JP2004522403A - Nucleic acid vaccine compositions having a mammalian CD80 / CD86 gene promoter driving antigen expression - Google Patents

Abstract

Provided are polynucleotides encoding at least one immunizing antigen whose expression is controlled by a promoter from the gene encoding the costimulatory molecule. The polynucleotide may also encode an adjuvant. Also provided is a composition comprising at least one immunizing agent and at least one cytokine, which enhances the stimulation and / or survival of dendritic cells. Methods for raising an immune response to an immunizing agent are also provided. The method includes the steps of administering a polynucleotide and, if necessary, co-administering an adjuvant.

Description

ｐＷＧＲ７１２８を、緩衝液＃３中でＳａｌ ＩおよびＳａｃ ＩＩを用いて消化した。この消化したベクターを１％のアガロースゲルにかけて、正確なベクターバンドをゲルから切除し、ＧｅｎＥｌｕｔｅ^Ｔｍエチジウムブロミドスピンカラムを使用して精製した。
【０１３１】
調製したベクター中にプロモーターをライゲーションし、２０ｎｇのベクターおよび１００ｎｇのインサートプロモーターを、６ワイスユニットＴ４ ＤＮＡリガンド（Ｎｅｗ Ｅｎｇｌａｎｄ Ｂｉｏｌａｂｓ）によって、１×Ｔ４ ＤＮＡリガーゼ緩衝液（５０ｍＭ Ｔｒｉｓ−Ｃｌ、１０ｍＭ ＭｇＣｌ_２、１０ｍＭ ＤＴＴ、１ｍＭ ＡＴＰ、２５μｇ／ｍｌ ＢＳＡ、ｐＨ ７．８）中で１５℃で一晩かけてライゲーションさせた。
【０１３２】
適切にライゲーションさせた構築物の断片に関して、このライゲーション混合物を、生理食塩水中で１：３に希釈した。５μｌの希釈混合物を、５０μｌのＭＡＸ Ｅｆｆｉｃｉｅｎｃｙ ＤＨ５α Ｃｏｍｐｅｔｅｎｔ Ｃｅｌｌｓ^ＴＭ（Ｇｉｂｃｏ−ＢＲＬ）に添加し、そして氷上で３０分間インキュベートした。形質転換した混合物を４２℃で４５分間かけて熱ショックをかけて、そして２分間のインキュベーションに迅速に戻した。１ｍｌのＳＯＣ培地をこの形質転換した細胞に添加し、そして３７℃で１時間振盪した。インキュベーション後に、この細胞を、手短に遠心分離し、そしてカナマイシン−ＬＢプレート上にプレート化した。このプレートを、３７℃で一晩かけてインキュベートした。
【０１３３】
首尾良くライゲーションを確認するために、単一のクローンを取り出し、そして３．０ｍｌのＬＢ／Ｋａｎ培地中にインキュベートし、そして３７℃で一晩攪拌しながら培養した。プラスミドを培地から単離し、Ｓａｌ ＩおよびＳａｃ ＩＩを用いて消化し、エチジウムブロミドで染色した１％アガロースゲル上で視覚化させた。
【０１３４】
（Ｂ．抗原の発現）
２ｍｌのＢ１６細胞を、２×１０^５細胞／ｍｌの６ウェルプレートに播種した。この細胞を、５％のＣＯ_２を用いて３７℃インキュベーションで一晩かけて培養し、６０〜８０％のコンフルエンシーに達した。２μｇの血清培地（Ｇｉｂｃｏ−ＢＲＬ）を減らしたエンドトキシンフリープラスミドＤＮＡ（１００μｌ Ｏｐｔｉ−ＭＥＭ（登録商標）１中）および１０μｌの血清培地を減らしたリポフェクチン（９０μｌ Ｏｐｔｉ−ＭＥＭ（登録商標）１中）を、室温で４５分間別々にインキュベートした。次いで、２つの溶液を混合し、そして１５分間室温でインキュベートした。このインキュベートの間に、Ｂ１６細胞を、血清フリー培地中で３回洗浄した。各トランスフェクションに関して、０．８ｍｌの血清培地を減らしたＯｐｔｉ−ＭＥＭ（登録商標）１を、混合溶液に添加し、そしてこの複合体をＢ１６細胞上に被せた。次いで、この細胞を３７℃で５時間インキュベートした。続いて、１．０ｍｌの２０％ ＦＢＳ培地を添加した。この培地を、抗原発現分析のために４８時間後に収集した。
【０１３５】
トランスフェクトしたＢ１６細胞中のＨＢｓＡｇの発現を、表２に示した。この細胞を、以下の３つの手段の一つで処理した：（１）Ｂ１６細胞中で抗原（すなわち、ＨＢｓＡｇ）を発現させたポジティブコントロール（すなわち、ｐＷＲＧ７１２８）を用いたトランスフェクション；（２）上記のようなプラスミドｐ５０２０およびｐ５０２１を用いたトランスフェクションおよび（３）トランスフェクトしない（ネガティブコントロール）。
【０１３６】
（表２．ウイルスを用いて細胞をトランスフェクトすることによるＨＢｓＡｇのインビトロ発現）
（プラスミド）
【０１３７】
【表２】

