JP4076766B2 - Broad spectrum antagonists and vaccines for pyrogenic exotoxins - Google Patents

Description

【００４１】
もう１つの具体例では、本発明のペプチドアンタゴニストはＳＥＢ、ＳＥＡおよびＴＳＳＴ−１に対してヒトリンパ性細胞を防御する抗体を惹起することができ、このことは発熱性毒素に対する広域な防御免疫を付与し得ることを示している。しかし、特定の他のＳＥＢタンパク質ドメインに由来するペプチドに対して生じた抗体は、毒素から防御するというよりももしろ、ＩＬ−２およびＩＦＮ−γｍＲＮＡのより高い誘導によって発現されるように、実際にはＳＥＢおよびＳＥＡに対するヒトＰＢＭＣの応答を高めた。これらＳＥＢアンタゴニストペプチドでマウスを免疫化すると、致死量のＳＥＢに対する防御を惹起し、その結果試験動物が生存した。これらのペプチドはまた発熱性外毒素により誘導される毒性ショックに対して長期免疫を付与するのに使用できることは明らかである。
【００４２】
このように第１の態様では、本発明は、配列番号１のアミノ酸配列を含んでなるペプチド、およびかかるペプチドの機能的誘導体に関するものであり、ここで該単離、精製ペプチドは毒素アドニスト活性を持たず、外毒素により誘導される毒性ショックに対して防御免疫を惹起することができる。本明細書で用いられる用語の誘導体および機能的誘導体とは、毒素アゴニスト活性を示さず、かつ、外毒素により誘導される毒性ショックに対して防御免疫を惹起するペプチドの能力を妨げず、および／または毒素により媒介されるＴ細胞の活性化に拮抗するペプチドに対する任意の、挿入、欠失、置換および修飾を伴ったペプチド（以下、「誘導体」と呼ぶ）を意味する。誘導体は該ドメインと最低の、例えば約３０％または３０％よりいくらか低い相同性を維持すべきである。
【００４３】
第２の態様では、本発明は、毒素アゴニスト活性を阻害せず、かつ、毒素により媒介されるＴ細胞の活性化に拮抗することができる配列番号１のアミノ酸配列を含んでなる単離・精製ペプチド、およびかかるペプチドの機能的誘導体に関する。本発明のペプチドは発熱性外毒素または発熱性外毒素の混合物により誘導される毒性ショックに対して防御することができる。
【００４４】
発熱性外毒素は通常、細菌外毒素、特に黄色ブドウ球菌または化膿連鎖球菌により産生される外毒素である。
【００４５】
本発明の単離・精製ペプチドは配列番号１で示されるアミノ酸配列（以下、ｐ１４またはｐ１４（１４８〜１６１）とも呼ぶ）、およびその機能的誘導体を含んでなる。ｐ１４およびその機能的誘導体は発熱性外毒素または発熱性外毒素混合物により誘導される毒性ショックに対して防御免疫を惹起し、および／または毒素により媒介されるＴ細胞の活性化に拮抗することができる。これらのペプチドはまた、急性毒性ショック、および例えば発熱性外毒素により誘導される偶発性食中毒によるものであり得る有害な作用のいずれもの応急処置のために、またかかる毒性ショックに対する長期免疫の付与のために使用することができる。上記に示されたように、ｐ１４ペプチドと同様、機能的誘導体も好ましくは該発熱性外毒素のタンパク質ドメインと少なくとも３０％の相同性を有する。ｐ１４（配列番号１）はその１４のアミノ酸残基のうち１１において、配列番号６で示される天然に存在するタンパク質の配列の１４８〜１６１位と相同である。
【００４６】
さらに、後記の実施例で示されるように、ｐ１４ペプチドはまたＴＳＳＴ−１に対して毒素アンタゴニスト活性を有するが、ＴＳＳＴ−１の対応領域とは３０％未満の相同性しかなく、１４のアミノ酸残基のうち４つを共有している。
【００４７】
直鎖ペプチドの構造の欠如は、それらをヒト血清中のプロテアーゼの作用を受けやすくし、可能性のあるコンホメーションのわずかしか活性ではないので、標的部位に対するそれらの親和性を低下させるように働く。従ってアンタゴニストペプチド構造を最適化することが望ましい。
【００４８】
ペプチド構造を改良するためには、本発明のペプチドをそれらのＮ末端を介してラウリル−システイン（ＬＣ）またはリシル−パルミトイル残基と、および／またはそれらのＣ末端を介してシステイン（Ｃ）残基と、あるいは以下でさらに詳細に記載されるが、ペプチドを免疫化のためのアジュバントと結合させるのに好適な他の残基に結合させることができる。プロテアーゼ耐性を向上させるには、Ｄ−ＡｌａをＮ末端またはＣ末端のいずれか、あるいは両者に結合させればよい。
【００４９】
本発明のペプチドならびにその誘導体は総て正電荷有していてもよいし、負電荷を有していてもよいし、あるいは中性であってもよく、また、ダイマー、マルチマーの形態であってもよいし、あるいは拘束されたコンホメーションであってもよい。
【００５０】
拘束コンホメーションは内部架橋、短範囲の環化、延長または他の化学修飾によって得られる。ダイマーおよびトリマーなどのオリゴマーも考えられる。
【００５１】
さらに本発明のペプチドをそのＮ末端および／またはＣ末端において種々の同一または異なるアミノ酸残基で延長してもよい。かかる延長の一例としては、ペプチドをそのＮ末端および／またはＣ末端において、天然に存在するアミノ酸残基であっても合成アミノ酸残基であってもよい同一または異なる疎水性アミノ酸残基で延長することができる。好ましい合成アミノ酸残基はＤ−アラニンであり、記載のように、これによりペプチドのプロテアーゼ耐性が増す。
【００５２】
合成アミノ酸残基で延長されたペプチドの具体例としては、発熱性外毒素により誘導される毒性ショックに対して防御免疫を惹起し、および／または毒素により媒介されるＴ細胞の活性化に拮抗することができる配列番号２で示されるアミノ酸配列を有するペプチド（以下、ｐ１４Ａとも呼ぶ）、およびその機能的誘導体である。一つ好ましい態様では、本発明のペプチドはそのＮ末端およびＣ末端の双方にＤ−Ａｌａ残基を有するｐ１４Ａである。
【００５３】
かかる延長のさらなる例は、そのＮ末端および／またはＣ末端の双方においてシステイン残基で延長したペプチドにより提供される。もちろんかかる延長はジスルフィド結合の形成から生じるＣｙｓ−Ｃｙｓ環化による拘束されたコンホメーションをもたらしてもよい。
【００５４】
もう１つの例は、Ｎ末端リシル−パルミトイルテールの組み込み（リシンはリンカーとして働き、パルミチン酸は疎水性アンカーとして働く）による誘導体ｐ１４Ａ−ＬＰ（配列番号３）の作製である。
【００５５】
さらに本ペプチドは天然に存在するアミノ酸残基であっても合成のアミノ酸残基であってもよい、延長された芳香族アミノ酸残基であってもよい。好ましい芳香族アミノ酸残基としては、チロシンおよびトリプトファンが挙げられる。あるいは、本ペプチドはそのＮ末端および／またはＣ末端において、天然に存在する発熱性外毒素のアミノ酸配列の対応する位置に存在するアミノ酸で延長することができる。
【００５６】
しかしながら、本発明によれば、本発明のペプチドはそのＮ末端および／またはＣ末端において、天然には存在しない、もしくは合成アミノ酸である種々の同一または異なる有機部分で延長することができる。かかる延長の一例として、本ペプチドはそのＮ末端および／またはＣ末端において、Ｎ−アセチル基またはＤ−アラニン基で延長してもよい。このようにして延長されたペプチドはもまた、本発明の他のペプチドとして、急性毒性ショック、およびそれにより引き起こされる有害な作用のいずれもの応急処置に、またかかる毒性ショックに対する長期免疫を付与するために使用することができる。
【００５７】
本発明のペプチドは後記の実施例に示されるように、ＩＬ−２、ＩＦＮ−γまたはＴＮＦ−β遺伝子によりコードされる発熱性毒素により誘導されるｍＲＮＡの発現を阻害することができる。
【００５８】
さらに本発明のペプチドは、免疫化した個体においてＴ細胞活性化を遮断する抗体の産生を惹起することができる。抗体の産生は好適な免疫アジュバントの存在下で高まる。好ましいアジュバントとしてはスカシガイ・ヘモシアニン（ＫＬＨ）、プロテオソームまたはミョウバンが挙げられる。
【００５９】
さらなる態様では、本発明は、少なくとも本発明のｐ１４ペプチドまたはその機能的誘導体の治療上有効量を有効成分として含んでなる、毒素により媒介されるＴ細胞の活性化を治療または予防するための医薬組成物に関する。上記のように、本ペプチドは上記発熱性外毒素のタンパク質ドメインのアミノ酸配列と実質的に相同なアミノ酸配列を含んでなる。本発明の医薬組成物はまた、発熱性外毒素または発熱性外毒素混合物により誘導される毒性ショックに対する防御に有用である。
【００６０】
本明細書の目的のための医薬上「有効量」とは、当技術分野で既知の考慮により決定されるものである。この量は毒素により媒介されるＴ細胞の活性化に十分拮抗するものでなければならない。
【００６１】
本願を通じて用いられる毒素により媒介される活性化とは、単一の発熱性外毒素またはかかる毒素の混合物により媒介されるＴ細胞の活性化を意味し得る。
【００６２】
実施例１〜３は１４のアミノ酸からなる短いペプチド（ｐ１４またはｐ１４Ａ、配列番号６に示される天然に存在するＳＥＢタンパクの配列の１４８〜１６１位に相当）は、対応するＴＳＳＴ−１との配列の相同性が比較的低くとも（３０％未満）、マウスにおいてＳＥＢにより、またＴＳＳＴ−１により誘導される毒性ショックに対して著しく有効なアンタゴニスト活性を示すことを示している。さらに、ｐ１４ＡはＳＥＢ抗原投与から７時間後というように遅れて投与した時でも、毒性ショックに対して防御免疫を惹起することができた。さらにｐ１４Ａは抗原投与して保護されたマウスに、種々の毒素に対して広スペクトル耐性を迅速に発展させることができた。
【００６３】
本発明の医薬組成物は単位投与形で調製することができ、製薬分野で周知のいずれの方法によって製造してもよい。さらに本発明の医薬組成物は医薬上許容される担体、賦形剤または安定剤などの医薬上許容される添加剤、および所望によりその他の治療成分をさらに含んでもよい。もちろん許容される担体、賦形剤または安定剤は使用する用量および濃度で受容者に無毒なものである。
【００６４】
本発明の組成物の治療上の用量はもちろん、患者群（年齢、性別など）、治療する症状の性質、および投与経路によって異なり、主治医により決定される。
【００６５】
さおさらなる態様では、本発明は、少なくとも１種の本発明のペプチドまたはその誘導体の免疫学的有効量を有効成分として含んでなる、発熱性外毒素により誘導される毒性ショックに対する免疫を付与するためのワクチンに関し、かかるペプチドおよび誘導体の混合物を含んでもよい。
【００６６】
用語「免疫学的有効量」とは発熱性外毒素または発熱性外毒素混合物により誘導される毒性ショックに対して免疫を付与するに十分な任意の量を意味する。
【００６７】
本発明のワクチンは所望により好適な免疫アジュバントまたはその混合物をさらに含んでもよい。好適なアジュバントとしては、プロテオソーム、ＫＬＨおよびミョウバン、ならびにプロテオソームとミョウバン、およびＫＬＨとミョウバンの組合せが挙げられる。
【００６８】
後記の実施例で示されるように、本発明のワクチンは発熱性外毒素に対して防御免疫を惹起することができる。
【００６９】
毒性ショック症候群に対して用いる抗体を開発する努力は、主としてモノクローナル抗体または可溶性受容体でＴＮＦの作用を阻害することによって毒性カスケードの下流の現象を遮断することに注がれていた。毒素に応答して産生される高レベルのサイトカインはこのアプローチを無効にする。本発明は、Ｔ細胞の活性化が起こる前に毒性カスケードの最上で毒素作用を阻害するアンタゴニストを用いて、全く異なる戦略で発熱性外毒素の作用を遮断することができるということを示す。
【００７０】
実施例では、毒素アンタゴニスト活性を評価するための、ＰＢＭＣにおける、ＩＦＮ−γ遺伝子（ならびに本明細書には示されていないがＩＬ−２およびＴＮＦ−β遺伝子）の発現による、発熱性外毒素により媒介されるヒト細胞免疫応答の活性化の分子的方法、解析を詳細に記載する。ヒトＰＢＭＣでの研究は毒素拮抗特性、免疫特性およびワクチン効力を評価する動物試験と組合せたが、これらの方法が発熱性外毒素系の他のメンバーに対してもヒトＰＢＭＣを中和するか、またはそれを保護する薬剤を考案する際に適用できることが示される。
【００７１】
ヒトはマウスよりも発熱性外毒素に対する感受性がはるかに高く、一方、霊長類モデルはコストなどのその他の制限要因を有することから、毒素により媒介される免疫応答の活性化および抑制のメカニズムを分析し得るヒトin vitro系が必要とされる。本発明はかかる系を提供し、それは以下のような主な利点がある。
【００７２】
（１）サイトカイン産生の調節に関与する細胞間相互作用を保護する、新しく調製されたヒトリンパ系細胞集団は身体の末梢免疫系に可能な限り近いものである。
【００７３】
（２）初期の免疫応答の結果はＩＬ−２、ＩＦＮ−γおよびＴＮＦ−β ｍＲＮＡの一時的および高度に調節された発現を追跡することにより正確かつ直接的に分析することができる。
【００７４】
（３）ＩＦＮ−γ（ならびに所望によりＩＬ−２およびＴＮＦ−β）遺伝子の発現はＳＥＢにより惹起される活性に鋭敏な感受性がある。
【００７５】
（４）この分子学的アプローチは一連の事象の累積した結果である細胞増殖または抗体産生などの生物学的応答の測定よりもはるかに直接的かつ特異的である。
【００７６】
（５）提示したアプローチは、ヒトＩＬ−２、ＩＦＮ−γおよびＴＮＦ−β遺伝子の活性化に不可欠なＳＥＢ中の機能的ドメインをマッピングする手段を与え、アンタゴニストおよびワクチン双方の開発を助ける働きをし得る。
【００７７】
このように本発明はまた、発熱性外毒素または発熱性外毒素混合物により誘導される毒性ショックを治療する方法に関する。該方法はかかる治療を必要とする患者に、治療上有効量の本発明の医薬組成物、または治療上有効量の少なくとも１種の本発明のペプチドもしくはその機能的誘導体を投与することを含んでなる。
【００７８】
さらなる態様では、発熱性外毒素または発熱性外毒素混合物により誘導される毒性ショックを予防する方法が提供され、該方法はかかる治療を必要とする患者に、治療上有効量の本発明の組成物、または治療上有効量の少なくとも１種の本発明のペプチドもしくはその機能的誘導体を投与することを含んでなる。
【００７９】
本発明はまた、発熱性外毒素により誘導される毒性ショックに対して患者を免疫化する方法に関し、該方法は患者に、有効免疫量の本発明のワクチン、または少なくとも１種の本発明のペプチドもしくはその機能的誘導体を投与することを含んでなる。
【００８０】
本発明のペプチドまたはワクチンの治療上の用量はもちろん、患者群（年齢、性別など）、治療する症状の性質、および投与経路によって異なり、主治医により決定される。
【００８１】
本発明のペプチドおよびワクチンは優良医療規範に従って投与および服用できる。特に本発明の免疫化法は本発明のペプチドまたはワクチンの１回の投与を含んでなる。投与は、静脈内、筋肉内または皮下注射をはじめ種々の方法で行える。しかし、鼻内投与などのその他の投与方法も可能である。
【００８２】
本明細書に記載の発熱性外毒素アンタゴニストペプチドの設計はこれまで軽視されていた領域、発熱性外毒素の予防および毒素に曝された個体の治療における新規な適用を見出すだけでなく、安全な発熱性外毒素ワクチンの開発をも促進する。定義されるペプチドワクチンは悪化させる特性がなく、トキソイドワクチンよりも優れていると考えられる。
