the subject.
The present invention provides a method of treating HIV-1 infection in a patient comprising administering to the patient the pharmaceutical composition described above.
The invention will be better understood by reference to the following experimental details. However, one of ordinary skill in the art will appreciate that the specific experiments detailed are merely illustrative and are intended to limit the invention described herein (as set forth in the claims that follow). It will be easily understood that it is not.
Experimental details
To study how β-chemokines inhibit HIV-1 replication, the env-deficient virus NL4 / 3Δenv (which also carries a luciferase reporter gene, supplemented by a trans-expressed envelope glycoprotein) A virus entry assay based on a single cycle of infection (10,11). In PM1 cells, a variant of HUT-78 that has a unique ability to aid replication between the primary and TCLA HIV-1 strains and allows comparison of envelope glycoprotein function against a common cell background, Various env supplement viruses were tested (2,12). Together, MIP-1α, MIP-1β and RANTES are most active against HIV-1 (2,3) and enter PM1 cells with enveloped viruses with envelopes from NS1 primary strains ADA and BaL Strongly inhibited the infection (Table 1a).

Explanation of Table 1:
PM1 cells were cultured as described by Lusso et al. (12). PBMCs isolated from lab workers (LW) in Ficoll / Hypack CD8 with anti-CD8 immunomagnetic beads (DYNAL, Great Neck, NY)⁺Stimulated with PHA for 72 hours prior to lymphocyte depletion. CD4 + lymphocytes were maintained in medium containing interleukin-2 (100 U / ml; Hofmann LaRoche, Nutley, NJ) as described in literature (3). The supernatant (10-50 ng of HIV-1 p24) from 293 cells co-transfected with NL4 / 3Δenv-luciferase vector and HIV-1 env expression vector was targeted to the target cell (1-2 × 10^Five(10,11). β-chemokine (R & D Systems, Minneapolis) was added to the target cells at the same time as the virus at the final concentration (ng / ml) indicated in parenthesis in the first column. Based on previous studies (2,3), a range of β-chemokine concentrations was selected. After 2 hours, the cells were washed twice with PBS, resuspended in medium containing β-chemokine and maintained for 48-96 hours. Luciferase activity in cell lysates was measured as described in the literature (10,11). The values shown represent the luciferase activity (cpm) / ng p24 / mg protein expressed relative to a virus control culture (100%) lacking β-chemokine, with an average of double or hexafold measurements is there. nd is not implemented. R / Mα / Mβ, RANTES + MIP-1α + MIP-1β.
RANTES and MIP-β show potent activity when added separately, while other β-chemokines (MIP-1α, MCP-1, MCP-2 and MCP-3 (references 13-15)) Was a weaker inhibitor (Table 1a). The combination of MIP-1α, MIP-1β and RANTES did not inhibit infection of PM1 cells by TCLA strains NL4 / 3 and HxB2 or by the amphotropic murine leukemia virus (MuLV-Ampho) pseudotype (Table 1a) . Thus, the phenotypic characteristics of HIV-1 envelope glycoproteins affect their sensitivity to β-chemokines in virus entry assays.
CD4 from two control individuals (LW4 and LW5) using the env supplement assay⁺HIV-1 entry into T cells was evaluated. MIP-1α, MIP-1β and RANTES are CD4 for LW4 and LW5⁺It strongly inhibited the infection of T cells with NSI primary strain JR-FL and slightly reduced the infection of Lx cells with HxB2 (Table 1b), indicating that activated CD4 by various virus strains⁺It suggests that there may be some overlap in the utilization of receptors on T cells. In addition, BaL env-mediated replication in normal PBL was inhibited by MIP-1α, MIP-1β and RANTES, although there was significant variation between donors in sensitivity (data not shown).
2 or 24 hours by indicating that complete inhibition of infection in PM1 cells requires continuous presence of β-chemokine up to 5 hours after addition of ADA or BaL env supplemented virus When cells were pretreated with β-chemokine and then washed prior to virus addition, no inhibitory effect was obtained. Furthermore, addition of β-chemokine 2 hours after virus addition had only a minimal effect on virus entry (FIG. 1a). A PCR-based assay was then used to detect HIV-1 early DNA reverse transcripts in PM1 cells 10 hours after infection. In the presence of MIP-1β and RANTES, reverse transcription of ADA but not NL4 / 3 could not be detected (FIG. 1b). Thus, inhibition by β-chemokines requires its presence in at least one of the early stages of HIV-1 replication (viral attachment, fusion and early reverse transcription).
First, test whether β-chemokines inhibit JR-FL or BRU gp120 binding to soluble CD4, or tetrameric CD4-IgG2 binding to HeLa-JR-FL cells expressing oligomeric envelope glycoproteins To identify these sites of action (17). None of the β-chemokines showed inhibition in either assay, but OKT4a CD4-Mab showed strong inhibition in both (data not shown). Thus, β-chemokines inhibit the post-CD4 binding step when conformational changes in the envelope glycoprotein cause fusion of the virus with the cell membrane (18). Cell-cell membrane fusion can also be directly monitored by resonance energy transfer (RET) between fluorescent dyes induced by gp120-CD4 interaction and incorporated into the cell membrane (17). In the RET assay, OKT4a completely inhibited membrane fusion of PM1 cells with HeLa cells expressing either JR-FL or BRU envelope glycoproteins (HeLa-JR-FL or HeLa-BRU, respectively). This proves the specificity of this process (17). RANTES, MIP-1β (and to a lesser extent MIP-1α) strongly inhibited membrane fusion between PM1 cells and HeLa-JR-FL cells, but fusion between PM1 cells and HeLa-BRU cells Was insensitive to these β-chemokines (Table 2a).
Table 2: HIV-1 envelope glycoprotein-mediated membrane fusion measured by RET assay

Explanation of Table 2:
CD4⁺Target cell (CD4 activated by mitogen⁺Lymphocytes or PM1 cells) labeled with octadecyl rhodamine (Molecular Probes, Eugene, OR) and HeLa-JR-FL cells, HeLa BRU cells (or control HeLa cells, not shown) with octadecyl fluorescein (Molecular Probes) Labeled overnight at 37 ° C. Equal numbers of labeled target cells and env expressing cells are mixed in a 96-well plate and β-chemokine (or CD4 MAb OKT4a) is added at the final concentration (ng / ml) indicated in parenthesis in the first column. added. Fluorescein luminescence was measured 4 hours after cell mixing (17). When cell fusion occurs, the dye is intimately bound within the combined membrane so that excitation of fluorescein at 450 nm causes resonance energy transfer (RET) and emission at 590 nm by rhodamine. Fusion rate (%) is 100 × [(Exp RET−minimum RET) / (maximum RET−minimum RET)] [where, maximum RET = HeLa-Env and CD4⁺RET (%) obtained when mixing with cells, Exp RET = HeLa Env and CD4 in the presence of fusion inhibitor compound⁺RET (%) obtained when mixed with cells, and minimum RET = HeLa cells (those lacking HIV-1 envelope glycoprotein) and CD4⁺RET (%) obtained when mixed with cells]]. The RET value (%) is defined by the calculation described in literature (17), and each is the average of triplicate measurements. These values are 11.5% and 10.5% PM1 cells for HeLa-JR-FL and HeLa-BRU cells, respectively; LW5 CD4⁺Cells, 6.0%, 10.5%; R / Mα / Mβ, RANTES + MIP-1α + MIP-1β.
The first CD4 from LW5⁺Similar results were obtained with T cells (Table 2b) (in order to inhibit membrane fusion in primary cells, higher concentrations of β-chemokines that inhibit membrane fusion in PM1 cells were required). Therefore, the action of β-chemokines is not limited to the PM1 cell line. The RET assay confirms that β-chemokines inhibit env-mediated membrane fusion.
The simplest interpretation of these results is that the binding of a β-chemokine to its receptor, either directly or otherwise,⁺It prevents fusion of T cells with HIV-1. HIV-1 is CD4⁺The need for a second receptor for entry into T cells has been known for decades (19-21). In the case of TCLA strains, this function is conferred by fusin (9). Several receptors for MIP-1α, MIP-1β and RANTES have been identified (6,7), and β-chemokines show considerable cross-reactivity in receptor utilization (4-8). However, CC CKR-1, and in particular CC CKR-5, appears to be the most promising candidate based on tissue expression patterns and their ability to bind to MIP-1α, MIP-1β and RANTES ( 4,7,8,15,22). C-C CKR-1, C-C CKR-5 and LESTR are expressed at the mRNA level in PM1 cells and primary macrophages, respectively (data not shown). Therefore, these and other β-chemokine receptors were PCR amplified, cloned and expressed.
In the env supplementation assay for HIV-1 entry, the expression of CC CKR-5 in HeLa-CD4 (human), COS-CD4 (monkey) and 3T3-CD4 (mouse) cells resulted in primary NSI strains ADA and BaL Became able to easily infect each of them