＊データは、２つの別個の培地から得られる上清に関して平均ＯＤ値（４９２．６波長）として示され、ＨＢＶ表面抗原キット（Ａｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ Ｄｉａｇｎｏｓｔｉｃ Ｄｉｖｉｓｉｏｎ （Ａｕｓｚｙｍｅ Ｍｏｎｏｃｌｏｎａｌ， Ｌｉｓｔ Ｎｏ．１９８０−２４）を使用して分析した。
【０１３８】
表２に示されるように、陽性コントロールプラスミドｐＷＲＧ７１２８と異なり、ＨＢＶ表面抗原の発現は、プラスミドｐ５０２０およびｐ５０２１でトランスフェクトされた培養Ｂ１６細胞中で検出されなかった。従って、ＣＤ８０プロモーターは、非抗原提示細胞中で抗原の発現を駆動しない。
【０１３９】
（Ｃ．コートされた微小粒子の調製）
プラスミドＤＮＡを、１〜３μｍ金粒子（Ｄｅｇｕｓｓａ Ｃｏｒｐ．，Ｓｏｕｔｈ Ｐｌａｉｎｆｉｅｌｄ，ＮＪ）上に、Ｅｉｓｅｎｂｒａｕｎら（１９９３）ＤＮＡ Ｃｅｌｌ Ｂｉｏｌ．１２：７９１−７９７によって記載される技術を用いてコートした。簡単に言うと、金粒子を、５０ｍＭ スペルミジン中に懸濁し、そして水中の等量のプラスミドと混合する。この溶液を、ボルテックス上で混合し、そしてこの金／ＤＮＡ混合物の半分の量の１Ｍ ＣａＣｌ_２を滴下した。この混合物を、室温で１０分間インキュベートし、そして遠心分離してこの粒子をペレットにした。この粒子を、１００％エタノールで３回洗浄し、そして０．０５〜０．５％のポリビニルピロリドン（ＰＶＰ）を含むエタノール中に再懸濁した。次いで、ＤＮＡコートされた金粒子を、ＭｃＣａｂｅに対する米国特許第５，５８４，８０７に記載されるようにＴｅｆｚｅｌ（登録商標）チューブにロードし、このチューブを１．２７ｃｍ長に切断してＰｏｗｄｅｒＪｅｃｔ（登録商標）ＸＲ−１粒子送達装置（ＰｏｗｄｅｒＪｅｃｔ Ｖａｃｃｉｎｅｓ，Ｉｎｃ．Ｍａｄｉｓｏｎ，ＷＩ）中のカートリッジとして提供する。ヘリウムパルスＸＲ−１粒子送達装置は、以前に記載されている（例えば、米国特許第５，５８４，８０７および５，８６５，７９６を参照のこと）。ワクチン接種において、各１．２７ｃｍのカートリッジは、２μｇのプラスミドＤＮＡでコートされた０．５ｍｇの金粒子を含んだ。
【０１４０】
（Ｄ．抗体応答）
上記のインビトロトランスフェクト研究で見出された陽性の結果に基づいて、ワクチン接種試験を、インビボ粒子媒介送達方法を用いて開始した。本研究において核酸免疫を受ける動物被験体は：（１）ｐＷＧＲ７１２８ポジティブコントロールを用いた表皮への粒子媒介送達によってワクチン接種された４匹のマウスの、第１の実験群；および（２）ＣＤ８０プロモーター駆動ＨＢｓＡｇプラスミドｐ５０２０およびｐ５０２１を用いた表皮への粒子媒介送達によってワクチン接種された６匹のマウスの、第２の実験群を含んだ。血液サンプルを、免疫の前に各動物から採取した（ナイーブマウス）。
【０１４１】
マウスを、プラスミドｐ５０２０およびｐ５０２１またはコントロールｐＷＧＲ７１２８で、金粒子上にプラスミドコートされた粒子媒介送達（２μｇＤＮＡ／ｍｇ金でコートされた０．５ｍｇの金／ショット）によって免疫（プライム）した。免疫に関して、マウスを剃り、この粒子を５００ｐｓｉのヘリウムで操作された研究バレルでＸＲ−１装置を用いて腹部の皮膚に送達した。２ショットを、１免疫につき各マウスに与えた。４週間後、プライム免疫に使用した同じ免疫プロトコールに従って、このマウスをブーストした。
【０１４２】
血清を、免疫前、プライム後２週間目および４週間目、ならびにブースト後２週間目にこの動物から収集した。血清抗体レベルを、ＡＵＳＡＢ ＥＩＡキット（Ａｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ Ｄｉａｇｎｏｓｔｉｃｓ Ｄｉｖｉｓｉｏｎ）を用いて、製造者によって供給された指示書に従って分析した。Ｂ型肝炎ウイルス（ＨＢＶ）表面抗原に対する抗体反応性は、プラスミドｐ５０２０およびｐ５０２１で免疫した動物から採取された血清サンプル中で検出されなかったが；ブースト後２週間目に、血清抗体力価は、ｐＷＧＲ７１２８で免疫したマウスにおいて＞３０００ｍｌＵ／ｍｌであった（表３）。
【０１４３】
（表３．ＣＤ８０プラスミドで免疫したマウス中のＨＢｓＡｇ特異的血清抗体力価）
【０１４４】
【表３】

^＊データを、示されたマウス群に対する平均ＨＢＶ表面抗原抗体力価（ｍｌＵ／ｍｌ）として示す。血清抗体力価を、Ａｂｂｏｔｔ Ｌａｂｏｒａｔｏｒｉｅｓ Ｄｉａｇｎｏｓｔｉｃｓ Ｄｉｖｉｓｉｏｎ（ＡＵＳＡＢ ＥＩＡ、リスト９００６−２４）からのアッセイキットを用いて決定した。
【０１４５】
従って、ＣＤ８０プロモーター駆動プラスミドは、血清中の平均抗体力価に上昇を引起さない。
【０１４６】
（Ｅ．細胞媒介免疫応答）
上記の血清抗体力価の分析に見出された陽性の結果に基づいて、免疫マウス中の細胞障害性Ｔ細胞活性の研究を実施した。細胞障害性Ｔ細胞（ＣＴＬ）活性を、ブースト後２週間目に屠殺した免疫マウスから得た脾細胞を用いて分析した。図８に示されるように、プラスミドｐ５０２０およびｐ５０２１での免疫によって誘発されたＣＴＬ応答は、プラスミドｐＷＧＲ７１２８によって誘発された応答よ類似していた。これらの応答は、ナイーブマウスから収集された脾細胞に見出されたものよりもはるかに大きかった。
【０１４７】