【００８３】
ヒトでは、発熱性外毒素に曝されることによる毒性ショックは、（１）活動不能と（２）死という異なる２つの要素を持つ。
【００８４】
致死濃度よりも数ｌｏｇ低い濃度でさえ、発熱性外毒素は著しく活動不能をもたらし、高い罹病率を示す[USAMRIID Manual, (1998) Eitzen E, Pavlin J, Cieslak T, Christopher G, Culpepper R, eds. Medical Management of Biological Casualties Handbook. 3rd ed. Fort Detrick, Maryland: United States Army Medical Research Institute of Infectious Diseases, 1998]。例えば食中毒（集団食中毒であり得る）で見られる活動不能応答は多数の人々に影響を及ぼす。さらに、活動不能応答は軍隊の脅威であり、国家の保安の脅威である。死に対する防御については、発明者らはペプチドアンタゴニストｐ１２Ａおよびｐ１４Ａがマウスにおいて毒素によりもたらされる死を回避することを示した。マウスにおいて嘔吐または下痢などの活動不能症状は見られなかった。活動不能を予防および／または治療する実験はブタにおいて行った（実施例７）。
【００８５】
吐気、すなわち、悪心、嘔吐および食欲不振は典型的には中脳（髄質）によって制御される一連の神経症状である。嚥下および嘔吐の血管運動・呼吸中枢および反射中枢は髄質にある。延髄の嘔吐中枢は、吐気を引き起こす最終の共通の経路の起点であり、髄質をよじることで嘔吐が起こる[Hamilton, (1956) Textbook of human anatomy. McMillan & Co Ltd, London]。嘔吐中枢は実際には嘔吐に関与する原動力、呼吸、血管および唾液分泌のプロセスのパターンを制御する絡み合った神経ネットワークである。真の嘔吐中枢はいくつかの神経学的経路を介し、ドーパミン、ヒスタミン、アセチルコリン、セロトニン（５ＨＴ）、ノルエピネフリン、およびグルタミンに応答する髄質の種々のタイプの神経伝達受容体により刺激される。制吐剤（主としてドーパミンおよびセロトニンアンタゴニスト）はこれらの受容体を遮断することによって働く。
【００８６】
真の嘔吐中枢に対するこれら複数の経路の作用は複雑である。マウスのＳＥＢへの経口暴露の数時間内に、腸管粘膜に存在するＴ細胞ではＩＬ−２およびＩＦＮ−γ遺伝子の強い発現が誘導される[Spiekermann and Nagler-Anderson, (1998) Spiekermann GM, Nagler-Anderson C (1998) J Immunol 161:5825-5831]。このように、結果としての粘膜Ｔ細胞の活性化および高レベルＴｈ１サイトカインに対する腸管粘膜の他の細胞の応答は脳への神経伝達物質の放出を誘発し、内臓感覚系の必須の役割を助ける。嘔吐に関しては胃からの刺激は必要とされないことに注目すべきである。胃は上記で説明したように種々の刺激に応答して産生される髄質からのシグナルを受容した際に嘔吐反射のエフェクターとして働く。大腸の刺激が小腸へ向けて移動し、最終的に肛門へ達すると下痢が起こる。
【００８７】
後記の実施例（特に実施例７ならびに図９および１０）で示されるように、本発明者らは、それが本発明の目的であるが、発熱性外毒素アンタゴニストが毒素により媒介される活動不能症状（ＳＥ中毒および初期致死ショックに対するヒト応答に類似）を著しく軽減することを見出した。毒素はＴｈ１サイトカインの誘導および放出を惹起し、次いで髄質のニューロンを直接的または間接的に活性化して活動不能症状を引き起こす細胞免疫応答を標的とすることで働くと考えられる。このようにＴ細胞の活性化を遮断する発熱性外毒素アンタゴニストの単一の作用様式は死および活動不能の双方を防ぐその能力を説明する。図９および１０のデータは、発熱性外毒素に対する免疫応答においてヒトに近い動物であるブタモデルにおいて、毒素による活動不能に対するアンタゴニストの保護作用の確実性を示す。
【００８８】
このように本発明はさらに、少なくとも１種の発熱性外毒素により誘導される活動不能の予防および／または治療のための医薬組成物に関し、該組成物は配列番号１のアミノ酸配列を有するペプチド、配列番号２のアミノ酸配列を有するペプチド、配列番号３のアミノ酸配列を有するペプチド、およびその任意の混合物から選択される単離・精製ペプチドと、所望により医薬上許容される担体、希釈剤、アジュバントおよび／または賦形剤をさらに含んでなる。
【００８９】
本発明はさらに、少なくとも１種の発熱性外毒素により誘導される活動不能を予防および／または治療する方法に関し、該方法はかかる治療を必要とする患者に、配列番号１のアミノ酸配列を有するペプチド、配列番号２のアミノ酸配列を有するペプチド、配列番号３のアミノ酸配列を有するペプチド、およびその任意の混合物から選択される少なくとも１種の治療上有効量の単離・精製ペプチド、またはそれを含んでなる組成物を投与することを含んでなる。有効量の単離ペプチドまたはそれを含んでなる組成物は所定の時間間隔で繰り返し患者に投与してもよい。毒性ショックになった時患者には通常塩の点滴を施すが、容易に水に溶けるアンタゴニストペプチドとの混合にはクリアランスを相殺するため、救急期のその持続投与を見込んでおく。このことは好ましく、極めて迅速なアンタゴニスト投与経路である。投与経路、用量および処置頻度は主治医により決定される。
【００９０】
実施例
材料および方法
細胞培養およびヒトサイトカイン遺伝子発現の誘導
健康なヒト提供者のＰＢＭＣをＦｉｃｏｌｌ Ｐａｑｕｅ(Pharmacia)で分離し、５０ｍｌのＲＰＭＩ１６４０培地で２回洗浄し、４×１０^６／ｍｌの濃度に再懸濁し、２％ウシ胎児血清、２ｍＭグルタミン、１０ｍＭ ＭＥＭ非特異的アミノ酸、１００ｍＭピルビン酸ナトリウム、１０ｍＭ Ｈｅｐｅｓ ｐＨ７．２、５×１０^−５Ｍ ２−メルカプト−エタノール、１００単位／ｍｌペニシリン、１００μｇ／ｍｌストレプトマイシンおよび５μｇ／ｍｌナイスタチンを添加した同培地で培養した。ＳＥＢ（ロット１４〜３０、the Department of Toxinology, U. S. Army Medical Research Institute of Infectious Diseasesから）、ＳＥＡまたはＴＳＳＴ−１(Sigma)を加えて１００ｎｇ／ｍｌとした。
【００９１】
ＲＮアーゼプロテクション解析
グアニジニウムイソチオシアネートで全ＲＮＡを抽出した[Chomczynski and Sacchi, Anal Biochem 162: 156 (1987)]。ＲＮアーゼプロテクション解析は、ｐＢＳ(Promega)に挿入したＤＮＡから、in vitroでα−［^３２Ｐ］ＵＴＰを用いて転写されたゲノムアンチセンスＲＮＡプローブを用いて行った[Arad et al. (1995)同上]。Ｔ７プロモーターから転写されたＩＬ−２プローブ（６００ヌクレオチド（ｎｔ））はＩＬ−２遺伝子の３番目のエキソンおよび３番目のイントロンの一部に相補的であり、８Ｍ尿素−ポリアクリルアミドゲルでは、ＩＬ−２ ｍＲＮＡで保護された１１７ｎｔのＲＮＡ断片が得られる。Ｔ３プロモーターから転写されたＩＦＮ−γプローブ（２７４ｎｔ）はＩＦＮ−γ遺伝子の３番目のエキソンおよび３番目のイントロンの一部に相補的であり、ＩＦＮ−γ ｍＲＮＡで保護された１８３ｎｔのＲＮＡ断片が得られる。 Ｔ３プロモーターから転写されたＴＮＦ−βプローブ（７００ｎｔ）はエキソン１の一部、エキソン２、エキソン３およびイントロン３の一部ならびにエキソン４に相補的であり、ＴＮＦ−β ｍＲＮＡは２７４および２６３ｎｔの２断片を保護する。ハイブリダイゼーションではセンスＲＮＡ転写物を検出するシグナルが得られなかった。１８Ｓ ｒＲＮＡ（９０ｎｔを保護する）またはβ−アクチン（４１５ｎｔを保護する）のアンチセンスＲＮＡプローブを負荷対照とした。
【００９２】
ＩＬ−２およびＩＦＮ−γ ＲＮＡの定量ドットブロットハイブリダイゼーション
１ｍｌ培養物からＰＢＭＣを回収し、７．５Ｍグアニジニウム−ＨＣｌに溶解した。エタノール中、−２０℃で一晩沈殿させたＲＮＡをホルムアルデヒドに溶解し、６０℃で１５分間インキュベートした。１０×クエン酸ナトリウム生理食塩水による４種の連続２倍希釈物を９６ウェルドットブロット装置を用いて２組、ニトロセルロースシートに塗布した。真空炉にて８０℃で焼き付けた後、シートをヒトＩＬ−２およびＩＦＮ−γ各々の^３２Ｐ標識アンチセンスＲＮＡプローブとそれぞれハイブリダイズした。感光させたオートラジオグラムをＥＬＩＳＡリーダーで６３０ｎｍで走査した。ＲＮＡレベルをＡ_６３０の単位で表した。所定のＲＮＡサンプルの連続２倍希釈物では、各サンプルに存在する特異的ＲＮＡの濃度に比例した遺伝子発現強度の２００倍レンジを超える直線的光学濃度応答が得られる[Arad et al. (1995) 同明細書参照; Gerez et al., Clin Immunol Immunopathol 58: 251 (1991); Kaempfer et al., J Clin Oncol 14: 1778 (1996)]。
【００９３】
ＳＥＢ関連ペプチドの合成
ペプチドは、一般に、引用することにより本明細書の一部とされるＷＯ９８／２９４４４に記載されるように合成した。
【００９４】
ｐ１４はフルオロニル−メトキシカルボニル化学を用いて合成し、切断し、その側鎖をトリフルオロ酢酸で脱保護した。トリフルオロ酢酸−ペプチドスレートは培地に可溶であった。同じ手法を用いてＤ−Ａｌａを結合した。
【００９５】
Ｎ末端ラウリルシステイン（ＬＣ−）およびリシル−パルミトイル（ＬＰ−）ならびにＣ末端システイン（Ｃ−）は他のアミノ酸に用いた同じ条件の下で加えた。
【００９６】
ペプチドはＨＰＬＣにより９５％を超える純度であった。
【００９７】
免疫化
実施例５に記載のようにマウスを免疫化した。
【００９８】
毒性ショックに対するマウスの保護
１０〜１２週齢の１０匹の雌ＢＡＬＢ／ｃマウス(Harlan, Jerusalem, Israel)群を腹腔内注射による毒素抗原投与時に２０ｍｇＤ−ガラクトサミン(Sigma)を腹腔内注射により免疫化した。
【００９９】
毒性ショックに対するブタの保護
５日齢混血ブ（主としてヨークシャー；２．５ｋｇ）を各試験条件について６匹の群に無作為化し、各群の各々個体にＳＥＡ（２５μｇ）を腹腔内注射した。アンタゴニストペプチドはＳＥＡの３０分前およびＳＥＡ後５時間まで図に示された時間に腹腔内投与した。嘔吐および下痢は８時間までは毎時、次いで１２時間までは３時間ごと、さらに２１時間目と２４時間目に評価した。
【０１００】
実施例１
致死抗原投与したマウスにおけるｐ１４Ａの有効な防御活性
図１の試験では、ＳＥＢ暴露から２０時間までに９０％の対照マウスが死に至った。しかしながら、ＳＥＢを致死抗原投与する直前にｐ１４Ａを投与した場合にはこれらのマウスの９０％が生存していた（図１）。ｐ１４Ａの防御効果は１５の試験にわたって再現性が認められた。生存している動物には苦痛である様子もなく、挙動においても正常な対照動物と見分けがつかないほどであった。それらはモニターした２週間に限っては生存していた。ｐ１４Ａ単独に暴露したマウスは十分に生存可能な状態であり、副作用もみられなかった。
【０１０１】
この防御効果はＢＡＬＢ／ｃマウス株に特有のものではなかった。Ａ／ＪマウスもまたＳＥＢによる死を防ぐＩＰｐ１２Ａにより防御された［ＷＯ９８／２９４４４］。このことから防御が厳密にＭＨＣ依存性でないことが示され、従ってアンタゴニストペプチドがＭＨＣクラスII分子と相互作用することが知られていない超抗原ドメインを標的化するという事実に一致している。
【０１０２】
実施例２
アンタゴニストペプチドでＳＥＢにより誘導される致死ショックからマウスを防御・救済する
アンタゴニストペプチド単独に暴露したマウスは生存可能な状態であり、副作用もみられなかった。ＳＥＢを抗原投与したマウスのうち、毒素暴露から２４時間の時点でまだ生存しているのは３０％だけで、後には２０％となった。しかしながら、毒素の３０分前にｐ１４を施した場合にはＳＥＢを抗原投与した総てのマウスが防御された（ｐ＝０．０００３）。生存している動物には苦痛である様子もなく、挙動においても正常な対照動物と見分けがつかないほどであった。それらはモニターした２週間に限っては生存していた。アンタゴニストペプチドの投与を、致死抗原投与の３時間（ｐ＝０．０５）、５時間（ｐ＝０．０８）または７時間後にまで遅らせた場合には部分的防御が得られた。２０〜４０時間の間にはｐ１４Ａの漸減的防御効果がみられ、それぞれ７０％、６０％および５０％の生存を得た。ｐ１４Ａによって、ＳＥＢ抗原投与前に施した場合に感染が完全に防御されるだけでなく、すでに毒性ショックに陥っているマウスを救うことができることに注目すべきである。これらの結果は図２に示されている。
【０１０３】
アンタゴニストペプチドがＳＥＢに対し２０〜４０倍モル過剰である場合に防御されるまたは救われることから、ペプチドがin vivoにおいて効力のある超抗原アンタゴニストであることがわかる。
【０１０４】
実施例３
致死抗原投与したマウスにおけるｐ１４Ａの有効な防御活性
超抗原の致死性研究用に許容される動物モデルのＤ−ガラクトサミン感作マウスを用いて、ｐ１２Ａおよびｐ１４Ａの防御活性を調査した。図３では、ＳＥＢを致死抗原投与する直前に、ｐ１２Ａで処置したマウスでは約７０％生存、未処置（ペプチドを投与しない）の動物ではたった４０％であるのに対して、ｐ１４を投与した場合にはこれらのマウスの約９０％が６０時間を超えた後でも生存していたことがわかる。ｐ１４Ａの防御効果は１５にわたる試験で再現性が認められた。生存している動物には苦痛である様子もなく、挙動においても正常な対照動物と見分けがつかないほどであった。それらはモニターした２週間に限っては生存していた。ｐ１４Ａまたはｐ１２Ａ単独に暴露したマウスは十分に生存可能な状態であり、副作用もみられなかった。
【０１０５】
実施例４
ＩＦＮ−γ遺伝子発現の誘導に対するｐ１４Ａの有効な拮抗活性
ＰＢＭＣ集団におけるＴＳＳＴ−１またはＳＥＢによるＩＦＮ−γの誘導の程度に対するｐ１４Ａおよびｐ１２Ａ（配列番号５）の効力を比較し、その結果を図４および５に示している。
【０１０６】
ｐ１４Ａはｐ１４の誘導体であり、そのＮおよびＣ両末端にＤ−Ａｌａ残基を有している。同様に、ｐ１２Ａはｐ１２の誘導体であり、そのＮおよびＣ両末端にＤ−Ａｌａ残基を有している。ＴＳＳＴ−１はＳＥＢの全配列とほんの６％しか相同性を示さないが、ｐ１２Ａおよびｐ１４Ａとも、この毒素により誘導されるＩＦＮ−γ発現を阻害した（ＴＳＳＴ−１、図４）。また、ｐ１２Ａおよびｐ１４Ａの両方がＳＥＢにより誘導される発現も阻害した（図５）。ｐ１４ＡがＴＳＳＴ−１またはＳＥＢのいずれに対しても、ｐ１２Ａよりも極めて有効な拮抗活性を示したことは驚くべきことであった。
【０１０７】
ｐ１２Ａおよびｐ１４ＡはＴＳＳＴ−１の関係のあるドメインとほとんど相同性を有していない。それにもかかわらず、両ペプチドともＴＳＳＴ−１により誘導されるＩＦＮ−γ発現を阻害した。またこの際、実際にｐ１４Ａがｐ１２Ａの４／１２に対して、たった４／１４アミノ酸残基しかＴＳＳＴ−１の関連ドメインと共有していないにもかかわらず、ｐ１４Ａで予期しない有利な結果が得られた。