Explanation of Table 3:
The chemokine receptor genes CC CKR-1, CC CKR-2a, CC CKR-3, CC CKR-4 and CC CKR-5 have no introns (4-8,15,22) and are healthy for 7 people They were isolated by PCR performed directly on a human genomic DNA pool derived from a PBMC from a non-donor donor. Oligonucleotides that overlap with ATG and stop codon and contain BamHI and XhoI restriction sites for directional cloning into pcDNA3.1 expression vector (Invitrogen Inc.) were used. LESTR (also known as fusin or HUMSTR) (4, 9, 24) was cloned by PCR performed directly on cDNA derived from PM1 cells using sequences derived from the NIH database. 5 'and 3' primer pairs used in the first (5-1 and 3-1) and second (5-2 and 3-2) round PCR amplifications of CSTR gene derived directly from human genomic DNA and LESTR from PM1 cDNA Are listed below. Only a single primer set was used for CKR-5 amplification.

Human CD4 expressing cell lines HeLa-CD4 (P42), 3T3-CD4 (sc6) and COS-CD4 (Z28T1) (23) were transfected with various pcDNA3.1-CKR constructs by the calcium phosphate method, and after 48 hours, Various reporter viruses (200 ng HIV-1 p24 / 10) in the presence or absence of β-chemokines (400 ng / ml RANTES, MIP-1α and MIP-1β, respectively)⁶Cells). After 48 hours, luciferase activity in the cell lysate was measured (10, 11). Since infections mediated by C-C CKR genes other than C-C CKR-5 were not strong enough to allow quantification of inhibition, β-chemokine blocking data are shown for C-C CKR-5 only. In the PCR-based HIV-1 entry assay, low levels of NL4 / 3 and ADA entry into C-C CKR-1-expressing cells were consistently observed (data not shown).
In this assay, neither LESTR nor C-C CKR-1, -2a, -3 or -4 could replace C-C CKR-5. Expression of LESTR in COS-CD4 and 3T3-CD4 did not tolerate HxB2 entry, and HxB2 readily entered untransfected (or transfected with a control plasmid) HeLa-CD4 cells ( Table 3). Invasion of BAL and ADA into a total of three CC CKR-5 expressing cell lines is almost completely inhibited by the combination of MIP-1α, MIP-1β and RANTES, while HxB2 entry into LESTR expressing cells is , Insensitive to β-chemokines (Table 3). These results suggest that C-C CKR-5 functions as a β-chemokine sensitive second receptor for the primary NSI HIV-1 strain.
The function of the second receptor of C-C CKR-5 was confirmed by an env-mediated membrane fusion assay. When CC CKR-5 is transiently expressed in COS and HeLa cell lines that permanently express human CD4, both cell lines are more potent than HeLa cells that express the JR-FL envelope glycoprotein. Fusion did not occur when the control plasmid was used (data not shown). When LESTR was expressed instead of CC CKR-5, fusion of COS-CD4 cells or HeLa-CD4 cells with HeLa-JR-FL cells was not possible, but COS-CD4 cells and HeLa-BRU Fusion with cells became possible (data not shown).
In addition, the fusion ability of β-chemokine receptor was tested in the RET assay. Expression of C-C CKR-5 but not C-C CKR-1, -2a, -3 or -4 allowed for strong fusion of HeLa-CD4 and HeLa-JR-FL cells. The degree of fusion between HeLa-JR-FL cells and C-C CKR-5 expressing HeLa-CD4 cells was greater than the constitutive level of fusion between HeLa-BRU cells and HeLa-CD4 cells (FIG. 2). Thus, the C-C CKR-5 fusion-conferring function for the first NSI HIV-1 strain has been confirmed in two independent fusion assays.
Experiment considerations
Taken together, the above results support that MIP-1α, MIP-1β and RANTES inhibit HIV-1 infection at the entry stage by preventing the viral cell fusion reaction following CD4 binding. CC CKR-5 can also serve as a second receptor for entry of HIV-1 primary NSI strains into CD4 + T cells, and the interaction between β-chemokines and CC CKR-5 Has been shown to inhibit the HIV-1 fusion reaction.
References in the previous section