上記研究の結果として、抗原発現が同時刺激分子から誘導されたプロモーターによって駆動される場合に、核酸免疫はＴ細胞特異的免疫応答を提供することが、見出され得る。さらに、Ｔ細胞応答は、ポジティブコントロールを用いて見出されたものに匹敵する。
【０１４８】
（実施例２：ＳＩＶ免疫応答）
ヒトＣＤ８０プロモーター駆動ＨＢｓＡｇ（ｈＣＤ８０−ＨＢｓＡｇ）をコードする発現ベクターがサル樹状細胞中で発現されるか否かを決定するために、以下の研究を実施する。サル樹状細胞（ＤＣ）を、本質的に、ｖａｎ ｄｅｒ Ｍｅｉｄｅら（１９９５）Ｊ．Ｍｅｄ．Ｐｒｉｍａｔｏｌ．２４：２７１−２８１に記載されるように、ＰＢＭＣから単離する。次いで、単離したＤＣを、本質的にＢ１６細胞に関して実施例１に記載されるように、ヒトＣＤ８０プロモーター駆動ＨＢｓＡｇをコードするプラスミドでトランスフェクトする。非ＡＰＣ株（例えば、サルＣＯＳ細胞）を、同様にトランスフェクトする。上清および／または細胞中のＨＢｓＡｇの発現を、免疫組織化学染色によって実施する。
【０１４９】
細胞特異的発現を考慮して、ＡＰＣ中でのＨＢｓＡｇ発現の程度を決定するための研究を実施する。プラスミドｈＣＤ８０−ＨＢｓＡｇを、ＰｏｗｄｅｒＪｅｃｔ（登録商標）ＸＲ粒子送達装置を用いてサルの皮膚の表皮に送達する。種々のさらなる表皮部位をまた、研究する。金をネガティブコントロールとして使用し、一方、ＣＭＶ−ＨＢｓＡｇをポジティブコントロールとして使用する。ＧＭ−ＣＳＦを、樹状細胞リクルートメントを試験するために投与する。生検を、２４または４８時間後に、投与部位において実施する。組織を断片化し、そしてＨＢｓＡｇの特異的抗体および樹状細胞表面マーカーを用いた免疫組織化学染色によって、樹状細胞中のＨＢｓＡｇ発現を評価する。樹状細胞およびランゲルハンス細胞におけるＨＢｓＡｇ発現を評価する。
【０１５０】
ｈＣＤ８０−ＳＩＶ発現ベクターを、例えば、ＨＢｓＡｇをコードする配列を適切なＳＩＶコード配列で置換することによって構築する。サルを、ｈＣＤ８０−ＳＩＶ構築物で免疫し、そしてＣＭＶ−ＳＩＶプラスミド（ポジティブコントロール）で免疫したサルと比較する。プラスミドの送達を、粒子送達装置を用いて実施する。抗原発現、抗体応答、およびＣＴＬ活性化を、本質的に上記の実施例１に記載のように評価する。次いで、このサルを、病原性ＳＩＶでチャレンジし、ＳＩＶの臨床的な発現に関してモニターする。
【０１５１】
（実施例３：サイトカインアジュバントの使用）
ＴＮＦ関連活性化誘導キナーゼ（ＴＲＡＮＣＥ）をコードする核酸が同時投与された抗原配列に対する免疫応答を増強する能力を評価するために、以下の研究を実行した。
【０１５２】
（Ａ．プラスミド調製物）
マウスＴＲＡＮＣＥのｃＤＮＡコード配列を、ｍＲＮＡ配列（ＧｅｎＢａｎｋ番号ＡＦ０１３１７０）から誘導し、そしてｐＦＬＡＧ−ＣＭＶ２発現ベクター（Ｓｉｇｍａ、カタログ番号Ｅ４０２６）の挿入部位にクローン化し、ＣＭＶ２プロモーターの転写制御下でＴＲＡＮＣＥコード配列を含む発現構築物を得た。このプラスミド構築物を、ｐＴＲＡＮＣＥと命名した。
【０１５３】
Ｂ型肝炎コア抗原（ＨＢｃＡｇ）およびＢ型肝炎表面抗原（ＨＢｓＡｇ）をコードする配列を含むプラスミドを、以下のように構築した。ＨＢｃＡｇおよびＢＨＢｓＡｇをコードする配列の両方を、ＨＢＶクローンｐＡＭ６（ＡＴＣＣ登録番号４５０２０）から得た。ＨＢｓＡｇコード領域を作製するために、ｐＡＭ６構築物をＮｃｏＩで切断し、そして緑豆ヌクレアーゼを用いて処理し、Ｘ抗原の開始コドンを取り除いた。次いで、生じるＤＮＡを、ＢａｍＨＩで切断し、Ｔ４ ＤＮＡポリメラーゼで処理してこのＤＮＡを平滑末端にし、ＨＢｓＡｇ発現カセットを作製する。ＨＢｓＡｇ発現カセットは、１．２ｋＢフラグメントで存在する。全長ヒトＣＭＶ（Ｔｏｗｎｅ種）最初期プロモーター（エンハンサーと共に）を含むプラスミド構築物ｐＰＪＶ７０７７（Ｓｃｈｍａｌｊｏｈｎら（１９９７）Ｊ．Ｖｉｒｏｌ．７１：９５６３−９５６９）を、ＨｉｎｄＩＩＩおよびＢｇｌＩＩで切断し、次いで、Ｔ４ ＤＮＡポリメラーゼおよびウシアルカリホスファターゼで処理して平滑末端ＤＮＡを作製し、そしてＨＢｓＡｇ発現カセットを、プラスミドに連結してｐＷＲＧ７１２８構築物を得た。
【０１５４】
ＨＢｃＡｇコード領域を作製するために、ｐＡＭ６構築物を切断してＨＢｃＡｇ発現カセットを作製し、この後、ＨＢｃＡｇ配列を部位指向型変異誘発によって短縮化し、Ｃ末端アルギニンリッチ領域をコア抗原粒子から取り除いた（この欠失は、粒子形成を妨害しない）。次いで、この短縮ＨＢｃＡｇ配列を、ヒト伸長因子プロモーター（「ｈＥＬＦ」、Ｍｉｚｕｓｈｉｍａら（１９９０）Ｎｕｃｌ．Ａｃｉｄｓ Ｒｅｓ．１８：５３２２）を含むプラスミド構築物にクローン化してＨＢｃＡｇベクター構築物を得た。
【０１５５】
（ａ）ＣＭＶプロモーター／エンハンサー、イントロンＡ−５’非翻訳領域、およびヒト組織プラスミノーゲンアクチベーター（ｈＴＰＡ）シグナルペプチド（「ＣＭＶ−ＩＡ−ＴＰＡ」）；または（ｂ）ウシ成長ホルモンポリＡ配列（ｂＧＨｐＡ）を含む発現カセットを、各々、ＪＷ４３０３ベクター構築物（マサチューセッツ大学のＨａｒｒｉｅｔ Ｒｏｂｉｎｓｏｎ博士の供与）から得て、プラスミド骨格に挿入した。生じる構築物を、ＮｈｅＩで切断し、ポリメラーゼで充填し、次いで、ＢａｍＨＩで切断して、ｐＵＣ１９複製起点、アンピシリン耐性遺伝子およびｂＧＨｐＡ配列を含むベクターフラグメントを作製した。このプラスミド骨格を、２度目はＳａｌＩで切断し、ポリメラーゼで充填し、ＢａｍＨＩで切断して、ＣＭＶ−ＩＡ−ＴＰＡベクターフラグメントを含むベクターフラグメントを遊離させた。この２つのベクターフラグメントを一緒に連結して、ｐＷＲＧ７０５４と命名する構築物を得た。