【０１０８】
実施例５
保護マウスには致死ショックに対する広スペクトル耐性が急速に展開する
アンタゴニストペプチドが広域防御活性を示すかどうかを調べるために、本発明者らは、ＳＥＢと全体で２７％のアミノ酸配列相同性しか示さない［Betley and Mekalanos 1988. J. Bacteriol. 170: 34-41］超抗原、ＳＥＡの致死抗原投与中にその効果を調べた。図６Ａからわかるように、ｐ１４ＡはまたＳＥＡの致死抗原投与からもマウスを保護した。ＷＯ９８／２９４４４では、アンタゴニストペプチドｐ１２ＡがＳＥＢ、連鎖球菌毒素ＳＰＥＡ（ＳＥＡと３０％、ＳＥＢと４８％相同性を有する）またはＳＥＡおよびＳＥＢ各々と全体で７％および６％の配列相同性しか示さないＴＳＳＴ−１による死からマウスを保護したことを示した。
【０１０９】
図６の試験では、ｐ１４Ａでの防御によりＳＥＡの致死抗原投与で生存した（図６Ａ）７匹のマウスに２週間後、ＳＥＢを再び抗原投与した。この際ペプチドアンタゴニスト不在下であった（図６Ｂ）。これらのマウスは総て生存していたが、１０匹の対照群ではＳＥＢ暴露で生存したものはいなかった。２週間後、ＳＥＢのこの第２の抗原投与で生存したマウスをもう一度さらに別の超抗原毒素、このときＴＳＳＴ−１に、再びペプチドアンタゴニスト不在下で暴露させた。ＴＳＳＴ−１を抗原投与した対照群では１０匹総てのマウスが死に至ったが、処置群では７匹のうち１匹だけが死に至った（図６Ｃ）。これらの６匹の生存個体に２週間後、そこでＳＰＥＡを４回抗原投与した。これら６匹のマウスのうち４匹が生存していたが、対照群では１０匹のマウスのうち１匹しかＳＰＥＡ暴露で生存していなかった。このように、ｐ１４Ａにより、ＳＥＡが媒介する致死ショックから保護されたマウスは異なる発熱性外毒素の連続的抗原投与に対する抵抗力を急速に獲得していた。
【０１１０】
実施例６
アンタゴニストペプチドでＳＥＡにより誘導される致死ショックからマウスを保護する
ＳＥＡを抗原投与する３０分前にマウスにｐ１４Ａを注射した。この結果は図７に示されている。わかるように、アンタゴニストペプチドによる前処置でＳＥＡ抗原投与に対して１００％の防御が得られた。これに対し、未処置のマウスでは６０時間後、１０％しか生存していなかった。ｐ１４ＡがＳＥＡの関係のあるドメインと１０／１４残基しか一致性を示していなくとも、ｐ１４ＡはＳＥＡの有利なアンタゴニストである。
【０１１１】
実施例６
アンタゴニストペプチドによる免疫化でＳＥＢにより誘導される致死ショックからマウスを防御する
本発明者らはアンタゴニストペプチドを細胞膜に固定することにより、その免疫原性が高まる可能性を考察した。ｐ１４Ａを、リジンがリンカーとして、パルミチン酸が疎水性アンカーとして作用する、Ｎ末端リシル−パルミトイルリーダーで合成してｐ１４Ａ−ＬＰを得た。図８はｐ１４Ａ−ＬＰおよびｐ１４Ａによるマウスの免疫化を示している。ｐ１４Ａ−ＬＰで免疫化した１１匹のマウスのうち１０匹が、ｐ１４Ａで免疫化した１０匹のマウスのうち７匹が致死ＳＥＢ抗原投与で生存していた。これに対し、アジュバントで免疫化した対照群では１０匹のマウスのうち３匹だけであった。免疫化した生存個体に２週間後、再び抗原投与すると、ｐ１４Ａ−ＬＰで免疫化した１０匹のマウスのうち７匹が、ｐ１４Ａで免疫化した７匹のマウスのうち４匹が２倍用量のＳＥＢでも生存していた。この用量は投薬を受けていない１０匹の対照マウスを２４時間内に１０匹とも死に至らしめることができる用量である（図８Ｂ）。ＳＥＢの最初の抗原投与から１週間後に採収した免疫化マウス血清の酵素結合免疫収着検定（ＥＬＩＳＡ）によりかなりのレベルの抗ＳＥＢ ＩｇＭ（０．８３５単位）が認められたが、ｐ１４Ａペプチドに対して向けられたＩｇＭは極めて低い値（０．０８単位）であった。さらにこれらの血清を漸増濃度のｐ１４Ａ、１０μｇ／ｍｌまで、とともに一晩プレインキュベーションしたが、それらのＳＥＢとの結合能力は低下しなかった（０．８４０単位）。アンタゴニストペプチドがＳＥＢ−結合抗体を完成していないことは明らかである。アンタゴニストペプチドは抗ＳＥＢ−抗体を誘導しているが、まだそれ自体に対する抗体が検出されていないことは注目すべきである。このことから、アンタゴニストペプチドのそれが誘導する抗体に対する親和性は毒素の親和性よりもはるかに低いことが示される。これらの結果は、アンタゴニストペプチドを用いる抗毒素ワクチンアプローチには、疎水性アンカーの利用に限定されるものではないが、生産性があることを示している。
【０１１２】
実施例７
ｐ１４ＡでＳＥＡにより誘導される活動不能からブタを保護する
ヒトＴ細胞はブドウ状球菌超抗原に対し、ネズミのものより少なくとも２オーダーの大きさで感作しやすい。ヒトは毒性ショック症候群を引き起こしやすいが、マウスは、ＴＣＲの最も高い反応性のあるＶβ鎖を示す細胞がネズミＴ細胞のすべての範囲から消失しているかまたは関連のＶβ遺伝子が欠失していることが明らかであり［Arad et al., 2000,同明細書参照］、耐性である。そのためマウスは超抗原毒素に対して自然耐性を有しており、それらが毒性ショックを受ける前に感作しているはずである。さらにマウスは体重および免疫系に関してヒトとはかけ離れている。よって人間により近い動物モデルでもアンタゴニストの効果を立証しておくことが重要である。
【０１１３】
本発明者らは両方の問題：毒素活動不能およびより高度な動物モデルの使用、に取り組んできた。ブタはヒトとよく似た免疫系を有している；例えば、ブタおよびヒト間の、ショックを媒介するＴｈ１サイトカインＩＬ−２およびＩＦＮ−γをコードするｍＲＮＡのヌクレオチド配列相同性は７５〜８５％のオーダーであるが、ヒトおよびマウスｍＲＮＡ間では類似性は認められない。
【０１１４】
ブタは感作の必要のない急性超抗原暴露に感受性であり、ヒトに見られるものとよく似た活動不能症状をひき起こす。悪心、嘔吐（ニューロン応答）ならびに重症の下痢（腸免疫応答）をはじめとする毒性ショックの早期活動不能症状の再現可能なブタモデルを確立した。図９からわかるように、アンタゴニストペプチドｐ１４Ａは毒素により誘導される活動不能を効果的に緩和している。
【０１１５】
５日齢混血ブタ（主としてヨークシャー；２．５ｋｇ）を、各試験条件について６匹の群に無作為し、各群の各々の個体にＳＥＡ（２５μｇ）により重症の活動不能を引き起こした。これは静脈または腹腔経路のいずれによって与えても類似した動態および強さで、２時間以内には目にも明らかとなり、２４〜３６時間で鎮静した。腹腔経路は静脈経路と同様に効果的であり、なお外傷が少なくてすむことから活動不能試験にはＳＥＡの腹腔内投与を用いた。この結果からはブタが特別にＳＥＡ感受性であることが確認される［Taylor et al., Infect Immun 36: 1263 (1982)］。この選択的感受性の理由には、ＳＥＡがそのβを捕らえて独立した第２の部位にあるＭＨＣII分子と結合し、それをいっそう強固に結合して、ＭＨＣII分子間に架橋をもたらしていることがある［Hudson et al., J Exp Med 182: 711 (1995); Schad et al., EMBO J 14: 3292 (1995); Abrahmsen et al., EMBO J 14: 2978 (1995)］。毒素アンタゴニストにより標的化されるドメインはＭＨＣIIの結合部位およびＳＥＡのＴＣＲ結合部位とも離れている。それにもかかわらず、アンタゴニストｐ１４Ａにより１０匹のマウスのうち７匹がＳＥＡによる死から保護され、かつ生存個体には広域防御免疫が惹起され（図６）、図７の試験ではＳＥＡ抗原投与した１０匹のマウスのうち１０匹にそれが現れた。
【０１１６】
次の基準を用いてブタの活動不能を評価した：嘔吐（評点４）；下痢、軽度（評点１）、中度（評点２）、重度（評点４）、および水下痢（評点６）（データなし）。長期食欲不振および雌ブタの乳首の代わりの吸水口の使用、無関心ならびによろめきがはっきりと観察されるが、これらは容易に定量できない；体温格差は正常な対照動物と平均１℃未満にとどまる。
【０１１７】
本発明者らはヒトＰＢＭＣ（図４および５）および致死毒性ショックから保護されたマウス（図３）においてin vitroでの超抗原の作用を阻害する能力に関し、ｐ１２Ａよりもｐ１４Ａの方が効果があることを示した。ｐ１４ＡアンタゴニストペプチドによりブタのＳＥＡ活動不能症状（嘔吐および下痢）が鎮静した。
【０１１８】
図９からわかるように、漸増用量のｐ１４Ａにより全般的な活動不能、ならびに嘔吐および下痢が目に見えて緩和された。これらの結果は、実施した６回のアンタゴニスト投与の最後の投与後に起こり、広く維持、発症する全般的活動不能（ｐ＝０．００２）および下痢（ｐ＜０．００１）に関してはかなり有意であり、ｐｏｓｔ ｈｏｃ検定で有意であった。また、嘔吐もアンタゴニスト用量に応じて緩和した。
アンタゴニストペプチド単独では副作用はみられなかった：ｐ１４Ａ単独を受けた混合群の１８匹のブタでは活動不能のいずれの症状も認められなかった（図９）。
本発明者らはアンタゴニストペプチドｐ１４Ａによる、対照動物では長期化し、たいてい５時間（試験群におけるアンタゴニストペプチドの最終投与時間）を超えて２４時間まで、発症する下痢の効果的な抑制を認めた；このことからアンタゴニストペプチドの長期にわたる効果が示される。
【０１１９】
ｐ１４Ａアンタゴニストペプチドｐ１４ＡによりブタのＳＥＡ活動不能症状（嘔吐および下痢）が抑制され、アンタゴニストの投与回数によってその効果が高まった（図１０）。この抑制は統計学的に有意である。ＳＥＡ暴露後にｐ１４Ａアンタゴニストペプチドの投与を４回受けたブタ群は、ＳＥＡ暴露後にアンタゴニストペプチドの投与を２回しか受けていない群よりも低い活動不能を示した。また後の両方の群はＳＥＡ暴露の３０分前に１用量のアンタゴニストペプチドを受けていたが、より多くの回数の毒素後処置を行うほどより効果の高い活動不能の抑制が得られる。これらの結果から、例えば、毒性ショックにあるおよび重症の食中毒にあるヒトで見られるような、活動不能症状の治療におけるアンタゴニストペプチドの可能性ある効用が示される。
【０１２０】
このように、アンタゴニストペプチドｐ１４Ａは、マウスだけでなく、免疫系がヒトのものとより密接に関係している動物であるブタにおいても、in vivoで毒素アゴニスト活性を全く検出することなく超抗原毒素、ＳＥＡの毒性ショック症状を止めることが可能である。
【０１２１】
これらの実施例は本発明の範囲の具体例をさらに記載し、例示するものである。これら実施例は本発明を限定するものではなく、単に例示を目的としたものであり、本発明の精神および範囲を逸脱することなくその多くの変法が可能である。
【０１２２】
本明細書において引用される総ての参考文献は、引用することにより本明細書の一部とされる。
【０１２３】
【配列表】

【図面の簡単な説明】
以下の図面により本発明をさらに詳細に説明する。
【図１】アンタゴニストペプチドがＳＥＢにより誘導される致死ショックからマウスを保護することを示す説明図。
１０匹のＢａｌｂ／ｃマウスに２０ｍｇＤ−ガラクトサミンを感作させ、同時に１０μｇＳＥＢを抗原投与した（○）。もう一方の１０匹のマウスにはＳＥＢ抗原投与３０分前に２５μｇのｐ１４Ａを施した（●）。生存個体をモニターした。
【図２】アンタゴニストペプチドがＳＥＢにより誘導される致死ショックからマウスを保護・救済することを示す説明図。
１０匹のＢＡＬＢ／ｃマウス群に２０ｍｇＤ−ガラクトサミンを感作させ、同時に１０μｇＳＥＢを抗原投与した。同時に注射した。１アリコートの２５μｇのｐ１４ＡをＳＥＢ抗原投与３０分前（▲）またはＳＥＢ投与後の３（黒四角）、５（○）もしくは７（●）時間後に注射した。ｐ１４Ａを受けなかった１０匹の感作無投薬マウスをＳＥＢ対照とした（□）。総ての注射は腹腔内により行った。
【図３】アンタゴニストペプチドｐ１２Ａおよびｐ１４ＡによるＳＥＢにより誘導される致死ショックからのマウスの保護を示す説明図。
１０匹のＢＡＬＢ／ｃマウス群に、２０ｍｇＤ−ガラクトサミンとともに５μｇの毒素を腹膜内注射してＳＥＢ(Sigma)を抗原投与した。示されたように２５μｇのｐ１２Ａまたはｐ１４ＡのいずれかをＳＥＢ投与３０分前に腹膜内注射した。生存個体をモニターした。
【図４】ＴＳＳＴ−１によるＩＦＮ−γ遺伝子発現誘導に対するアンタゴニストペプチドｐ１２Ａおよびｐ１４Ａの効力を示す説明図。
健康なヒト提供者のＰＢＭＣをＦｉｃｏｌｌ Ｐａｑｕｅ(Pharmacia)で分離し、５０ｍｌのＲＰＭＩ１６４０培地で２回洗浄し、４×１０^６／ｍｌの濃度に再懸濁し、２％ウシ胎児血清、２ｍＭグルタミン、１０ｍＭ ＭＥＭ非特異的アミノ酸、１００ｍＭピルビン酸ナトリウム、１０ｍＭ Ｈｅｐｅｓ、ｐＨ７．２、５×１０^−５Ｍ ２−ＭＥ、１００単位／ｍｌペニシリン、１００μｇ／ｍｌストレプトマイシンおよび５μｇ／ｍｌナイスタチンを添加した同培地で培養した。一晩静置した後、１ｍｌ中４×１０^６ＰＢＭＣのアリコートを、１００ｎｇ／ｍｌのＴＳＳＴ−１(Sigma)単独の存在下または示された１０μｇ／ｍｌのｐ１２Ａもしくはｐ１４Ａの存在下で、示された回数培養した。全ＲＮＡを示された回数抽出し、連続２倍希釈物を^３２Ｐ標識ＩＦＮ−γアンチセンスＲＮＡプローブを用いるブロットハイブリダイゼーション解析[Arad et al., (2000) 同上]に付した。オートラジオグラムを濃度計で６３０ｎｍにて定量し、６３０ｎｍの吸光度をプロットする。
【図５】ＳＥＢによるＩＦＮ−γ遺伝子発現誘導に対するアンタゴニストペプチドｐ１２Ａおよびｐ１４Ａの効力を示す説明図。
１ｍｌ中４×１０^６ＰＢＭＣのアリコートを、１００ｎｇ／ｍｌのＳＥＢ（ロット１４〜３０、the Department of Toxinology, United States Army Medical Research Institute of Infectious Diseases(USAMRIID, Frederick, Maryland)から）単独の存在下または指示された１０μｇ／ｍｌのｐ１２Ａもしくはｐ１４Ａの存在下で、示された回数培養した。全ＲＮＡを示された回数抽出し、連続２倍希釈物を^３２Ｐ標識ＩＦＮ−γアンチセンスＲＮＡプローブを用いるブロットハイブリダイゼーション解析に付した。オートラジオグラムを濃度計で６３０ｎｍにて定量し、６３０ｎｍの吸光度をプロットする。図４の場合と同じＰＢＭＣ集団を用いた。
【図６】アンタゴニストにより保護したマウスには致死ショックに対する広域免疫が急速に現れることを示す説明図。
（Ａ）２０ｍｇＤ−ガラクトサミンによる感作および１０μｇＳＥＡによる同時抗原投与３０分前に、１０匹のＢＡＬＢ／ｃマウスにｐ１４Ａを施した（●）。さらにｐ１４Ａの注射は行わずに、７匹の生存個体に２週間後、１０μｇＳＥＢ（▲；Ｂ）、続いて２週間後に１０μｇＴＳＳＴ−１（黒四角；Ｃ）、さらに２週間後に１５μｇＳＰＥＡ（▲；Ｄ）を再び抗原投与した。各抗原投与では、１０匹の感作無投薬マウスを毒素対照とした（○、△、□）。総ての注射は腹腔内により行った。
【図７】アンタゴニストペプチドがＳＥＡにより誘導される致死ショックからマウスを保護することを示す説明図。