Second series of experiments
experimental method
Source of reagents: Recombinant human β-chemokine was purchased from R & D Systems (Minneapolis)¹²⁵I-MIP-1β (specific activity 2,200 Ci nmol^-1) Was purchased from Dupont-NEN. Anti-CD4 monoclonal antibody was obtained from Q. Sattentau (16). However, 5A8 was obtained from L. Burkly (Biogen) (27) and L120 (UK MRC AIDS Reagent Repository) (16). Soluble CD4 has been described in the literature (17). Monoclonal antibodies against gp120 are described in the literature (19,19) except 23A (gp120C terminus, from J. Robinson), 447-D and 697-D (ref 28; Cellular Products Inc.) and 83.1 (ref 29; Repligen). It was obtained from the donors listed in 20). Recombinant gp41 (IIIB) ectodomain was obtained from Viral Therapeutics Inc. and V3 peptide was obtained from Repligen or UK MRC AIDS Reagent Repository. Recombinant MN gp120 (Genentech), SF-2 gp120 (Chiron) and CM243 gp120 were provided by the NIAID AIDS Reagents Repository, and W61D gp120 (SmithKline Beecham, Belgium) was obtained from the UK MRC AIDS Reagent Repository.
Recombinant monomeric JR-FL and LAI gp120 (both full length and lacking V3 loop) using vectors developed by Progenics Pharmaceuticals containing dihydrofolate reductase expression cassette I let you. The expression of the gene is placed under the control of the cytomegalovirus immediate early promoter. In the case of Δ-V3 gp120, the V3 loop is excised by splicing-by-overlap-extension technique, and the cysteine defining the loop is retained and cross-linked by the peptide sequence TGAGH. (Spanned). All constructs were sequenced to confirm that no mutations were introduced during the cloning operation. The protein was expressed in stably transfected Chinese hamster ovary cells (DXB-11), selected in medium without nucleosides, and amplified using methotrexate (according to the method described in literature (17)) Done). Secreted proteins were purified to> 95% homogeneity by non-denaturing methods including ion exchange, Galanthus nivalus lectin affinity and gel filtration chromatography. The purified protein bound to sCD4 with nanomolar affinity (18).
For the expression of SF162 gp160, a 3.5 kb EcoRI-BamHI fragment containing the env gene was excised from pSM, a vector based on SV40, and placed in the R1 / Bg1lll site of pCAGGS, an expression vector based on β-actin. Subcloned. In the case of SF170 gp160 expression, a 3.8 kb fragment containing the env gene is replaced with pBSKS.⁺The plasmid was excised from the plasmid, blunt-ended by treatment with T4 DNA polymerase, and subcloned into the RV / Xho1 site of pCAGGS. The expression plasmid was transfected into 293T cells by the calcium phosphate coprecipitation method. Three days later, soluble gp120 in the culture supernatant was collected, filtered through a 0.2 μm filter, and concentrated on an Amicon 1000 membrane.
Preparation of soluble oligomeric forms of JR-FL and 94RW020 envelope glycoproteins (and monomeric gp120 from 92TH014) was performed as follows. The JR-FL env gene was provided by I. Chen (UCLA), and the env genes for 92TH014 and 92RW020 were obtained from the NIAID AIDS Reagent Repository (30). A soluble expression plasmid encoding only gp120 and gp41 ectodomain (from JR-FL and 92RW020), 92TH014 gp120 was constructed as described in the literature (30) and transfected into Chinese hamster ovary cells by the calcium phosphate method . A cleavage site between the JR-FL and 92TH014 gp120 and gp41 moieties was retained and the protein was secreted as an oligomer (J.A., J.M.B. and J.P.M., unpublished data). The envelope glycoprotein was partially purified from the culture supernatant by immobilized metal affinity chromatography. A control preparation 93MW959 (c) containing a gp120 / gp41 molecule in which CD40 binding was disabled by a single point mutation at residue 457,¹²⁵It did not compete with I-MIP-1β. Estimating the concentration of monomeric gp120 or oligomeric gp120 / gp41 in the unpurified culture supernatant was performed by denaturing the protein, dot blotting on a nitrocellulose membrane, a mixture of mouse monoclonal antibodies against a continuous epitope (19), and then This was done by detecting gp120 with an anti-mouse IgG-HRP conjugate and ECL chemiluminescence system (Amersham). Purified monomeric JR-FL gp120 was used as a concentration standard (17). The concentration of oligomeric gp120 / gp41 complex was defined as the total concentration of monomeric gp120 subunit within the preparation. Enzyme-linked antibody immunoassay (ELISA) confirmed high affinity CD4 binding to gp120 (19).
Cells and cell lines
PBMCs were isolated from blood donors by Ficoll-Hypac centrifuge and phytohemagglutinin (5 μg ml^-1) And IL-2 (100 U ml)^-1) (Roche) for 2-3 days. CD4 from activated PBMC by positive selection using anti-CD4 immunomagnetic beads (Dynal Inc)⁺T cells were purified. Purified lymphocytes¹²⁵Before using in the I-MIP-1β binding assay, IL-2 (200 U ml^-12 × 10 in medium containing⁶Cultured at / ml for at least 3 days. The cells were screened for CCR-5 deficient alleles (14) and only cells from wild type donors were used (unless otherwise indicated). PcDNA3.1-ckr-5 (Reference 1) was transfected into 293 cells using the calcium phosphate method and 1 μgml^-1Resistant clones were selected in medium containing neomycin (G418; Sigma). Subcloning resistant cells,¹²⁵Tested for CCR-5 expression in a binding assay using I-MIP-1β.
MIP-1β binding assay and gp120 competition
Purified CD4⁺T cells were washed twice in ice cold binding buffer (RPMI 1640 medium containing 1% BSA, 25 mM HEPES, 0.05% sodium azide). Duplicate sample (2 x 10 in 200 μl⁶Cells), 0.1nM₁₂₅I-MIP-1β (2,200Ci nmol^-1; 0.25μCml^-1) On ice for 2 hours. Immediately before adding the radiolabeled ligand, unlabeled ligand or gp120 (mixed with monoclonal antibody as appropriate) was added to the cells. Anti-CD4 monoclonal antibody was added to the cells simultaneously with gp120. The cells are then separated from unbound ligand by centrifugation (60 seconds, 14,000 g) with oil (80% silicone oil, Aldrich; 20% mineral oil, Sigma), and the radioactivity in the cell pellet is determined by gamma counter. It was measured. By adding 100 times excess unlabeled β,¹²⁵Specific binding of I-MIP-1β was estimated. Each experiment was repeated at least twice using cells from different donors. In experiments with 293-CCR-5 cells, the cells were dissociated with 1 mM EDTA and then washed twice with binding buffer. Sample (5 × 10^FiveCells) 0.5nM¹²⁵Incubated with I-MIP-1β and then processed as described above.¹²⁵No specific binding was detected when the concentration of I-MIP-1β was decreased to 0.1 nM.
Summary
The β-chemokine receptor CCR-5 is a non-syncytium-inducing (NSI) phenotype of HIV-1 and CD4⁺It is an essential cofactor for fusion with T cells (1-5). The initial binding site of human immunodeficiency virus (HIV) -1 is the CD4 molecule, whose interaction is mediated by the viral surface glycoprotein gp120 (6,7). Although the mechanism of CCR-5 function during HIV-1 entry has not been elucidated, we have already shown that its β-chemokine ligand prevents fusion of HIV-1 with the cell Yes (1). Therefore, the present inventors examined whether CCR-5 acts as a second binding site for HIV-1 simultaneously with or after the interaction between gp120 and CD4. The present inventors have shown that CCR-5 ligand, macrophage inflammatory protein (MIP) -IP is activated CD4⁺A competition assay based on the inhibition of gp120 binding to its receptor on T cells or CCR-5 positive CD4 cells was used. We conclude that CD4 binding is not absolutely required for gp120-CCR-5 interaction, but significantly increases its efficiency. Neutralizing monoclonal antibodies against several sites on gp120, including the V3 loop and CD4-induced epitopes, inhibit the interaction between gp120 and CCR-5 without affecting gp-120-CD4 binding did. Inhibition of HIV-1 binding to one or both HIV-1 receptors (CD4 and CCR-5) may be an important mechanism of virus neutralization.
MIP-1β is the most specific ligand for CCR-5 (8-10). This is because MIP-1α and RANTES bind with high affinity to other members of the β-chemokine receptor family on lymphoid cells (8-11). Therefore, we used MIP-1β as the CCR-5 ligand in the competition assay. Like other members of this receptor family (12), CCR-5 is a mitogen-responsive gene. Purified CD4 in stationary phase⁺Its expression in T cells is usually minimal, but three days after activation of the cells by phytohemagglutinin and interleukin (IL) -2, we have found that CCR-5 messenger RNA and¹²⁵A significant increase with I-labeled MIP-1β binding was observed (data not shown). As a specificity control, we have determined that CD4 from individuals homozygous for the defective CCR-5 allele⁺T cells were used (13, 14). Specific for cells from three such individuals (ie, competed with non-radioactive MIP-1β)¹²⁵The amount of I-MIP-1β (0.1 nM) binding is 2 × 10⁶92 ± 12 c.p.m. (average ± s.d.) Per cell. In contrast, the average binding to cells from 21 control individuals is 2 × 10⁶1,044 ± 1,073 per cell (range, 222-4,846 c.p.m.). Therefore, activated CD4⁺With T cells¹²⁵Most of the reactivity of I-MIP-1β comes from binding to CCR-5.
Recombinant monomer gp120¹²⁵Activated CD4 with I-MIP-1β⁺When added to T cells, we found that gp120 from NSI strain JR-FL [which used CCR-5 for invasion (1)] potentiated MIP-1β binding. Inhibition was found (Figure 3a: Table 4).