【０１５６】
ｐＷＲＧ７０５４構築物を、ＮｈｅＩで切断し、ポリメラーゼで充填し、ＢａｍＨＩで切断して、ベクターフラグメントを作製した。ＨＢｃＡｇベクター構築物を、ＮｃｏＩで切断し、ポリメラーゼで充填し、ＢａｍＨＩで切断して、挿入フラグメントを作製した。次いで、この２つのフラグメントを一緒に連結して、ｐＷＲＧ７０６３と命名する構築物を得た。
【０１５７】
ＰＥＬ−ＢｏｓをＥｃｏＲＩで切断し、ウシ腸ホスファターゼで脱リン酸化して、ベクターフラグメントを作製した。ｐＷＲＧ７０６３プラスミドをＨｉｎｄＩＩＩで切断し、ポリメラーゼで充填し、ＥｃｏＲＩで切断して、ｈＴＰＡシグナルペプチド、ＨＢｃＡｇ抗原配列およびｂＧＨｐＡ領域を含む挿入フラグメントを作製した。これらの２つのフラグメントを一緒に連結して、ｐＷＲＧ７１４５と命名する構築物を得た。
【０１５８】
ｐＷＲＧ７１２８構築物を、ＥｃｏＲＩで切断し、ウシ腸ホスファターゼで脱リン酸化して、ｈＣＭＶプロモーターの転写制御下でＨＢｓＡｇコード領域を含むベクターフラグメントを作製した。このｐＷＲＧ７１４５構築物を、ＭｆｅＩおよびＥｃｏＲＩで切断して、ｈＥＬＦプロモーター／イントロン、ｈＴＰＡシグナルペプチド配列、ＨＢｃＡ抗原配列およびｂＧＨｐＡ領域を含む挿入フラグメントを作製した。次いで、これらの２つのフラグメントを一緒に連結して、ＨＢｃＡｇおよびＨＢｓＡｇコード配列を含むｐＰＪＶ７１９３プラスミド構築物を得た。
【０１５９】
（Ｂ．ワクチン調製および免疫）
ＤＮＡワクチン組成物のパネルを、以下のＤＮＡプラスミドの種々の組合せを用いて整理した：ｐＰＪＶ７１９３構築物（Ｂ型肝炎表面抗原およびＢ型肝炎コア抗原をコードする）；ｐＰＪＶ７０４６構築物（空のＤＮＡプラスミド）；およびｐＴＲＡＮＣＥ構築物（ＴＲＡＮＣＥサイトカインをコードする）。各ワクチン組成物中のＤＮＡの終濃度は、２．０μｇ／ｍｇ金であり、そして、抗原構築物（ｐＰＪＶ７１９３）の濃度を、全組成物中で一定に保ち、一方で、ＴＲＡＮＣＥ構築物の濃度を変化させた。ＤＮＡワクチン組成物のパネル中に存在する各構築物の実際の濃度を、以下の表４に報告する。
【０１６０】
（表４．ＴＲＡＮＣＥ ＤＮＡワクチン組成物）
【０１６１】
【表４】

プラスミドＤＮＡ（ｐＰＪＶ７１９３、ｐＰＪＶ７０４６およびｐＴＲＡＮＣＥ）を、上記表４に報告される比率であわせ、次いで、このプラスミド混合物を、上記実施例１に記載される技術を用いて１〜３μｍ金粒子上にコートして、２μｇ ＤＮＡ／ｍｇ金の終濃度を得た。ＤＮＡコートされた金粒子を、次いで、上記実施例１のようにＴｅｆｚｅｌ（登録商標）チューブにロードし、ＰｏｗｄｅｒＪｅｃｔ（登録商標）ＸＲ粒子送達装置のカートリッジとして提供するされる長さに切断する。各カートリッジは、０．５ｍｇの金粒子を含んだ。
【０１６２】
４匹のＢａｌｂ／ｃマウスの６実験群を各々集合させ、そして上記表４に列挙されるＤＮＡワクチン組成物を用いて、金粒子上にコートされたプラスミドＤＮＡの粒子媒介送達によって免疫（プライム）した。免疫のために、マウスを剃り、この粒子を５００ｐｓｉのヘリウムで操作された粒子送達装置を用いて単一ショットで腹部の皮膚に送達した。これらのマウスを、免疫２週間後に屠殺し、そして脾臓をＥＬＩＳＰＯＴ分析のために取り除いた。
【０１６３】
（Ｃ．ＥＬＩＳＰＯＴ分析）
ＥＬＩＳＰＯＴフィルタープレートを、ラット抗マウスＩＬ−４抗体を用いて０．１Ｍ カーボネート緩衝液中０．７５μｇ／ウェルの濃度でコートした。このプレートを一晩４℃でインキュベートした。リン酸緩衝化生理食塩水（ＰＢＳ）を用いた２回の洗浄後、このプレートを、１０％ 胎仔ウシ血清（ＦＢＳ）を補充した１００μｌのＲＰＭＩ培地を用いて１時間３７℃でブロックした。この培地をブロッキング後に捨てた。溶解されそしてＲＰＭＩ（１０％ ＦＢＳ、ピルビン酸ナトリウム、および非必須アミノ酸で補充されている）に１×１０^７細胞／ｍｌで再懸濁された赤血球を有する脾細胞を、１ウェルにつき１×１０^６細胞または１ウェルにつき０．５×１０^６細胞の濃度で添加した。１０％ＦＢＳで補充されたＲＰＭＩ中に２０μｇ／ｍｌに希釈された、１００μｌの全Ｂ型肝炎ウイルスコア抗原（ＢｉｏＤｅｓｉｇｎ）を、ウェルに添加し、そしてプレートを、３７℃で４８時間インキュベートした。インキュベーション後、細胞および培地を捨て、プレートを２回ＰＢＳで洗浄し、続いて、任意の残存する細胞を溶解するために脱イオン水で洗浄し、次いで、ＰＢＳを用いてさらに２回洗浄した。検出抗体（ビオチン化されたラット抗マウスＩＬ−４）を、ＰＢＳ中１μｇ／ｍｌに希釈し、そして５０μｌを、各ウェルに添加し、そして室温で１時間インキュベートした。次いで、このプレートを、５回ＰＢＳで洗浄し、そして５０μｌのストレプトアビジンアルカリホルファターゼ結合体（ＰＢＳ中１：１０００希釈）を、各ウェルに添加した。室温で１時間のインキュベーション後、このプレートをＰＢＳで５回洗浄し、そして５０μｌのクロマゲン（ｃｈｒｏｍａｇｅｎｉｃ）アルカリホスファターゼ基質を、各ウェルに添加した。スポットが現れると直ぐに、この反応を、タップ水を用いてプレートをリンスすることによって停止した。スポットを、計数し、この結果を、図９に、１００万個の脾細胞当たりの特定のスポットとしてグラフにした。図９に示されるデータを再検討することによって見出されるように、ＨＢｃＡｇに特異的なＩＬ−４産生は、コントロール（非ｐＴＲＡＮＣＥ含有組成物）に比較して増加し、そして特定のスポットに用量関連の減少が示された。