１０匹のＢＡＬＢ／ｃマウス群に１０μｇＳＥＡを腹腔内により抗原投与し、ＳＥＡ投与３０分前に２５μｇのｐ１４Ａを腹腔内注射した。
【図８】アンタゴニストペプチドでの免疫化でＳＥＢにより誘導される致死ショックからマウスを保護することを示す説明図。
Ａ：１０匹のＢＡＬＢ／ｃマウス群（ｐ１４Ａ−ＬＰについては１１匹のマウス）に、フロイントアジュバント（ＦＡ）中の示されたペプチド１０μｇを３回注射（０、２および４週間目）して免疫化し、その後６週間目に１０μｇＳＥＢおよび２０ｍｇＤ−ガラクトサミンを腹腔内注射して抗原投与した。生存個体をモニターした。Ｂ：最初の抗原投与から２週間後、Ａの生存個体（ｐ１４Ａ−ＬＰ、ｎ＝１０；ｐ１４Ａ、ｎ＝７）に２０μｇＳＥＢおよび２０ｍｇＤ−ガラクトサミンを再び抗原投与した。ＬＰ、リシル−パルミトイル。ＦＡおよびｐ１４Ａ−ＬＰ（▲）、ＦＡおよびｐ１４Ａ（□）またはＦＡ単独（黒四角）で免疫化したマウス；ＳＥＢを抗原投与した無投薬対照マウス（○）。
【図９】発熱性外毒素アンタゴニストでＳＥＡにより用量依存的に起こる浮力かからブタを保護することを示す説明図。
ブタ群（全体の群の大きさ、ｎ）に、０時間に２５μｇ／ｋｇＳＥＡを腹腔内により抗原投与した。示されたようにｐ１４Ａ（２５、５０または１００μｇ／ｋｇ）をＳＥＡ投与から〜０．５時間および１、２、３、４および５時間後に腹腔内により施した。それぞれのブタを、１１時点を２４時間にわたって観察した。これらの時点の曲線の下にある面積を全般的活動不能、嘔吐および下痢についてプロットする。全般的活動不能のスコアは嘔吐および下痢スコアの合計である。群間の反復測定解析の結果、ｐ値を得る。統計解析はＷｉｎｄｏｗｓ９．０ソフトウェアのＳＰＳＳを用いてＴｕｋｅｙ ＨＳＤ ｐｏｓｔ ｈｏｃ検定によりデータ集合のＡＮＯＶＡを行った（なお、ｐ≦０．０５には１つのアステリスクを付けている）。毒素投与なしのｐ１４Ａ対照を右に示す。
【図１０】アンタゴニストペプチドの反復投与によるＳＥＡにより誘導される活動不能からのブタの保護を示す説明図。
６匹の子ブタ群に２５μｇ／ｋｇＳＥＡを腹腔内により抗原投与した。ｐ１４Ａ（５０μｇ／ｋｇ）を０、１．５および２．５時間（×３）にまたは０、１．５、２．５、３．５および４．５時間（×５）にＩＰにより施した。嘔吐および下痢の活動不能スコアをそれぞれ示す。生理食塩水対照動物にはＳＥＡもｐ１４Ａも与えなかった。曲線の下にある面積を、評価した全般的活動不能、嘔吐および下痢について、１０時点を２４時間にわたってプロットする。統計解析はＷｉｎｄｏｗｓ９．０ソフトウェアのＳＰＳＳを用いてＴｕｋｅｙ ＨＳＤ ｐｏｓｔ ｈｏｃ検定によりデータ集合のＡＮＯＶＡを行った（なお、ｐ≦０．０５には１つのアステリスクを付けている）。[0001]
Field of Invention
The present invention relates to peptides structurally related to protein domains in pyrogenic exotoxins. The peptides of the present invention can antagonize T cell activation mediated by the pyrogenic exotoxin and elicit protective immunity against toxic shock induced by the exotoxin. The present invention further comprises a pharmaceutical preparation containing the peptide for the treatment or prevention of toxic shock, which can elicit protective immunity against the toxic shock induced by the exotoxin, and the peptide Regarding vaccines.
[0002]
Background of the Invention
The pyrogenic exotoxins are also called superantigen toxins andStaphylococcus aureus) And Streptococcus pyogenes (Streptococcus pyogenes). Exotoxins consisting of Staphylococcus aureus exotoxin (SE) cause major human food poisoning cases as a sign of vomiting and diarrhea after ingestion [Schlievert, J Infect Dis 167: 997 (1993)]. Staphylococcus aureus is widely found in nature and is often associated with humans. Of the five major SE serotypes (designated SEA-SEE and SEG), SEB is the most prevalent [Marrack and Kappler, Science 248: 705 (1990)]. SEB has also been recognized as a major cause of viral infections in the airways of non-menstrual toxic shock syndrome, which can be associated with surgical or injured wounds, and particularly influenza patients who are vulnerable to children [Schlievert ( 1993) ibid; Tseng et al., Infect Immun 63: 2880 (1995)]. Toxic shock syndrome, in its most severe form, results in shock and offspring [Murray et al., ASM News 61: 229 (1995); Schlievert (1993) ibid.]. More generally, in atopic dermatitis [Schlievert (1993) supra] and Kawasaki disease syndrome [Bohach et al., Crit Rev Microbiol 17: 251 (1990)], SEA to SEE and Toxic Shock Syndrome Toxin 1 (TSST-1) The membranes of members of the staphylococcal exotoxin family including) have been associated with toxic shock syndrome.
[0003]
There is a view that SEB can be used as a biological weapon because it can cause lethal shock in humans after aerosol exposure and SEB can be mass produced relatively easily [Lowell et al., Infet Immun 64: 1706 (1996)]. SEB is considered to be a potential biological weapon primarily in terms of its lethality. But SEB also has an incapacitating agent that, due to its powerful vomiting and diarrhea-inducing ability, can significantly impair the effectiveness of combat power, even temporarily, thereby making it susceptible to attack by normal military means. It is. Needless to say, the harmful effects of SEBs must generally attack, not only with respect to military aspects.
[0004]
SEB is a toxic mitogen that induces a paradoxical response in infected organisms, on the one hand it is a powerful stimulator of the immune system and on the other hand it can allow the growth of infectious bacteria that are not disturbed by the immune response It is a huge immunosuppressant [Hoffman, Science 248: 685 (1990); Smith and Johnson J Immunol 115: 575 (1975); Marrack et al., J Exp Med 171: 455 (1990); Pinto et al., Transplantation 25: 320 (1978)]. Activation of Th1-type cytokine gene expression exemplified by interleukin-2 (IL-2), interferon-γ (IFN-γ) and tumor necrosis factor-β (TNF-β) during cellular immune response; On the other hand, CD8 cells and other cell subsets [Ketzinel et al., Scand J Immunol 33: 593 (1991); Arad et al., Cell Immunol 160: 240 (1995)], and also Th2 cells, IL-4 and IL -10-derived inhibitory cytokines [Mosmann and Coffman, Annu Rev Immunol 7: 145 (1989)] induced dynamic interactions with antigens or mitogens between their cell-mediated suppression. The
[0005]
SEB is a member of the pyrogenic exotoxin family [Herman et al., Ann Rev Immunol 9: 745 (1991)] and consists of bacterial exotoxins and Mls proteins. These antigen toxins stimulate rodent or human T cell production 20,000 times higher than normal antigens. Thus, SEB activates 30-40% of total T cells in some mice [Marrack and Kappler (1990) supra]. In fact, SEB toxicity requires T cells, and mice lacking T cells or SEB-reactive T cells are not affected by SEB doses resulting in weight loss and death in normal animals [Marrack et al. (1990) ibid .; Marrack and Kappler (1990) ibid.]. Unlike normal antigens, SEB and related toxic mitogens do not require processing and antigen presentation [Janeway et al., Immunol Rev 107: 61 (1989)], but the T cell receptor β chain (V- T cells are activated by binding to specific sites in the variable part of β) [Choi et al., Nature 346: 471 (1990)]. The region important for the interaction of the T cell receptor with the toxin is the outer surface of the V-β domain, which is not involved in normal antigen recognition [Choi et al., Proc Natl Acad Sci USA 86: 8941 (1989) ]. At the same time, pyrogenic exotoxins bind directly to MHC class II branches [Scholl et al., Proc Natl Acad Sci U.S.A. 86: 4210 (1989)], and thus mainly CD4.⁺Acts on T cells but CD8⁺Cells are also activated [Fleischer and Schrezenmeier, J Exp Med 167: 1697 (1988); Fraser, Nature 339: 221 (1989); Misfeldt, Infect Immun 58: 2409 (1990)]. At present, there is a consensus that pyrogenic exotoxin activates T cells very effectively because it bypasses the normal interaction of antigens with class II MHC and T cell receptors [ Janeway, Cell 63: 659 (1990)]. Another view is that pyrogenic exotoxin acts as a co-ligand that helps the action of minimal amounts of normal antigens and thus significantly overloads [Janeway (1990) supra].