Explanation of Table 4:
Recombinant protein, 0.1 nM¹²⁵Titration in the presence of I-MIP-1β and activation of CD4⁺Added to T cells. At each gp120 concentration¹²⁵Percentage inhibition of I-MIP-1β binding is shown, which is the mean ± sd of 2-4 independent experiments. The absence of a value indicates that the gp120 molecule was not tested at that concentration (some molecules> 1 μg ml^-1Not available).
^*Oligomer gp120 / gp41 complex
Half-maximal inhibition is similar to the association constant for gp120-CD4 interaction (7) 0.1-1.0 μg ml^-1(0.8-8 nM) of gp120. In contrast, gp120 from T-cell-line adapted (TCLA) SI strain LAI was ineffective (FIG. 3a; Table 4). The virus uses fusine (CXCR-4) for entry instead of CCR-5 (1-5). Mutants of JR-FL and LAI gp120 lacking the V3 loop (Δ-V3 gp120) but binding to CD4 with high affinity did not block MIP-1β binding (FIG. 3a). However, peptides from the V3 loop of the following strains (15 residues unless otherwise indicated) were also inactive: JR-FL (32 residues), RA, VS, Case-B (each NSI); HxB2, MN, SF-2 (each TCLA) (60μml^-1 Approximate molar equivalent of gp120, 1 μg ml^-1The peptide was added at The oligomeric complex of JR-FL gp120, noncovalently linked to the gp41 ectodomain, was an effective inhibitor of MIP-1β binding, but not the recombinant molecule containing only the gp41 ectodomain ( (Table 4) (The latter molecule may not fold into its native structure (15)).
HIV-1 strains from gene subtypes A, B, C and E utilize CCR-5 for invasion (3) and we have determined that MIP-1β is from subtypes A-E. It has been found to inhibit replication of most primary NSI HIV-1 strains. This breadth of HIV-1 reactivity with CCR-5 is¹²⁵It extends to the I-MIP-1β competition assay. Recombinant gp120 from the following NSI primary strains is competitive, with half-maximal inhibition of MIP-1β of about 0.1-0.5 μg ml^-1Occurred at concentrations of: JR-FL (subtype B), SF162 (B), W61D (B), 92TH014 (B), SF170 (A), 92RW020 (A) and CM243 (E) (Table 4). In contrast, no competition with gp120 from TCLA subtype B strains LAI, MN and SF-2 was observed (Table 4) (but each of them binds to CD4 with high affinity (shown) Not)). Therefore, in determining the interaction with CCR-5, the phenotype of the virus from which the gp120 molecule is derived is more important than the viral genotype.
We evaluated the role of CD4 in the competition between NSI gp120 and MIP-1β by using antagonists of gp120-CD4 interaction. Both monoclonal antibodies Q4120 and L77 (16), which react with domain 1 of CD4 and inhibit gp120 binding, and soluble CD4 (sCD4) (17), which react with gp120 and inhibit CD4 binding, are both MIP- for CCR-5 Inhibition of 1β binding by JR-FL gp120 was reversed (FIG. 3b; Table 5).