【０１６４】
（Ｄ．抗体分析（ＥＬＩＳＡ））
上記研究を、前出のように調製されたコートＤＮＡ粒子カートリッジを用いて反復したが、以下の改変を用いた：ｐＰＪＶ７１９３構築物（Ｂ型肝炎表面抗原およびＢ型肝炎コア抗原をコードする）およびｐＴＲＡＮＣＥ構築物（ＴＲＡＮＣＥサイトカインをコードする）を、別のバッチの金粒子上にコートした（ｐＴＲＡＮＣＥおよびｐＰＪＶ７０４６プラスミドは、上記のようにあわせ、この組合せを金粒子上にコートしたが、ｐＴＲＡＮＣＥプラスミドは、別のバッチの金粒子上にコートした）。次いで、２つのバッチのコートされた金粒子を、Ｔｅｆｚｅｌ（登録商標）チューブをコードする前に一緒に混合した。上記表４に報告されるような同じ濃度のＤＮＡを、６２５：１の比率をこの第２の研究に含めなかったことを１つの例外として、使用した。４匹のＢａｌｂ／ｃマウスの各々の実験群を、前出のように免疫し、そしてマウスを、免疫の２週間後に屠殺し、そして血清を、ＥＬＩＳＡによる抗体分析のために収集した。
【０１６５】
抗体分析のために、ＥＬＩＳＡプレートを１００μｌの全Ｂ型肝炎ウイルスコア抗原（ＢｉｏＤｅｓｉｇｎ）でコートし、ＰＢＳ中０．１μｇ／ｍｌに希釈し、そして４℃で一晩インキュベートした。このプレートを、０．０５％ Ｔｗｅｅｎ２０を有するＰＢＳ（ＰＢＳ−Ｔ）を用いて１回洗浄し、次いで、３００μｌのブロッキング溶液（ＰＢＳおよび５％乾燥ミルク）を用いて室温で１時間ブロックした。このブロッキング溶液を捨て、そして１００μｌの連続希釈血清を添加した。このプレートを室温で１時間インキュベートし、続いて、ＰＢＳ−Ｔを用いて３回洗浄した。結合体化抗体（ヤギ抗マウスＩｇＧ−西洋ワサビペルオキシダーゼ）を、ＰＢＳおよび２％乾燥ミルク中で１：５０００希釈した。１００μｌの結合体化抗体を各ウェルに添加し、プレートを１時間室温でインキュベートした。次いで、プレートをＰＢＳ−Ｔで５回洗浄し、そして１００μｌのＴＭＢ基質を各ウェルに添加した。１５分後、この反応を、１００μｌの１Ｎ Ｈ_２ＳＯ_４で停止した。吸光度を、４５０ｎｍで読み取った。各実験群の相乗平均を、次いで算出し、そして図１０に、希釈に対してグラフ化した。図１０に示されるデータを再検討することによって見出されるように、２５：１の比率を含む組成物に関して以外、ｐＴＲＡＮＣＥアジュバント組成物を受けた全ての群において、抗ＨＢｃＡｇ抗体応答が増加した。
【０１６６】
従って、肝炎ＤＮＡワクチン組成物中のプラスミド構築物をコードするｐＴＲＡＮＣＥサイトカインの添加は、目的の抗原に対する細胞性免疫応答および体液性免疫応答の両方を増強した。
【０１６７】
従って、免疫を誘発する新規組成物が、記載される。これらの組成物を用いる方法がまた記載される。本発明の好ましい実施形態はいくらか詳細に記載されるが、明らかな改変は、本発明の精神および範囲から逸脱することなく添付の特許請求の範囲によって規定されるようになされえることが理解される。
【図面の簡単な説明】
【図１】
図１は、ＣＤ８０プロモーター駆動発現ベクターｐ５０２０の概略図である。このプラスミドベクターは、ｐＵＣ１９骨格に基づくｐＷＲＧ７１２８（哺乳動物発現ベクター）から構築された。ＣＭＶプロモーターが除去され、そしてマウスＣＤ８０プロモーター（Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ，Ｇｉｂｃｏ ＢＲＬから得られた）の増幅によって得られた２５４塩基対ＰＣＲフラグメントで置換した。このプラスミドはまた、調節エレメント、およびウシ成長ホルモンのポリＡシグナルを含み、Ｂ型肝炎表面抗原をコードする全長ｃＤＮＡに作動可能に連結される。
【図２】
図２は、ＣＤ８０プロモーター駆動発現ベクターｐ５０２１の概略図である。このプラスミドベクターは、ｐＵＣ１９骨格に基づくｐＷＲＧ７１２８（哺乳動物発現ベクター）から構築された。ＣＭＶプロモーターが除去され、そしてマウスＣＤ８０プロモーター（Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ，Ｇｉｂｃｏ ＢＲＬから得られた）の増幅によって得られた４８９塩基対ＰＣＲフラグメントで置換した。このプラスミドはまた、調節エレメント、およびウシ成長ホルモンのポリＡシグナルを含み、Ｂ型肝炎表面抗原をコードする全長ｃＤＮＡに作動可能に連結される。
【図３】
図３は、ＣＤ８０プロモーター駆動発現ベクターｐ５０２２の概略図である。このプラスミドベクターは、ｐＵＣ１９骨格に基づくｐＷＲＧ７１２８（哺乳動物発現ベクター）から構築された。ＣＭＶプロモーターが除去され、そしてマウスＣＤ８０プロモーター（Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ，Ｇｉｂｃｏ ＢＲＬから得られた）の増幅によって得られた３１２３塩基対ＰＣＲフラグメントで置換した。このプラスミドはまた、調節エレメント、およびウシ成長ホルモンのポリＡシグナルを含み、Ｂ型肝炎表面抗原をコードする全長ｃＤＮＡに作動可能に連結される。