[0006]
The toxicity of SEB and related exotoxins is thought to be related to the ability of these molecules to stimulate rapid and excessive production of cytokines, particularly IL-2, IFN-γ and tumor necrosis factor (TNF). IL-2, IFN-γ and TNF-β are secreted from activated T helper type 1 (Th1) cells, whereas TNF-β is secreted by Th1 cells, monocytes and macrophages. Suddenly produced high levels of these cytokines have been implicated as a central etiology in toxin-related toxicities [Schard et al., EMBO J 14: 3292 (1995)], resulting in a rapid decrease in blood pressure that causes toxic shock. It is thought to cause.
[0007]
Although the study provides a plausible explanation for the strong stimulation of T cells by SE, it is not yet clear why these toxins also have strong immunosuppressive properties. They cause a decrease in both primary T and B cell responses, including antibody production and plaque-forming cell development [Hoffman (1990) ibid; Smith and Johnson (1975) ibid; Marrack et al. (1990) Pinto et al. (1978) ibid .; Ikejima et al., J Clin Invest 73: 1312 (1984); Poindexter and Schlievert, J Infect Dis 153: 772 (1986)].
[0008]
Human staphylococcal toxin sensitivity is 100 times better than mice. Another pyrogenic exotoxin from Staphylococcus aureus, Toxic Shock Syndrome Toxin 1, TSST-1, stimulates human T cells to reduce the key cytokines IL-2, IFN-γ and TNF-β to 0 .. Less than 1 pg / ml is expressed, whereas murine cells require approximately 10 pg / ml [Uchiyama et al., J Immunol 143: 3173 (1989)]. Mice are relatively resistant to toxic mitogens from their T cell repertoire by lacking cells that display the most reactive V-β chain or by deleting these V-β genes. [Morrack and Kappler (1990) same as above]. Such defects are not detected in humans and are quite inconsistent.
[0009]
The effects of SEB (and the same superantigen exotoxins) in inactive and potentially fatal humans, whether civilian or military, prevent SEB, treat people exposed to SEB and safe SEB vaccines Need.
[0010]
Despite this urgent need, there is still no protection or cure. Thus, in murine models of SEB poisoning sensitized to D-galactosamine (some based on intramuscular challenge with SEB toxin and some based on intranasal challenge with mucosal SEB exposure). The toxoid vaccine was able to protect mice, but here the SEB toxoid component was prepared by treating a biologically active complete protein molecule with formalin for 30 days [Lowell et al. (1996) ibid. ]. The inventors have shown that antibodies formed against specific peptide domains within the SEB molecule enhance the ability of SEB to stimulate human T cells rather than protecting against toxins [WO 98/29444; Arad et al., Nature Medicine, 6 (4): 414-421 (2000)]. In view of the risk of eliciting specific SEB-sensitized antibodies that not only can not confer protective immunity in people exposed to SEB, but can also result in a significant deterioration of the toxic response, this discovery is a vaccine for SEB toxoids. Use is limited.
[0011]
Other researchers sought to rely on using fragments rather than complete SEB proteins by synthesizing a series of overlapping SEB peptides, each on the order of 30 amino acids long [Jett et al., Infect Immun 62: 3408 (1994)]. These peptides were used to generate antisera in rabbits and then examined for their ability to inhibit the growth of a mixture of human T cells and macrophages induced by SEB. This effort did not yield an effective or specific inhibitory response. Thus, peptide pSEB (113-144), which contains amino acids 113-144 of the SEB protein molecule, and peptides spanning amino acids 130-160, 151-180, and 171-200, respectively, exhibited some lymphocyte proliferation induced by SEB. Inhibited antisera were raised up to 2.5-fold [Jett et al. (1994) supra].
[0012]
Several researchers have attempted to create peptide vaccines. For example, Mayordomo et al. [J Exp Med 183: 1357 (1996)] used p53-derived peptide variants as vaccines for the treatment of murine tumors. Huges and Gilleland [Vaccine 13: 1750 (1995)]Pseudomonas aeruginosa) Was able to protect against P. aeruginosa infection in a murine acute pneumonia model. In an attempt to use peptide immunization in humans, Brander et al. [Clin Exp Imunol 105: 18 (1996)]⁺/ CD4⁺T cell targeted conjugate vaccine is memory CD4⁺It was shown that restimulation of the T cell response failed to induce cytotoxic T lymphocytes.
[0013]
The main sources of exotoxins are Staphylococcus aureus and Streptococcus pyogenes as already described. The carnivorous bacterium Streptococcus pyogenes produces a series of different toxins in a mode of action that mimics excessive activation of T cells. S. aureus also produces SEA, SECs, SEE and TSST-1 (toxic shock syndrome toxin 1), followed by SEB as the main component, and Streptococcus pyogenes is the main toxin in addition to SPE A and other pyrogenic Produces exotoxins. Thus, toxin mixtures are encountered in staphylococcal food poisoning and in more serious biological warfare or toxic shock caused by Streptococcus pyogenes. The composition of such a mixture cannot be predicted with certainty. In the worst-case scenario of biological warfare, it is not a single purified pyrogenic exotoxin as preferred for immunological studies, but is readily available and unpurified, such as produced by culturing S. aureus Naturally, a natural toxin mixture is used.
[0014]
Clearly this complexity requires the development of a wide range of pyrogenic exotoxin antagonists as well as a wide range vaccine. Antagonists have been found by the inventors [WO 98/29444 and Arad et al., (2000) supra] that are not sensitizing and can protect test animals from lethal doses of toxin. These antagonists are peptides that are structurally related to a domain of pyrogenic exotoxin, which has a central folding point within the exotoxin, starting with β chain 7 based on the domain number of SEB, The β chain 7 is connected to the α-helix 4 via the short β chain 8 and ends in the α-helix 4. This domain is not known to interact with MHC class II molecules.
[0015]
These antagonist peptides are described in detail in WO98 / 29444 and Arad et al., Nature Medicine, 6 (4): 414-421 (2000) owned by the same person, the contents of which are incorporated herein by reference in their entirety. To be part of
[0016]
Thus, WO 98/29444 particularly discloses a vaccine that can confer protection not only against SEB but also against a wide range of SE venom systems such as SEA, SEC, TSST-1. Further, this publication WO 98/29444 discloses the prevention of toxic shock induced by SEB or any other pyrogenic exotoxin and the treatment of exposed patients. Prior art by the inventors has shown the design of drugs that antagonize the effects of SEB and any other pyrogenic exotoxins, which are used in medical treatment of acute food poisoning, in toxic shock and related pathologies. It also has great value in protecting life. The peptides developed so far by the inventors are further used in and for long-term treatment of acute toxic shock and the immediate or short-term and emergency prevention of the harmful effects of such toxins, which may be due to accidental food poisoning, for example. It has been shown to be an antipyretic exotoxin antagonist used in vaccines for immunization against febrile exotoxin poisoning for prevention.
[0017]
In studies of further improved toxin antagonists, the inventors have shown that blocking pyrogenic exotoxin action in human immune responses in vitro, strictly inhibiting toxin-mediated induction of IFN-γ mRNA, and many fevers A novel 14-mer peptide, which is superior to the known peptide antagonists in inducing protective immunity against the response mediated by sex exotoxin, is newly developed. This is the main object of the present invention, and can be developed as a broad-area vaccine. It suggests sex.
[0018]
This and other objects of the present invention will become apparent from the following description.
[0019]
Summary of the Invention
The present invention relates to an isolated and purified peptide having the amino acid sequence of SEQ ID NO: 1, and is also referred to herein as p14. The p14 peptides of the present invention can antagonize toxin-mediated T cell activation and / or protect against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins.
[0020]
The invention further relates to derivatives of p14 that antagonize toxin-mediated T cell activation and can protect against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins. Such derivatives are referred to herein as functional derivatives. Specific functional derivatives of the peptides of the invention include p14 (SEQ ID NO: 2, also referred to herein as p14A) having D-Ala at both its N-terminus and C-terminus, and lysyl-palmitoyl at its N-terminus It is a derivative of p14 having a residue (SEQ ID NO: 3, also called p14-LP).
[0021]
Peptides and derivatives of the present invention antagonize toxin-mediated T cell activation and, for example, from SEB, SEA, SEC, TSST-1, SPEA, SPEC, SED, SEE, SEH, SEC1, SEC2, SEC3 It can protect against toxic shock induced by a selected pyrogenic exotoxin or mixture of pyrogenic exotoxins. These exotoxins share the structural domain targeted by the peptides and derivatives of the present invention, but this domain starts within β chain 7 based on the SEB domain number and β chain 7 is short. It is connected to α-helix 4 via β chain 8 and ends in α-helix 4. The peptides and derivatives of the invention have at least 30% homology with the sequences of these domains in various toxins.
[0022]
Thus, the pyrogenic exotoxin can be a bacterial exotoxin produced by Staphylococcus aureus or Streptococcus pyogenes.
[0023]
According to the present invention, the peptide comprises a lauryl-cysteine (LC) or lysyl-palmitoyl residue via its N-terminus and / or a cysteine (C) residue via its C-terminus, or the peptide It may be further coupled with other residues suitable for coupling with adjuvants for immunization. There are several modifications that can be used to obtain functional derivatives.
[0024]
Furthermore, the peptide may be in the form of an oligomer, in particular a dimer, a trimer, or may be in a constrained conformation, where the constrained conformation is an internal cross-link, small range cyclization, extension. Or obtained by other chemical modifications.
[0025]
The peptides and derivatives of the present invention can inhibit the expression of pyrogenic toxin-induced mRNA encoded by IL-2, IFN-γ or TNF-β genes.
[0026]
Furthermore, the peptides and derivatives of the invention can elicit the production of antibodies that block pyrogenic toxin-mediated T cell activation in the presence of a suitable immune adjuvant in immunized individuals. .
[0027]
In a second aspect, the present invention provides toxin-mediated activation of T cells comprising as an active ingredient a therapeutically effective amount of at least a peptide of the present invention or a functional derivative thereof, or any mixture thereof. A pharmaceutical composition for the treatment or short-term prevention of is provided. The peptides and derivatives of the present invention can protect against toxic shock induced by pyrogenic exotoxins or mixtures of pyrogenic exotoxins.
[0028]
The present invention further relates to a toxic shock induced by at least one pyrogenic exotoxin comprising as an active ingredient an immunologically effective amount of at least the peptide of the present invention or a functional derivative thereof, or a mixture thereof. Provide vaccines that confer long-term immunity.
[0029]
The invention further relates to a composition for the prevention and / or treatment of inactivity induced by at least one pyrogenic exotoxin comprising an isolated, purified peptide or derivative of the invention.
[0030]
Also provided by the present invention is a method for first-aid or short-term prevention of an acute toxic shock and adverse effects that may be due to, for example, accidental food poisoning induced by at least one pyrogenic exotoxin, said method Comprises administering to a patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition of the invention, or a therapeutically effective amount of a peptide of the invention or a functional derivative thereof or a mixture thereof.
[0031]
The present invention further provides a method of conferring long-term immunity against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins comprising administering to a patient an effective immunizing amount of the vaccine of the present invention. provide.
[0032]
Still further, the present invention provides a method for preventing or treating inactivity induced by at least one pyrogenic exotoxin comprising the isolated or purified peptide or derivative of the present invention.
[0033]
Detailed Description of the Invention
The present invention is directed to agents effective for the long-term and short-term treatment and prevention of toxic shock induced by pyrogenic exotoxin, particularly a 14-mer peptide having the amino acid sequence of SEQ ID NO: 1, and pyrogenic It relates to specific domains within exotoxins and functional derivatives thereof. The biological properties of a peptide can be evaluated by several criteria:
[0034]
1. Lack of SEB agonist activity
By the ability to induce expression of mRNA encoded by IL-2 and IFN-γ genes in peripheral blood mononuclear cells (PBMC) from normal human blood donors in the absence of any other inducer Assayed.
[0035]
2. Pyrogenic exotoxin antagonist activity
In PBMC, it is assayed by its ability to inhibit the expression of mRNA encoded by IL-2, IFN-γ and TNF-β genes induced by pyrogenic exotoxins such as SEB.
[0036]
3. Immunogenicity
In immunized rabbits, it is assayed by the ability of the peptide to elicit the production of immunoglobulin G (IgG) antibodies that bind SEB.
[0037]
4). Immunogenicity
In immunized rabbits, assayed by the ability of peptides to elicit the production of antibodies that block the deleterious effects of pyrogenic exotoxins such as SEB on the human cellular immune response, by SEB in PBMC, or TSST-1 or more related This is monitored by the ability of the rabbit serum generated against the peptide to inhibit the induction of IL-2 and IFN-γ mRNA by other pyrogenic exotoxins such as the SEA toxin SEA.
[0038]
5. Activity of vaccines such as anti-SEB vaccine
In the D-galactosamine mouse model, it is assayed by the ability of the peptide to protect immunized animals against lethal doses of SEB.
We have obtained peptides that meet each of these five criteria. Some of such peptides are disclosed in WO 98/29444. The inventors have now found that certain 14-mer peptides and derivatives thereof show improved properties compared to prior art peptides. Thus, in certain embodiments of the invention, 14-mer peptide antagonists were designed. This peptide blocks the effects of SEB and other pyrogenic exotoxins on the human immune response in vitro, and strictly inhibits SEB-mediated induction of IL-2, IFN-γ and TNF-β mRNA. It is clear that this peptide can be used for the treatment of acute toxic shock and adverse effects that may be due to, for example, accidental food poisoning induced by pyrogenic exotoxins.
[0039]
The peptide of the present invention has an amino acid sequence that is homologous to the amino acid sequence of the pyrogenic exotoxin domain (this domain forms a central fold in the exotoxin and is based on the domain number of SEB and is in β chain 7 And β chain 7 is linked to α-helix 4 via short β chain 8 and ends in α-helix 4), has no toxin agonist activity and is mediated by toxin-mediated T lymphocytes. Can antagonize activation. Homology in this region is as low as no more than about 30% or even less than the corresponding region in other pyrogenic exotoxins. Specifically, the peptide p14 of the present invention has the amino acid sequence VQYNKKKATVQELD (SEQ ID NO: 1), and two additional amino acids of the SEB sequence at the N-terminus before the sequence YNKKKATVQELD of the prior art p12 (SEQ ID NO: 4) (VQ). p14A (SEQ ID NO: 2) is flanked on both ends by D-alanine for stronger protease resistance, similar to the prior art p12A (SEQ ID NO: 5) (WO 98/29444).