Explanation of Table 5:
Activated CD4⁺Against T cells¹²⁵JR-FL gp120 with I-MIP-1β binding (2 μg ml^-1SCD4 (50 μg ml)^-1) Or monoclonal antibody against CD4 (50 μg ml^-1) Or antibody against gp120 (20 μg ml^-1) In the presence or absence. The mean reversal rate (%) of the competitive effect of gp120 in the presence of each antibody (± s.d; n = 2 to 4 independent experiments) is shown. Specific recorded in the presence of gp120 and in the absence of antibody¹²⁵The level of I-MIP-1β binding (c.p.m.) was set to 0%. The level recorded in the absence of both gp120 and antibody was set as 100%. Negative reversal rate (%)¹²⁵It shows that the competitive effect of gp120 on I-MIP-1β binding was increased in the presence of the antibody. It also describes the approximate location of antibody epitopes on gp120 as defined in the literature (19, 20). References to the origin of the antibody can be found in the literature (19, 20) or in the “Methods” section.
^*HIV-1_JR-FLAnti-gp120 monoclonal antibody (or sCD4) that can neutralize
⁺Monoclonal antibodies with neutralizing activity against other HIV-1 strains (primary or TCLA)
Monoclonal antibodies against other domains of CD4 [which does not block gp120-CD4 binding (16)] are ineffective (Table 5), and in the absence of gp120, sCD4 (50 μg ml^-1) Did not cause inhibition of MIP-1β (data not shown). Thus, interaction with cell surface CD4 is important for gp120 to interact efficiently with CCR-5 and block MIP-1β binding. To ascertain whether CD4 is an absolute requirement, we have identified stable human CD4^- CCR-5⁺A 293 cell line was generated. These cells are¹²⁵I-MIP-1β (5 × 10^FiveSpecific binding (up to 2,500 c.p.m. per cell), whereas non-transfected 293 cells do not bind (<50 c.p.m. specific binding). CD4^- CCR-5⁺Against 293 cells¹²⁵I MIP-1β binding was inhibited only once by JR-FL gp120, which was observed only at the highest gp120 concentration tested (50-100 μg ml^-1). The strongest competition observed on these cells is 50 μg ml^-1Of JR-FL gP120 was 73% inhibition of MIP-1β binding (comparable inhibition was observed in the other two experiments), but often no competition was detected, the inventors No inhibition of MIP-1β binding was observed at lower gp120 concentrations. CD4^- CCR-5⁺Adding excess sCD4 to the cells did not reduce or increase the inhibitory effect of JR-FL gp120 (data not shown).
The interaction between JR-FL gp120 and CCR-5 is CD4^-CD4 on cells⁺Requires a gp120 concentration of at least 100 times higher than on the cells. This suggests that the binding of gp120 to CD4 on the cell surface is due to an increased probability of gp120-CCR-5 interaction. That is, either a gp120-CD4-CCR-5 triple complex is formed or a sequential interaction between gp120 and CD4 and then CCR-5 occurs. One possibility is that a high affinity association between gp120 and CD4 increases the probability of a lower affinity interaction between gp120 and CCR-5 (proximity effect). This is consistent with the finding that sCD4 does not replace cell surface CD4 (at least with JR-FL gp120). Alternatively, binding to CD4 may be necessary to (better) expose the CCR-5 binding site on gp120. This is particularly important in the case of virions where some of the regions of the oligomeric envelope glycoprotein accessible on monomeric gp120 (including the V3 loop) are not optimally exposed prior to CD4 binding (18).
gp120-CD4 complex is activated CD4⁺In order to gain further insights on how to interact with CCR-5 on T cells, we have identified HIV-1, each of which has been confirmed to be able to bind to JR-FL gp120. A panel of neutralizing and non-neutralizing anti-gp120 monoclonal antibodies (19,20) was used. The antibodies were tested for reversal of the competitive effect of gp120 on the MIP-1β binding site (Table 5). As with sCD4, antibodies against conformational epitopes (15e and IgG1b12) or linear epitopes (G3-508) that overlap with the CD4 ginding site (20), JR-FL gp120 is MIP-1β Prevented from competing with. However, some antibodies that did not affect the binding of monomeric gp120 to CD4 (20) also inhibited the gp120-CCR-5 interaction (Table 5). These are 3 for the V3 loop (447-D, 10b and 83.1), 1 for the conserved epitope within the C3-V4 region (2G12), and 2 for the conserved CD4 inducible epitope ( 48d and 17b) included. All of these except A32 map to the position (20) that we defined as the gp120 neutralizing surface. The other 8 monoclonal antibodies that did not prevent JR-FL gp120 from blocking MIP-1β binding (Table 2) clustered at the position we defined as the gp120 non-neutralizing surface (20). To do. These epitopes can be accessed by monomeric gp120, but in the case of oligomers they are blocked by other gp120 subunits or gp41 molecules (19,20). These ineffective antibodies contain 2 / 11c against an epitope that overlaps that of A32. For this reason, and since A32 does not strongly neutralize the HIV-1 strain, the significance of the partial inhibitory action of A32 on the gp120-CCR-5 interaction is not clear. Two monoclonal antibodies against the V2 loop (697-D and SC259) were also ineffective. Although the V2 loop structure is modeled on the gp120 neutralization surface (20), these two antibodies are HIV-1_JR-FLDoes not have the ability to neutralize Monoclonal antibodies 2G12, 17b, 447-D, 48d, IgG1b12, G3-508 and 697-D were also tested against oligomeric JR-FL gp120 / gp41 protein, all but 697-D Inhibited the interaction between CCR-5 and CCR-5 (not shown).
Thus, most of these antibodies to the neutralizing surface of gp120 prevented binding of gp120 to CD4 or subsequent interaction with the CCR-5 second receptor. Since primary viruses resist neutralization and studies with recombinant proteins can only accurately predict neutralization potency (18), all antibodies against this aspect of gp120 are not HIV-1_JR-FLDoes not actually neutralize. However, our findings may suggest an understanding of how HIV-1 is neutralized by antibodies. Blocking the viral first or second receptor interaction may be particularly important.
The simplest interpretation that Δ-V3 JR-FL gp120 does not have the ability to block MIP-1β binding (Figure 3a) is that the CCR-5 binding site is contained within the V3 loop. Is. This is consistent with numerous observations that the V3 loop contains important determinants of HIV-1 phenotype and tropism (18, 21) and can affect the utilization of secondary receptors (3). However, we believe that the CCR-5 binding site is not limited to the V3 loop. The V3 sequences of gp120 of subtypes A, B and E that interact with CCR-5 are fairly variable (Table 4). In addition, some simian immunodeficiency virus (SIV) strains can also use human CCR-5 as a second receptor (ZW Chen and P. Marx, personal communication), but the V3 region of HIV-1 and SIV is , Has little primary sequence homology. Is it possible for each of all these sequences to form a binding site for the same conserved cellular protein if a similar V3 sequence from the TCLA HIV-1 strain cannot do so (Table 4)? The twin-site models of ligand-chemokine receptor interaction (8) show that the relatively conserved V3 loop is multi-point binding to CCR-5 on gp120. There still remains the possibility of being a component of the site. However, we suggest that the CCR-5 binding site must contain a gp120 region that is strongly conserved among primate immunodeficiency viruses as well as between HIV-1 gene subtypes. Whether this applies to HIV-1 interaction with CXCR-4 is still unclear.
The structure of the V3 loop may affect the nature of the complex binding site for CCR-5 on gp120. A region of gp120 that overlaps (but is not necessarily identical to) the CD4 inducible epitope of monoclonal antibodies 48d and 17b is an excellent candidate for such a site. These antibodies recognize similar conformationally sensitive structures that are probably located around the bases of the V1, V2, and V3 loops (22,23). Deletion of the V3 loop from both HxBc2 gp120 and JR-Fl gp120 disrupts the 48d and 17b epitopes (22,23) (unpublished data), indicating that Δ-V3 JR-FL gp120 is CCR-5 May be associated with not having the ability to interact with HIV-1 (Figure 5A)_LAIChanges in single amino acids within the V3 and C4 regions of these may also have a major impact on the structure of these epitopes (24).
Further research will be required to deepen our understanding of the CCR-5 binding site. The efficiency of β-chemokines to inhibit the replication of NSI primary isolates in peripheral blood mononuclear cells (PMBC) depends on the strain, not the subtype (unpublished data), which is probably gp120 and CCR- This suggests that the degree of overlap with the β-chemokine binding site on 5 differs between gp120. If so, the CCR-5 binding site on gp120 may be more flexible than the CD4 binding site. Finally, in some strains of HIV-1 and (especially) HIV-2 and SIV, sCD4 is CD4⁺Although the interaction between JR-FL gp120 and CCR-5 on cells was inhibited, sCD4 enhanced the efficiency of the second receptor interaction, CD4 or CD4⁺It may facilitate the entry of these primate immunodeficiency viruses into cells (25,26).
References for the second series of experiments