【図４】
図４は、ＣＤ８０プロモーター駆動発現ベクターｐ５０２３の概略図である。このプラスミドベクターは、ｐＵＣ１９骨格に基づくｐＷＲＧ７１２８（哺乳動物発現ベクター）から構築された。ＣＭＶプロモーターが除去され、そしてマウスＣＤ８０プロモーター（Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ，Ｇｉｂｃｏ ＢＲＬから得られた）の増幅によって得られた３３５７塩基対ＰＣＲフラグメントで置換した。このプラスミドはまた、調節エレメント、およびウシ成長ホルモンのポリＡシグナルを含み、Ｂ型肝炎表面抗原をコードする全長ｃＤＮＡに作動可能に連結される。
【図５】
図５は、ＣＤ８０プロモーター駆動発現ベクターｐ５０２４の概略図である。このプラスミドベクターは、ｐＵＣ１９骨格に基づくｐＷＲＧ７１２８（哺乳動物発現ベクター）から構築された。ＣＭＶプロモーターが除去され、そしてマウスＣＤ８０プロモーター（Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ，Ｇｉｂｃｏ ＢＲＬから得られた）の増幅によって得られた５７８塩基対ＰＣＲフラグメントで置換した。このプラスミドはまた、調節エレメント、およびウシ成長ホルモンのポリＡシグナルを含み、Ｂ型肝炎表面抗原をコードする全長ｃＤＮＡに作動可能に連結される。
【図６】
図６は、ＣＤ８０プロモーター駆動発現ベクターｐ５０２５の概略図である。このプラスミドベクターは、ｐＵＣ１９骨格に基づくｐＷＲＧ７１２８（哺乳動物発現ベクター）から構築された。ＣＭＶプロモーターが除去され、そしてマウスＣＤ８０プロモーター（Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ，Ｇｉｂｃｏ ＢＲＬから得られた）の増幅によって得られた２０２塩基対ＰＣＲフラグメントで置換した。このプラスミドはまた、調節エレメント、およびウシ成長ホルモンのポリＡシグナルを含み、Ｂ型肝炎表面抗原をコードする全長ｃＤＮＡに作動可能に連結される。
【図７】
図７は、ＣＤ８０プロモーター駆動発現ベクターｐ５０２６の概略図である。このプラスミドベクターは、ｐＵＣ１９骨格に基づくｐＷＲＧ７１２８（哺乳動物発現ベクター）から構築された。ＣＭＶプロモーターが除去され、そしてマウスＣＤ８０プロモーター（Ｌｉｆｅ Ｔｅｃｈｎｏｇｉｅｓ，Ｇｉｂｃｏ ＢＲＬから得られた）の増幅によって得られた２９４塩基対ＰＣＲフラグメントで置換した。このプラスミドはまた、調節エレメント、およびウシ成長ホルモンのポリＡシグナルを含み、Ｂ型肝炎表面抗原をコードする全長ｃＤＮＡに作動可能に連結される。
【図８】
図８は、ＣＭＶプロモーターまたはＣＤ８０プロモターの制御下で、Ｂ型肝炎抗原（ＨＢｓＡｇ）をコードするプラスミドで免疫されたマウスにおいて惹起された細胞障害性Ｔ細胞（ＣＴＬ）応答を示すグラフである。
【図９】
図９は、Ｂ型肝炎コア／表面抗原をコードするプラスミドおよびＴＲＡＮＣＥサイトカインアジュバントで免疫されたマウスからの脾細胞における抗Ｂ型肝炎コア抗原特異的ＩＬ−４産生を示すヒストグラムである。
【図１０】
図１０は、Ｂ型肝炎コア／表面抗原をコードするプラスミドおよびＴＲＡＮＣＥサイトカインアジュバントで免疫されたマウスにおける抗Ｂ型肝炎コア抗原特異的抗体産生を示すグラフである。[0001]
(Field of the Invention)
The present invention generally relates to vaccine compositions and methods of using the same. More specifically, the present invention relates to polynucleotides encoding at least one immunizing antigen whose expression is controlled by a promoter from a costimulatory molecule. Immunization methods using these polynucleotides are also provided. Also provided is a composition comprising at least one immunizing agent and at least one cytokine (associated with antigen presenting cell maturation). Methods of raising an immune response using these compositions are also provided.
[0002]
(background)