[0040]
Table 1 summarizes the specific peptides described in this application and their SEQ ID NOs.
[Table 1]

[0041]
In another embodiment, the peptide antagonists of the invention can elicit antibodies that protect human lymphoid cells against SEB, SEA and TSST-1, which confers broad protective immunity against pyrogenic toxins It shows you can. However, antibodies raised against peptides derived from certain other SEB protein domains actually do not protect against toxins, but are expressed by higher induction of IL-2 and IFN-γ mRNA. Enhanced the response of human PBMC to SEB and SEA. Immunization of mice with these SEB antagonist peptides elicited protection against lethal doses of SEB, resulting in survival of the test animals. It is clear that these peptides can also be used to confer long-term immunity against toxic shock induced by pyrogenic exotoxins.
[0042]
Thus, in a first aspect, the present invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1 and a functional derivative of such peptide, wherein the isolated and purified peptide exhibits toxin adonist activity. Without it, it can elicit protective immunity against toxic shock induced by exotoxins. As used herein, the term derivatives and functional derivatives do not exhibit toxin agonist activity and do not interfere with the ability of the peptide to elicit protective immunity against toxic shock induced by exotoxin and / or Alternatively, it means any peptide with insertions, deletions, substitutions and modifications (hereinafter referred to as “derivatives”) to peptides that antagonize toxin-mediated T cell activation. Derivatives should maintain a minimum of homology with the domain, for example about 30% or somewhat less than 30%.
[0043]
In a second aspect, the present invention provides an isolation and purification comprising the amino acid sequence of SEQ ID NO: 1 that does not inhibit toxin agonist activity and is capable of antagonizing toxin-mediated T cell activation. It relates to peptides and functional derivatives of such peptides. The peptides of the present invention can protect against toxic shock induced by pyrogenic exotoxins or mixtures of pyrogenic exotoxins.
[0044]
Pyrogenic exotoxins are usually exotoxins produced by bacterial exotoxins, particularly S. aureus or Streptococcus pyogenes.
[0045]
The isolated / purified peptide of the present invention comprises the amino acid sequence represented by SEQ ID NO: 1 (hereinafter also referred to as p14 or p14 (148-161)), and a functional derivative thereof. p14 and functional derivatives thereof may elicit protective immunity against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins and / or antagonize toxin-mediated T cell activation. it can. These peptides are also used for first-aid treatment of any acute effects of toxic shock and adverse effects that may be due to, for example, fever exotoxin-induced food poisoning, and the provision of long-term immunity against such toxic shock. Can be used for. As indicated above, like the p14 peptide, the functional derivative preferably also has at least 30% homology with the protein domain of the pyrogenic exotoxin. p14 (SEQ ID NO: 1) is homologous to positions 148-161 of the sequence of the naturally occurring protein shown in SEQ ID NO: 6 at 11 of its 14 amino acid residues.
[0046]
In addition, as shown in the Examples below, the p14 peptide also has toxin antagonist activity against TSST-1, but has less than 30% homology with the corresponding region of TSST-1, leaving 14 amino acid residues. Share 4 of the groups.
[0047]
The lack of structure of the linear peptides makes them susceptible to proteases in human serum and reduces their affinity for the target site as only a few of the possible conformations are active work. It is therefore desirable to optimize the antagonist peptide structure.
[0048]
In order to improve the peptide structure, the peptides of the invention can be combined with lauryl-cysteine (LC) or lysyl-palmitoyl residues via their N-terminus and / or cysteine (C) residues via their C-terminus. As described in more detail below, or the group, the peptide can be linked to other residues suitable for coupling with adjuvants for immunization. In order to improve protease resistance, D-Ala may be bound to either the N-terminus or the C-terminus, or both.
[0049]
The peptides and derivatives thereof of the present invention may all have a positive charge, may have a negative charge, or may be neutral, and may be in the form of a dimer or multimer. Or a constrained conformation.
[0050]
The constrained conformation can be obtained by internal cross-linking, short range cyclization, extension or other chemical modifications. Also contemplated are oligomers such as dimers and trimers.
[0051]
Furthermore, the peptides of the present invention may be extended at the N-terminus and / or C-terminus with various identical or different amino acid residues. As an example of such extension, the peptide is extended at its N-terminus and / or C-terminus with the same or different hydrophobic amino acid residues, which may be naturally occurring or synthetic amino acid residues. be able to. A preferred synthetic amino acid residue is D-alanine, which increases the protease resistance of the peptide as described.
[0052]
Specific examples of peptides extended with synthetic amino acid residues elicit protective immunity against toxic shock induced by pyrogenic exotoxin and / or antagonize toxin-mediated T cell activation A peptide having the amino acid sequence represented by SEQ ID NO: 2 (hereinafter also referred to as p14A), and a functional derivative thereof. In one preferred embodiment, the peptide of the invention is p14A with D-Ala residues at both its N-terminus and C-terminus.
[0053]
A further example of such extension is provided by a peptide extended with cysteine residues at both its N-terminus and / or C-terminus. Of course, such extension may result in a constrained conformation due to Cys-Cys cyclization resulting from disulfide bond formation.
[0054]
Another example is the generation of the derivative p14A-LP (SEQ ID NO: 3) by incorporation of an N-terminal lysyl-palmitoyl tail (lysine serves as a linker and palmitic acid serves as a hydrophobic anchor).
[0055]
Furthermore, the peptide may be a naturally occurring amino acid residue or a synthetic amino acid residue, or an extended aromatic amino acid residue. Preferred aromatic amino acid residues include tyrosine and tryptophan. Alternatively, the peptide can be extended at its N-terminus and / or C-terminus with an amino acid present at the corresponding position in the amino acid sequence of a naturally occurring pyrogenic exotoxin.
[0056]
However, according to the present invention, the peptides of the present invention can be extended at the N-terminus and / or C-terminus with various identical or different organic moieties that are not naturally occurring or are synthetic amino acids. As an example of such extension, the peptide may be extended with an N-acetyl group or a D-alanine group at its N-terminus and / or C-terminus. Peptides extended in this way are also, as other peptides of the present invention, to confer first-aid treatment of any acute toxic shock and any adverse effects caused thereby, and long-term immunity against such toxic shock. Can be used for
[0057]
The peptides of the present invention can inhibit the expression of mRNA induced by pyrogenic toxins encoded by IL-2, IFN-γ or TNF-β genes, as shown in the Examples below.
[0058]
Furthermore, the peptides of the invention can elicit the production of antibodies that block T cell activation in immunized individuals. Antibody production is increased in the presence of a suitable immune adjuvant. Preferred adjuvants include mussel hemocyanin (KLH), proteosome or alum.
[0059]
In a further aspect, the present invention provides a medicament for treating or preventing toxin-mediated activation of T cells, comprising as an active ingredient a therapeutically effective amount of at least the p14 peptide of the present invention or a functional derivative thereof. Relates to the composition. As described above, the peptide comprises an amino acid sequence that is substantially homologous to the amino acid sequence of the protein domain of the pyrogenic exotoxin. The pharmaceutical compositions of the present invention are also useful for protection against toxic shock induced by pyrogenic exotoxins or mixtures of pyrogenic exotoxins.
[0060]
A pharmaceutically "effective amount" for purposes herein is determined by considerations known in the art. This amount must be sufficient to antagonize toxin-mediated T cell activation.
[0061]
Toxin mediated activation as used throughout this application may mean T cell activation mediated by a single pyrogenic exotoxin or a mixture of such toxins.
[0062]
In Examples 1 to 3, a short peptide consisting of 14 amino acids (p14 or p14A, corresponding to positions 148 to 161 of the sequence of the naturally occurring SEB protein shown in SEQ ID NO: 6) is the sequence with the corresponding TSST-1 Even with a relatively low homology of (less than 30%), it has been shown to show markedly effective antagonist activity in mice against toxic shock induced by SEB and TSST-1. In addition, p14A was able to elicit protective immunity against toxic shock even when administered as late as 7 hours after SEB antigen administration. Furthermore, p14A was able to rapidly develop broad spectrum resistance against various toxins in challenged and protected mice.
[0063]
The pharmaceutical composition of the present invention can be prepared in unit dosage form, and may be produced by any method well known in the pharmaceutical field. Furthermore, the pharmaceutical compositions of the present invention may further comprise pharmaceutically acceptable additives such as pharmaceutically acceptable carriers, excipients or stabilizers, and optionally other therapeutic ingredients. Of course, acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed.
[0064]
The therapeutic dose of the composition of the invention will of course depend on the patient group (age, sex, etc.), the nature of the condition to be treated and the route of administration and will be determined by the attending physician.
[0065]
In a still further aspect, the present invention confers immunity against toxic shock induced by pyrogenic exotoxin comprising as an active ingredient an immunologically effective amount of at least one peptide of the present invention or derivative thereof. For vaccines, a mixture of such peptides and derivatives may be included.
[0066]
The term “immunologically effective amount” means any amount sufficient to confer immunity against toxic shock induced by pyrogenic exotoxins or mixtures of pyrogenic exotoxins.
[0067]
The vaccine of the present invention may further comprise a suitable immune adjuvant or a mixture thereof as desired. Suitable adjuvants include proteosome, KLH and alum, and proteosome and alum, and a combination of KLH and alum.
[0068]
As shown in the Examples below, the vaccine of the present invention can elicit protective immunity against pyrogenic exotoxin.
[0069]
Efforts to develop antibodies for use against toxic shock syndrome have been focused on blocking events downstream of the toxic cascade, primarily by inhibiting the action of TNF with monoclonal antibodies or soluble receptors. High levels of cytokines produced in response to toxins negate this approach. The present invention shows that antagonistic action of pyrogenic exotoxin can be blocked with a completely different strategy using antagonists that inhibit toxin action at the top of the toxic cascade before T cell activation occurs.
[0070]
In the examples, pyrogenic exotoxin by expression of the IFN-γ gene (and IL-2 and TNF-β genes not shown here) in PBMC to assess toxin antagonist activity. The molecular methods and analysis of the activation of mediated human cellular immune responses are described in detail. Studies with human PBMC have been combined with animal studies to assess toxin antagonism, immune properties and vaccine efficacy, but whether these methods neutralize human PBMC against other members of the pyrogenic exotoxin system, Or it is shown that it can be applied in devising drugs that protect it.
[0071]
Humans are much more sensitive to pyrogenic exotoxins than mice, while primate models have other limiting factors, such as cost, to analyze the mechanism of activation and suppression of toxin-mediated immune responses A possible human in vitro system is needed. The present invention provides such a system, which has the following main advantages.
[0072]
(1) A freshly prepared human lymphoid cell population that protects the cell-cell interactions involved in the regulation of cytokine production is as close as possible to the body's peripheral immune system.
[0073]
(2) The results of the initial immune response can be accurately and directly analyzed by following the transient and highly regulated expression of IL-2, IFN-γ and TNF-β mRNA.
[0074]
(3) Expression of the IFN-γ (and optionally IL-2 and TNF-β) gene is sensitive to the activity elicited by SEB.
[0075]
(4) This molecular approach is much more direct and specific than measuring biological responses such as cell proliferation or antibody production that are the cumulative result of a series of events.
[0076]
(5) The presented approach provides a means to map functional domains in SEB essential for the activation of human IL-2, IFN-γ and TNF-β genes, and serves to help develop both antagonists and vaccines Can do.
[0077]
Thus, the present invention also relates to a method of treating toxic shock induced by pyrogenic exotoxins or mixtures of pyrogenic exotoxins. The method comprises administering to a patient in need of such treatment a therapeutically effective amount of a pharmaceutical composition of the invention, or a therapeutically effective amount of at least one peptide of the invention or a functional derivative thereof. Become.
[0078]
In a further aspect, there is provided a method of preventing toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins, said method comprising a therapeutically effective amount of a composition of the invention for a patient in need of such treatment. Or administering a therapeutically effective amount of at least one peptide of the invention or a functional derivative thereof.
[0079]
The present invention also relates to a method of immunizing a patient against toxic shock induced by pyrogenic exotoxin, said method comprising immunizing the patient with an effective immunizing amount of the vaccine of the invention, or at least one peptide of the invention. Or administering a functional derivative thereof.
[0080]
The therapeutic dose of the peptide or vaccine of the present invention will of course depend on the patient group (age, sex, etc.), the nature of the condition to be treated and the route of administration and will be determined by the attending physician.
[0081]
The peptides and vaccines of the present invention can be administered and taken according to good medical practice. In particular, the immunization method of the invention comprises a single administration of the peptide or vaccine of the invention. Administration can be accomplished in a variety of ways including intravenous, intramuscular or subcutaneous injection. However, other administration methods such as intranasal administration are possible.
[0082]
The design of pyrogenic exotoxin antagonist peptides described herein not only finds new applications in previously neglected areas, the prevention of pyrogenic exotoxins and the treatment of individuals exposed to toxins, but is also safe. It will also facilitate the development of febrile exotoxin vaccines. The defined peptide vaccine has no exacerbating properties and is considered superior to the toxoid vaccine.
[0083]
In humans, toxic shock from exposure to pyrogenic exotoxins has two distinct components: (1) inactivity and (2) death.
[0084]
Even at concentrations several logs below the lethal concentration, pyrogenic exotoxins can be extremely inactive and show high morbidity [USAMRIID Manual, (1998) Eitzen E, Pavlin J, Cieslak T, Christopher G, Culpepper R, eds Medical Management of Biological Casualties Handbook. 3rd ed. Fort Detrick, Maryland: United States Army Medical Research Institute of Infectious Diseases, 1998]. For example, the inactivity response seen in food poisoning (which can be mass food poisoning) affects many people. Furthermore, the inactivity response is a military threat and a national security threat. For protection against death, the inventors have shown that the peptide antagonists p12A and p14A avoid death caused by toxins in mice. There were no inactive symptoms such as vomiting or diarrhea in mice. Experiments to prevent and / or treat inactivity were conducted in pigs (Example 7).
[0085]
Nausea, ie nausea, vomiting and anorexia, is a series of neurological symptoms that are typically controlled by the midbrain (medulla). The vasomotor / respiratory and reflex centers of swallowing and vomiting are in the medulla. The vomit center of the medulla is the origin of the last common pathway that causes nausea, and the medulla causes vomiting [Hamilton, (1956) Textbook of human anatomy. McMillan & Co Ltd, London]. The vomiting center is actually an intertwined neural network that controls the patterns of motive, respiratory, vascular and salivary processes involved in vomiting. The true vomiting center is stimulated through several neurological pathways and by various types of neurotransmitter receptors in the medulla that respond to dopamine, histamine, acetylcholine, serotonin (5HT), norepinephrine, and glutamine. Antiemetics (primarily dopamine and serotonin antagonists) work by blocking these receptors.