Third series of experiments
Summary
C-C chemokine receptor CCR5 is CD4⁺It is required for efficient fusion of M-directed HIV-1 strains with the cell plasma membrane (1-5) and interacts directly with the viral surface glycoprotein gp120 (6,7). Receptor chimera studies provide useful information (8-10), but the domain of CCR5 that functions for HIV-1 entry, including the site of gp120 interaction, has not been clearly identified . Only substitutions of the negatively charged residues Asp-2 (D2), Asp-11 (D11) and Glu-18 (E18), alone or together, can be combined with ADA and JR / FL M-directed strains and DH123. To demonstrate that CCR5-mediated HIV-1 entry into dual-tropic strains is impaired or disrupted, we now use site-specific alanine scanning mutagenesis of CCR5 . These mutations also impair env-mediated membrane fusion and gp120-CCR5 interaction. Of these three residues, only D11 is required for CC-chemokine-mediated inhibition of HIV-1 entry, but it also depends on other extracellular CCR5 residues. Thus, the gp120 and CC-chemokine binding sites (on CCR5) only partially overlap and the former requires a negatively charged residue in the N-terminal CCR5 domain.
result
To identify the region of CCR5 involved in gp120 binding and HIV-1 entry, we based on the pre-involvement of charged residues in the interaction of CC-chemokines with their receptors. Alanine scanning mutagenesis of negatively (D, E) or positive (K, R, H) charged residues in the N-terminal (Nt) and three extracellular loops (ECL1-3) was performed (11 12). We have also identified any residues that differ between human CCR5 and its mouse homolog (which is non-functional for HIV-1 entry) (9,10) Modifications were made each time there was a charge change. In total, 15 single mutants, 4 double mutants and 1 triple mutant were tested in these studies (Figure 4).
Wild-type and mutant CCR5 proteins (HA-labeled at the C-terminus for ease of detection) are transiently expressed in U87MG-CD4 and SCL-1-CD4 cells and HIV-1 envelope sugars The ability of them to assist protein-mediated invasion was measured using an env supplement assay that reads with luciferase (FIG. 5) (1, 2). These non-lymphoid human cell lines were selected because they lack CCR5, CCR3 and CXCR4 co-receptors and resist infection by HIV-1 in the absence of transfected co-receptors (2,4,13,14) (a few exceptional HIV-1 strains enter U87MG-CD4 cells by unknown pathway (15), but we did not use them) ). Nearly the same results were obtained with both cell lines. Two M-directional viruses that use CCR5 instead of CXCR4 (ADA and JR-FL) (1-5), and one dual-directional virus that uses both CCR5 and CXCR4 equally well (DH123) ( 15) was used to test whether mutant CCR5 protein could assist HIV-1 entry. The expression level of each transfected CCR5 mutant was assessed by Western blotting and considered in determining the efficiency of the co-receptor.
Only 3 of the 15 single mutations had a significant inhibitory effect on CCR5 co-receptor function (FIG. 5). These were D2A, D11A and E18A, all located within the Nt domain of CCR5 (FIG. 4). Only the E18A substitution was sufficient to reduce CCR5 function by 15-20 fold. The double mutant D2A / D11A was less active than any single mutant and the triple mutant (D2A / D11A / E18A) was almost completely inactive (compared to the wild type> 50-fold reduction in invasion) (Figure 5A). None of the remaining substitutions had a significant effect on CCR5 function in this assay (FIGS. 5B, C). Similar results were obtained with both M-directed envelope glycoproteins. The only difference seen with the bi-directional DH123 envelope was a significant increase in sensitivity to D11A substitution (FIG. 5A). Thus, negatively charged residues in CCR5 Nt have a profound effect on the co-receptor function of this molecule.
To study the effects of D2A, D11A and E18A substitutions in an independent assay for HIV-1 env function, we have developed a HeLa cell that stably expresses the JR-FL env gene and wild type or mutant. A membrane fusion assay was used in which HeLa-CD4 cells transiently transfected with CCR5 were mixed (FIG. 6) (1,18). These two cell types are labeled with various fluorescent probes and fusion is monitored by resonance energy transfer (RET) (1,18) that occurs only when the two dyes are present in the same membrane. We tested D2A, D11A and E18A single mutants and double and triple mutants compared to wild type CCR5 in the RET assay. Each mutant had the same phenotype in this fusion assay as observed in the virus entry assay (FIGS. 5A, 6). E18A and double and triple mutants were completely unable to support env-mediated membrane fusion. However, if we have enhanced the expression of the co-receptor by about 100-fold using the vaccine-T7 polymerase (vFT7-pol) system (1,4.5,13), each of the CCR5 mutants Was able to assist in membrane fusion, although less efficient than the wild-type protein (FIG. 6). We have already observed that CCR overexpression disrupts the ability of CCR's CC-chemokine ligand to inhibit membrane fusion, indicating that if CCR5 expression is too high several phenotypes This suggests that there may be no change (1). However, the results with the vac-T7pol system show that even the triple mutant (D2A / D11A / E18A) is not completely inactive as a co-receptor and is only very strongly impaired. Show.
Next, we have determined that CCR5 mutants that assist HIV-1 entry are sensitive to the inhibitory effects of CCR5 CC-chemokine ligands (MIP-1α, MIP-1β and RANTES). (Table 1) (19-21).