Vaccines that induce cell-mediated immune responses represent an important strategy in combating parasites, autoimmune diseases, allergic diseases and cancer. Conventional vaccination strategies generally involve the administration of either "live" or "dead" vaccines. Ertl et al. (1996) J. Mol. Immunol. 156: 3579-3582. So-called live vaccines include recombinant molecules based on attenuated microorganisms and live vectors. Dead vaccines include those based on the entire pathogen killed, as well as subunit vaccines (eg, soluble pathogen subunits or protein subunits). Live vaccines have generally been successful in providing an effective immune response in immunized subjects; however, such vaccines have been shown to be dangerous in immunocompromised or pregnant subjects. Can be, return to pathogenic organisms, or be contaminated with other pathogens. Hassett et al. (1996) Trends in Microbiol. 8: 307-312. Dead vaccines circumvent the safety issues associated with live vaccines; however, such vaccines often provide a suitable and / or effective immune response in ￥ of immunized subjects. Fail.
[0003]
More recently, direct injection of plasmid DNA by intramuscular injection (Wolff et al. (1990) Science 247: 1465: 1468) or intradermal injection using a needle and syringe (Raz et al. (1994) PNAS UAS 91: 9519-9523) has been reported. Has been described. Another approach, referred to as bombardment or particle-mediated DNA delivery, uses a needle particle delivery device to administer microscopic gold beads coated with DNA directly to epidermal cells. Accordingly, a number of delivery techniques can be used to deliver nucleic acids for immunization, including particle-mediated techniques for delivering nucleic acid-coated microparticles to target tissues (see, eg, co-owned US Pat. No. 5,865,865). No. 796 (released on February 2, 1999). Particle-mediated nucleic acid immunization techniques have been shown to elicit both humoral and / or cytotoxic T lymphocyte immune responses following epidermal delivery of nanogram DNA. Pertmer et al. (1995) Vaccine 13: 1427-1430. Such particle-mediated delivery techniques have been compared to other types of nucleic acid inoculation and have been found to be significantly superior. Fynan et al. (1995) Int. J. Immunopharmacology 17: 79-83; Fynan et al. (1993) Proc. Natl. Acad. Sci. USA 90: 11478-11482, and Razs et al. (1994) Proc. Natl. Acad. Sci. USA 91: 9519-9523.