[0086]
The effects of these multiple pathways on the true vomiting center are complex. Within hours of oral exposure to SEB in mice, strong expression of IL-2 and IFN-γ genes is induced in T cells present in the intestinal mucosa [Spiekermann and Nagler-Anderson, (1998) Spiekermann GM, Nagler -Anderson C (1998) J Immunol 161: 5825-5831]. Thus, the resulting activation of mucosal T cells and the response of other cells of the intestinal mucosa to high levels of Th1 cytokines induces the release of neurotransmitters into the brain, helping the essential role of the visceral sensory system. It should be noted that no stomach irritation is required for vomiting. The stomach acts as an effector of the vomiting reflex when it receives signals from the medulla produced in response to various stimuli as described above. Diarrhea occurs when the irritation of the large intestine moves towards the small intestine and eventually reaches the anus.
[0087]
As shown in the examples below (especially in Example 7 and FIGS. 9 and 10), we have found that it is the object of the present invention, but the pyrogenic exotoxin antagonist is incapacitated by the toxin. It was found that symptoms (similar to human response to SE poisoning and early lethal shock) are significantly reduced. Toxins are thought to work by triggering the induction and release of Th1 cytokines and then targeting cellular immune responses that directly or indirectly activate medullary neurons causing inactive symptoms. Thus, the single mode of action of a pyrogenic exotoxin antagonist that blocks T cell activation explains its ability to prevent both death and inactivity. The data in FIGS. 9 and 10 show the certainty of the protective effect of antagonists against inactivity by toxins in a porcine model, an animal close to humans in the immune response to pyrogenic exotoxins.
[0088]
Thus, the present invention further relates to a pharmaceutical composition for the prevention and / or treatment of inactivity induced by at least one pyrogenic exotoxin, said composition comprising a peptide having the amino acid sequence of SEQ ID NO: 1, An isolated and purified peptide selected from a peptide having the amino acid sequence of SEQ ID NO: 2, a peptide having the amino acid sequence of SEQ ID NO: 3, and any mixture thereof, and optionally a pharmaceutically acceptable carrier, diluent, adjuvant and And / or further excipients.
[0089]
The invention further relates to a method for preventing and / or treating inactivity induced by at least one pyrogenic exotoxin, said method comprising a peptide having the amino acid sequence of SEQ ID NO: 1 for a patient in need of such treatment. At least one therapeutically effective amount of an isolated / purified peptide selected from a peptide having the amino acid sequence of SEQ ID NO: 2, a peptide having the amino acid sequence of SEQ ID NO: 3, and any mixture thereof, or Administering a composition comprising. An effective amount of the isolated peptide or a composition comprising it may be repeatedly administered to the patient at predetermined time intervals. When a toxic shock occurs, the patient is usually given a salt infusion, but admixing with an easily water soluble antagonist peptide will allow for clearance during the emergency phase to compensate for clearance. This is preferred and is a very rapid route of administration of antagonists. The route of administration, dose and frequency of treatment will be determined by the attending physician.
[0090]
Example
Materials and methods
Induction of cell culture and human cytokine gene expression
Healthy human donor PBMCs were separated with Ficoll Paque (Pharmacia), washed twice with 50 ml RPMI 1640 medium, 4 × 10 4⁶2% fetal bovine serum, 2 mM glutamine, 10 mM MEM non-specific amino acid, 100 mM sodium pyruvate, 10 mM Hepes pH 7.2, 5 × 10^-5The cells were cultured in the same medium supplemented with M 2-mercapto-ethanol, 100 units / ml penicillin, 100 μg / ml streptomycin and 5 μg / ml nystatin. SEB (Lot 14-30, from the Department of Toxinology, U.S. Army Medical Research Institute of Infectious Diseases), SEA or TSST-1 (Sigma) was added to give 100 ng / ml.
[0091]
RNase protection analysis
Total RNA was extracted with guanidinium isothiocyanate [Chomczynski and Sacchi, Anal Biochem 162: 156 (1987)]. RNase protection analysis was carried out by in vitro α- [³²P] was performed using a genomic antisense RNA probe transcribed with UTP [Arad et al. (1995) supra]. The IL-2 probe (600 nucleotides (nt)) transcribed from the T7 promoter is complementary to the third exon and part of the third intron of the IL-2 gene. In an 8M urea-polyacrylamide gel, IL-2 -2 A 117 nt RNA fragment protected with mRNA is obtained. The IFN-γ probe (274 nt) transcribed from the T3 promoter is complementary to the third exon and part of the third intron of the IFN-γ gene, and an 183 nt RNA fragment protected with IFN-γ mRNA can get. The TNF-β probe (700 nt) transcribed from the T3 promoter is complementary to part of exon 1, part of exon 2, exon 3 and intron 3 and exon 4, and TNF-beta mRNA is 2 of 274 and 263 nt. Protect the fragments. Hybridization did not provide a signal to detect the sense RNA transcript. Antisense RNA probes of 18S rRNA (protecting 90 nt) or β-actin (protecting 415 nt) served as loading controls.
[0092]
Quantitative dot blot hybridization of IL-2 and IFN-γ RNA
PBMCs were collected from 1 ml culture and dissolved in 7.5M guanidinium-HCl. RNA precipitated overnight at −20 ° C. in ethanol was dissolved in formaldehyde and incubated at 60 ° C. for 15 minutes. Two sets of 4 serial 2-fold dilutions with 10 × sodium citrate saline were applied to nitrocellulose sheets using a 96 well dot blot apparatus. After baking in a vacuum oven at 80 ° C., the sheets were each of human IL-2 and IFN-γ³²Each hybridized with a P-labeled antisense RNA probe. The exposed autoradiogram was scanned at 630 nm with an ELISA reader. RNA level A₆₃₀Expressed in units. A serial 2-fold dilution of a given RNA sample gives a linear optical density response that exceeds the 200-fold range of gene expression intensity proportional to the concentration of specific RNA present in each sample [Arad et al. (1995). See the same specification; Gerez et al., Clin Immunol Immunopathol 58: 251 (1991); Kaempfer et al., J Clin Oncol 14: 1778 (1996)].
[0093]
Synthesis of SEB-related peptides
The peptides were generally synthesized as described in WO 98/29444, which is hereby incorporated by reference.
[0094]
p14 was synthesized using fluoronyl-methoxycarbonyl chemistry, cleaved and the side chain deprotected with trifluoroacetic acid. Trifluoroacetic acid-peptide slate was soluble in the medium. D-Ala was bound using the same technique.
[0095]
N-terminal laurylcysteine (LC-) and lysyl-palmitoyl (LP-) and C-terminal cysteine (C-) were added under the same conditions used for other amino acids.
[0096]
The peptide was> 95% pure by HPLC.
[0097]
Immunization
Mice were immunized as described in Example 5.
[0098]
Protection of mice against toxic shock
Groups of 10 female BALB / c mice (Harlan, Jerusalem, Israel), 10-12 weeks old, were immunized by intraperitoneal injection with 20 mg D-galactosamine (Sigma) upon administration of toxin antigen by intraperitoneal injection.
[0099]
Protection of pigs against toxic shock
Five day old mixed blood (primarily Yorkshire; 2.5 kg) was randomized into groups of 6 for each test condition, and each individual in each group was injected intraperitoneally with SEA (25 μg). The antagonist peptide was administered intraperitoneally 30 minutes before SEA and at the times indicated in the figure up to 5 hours after SEA. Vomiting and diarrhea were evaluated every hour for up to 8 hours, then every 3 hours for up to 12 hours, and at 21 and 24 hours.
[0100]
Example 1
Effective protective activity of p14A in lethal challenged mice
In the test of FIG. 1, 90% of control mice died by 20 hours after SEB exposure. However, 90% of these mice survived when p14A was administered just before SEB was lethal challenged (FIG. 1). The protective effect of p14A was reproducible over 15 tests. Surviving animals did not appear to be painful, and their behavior was indistinguishable from normal control animals. They survived only for the 2 weeks they were monitored. Mice exposed to p14A alone were fully viable and had no side effects.
[0101]
This protective effect was not unique to the BALB / c mouse strain. A / J mice were also protected by IPp12A which prevents SEB death [WO 98/29444]. This indicates that protection is not strictly MHC-dependent, thus consistent with the fact that antagonist peptides target superantigen domains that are not known to interact with MHC class II molecules.
[0102]
Example 2
Protect and rescue mice from lethal shock induced by SEB with antagonist peptides
Mice exposed to the antagonist peptide alone were viable and had no side effects. Of the mice challenged with SEB, only 30% were still alive at 24 hours after toxin exposure and later 20%. However, when p14 was given 30 minutes before the toxin, all mice challenged with SEB were protected (p = 0.0003). Surviving animals did not appear to be painful, and their behavior was indistinguishable from normal control animals. They survived only for the 2 weeks they were monitored. Partial protection was obtained when administration of the antagonist peptide was delayed until 3 hours (p = 0.05), 5 hours (p = 0.08) or 7 hours after lethal challenge. Between 20 and 40 hours there was a gradual protective effect of p14A, with 70%, 60% and 50% survival, respectively. It should be noted that p14A not only provides complete protection against infection when given prior to SEB challenge, but can also save mice already in toxic shock. These results are shown in FIG.
[0103]
The peptide is protected or rescued when it is in a 20-40 fold molar excess over SEB, indicating that the peptide is a potent superantigen antagonist in vivo.
[0104]
Example 3
Effective protective activity of p14A in lethal challenged mice
The protective activity of p12A and p14A was investigated using D-galactosamine sensitized mice of an acceptable animal model for superantigenic lethality studies. In FIG. 3, just prior to lethal challenge with SEB, mice treated with p12A survived approximately 70%, while only 40% in untreated (no peptide) animals compared to p14 It can be seen that about 90% of these mice survived after more than 60 hours. The protective effect of p14A was reproducible in 15 tests. Surviving animals did not appear to be painful, and their behavior was indistinguishable from normal control animals. They survived only for the 2 weeks they were monitored. Mice exposed to p14A or p12A alone were fully viable and had no side effects.
[0105]
Example 4
Effective antagonistic activity of p14A against induction of IFN-γ gene expression
The efficacy of p14A and p12A (SEQ ID NO: 5) on the extent of induction of IFN-γ by TSST-1 or SEB in the PBMC population was compared and the results are shown in FIGS.
[0106]
p14A is a derivative of p14 and has D-Ala residues at both the N and C ends. Similarly, p12A is a derivative of p12 and has D-Ala residues at both the N and C ends. TSST-1 showed only 6% homology with the entire SEB sequence, but both p12A and p14A inhibited IFN-γ expression induced by this toxin (TSST-1, FIG. 4). Both p12A and p14A also inhibited expression induced by SEB (FIG. 5). It was surprising that p14A showed much more effective antagonistic activity than either pSTA or TSST-1 or SEB.
[0107]
p12A and p14A have little homology with the relevant domains of TSST-1. Nevertheless, both peptides inhibited IFN-γ expression induced by TSST-1. Also, at this time, p14A has unexpectedly advantageous results with p14A even though only 4/14 amino acid residues are shared with the related domain of TSST-1 compared to 4/12 of p12A. It was.
[0108]
Example 5
Protected mice rapidly develop broad spectrum resistance to lethal shock
In order to investigate whether antagonist peptides show broad protective activity, we show only 27% amino acid sequence homology overall with SEB [Betley and Mekalanos 1988. J. Bacteriol. 170: 34-41 The effects of the superantigen and SEA during the lethal antigen administration were examined. As can be seen from FIG. 6A, p14A also protected mice from lethal challenge with SEA. In WO 98/29444, the antagonist peptide p12A exhibits only 7% and 6% overall sequence homology with SEB, streptococcal toxin SPEA (30% with SEA, 48% homology with SEB) or SEA and SEB respectively. It was shown that mice were protected from death by TSST-1.
[0109]
In the study of FIG. 6, 7 mice that survived a lethal challenge with SEA due to protection with p14A (FIG. 6A) were challenged again with SEB after 2 weeks. At this time, there was no peptide antagonist (FIG. 6B). All of these mice were alive, but none of the 10 control groups survived SEB exposure. Two weeks later, mice that survived this second challenge of SEB were once again exposed to another superantigen toxin, now TSST-1, again in the absence of a peptide antagonist. In the control group challenged with TSST-1, all 10 mice died, while in the treated group only 1 out of 7 mice died (FIG. 6C). These 6 survivors were challenged with SPEA four times after 2 weeks. Of these 6 mice, 4 were alive, but only 1 of 10 mice in the control group survived SPEA exposure. Thus, with p14A, mice protected from SEA-mediated lethal shock rapidly acquired resistance to sequential challenge with different pyrogenic exotoxins.
[0110]
Example 6
Protect mice from lethal shock induced by SEA with antagonist peptides
Mice were injected with p14A 30 minutes prior to SEA challenge. The result is shown in FIG. As can be seen, pretreatment with antagonist peptide provided 100% protection against SEA challenge. In contrast, only 10% of untreated mice survived after 60 hours. Even though p14A shows only 10/14 residues of identity with the relevant domain of SEA, p14A is an advantageous antagonist of SEA.
[0111]
Example 6
Immunization with antagonist peptides protects mice from lethal shock induced by SEB
The present inventors have considered the possibility that the immunogenicity is enhanced by immobilizing the antagonist peptide on the cell membrane. p14A-LP was obtained by synthesizing p14A with an N-terminal lysyl-palmitoyl leader in which lysine acts as a linker and palmitic acid acts as a hydrophobic anchor. FIG. 8 shows immunization of mice with p14A-LP and p14A. Ten out of 11 mice immunized with p14A-LP and 7 out of 10 mice immunized with p14A survived the lethal SEB challenge. In contrast, only 3 of 10 mice were in the control group immunized with adjuvant. When immunized survivors were challenged again after 2 weeks, 7 out of 10 mice immunized with p14A-LP and 4 out of 7 mice immunized with p14A received double doses. Even SEB survived. This dose is the dose that allows 10 untreated mice to die within 10 hours (FIG. 8B). Significant levels of anti-SEB IgM (0.835 units) were observed by enzyme-linked immunosorbent assay (ELISA) of immunized mouse sera collected one week after the first challenge with SEB. The directed IgM was very low (0.08 units). In addition, these sera were preincubated overnight with increasing concentrations of p14A, 10 μg / ml, but their ability to bind SEB was not reduced (0.840 units). Clearly, the antagonist peptide does not complete the SEB-binding antibody. It should be noted that the antagonist peptide induces an anti-SEB-antibody but no antibody against itself has been detected yet. This indicates that the affinity of the antagonist peptide for the antibody it derives is much lower than the affinity of the toxin. These results indicate that the anti-toxin vaccine approach using antagonist peptides is productive, although not limited to the use of hydrophobic anchors.