Table 6:Inhibition of co-receptor function by CC-chemokines
U87MG-CD4 cells were transiently lipofected with wild type or mutant CCR5 and then infected with Nlluc / JR-FL (in the presence or absence of 2 μg / ml MIP-1α, MIP-1β and RANTES). In the presence). After 72 hours, luciferase activity was measured (1,2). The relative inhibition rate (%) by CC-chemokine for each mutant was expressed as [1- (luciferase cps in the presence of chemokine / luciferase cps in the absence of chemokine)] / [1- (wild type luciferase cps in the presence of chemokine). / Wild-type luciferase cps in the absence of chemokine)] x 100%. Each value is the average of three independent experiments each performed in quadruplicate. Mutant co-receptors with a relative% inhibition of <50% of that observed with wild-type CCR5 are shaded.
(Note that the D2A, D11A and E18A mutants are impaired with respect to HIV-1 entry, but they helped the entry to a degree sufficient to measure sensitivity to inhibition. Nt double and triple mutants did not apply). In U87MG-CD4 cells, as with other non-lymphoid cells (1,2,22), CC-chemokines do not completely block HIV-1 infection and are Requires concentration. We therefore compared the degree of inhibition obtained by CC-chemokines on mutant and wild type CCR5 receptors (40-60% depending on the individual ligand, each potency is RANTES> MIP -1β> MIP-1α). The following mutants were relatively insensitive to one or more actions of CC-chemokines: D11A, K22A, R31A (Nt), H181A, Y184A, K171A / E172A, K191A / N192A (ECL2) R274A / D276A (ECL3). Of these, only D11A was impaired for both HIV-1 entry and CC-chemokine inhibition of entry. Thus, amino acid substitutions at certain positions (mostly within Nt and ECL2) do not affect the HIV-1 co-receptor function of CCR5, but do affect CC-chemokine-mediated inhibition of this process ( Table 6). It is not clear how these substitutions affect the action of CC-chemokines. However, the simplest interpretation is that the CC-chemokine binding site and HIV-1 interaction site on CCR5 are not identical, and certain substitutions in ECL2 and ECL3 only affect the CC-chemokine binding site Is.
To understand how Nt substitutions affect the HIV-1 co-receptor function of CCR5, we determined whether they affect gp120 binding. We could not directly measure the binding of labeled gp120 to CCR5. This is because the level of CCR5 expression on transiently transfected cells was too low to obtain a reproducible signal in any of the several binding assays tested. Thus, we have developed a competitive assay that measures the ability of gp120 (JR-FL) (7) to inhibit binding to phycoerythrin (PE) labeled CCR5-specific MAb (2D7-PE) (23-25). Using. The epitope for this MAb is located within ECL2, and we have found that it can efficiently bind to HeLa cells co-transfected with CD4 and CCR5 Nt mutants.
Independent studies show that 2D7 inhibits the binding of (125) I-labeled gp120 to CCR5 on mouse L1.2 cell line (25) overexpressing CCR5 to the extent that it allows detection of gp120 binding (6,25). Here we show that 2D7-PE binding to wild-type CCR5 was strongly (70%) inhibited by pre-addition of gp120. This indicates that the interactions between gp120 and 2D7 and the receptor are mutually exclusive (FIG. 7). However, gp120 only partially (40%) inhibited binding of 2D7-PE to D2A, D11A and E18A mutants, with respect to blocking 2D7-PE binding to double and triple Nt mutants. Almost ineffective (25% and 15% inhibition, respectively) (Figure 7). Of note, the most impaired mutant for HIV-1 entry (FIG. 5) was at the same time that 2D7-PE binding was also the least sensitive to gp120 inhibition (FIG. 5). 7). The most reliable interpretation of this result is that gp120 binds to the wild-type CCR5 molecule so as to sterically hinder the binding of 2D7-PE to ECL2, but binds incompletely to the Nt mutant, That's it. Less likely is that gp120 binds efficiently to Nt mutants, but that binding occurs in an unusual orientation that reduces the likelihood of inhibiting 2D7-PE binding to ECL2. In the latter case, the interdomain interaction geometry within CCR5 has been altered by Nt substitutions that impair CCR5 co-receptor function.
In this study, we have identified three negatively charged residues in the amino-terminal domain that affect CCR5 co-receptor function without necessarily preventing CC-chemokine inhibition of co-receptor function. Identified point substitutions. The same substitution affects the ability of gp120 to interact correctly with CCR5, possibly by reducing the affinity of the gp120-CCR5 interaction. This may be sufficient to explain the co-receptor deficient phenotype. The loss of affinity for gp120 caused by Nt substitution in CCR5 can be partially compensated by overexpression of the mutant co-receptor (FIG. 6). This is likely to be a successful gp120-CCR5 interaction quickly enough that an increase in the number of low affinity co-receptors is sufficient to accommodate conformational changes in the envelope glycoprotein that initiate membrane fusion. It may be caused (26-28).
Thus, the gp120 binding site on CCR5 depends on the residues in Nt, and in fact, a separate gp120 binding domain may be confined to Nt. Previous studies using chimeric receptors or deletion mutants have shown the importance of CCR5 and CXCR4 Nt for co-receptor function (8-10, 29). The chimera studies also suggest that the site of interaction between CCR5 and HIV-1 is relatively wide and flexible to some extent (8-10). Although this possibility should not be ignored, modifications within the extracellular loop of the receptor chimera may indirectly affect the orientation of CCR5 Nt (and hence its ability to interact correctly with gp120) There is also. In contrast to the gp120 binding site, the CC-chemokine binding site on CCR5 depends on both residues in the Nt and in the extracellular loop (ECL is prominent although not exclusive). Thus, although there is some overlap between gp120 and the CC-chemokine binding site (as shown by gp120 inhibition of CC-chemokine binding to CCR5) (6,7), they are not identical and signal Conclusions are consistent with studies showing that transmission and co-receptor activity are separable functions of CCR5 (10, 30, 31).
To elucidate how HIV-1 uses CCR5 for HIV-1 entry into target cells, and possibly to develop inhibitors of this process, on CCR5 against gp120 and CC-chemokines A more detailed understanding of the interaction sites (and on these molecules for CCR5) will be needed. Also, the negatively charged residues we have identified in CCR5 Nt interact directly or indirectly with positively charged amino acids in gp120, in the V3 loop and / or elsewhere It will be important to check whether or not.
Method
Lipofection and reporter gene assays
The mutated cDNA was subcloned into the pcDNA3.1 (Stratagene) expression vector. U87MG-CD4 and SCL-1-CD4 cells were transformed into lipofectin (5 μg / ml) and pSVlacZ (5 μg / ml), or mutant DNA (4 μg / ml) + pSVlacZ (1 μg / ml) (OPTI-MEM (Gibco BRL) And at 5 ° C. for 5 hours. After 24 hours, the cells were infected with NLluc / Env supernatant containing 200-500 ng / ml p24 at 37 ° C. for 2 hours. Add 2 μg / ml MIP-1α, MIP-1β or RANTES (R & D System) simultaneously with HIV-1 (50-100 ng / ml p24) for blocking HIV-1 entry by CC-chemokines And maintained in the culture for 12 hours. 72 hours after infection, cell samples were treated with 100 μl lysis buffer (Promega), and luciferase (luc) and β-galactosidase activity (OD₄₂₀) Was measured (1, 2). Standardized luciferase activity was determined as (luc c.p.s / ng / ml p24 / (OD₄₂₀÷ Control OD₄₂₀) / (R.f.e. For mutant CCR5 band ÷ r.f.e. for wild-type CCR5 band)) (see below). Luc c.p.s.value is 5 × 10 for wild type CCR5^Five~ 2 × 10⁶Range.
Immunoblot analysis of CCR5 expression in whole cell extracts
All CCR5 molecules used in this study had a 9-residue hemagglutinin (HA) -tag as the C-terminal extension to facilitate detection. Lipofected U87MG-CD4 cells from a 60mm tissue culture plate were treated with 1% sodium dodecyl maltoside, 10 mM Tris-HCl (pH 6.8), 50 mM NaCl, 1 mM CaCl.₂, 0.1 mM PMSF, 5 μg / ml leupeptin, 10 μg / ml aprotinin, 0.7 μg / ml pepstatin and 10 mM EDTA. The suspension was incubated at 4 ° C. for 30 minutes and the supernatant fraction was collected. Total protein concentration was measured using the Bio-Rad DC Protein Assay. The protein (15 μg) was then fractionated on an SDS-polyacrylamide gel without prior boiling. Protein transferred to Immobilon-P membrane (Millipore), rabbit anti-HA tag antibody (1: 500 dilution; Berkeley Antibody Company) and AP-labeled goat anti-rabbit IgG (1:10) for CCR5^FourDiluted; Amersham) and then incubated with a chemiluminescent substrate (Vistra ECF, Amersham). Relative fluorescence emission (r.f.e.) of immunoreactive bombos excited at 450 nm was detected on a laser-based scanner (Molecular Dynamics Storm 860). The same expression pattern was obtained with whole cell extract and plasma membrane extract. The expression level of CCR5 mutant varied between 20% and 100% of the wild type protein. The relationship between the wild-type CCR5 expression level and HIV-1 entry efficiency was confirmed to be linear over a 10-fold range (data not shown).
Competition between gp120 and 2D7MAb for CCR5 binding
HeLa cells (2 x 10) with lipofectin (10 μg / ml) and pCDM8 CD4 expression vector (3.75 μg / ml) + wild type or mutant CCR5 plasmid (1.25 μg / ml)⁶Were incubated in OPTI-MEM for 5 hours. The cells are then given 2 × 10 6 to enhance CCR5 expression.⁷p.f.u. vFT7 was infected for 12 hours, the cells were dissociated with 2 mM EDTA / PBS and washed once with binding buffer (1% BSA in DPBS, 0.05% azide). Cells (1 × 10⁶Were incubated for 1 hour at 37 ° C. in the presence or absence of 10 μg / ml gp120 (JR-FL) (7), and then PE-labeled 2D7 MAb (23,24) (20 ng / ml) Added for 30 minutes at ° C. The cells were washed once with binding buffer and once with PBS, resuspended in 1% formaldehyde / PBS and analyzed by FACS to determine the mean fluorescence intensity (m.f.i.). CD4 expression was monitored by staining with Leu3A. The expression varied between samples by ± 10% or less. Expression levels of CCR5 mutants ranged from 20% to 100% of wild-type CCR5 expression levels
References for the third series of experiments

Fourth experiment
CCR5 ⁺ CD4 ^- HIV-1 to cells _JR-FL Direct binding of gp120
CCR5⁺CD4^-HIV-1 to cells_JR-FLDirect binding of gp120 was demonstrated. In this case, it is necessary to preincubate gp120 with sCD4 or other CD4-based molecules, presumably because this results in a conformational change in gp120 that exposes the chemokine receptor binding site. I think that the. FIG. 8 represents the use of flow cytometry to measure the direct binding of the sCD4 / gp120 complex to mouse L1.2 cells with human CCR5. The background level of binding is HIV-1 if using only biotinylated protein or a laboratory adapted HIV-1LAI strain._JR-FLObserved by either gp120 substitute (not shown in the figure).
The assay uses CCR5 of the sCD4 / gp120 complex for drug screening purposes.⁺/ CD4^-Cell binding was adapted to a 96-well microplate format measured using a fluorescent plate reader. One method is as follows.
1) L1.2-CCR5⁺Seed cells (approximately 500,000 / well)
2) Add inhibitor for 1 hour at room temperature
3) Washed, biotinylated sCD4 (2.5 μg / mL) and biotinylated HIV-1_JR-FLAdd gp120 (5 μg / mL), then incubate for 2 hours at room temperature
4) Wash and incubate with streptavidin-phycoerythrin (100ng / nL)
5) Wash and measure the amount of bound gp120 / sCD4 using a fluorescence plate reader with excitation at 530 nm and measurement fluorescence at 590 nm
Using this method, inhibition of CCR5 to gp120 / sCD4 by CC chemokines (FIG. 9) and antibodies against CCR5 that block HIV-1 infection (not shown in the figure) were demonstrated.
Inhibition of HIV-1 envelope-mediated membrane fusion by the extracellular domain of CCR5
Synthetic peptides corresponding to the four extracellular domains of human CCR5 were generated by Quality Controlled Biochemicals (Hopkinton, Mass.) And HIV-1 using the resonance energy transfer (RET) assay described above. The ability to inhibit membrane fusion mediated by the envelope glycoproteins of LAI or JR-FL strains was tested. Using the ECL2 peptide, specific inhibition of fusion mediated by the JR-FL envelope was seen, but not with other peptides. ECL2 is 97% at 100 μg / mL, 65% at 33 μg / mL, and 15% at 11 μg / mL, HeLa-env_JR-FLInhibition of fusion of cells with PM1 cells (average of two assays). ECL2 is HeLa-env_LAIFusion with PMI cells or HeLa-env_LAIAnd did not inhibit the fusion of HeLa-CD4 cells. These results strongly suggest that CCR5 ECL2 specifically inhibits fusion, probably by blocking the interaction between HIV-1 JR-FL gp120 and CCR5. Other peptides tested did not give significant levels of specific fusion inhibition.
Inhibition of membrane fusion via HIV-1 envelope by bicyclam, JM3100
A bicyclam obtained from Dr. J. Moore (Aaron Diamond AIDS Research Center, NY) uses the above-described resonance energy transfer (RET) assay to envelope the HIV-1 LAI or JR-FL strains. The ability to inhibit membrane fusion mediated by glycoproteins was tested. As shown in FIG. 10, the molecule is HIV-1_LAISpecifically and potently inhibits fusion mediated by gp120 / gp41 from HIV-1_JR-FLFusions mediated by strain-derived gp120 / gp41 are not inhibited. These data are HIV-1_LAIBlocking the interaction between gp120 and CXCR4 suggests that the molecule specifically inhibits HIV fusion.
Sequence listing
(1) General information:
(I) Applicant: Alaway, Graham P
Drazik, Tatiana
Litwin, Virginia A
Madon, Paul J
Moore, John P
(Ii) Title of invention: Use of chemokine receptor to inhibit HIV-1 infection
(Iii) Number of sequences: 30
(Iv) Communication destination:
(A) Addressee: Cooper and Dunham LLP
(B) Street: 1185 Avenue of the Americas
(C) City: New York
(D) State: New York
(E) Country: United States
(F) Zip code: 10036
(V) Computer readable form:
(A) Media type: floppy disk
(B) Computer: IBM / PC compatible
(C) Operating system: PC-DOS / MS-DOS
(D) Software: Patent In Release # 1.0, Version # 1.30
(Vi) Current application data:
(A) Application number:
(B) Application date:
(C) Classification:
(Viii) Atony / Agent information:
(A) Name: White, John
(B) Registration number: 28,678
(C) Reference / Docket number: 51320
(Ix) Telegraph information:
(A) Telephone: (212) 278-0400
(B) Telefax: (212) 391-0525
(2) Information for sequence recognition number: 1
(I) Sequence characteristics:
(A) Length: 38 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 1:

(2) Information for sequence recognition number: 2:
(I) Sequence characteristics:
(A) Length: 25 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 2:

(2) Information for sequence recognition number: 3:
(I) Sequence characteristics:
(A) Length: 23 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 3:

(2) Information for sequence recognition number: 4:
(I) Sequence characteristics:
(A) Length: 33 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 4:

(2) Information for sequence recognition number: 5:
(I) Sequence characteristics:
(A) Length: 31 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: peptide
(Xi) Sequence description: Sequence recognition number: 5:

(2) Information for sequence recognition number: 6:
(I) Sequence characteristics:
(A) Length: 15 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: peptide
(Xi) Sequence description: Sequence recognition number: 6:

(2) Information for sequence recognition number: 7:
(I) Sequence characteristics:
(A) Length: 32 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: peptide
(Xi) Sequence description: Sequence recognition number: 7:

(2) Information for sequence recognition number: 8:
(I) Sequence characteristics:
(A) Length: 17 amino acids
(B) Type: amino acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: peptide
(Xi) Sequence description: Sequence recognition number: 8:

(2) Information for sequence recognition number: 9:
(I) Sequence characteristics:
(A) Length: 36 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 9:

(2) Information for sequence recognition number: 10:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 10:

(2) Information for sequence recognition number: 11:
(I) Sequence characteristics:
(A) Length: 41 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Description of sequence: Sequence recognition number: 11:

(2) Information for sequence recognition number: 12:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 12:

(2) Information for sequence recognition number: 13:
(I) Sequence characteristics:
(A) Length: 32 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 13:

(2) Information for sequence recognition number: 14:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 14:

(2) Information for sequence recognition number: 15:
(I) Sequence characteristics:
(A) Length: 32 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 15:

(2) Information for sequence recognition number: 16:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 16:

(2) Information for sequence recognition number 17:
(I) Sequence characteristics:
(A) Length: 32 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 17:

(2) Information for sequence recognition number: 18:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 18:

(2) Information for sequence recognition number: 19:
(I) Sequence characteristics:
(A) Length: 31 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 19:

(2) Information for sequence recognition number: 20:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 20:

(2) Information for sequence recognition number: 21:
(I) Sequence characteristics:
(A) Length: 29 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 21:

(2) Information for sequence recognition number: 22:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 22:

(2) Information for sequence recognition number: 23:
(I) Sequence characteristics:
(A) Length: 33 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 23:

(2) Information for sequence recognition number: 24:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 24:

(2) Information for sequence recognition number: 25:
(I) Sequence characteristics:
(A) Length: 34 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 25:

(2) Information for sequence recognition number: 26:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 26:

(2) Information for sequence recognition number: 27:
(I) Sequence characteristics:
(A) Length: 34 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 27:

(2) Information for sequence recognition number: 28:
(I) Sequence characteristics:
(A) Length: 30 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 28:

(2) Information for sequence recognition number: 29:
(I) Sequence characteristics:
(A) Length: 39 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 29:

(2) Information for sequence recognition number: 30:
(I) Sequence characteristics:
(A) Length: 39 base pairs
(B) Type: Nucleic acid
(C) Number of chains: single chain
(D) Topology: Linear
(Ii) Sequence type: DNA (genomic)
(Xi) Sequence description: Sequence recognition number: 30:

Claims (5)

( 1) A polypeptide having a sequence present in the extracellular domain of a human CCR5 chemokine receptor, and (2) a sequence comprising the sequence set forth in SEQ ID NO: 7, wherein HIV-1 is fused to CD4 ⁺ cells. A polypeptide that inhibits. The polypeptide of claim 1, wherein the polypeptide sequence is the sequence of SEQ ID NO: 7. A composition comprising the polypeptide according to claim 1 or 2 and a carrier. A pharmaceutical composition for inhibiting fusion of HIV-1 and CD4 ⁺ cells, according to claim 1 or 2 in an amount effective to inhibit the fusion of HIV-1 and CD4 ⁺ cells A pharmaceutical composition comprising a polypeptide and a pharmaceutically acceptable carrier. (1) HIV-1 CD4 comprising a polypeptide having a sequence present in the extracellular domain of the human CCR5 chemokine receptor and (2) comprising the sequence of SEQ ID NO: 7. ⁺ A fusion inhibitor for inhibiting fusion to cells.

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