[0112]
Example 7
Protect pigs from inactivity induced by SEA at p14A
Human T cells are at least two orders of magnitude more susceptible to staphylococcal superantigens than murine ones. While humans are prone to toxic shock syndrome, mice exhibit either the most reactive Vβ chain of the TCR disappeared from the entire range of murine T cells or the associated Vβ gene is deleted. [Arad et al., 2000, see the same specification] and is resistant. As such, mice are naturally resistant to superantigen toxins and should have been sensitized before they received a toxic shock. Furthermore, mice are far from humans in terms of weight and immune system. Therefore, it is important to prove the effects of antagonists even in animal models closer to humans.
[0113]
The inventors have addressed both problems: the inability of toxin activity and the use of more sophisticated animal models. Pigs have a similar immune system to humans; for example, the nucleotide sequence homology of mRNA encoding Th1 cytokines IL-2 and IFN-γ that mediates shock between pigs and humans is 75-85% However, there is no similarity between human and mouse mRNA.
[0114]
Pigs are susceptible to acute superantigen exposure that does not require sensitization, and cause inactivity symptoms similar to those seen in humans. We established a reproducible porcine model of early inactivity symptoms of toxic shock, including nausea, vomiting (neuronal response) and severe diarrhea (intestinal immune response). As can be seen from FIG. 9, the antagonist peptide p14A effectively alleviates the inactivity induced by the toxin.
[0115]
Five-day-old mixed-breed pigs (mainly Yorkshire; 2.5 kg) were randomized into groups of 6 for each test condition, and each individual in each group caused severe inactivity by SEA (25 μg). This was similar in kinetics and strength, given either by the venous or peritoneal route, and was evident in the eyes within 2 hours and sedated in 24-36 hours. The intraperitoneal route is as effective as the venous route, and less trauma is required, so intraperitoneal administration of SEA was used for the inactivity test. This result confirms that pigs are particularly sensitive to SEA [Taylor et al., Infect Immun 36: 1263 (1982)]. The reason for this selective sensitivity is that SEA captures its β and binds to an MHCII molecule in an independent second site and binds it more tightly, resulting in cross-linking between the MHCII molecules. [Hudson et al., J Exp Med 182: 711 (1995); Schad et al., EMBO J 14: 3292 (1995); Abrahmsen et al., EMBO J 14: 2978 (1995)]. The domain targeted by the toxin antagonist is also separate from the MHCII binding site and the SEA TCR binding site. Nonetheless, antagonist p14A protected 7 out of 10 mice from death by SEA, and surviving individuals elicited broad protective immunity (FIG. 6), and in the study of FIG. It appeared in 10 of the mice.
[0116]
The following criteria were used to assess pig inactivity: vomiting (score 4); diarrhea, mild (score 1), moderate (score 2), severe (score 4), and water diarrhea (score 6) (data None). Long-term anorexia and the use of water intakes instead of sows nipples, indifference and staggering are clearly observed, but these are not readily quantifiable; body temperature disparities remain below 1 ° C on average with normal control animals.
[0117]
We have seen that p14A is more effective than p12A in terms of its ability to inhibit the action of superantigens in vitro in human PBMC (FIGS. 4 and 5) and mice protected from lethal toxic shock (FIG. 3). It showed that there is. The p14A antagonist peptide sedated swine SEA inactivity symptoms (vomiting and diarrhea).
[0118]
As can be seen from FIG. 9, increasing doses of p14A visibly alleviated general inactivity and vomiting and diarrhea. These results are quite significant with respect to general inactivity (p = 0.002) and diarrhea (p <0.001) that occur after the last administration of the 6 antagonist administrations performed and are widely maintained , Post hoc test was significant. Vomiting also relieved according to the antagonist dose.
No side effects were seen with the antagonist peptide alone: none of the inactive symptoms were observed in 18 pigs in the mixed group receiving p14A alone (FIG. 9).
We have found effective suppression of diarrhea that develops with antagonist peptide p14A, which is prolonged in control animals, usually over 5 hours (last dose of antagonist peptide in the test group) and up to 24 hours; This shows the long-term effect of the antagonist peptide.
[0119]
The p14A antagonist peptide p14A suppressed SEA inactivity symptoms (vomiting and diarrhea) in pigs, and the effect increased with the number of administrations of the antagonist (FIG. 10). This suppression is statistically significant. The pig group that received four doses of p14A antagonist peptide after SEA exposure showed less inactivity than the group that received only two doses of antagonist peptide after SEA exposure. Both later groups also received a dose of antagonist peptide 30 minutes prior to SEA exposure, but the more effective post-toxin treatment, the more effective inhibition of inactivity. These results indicate a potential utility of antagonist peptides in the treatment of inactivity symptoms, such as found in humans who are in toxic shock and severe food poisoning.
[0120]
Thus, the antagonist peptide p14A is a superantigen toxin without detecting any toxin agonist activity in vivo, not only in mice, but also in pigs where the immune system is more closely related to that of humans. It is possible to stop the toxic shock symptoms of SEA.
[0121]
These examples further describe and illustrate specific examples within the scope of the present invention. These examples are not intended to limit the invention, but are intended to be exemplary only, and many variations thereof are possible without departing from the spirit and scope of the invention.
[0122]
All references cited herein are hereby incorporated by reference.
[0123]
[Sequence Listing]

[Brief description of the drawings]
The invention is explained in more detail in the following figures.
FIG. 1 is an illustration showing that antagonist peptides protect mice from lethal shock induced by SEB.
Ten Balb / c mice were sensitized with 20 mg D-galactosamine and simultaneously challenged with 10 μg SEB (◯). The other 10 mice received 25 μg of p14A 30 minutes before SEB antigen administration (●). Surviving individuals were monitored.
FIG. 2 is an explanatory diagram showing that an antagonist peptide protects and rescues mice from lethal shock induced by SEB.
Groups of 10 BALB / c mice were sensitized with 20 mg D-galactosamine and simultaneously challenged with 10 μg SEB. Injections were made at the same time. One aliquot of 25 μg of p14A was injected 30 minutes before SEB antigen administration (▲) or 3 (black squares), 5 (◯) or 7 (●) hours after SEB administration. Ten sensitized untreated mice that did not receive p14A served as SEB controls (□). All injections were made intraperitoneally.
FIG. 3 is an illustration showing protection of mice from lethal shock induced by SEB by antagonist peptides p12A and p14A.
A group of 10 BALB / c mice was challenged with SEB (Sigma) by intraperitoneal injection of 5 μg toxin with 20 mg D-galactosamine. As indicated, 25 μg of either p12A or p14A was injected intraperitoneally 30 minutes prior to SEB administration. Surviving individuals were monitored.
FIG. 4 is an explanatory diagram showing the efficacy of antagonist peptides p12A and p14A for induction of IFN-γ gene expression by TSST-1.
Healthy human donor PBMCs were separated with Ficoll Paque (Pharmacia), washed twice with 50 ml RPMI 1640 medium, 4 × 10 4⁶2% fetal calf serum, 2 mM glutamine, 10 mM MEM non-specific amino acid, 100 mM sodium pyruvate, 10 mM Hepes, pH 7.2, 5 × 10^-5The cells were cultured in the same medium supplemented with M 2-ME, 100 units / ml penicillin, 100 μg / ml streptomycin and 5 μg / ml nystatin. After standing overnight, 4 x 10 in 1 ml⁶An aliquot of PBMC was cultured as indicated in the presence of 100 ng / ml TSST-1 (Sigma) alone or in the presence of the indicated 10 μg / ml p12A or p14A. Extract total RNA as indicated and serially double dilutions³²Blot hybridization analysis using P-labeled IFN-γ antisense RNA probe [Arad et al., (2000) same as above] was performed. The autoradiogram is quantified with a densitometer at 630 nm and the absorbance at 630 nm is plotted.
FIG. 5 is an explanatory diagram showing the efficacy of antagonist peptides p12A and p14A for induction of IFN-γ gene expression by SEB.
4 x 10 in 1 ml⁶PBMC aliquots in the presence of 100 ng / ml SEB (Lot 14-30, from the Department of Toxinology, United States Army Medical Research Institute of Infectious Diseases (USAMRIID, Frederick, Maryland)) alone or indicated 10 μg / ml In the presence of p12A or p14A. Extract total RNA as indicated and serially double dilutions³²Blot hybridization analysis using P-labeled IFN-γ antisense RNA probe was performed. The autoradiogram is quantified with a densitometer at 630 nm and the absorbance at 630 nm is plotted. The same PBMC population as in FIG. 4 was used.
FIG. 6 is an explanatory diagram showing that global immunity against lethal shock appears rapidly in mice protected with an antagonist.
(A) Ten BALB / c mice were given p14A 30 minutes before sensitization with 20 mg D-galactosamine and simultaneous antigen administration with 10 μg SEA (●). Furthermore, without injection of p14A, 7 survivors were given 2 weeks later, 10 μg SEB (▲; B), 2 weeks later 10 μg TSST-1 (black squares; C), and 2 weeks later, 15 μg SPEA (▲; D). ) Was challenged again. For each antigen administration, 10 sensitized untreated mice were used as toxin controls (◯, Δ, □). All injections were made intraperitoneally.
FIG. 7 is an illustration showing that antagonist peptides protect mice from lethal shock induced by SEA.
A group of 10 BALB / c mice was challenged intraperitoneally with 10 μg SEA and 25 μg p14A was injected intraperitoneally 30 minutes before SEA administration.
FIG. 8 is an illustration showing that immunization with an antagonist peptide protects mice from lethal shock induced by SEB.
A: A group of 10 BALB / c mice (11 mice for p14A-LP) were injected three times (0, 2 and 4 weeks) with 10 μg of the indicated peptide in Freund's adjuvant (FA). Immunized and challenged 6 weeks later by intraperitoneal injection of 10 μg SEB and 20 mg D-galactosamine. Surviving individuals were monitored. B: Two weeks after the first challenge, survivors of A (p14A-LP, n = 10; p14A, n = 7) were re-challenge with 20 μg SEB and 20 mg D-galactosamine. LP, lysyl-palmitoyl. Mice immunized with FA and p14A-LP (▲), FA and p14A (□) or FA alone (black squares); untreated control mice challenged with SEB (○).
FIG. 9 is an illustration showing protection of pigs from buoyancy that occurs in a dose-dependent manner by SEA with a pyrogenic exotoxin antagonist.
The pig group (total group size, n) was challenged intraperitoneally with 25 μg / kg SEA at time zero. As indicated, p14A (25, 50 or 100 μg / kg) was administered intraperitoneally ˜0.5 hours and 1, 2, 3, 4 and 5 hours after SEA administration. Each pig was observed for 11 time points over 24 hours. The area under the curve at these time points is plotted for general inactivity, vomiting and diarrhea. The overall inactivity score is the sum of the vomiting and diarrhea scores. As a result of repeated measurement analysis between groups, a p-value is obtained. For statistical analysis, ANOVA of the data set was performed by Tukey HSD post hoc test using SPSS of Windows 9.0 software (note that one asterisk is attached to p ≦ 0.05). The p14A control without toxin administration is shown on the right.
FIG. 10 is an illustration showing the protection of pigs from SEA-induced inactivity due to repeated administration of antagonist peptides.
A group of 6 piglets was challenged intraperitoneally with 25 μg / kg SEA. p14A (50 μg / kg) was applied by IP at 0, 1.5 and 2.5 hours (× 3) or at 0, 1.5, 2.5, 3.5 and 4.5 hours (× 5) . Inactivity scores for vomiting and diarrhea are shown, respectively. Saline control animals received neither SEA nor p14A. The area under the curve is plotted for 10 time points over 24 hours for assessed general inactivity, vomiting and diarrhea. For statistical analysis, ANOVA of the data set was performed by Tukey HSD post hoc test using SPSS of Windows 9.0 software (note that one asterisk is attached to p ≦ 0.05).

Claims (10)

An isolated and purified peptide consisting of the amino acid sequence of SEQ ID NO: 1. An isolated and purified peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 3.   Can elicit protective immunity against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins and / or inhibit activation of T lymphocytes mediated by pyrogenic exotoxin; 3. A functional derivative of the peptide of claim 1 that is capable of protecting against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins, the peptide of claim 2. A composition that inhibits T lymphocyte activation mediated by pyrogenic exotoxin and that protects against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins, comprising the amino acid of SEQ ID NO: 2 peptide consisting of the sequence, a peptide comprising the amino acid sequence of SEQ ID NO: 3, and isolation chosen from any mixture thereof, and the purified peptide, a pharmaceutically acceptable carrier optionally diluents, adjuvants and / or excipients A composition further comprising an agent. An immunogenic composition to elicit protective immunity against toxic shock induced by a pyrogenic exotoxin, a peptide comprising the amino acid sequence of SEQ ID NO: 2, a peptide comprising the amino acid sequence of SEQ ID NO: 3, and their An immunogenic composition further comprising an isolated and purified peptide selected from any mixture and optionally a pharmaceutically acceptable carrier, diluent, adjuvant and / or excipient. A pharmaceutical composition for the incapacitating therapy induced by at least one pyrogenic exotoxin, a peptide comprising the amino acid sequence of SEQ ID NO: 2, a peptide comprising the amino acid sequence of SEQ ID NO: 3, and combinations of any A pharmaceutical composition further comprising an isolated, purified peptide selected from a mixture of: and optionally a pharmaceutically acceptable carrier, diluent, adjuvant and / or excipient. SEQ ID NO: for the manufacture of a medicament for inhibiting the activation of T lymphocytes mediated by pyrogenic exotoxin and protecting against toxic shock induced by pyrogenic exotoxin or a mixture of pyrogenic exotoxins peptide consisting of the amino acid sequence, a peptide comprising the amino acid sequence of SEQ ID NO: 3, and use of an isolated, purified peptides or the composition comprising the same, which is selected from any mixture thereof. For the manufacture of a medicament for raising an immune protection against toxic shock induced by a pyrogenic exotoxin, a peptide comprising the amino acid sequence of SEQ ID NO: 2, a peptide comprising the amino acid sequence of SEQ ID NO: 3, and their Use of an isolated, purified peptide selected from any mixture, or an immunogenic composition comprising it. For preventing and / or treating cripple induced by at least one pyrogenic exotoxin for the manufacture of a medicament, a peptide comprising the amino acid sequence of SEQ ID NO: 2, a peptide comprising the amino acid sequence of SEQ ID NO: 3, And the use of an isolated, purified peptide selected from any mixture thereof, or a composition comprising it.   The use according to claim 9, wherein the medicament is for repeatedly administering an effective amount of the isolated / purified peptide or a composition comprising the same to a patient at a predetermined interval time.

Images