[0004]
A novel transdermal drug delivery system has been described that requires the use of a needleless syringe to deliver solid drug-containing particles to and through intact skin at a controlled dose. In particular, co-owned US Pat. No. 5,630,796 (Bellhouse et al.) Describes a particle delivery device (needleless syringe) that delivers pharmaceutical particles in synchrony with a supersonic gas flow. I do. This particle delivery device is used for transdermal delivery of powdered drug compounds and compositions, delivering genetic material in living cells (eg, gene therapy), and to the skin, muscle, blood, or lymph. Used for the delivery of biopharmaceuticals. The device can also be used in conjunction with surgery to deliver drugs and biopharmaceuticals to tissue surfaces, solid tumors and / or surgical cavities (eg, tumor beds or cavities after tumor resection). Pharmaceutical agents, which can be prepared to suit a substantially solid particulate form, can be delivered safely and easily using such devices.
[0005]
One particular particle delivery device is generally an elongated tubular, initially obstructing the passageway through the nozzle and having a modified rupturable membrane substantially adjacent the upstream end of the nozzle. Includes nozzle. The particles of the therapeutic agent to be delivered are placed adjacent to the rupturable membrane and rupture the membranes and apply a supersonic gas flow (including pharmaceutical particles) through a nozzle for delivery from their downstream end. It is delivered using pressurized means to apply gas pressure upstream of the membrane sufficient to produce. Thus, particles can be delivered from a needleless syringe at a delivery rate between Mach 1 and Mach 8 that can easily obtain a rupturable membrane rupture.
[0006]
Instead of having the pharmaceutical particles tuned during the supersonic gas flow, the downstream end of the nozzle is placed in a stationary “inverted” position (here, the septum is concave on the downstream surface to contain the pharmaceutical particles). Has) and an active "inside-out" position (where the septum has an outwardly convex surface on the downstream face of the septum as a result of an ultrasonic shockwave applied to the upstream face of the septum). Other needleless syringe configurations generally include elements similar to those described above, except that a possible bistable septum is provided. In this manner, the pharmaceutical particles contained within the concave surface of the septum are launched at a supersonic initial velocity from the device for their transdermal delivery to the intended skin or mucosal surface.
[0007]
Transdermal delivery using the device devices described above is generally performed using particles having an approximate size in the range between about 0.1-250 μm. Particles larger than about 250 μm can also be delivered from the device at the upper limit particle size, where the particle size causes harmful damage to skin cells. The actual distance through which the delivered particles penetrate depends on the particle size (e.g., nominal particle diameter assumed from approximate spherical particle geometry), particle density, initial velocity at which the particles strike the skin surface, And it depends on skin density and kinematic viscosity. Target particle densities for general use in needleless syringes are about 0.1 and 25 g / cm ³ And the injection speed is generally in the range between about 150 and 3,000 m / sec.
(Summary of the Invention)
The present invention relates to a polynucleotide comprising a first promoter from a gene encoding a costimulatory molecule and a first sequence encoding at least one antigen, wherein the first sequence is operable on the first promoter. Linked as possible. In certain embodiments, the promoter is from the CD80 (B7-1) or CD86 (B7-2) gene.
[0008]
In a further embodiment, the polynucleotide further comprises a second sequence encoding at least one cytokine operably linked to the first promoter or the second promoter. The promoter can be a constitutive promoter.
[0009]
In another embodiment, the invention relates to a core carrier coated with a polynucleotide as described above, and a pharmaceutical composition comprising the polynucleotide and a pharmaceutically acceptable excipient. The pharmaceutical composition optionally further comprises a cytokine.
[0010]
In yet a further embodiment, the present invention provides a method comprising: (a) an expression vector comprising a polynucleotide encoding at least one antigen; and (b) a CD40 ligand (CD40L), a tumor necrosis factor-related activation-inducing cytokine (TRANCE) and Flt3. A vaccine composition comprising a cytokine selected from the group consisting of ligand (flt-3L).
[0011]
In another embodiment, the present invention consists of (a) at least one peptide antigen; and (b) CD40 ligand (CD40L), a tumor necrosis factor-related activation-inducing cytokine (TRANCE), and Flt3 ligand (flt-3L). A vaccine composition comprising an expression vector comprising a polynucleotide encoding a cytokine selected from the group.
[0012]
In yet another embodiment, the present invention provides a method comprising: (a) at least one peptide antigen; and (b) a CD40 ligand (CD40L), a tumor necrosis factor-related activation-inducing cytokine (TRANCE), and a Flt3 ligand (flt-3L). A vaccine composition comprising a cytokine selected from the group consisting of:
[0013]
Also provided is a method for raising an immune response in a vertebrate subject comprising the step of administering the vaccine.
[0014]
The various components of the vaccine composition can be coated on a core carrier and used in a method for raising an immune response in a vertebrate subject. In this context, the method involves administering the composition to the subject using particle mediated delivery technology.
[0015]
In another embodiment, the invention is directed to a method for raising an immune response in a vertebrate subject. The method comprises the steps of: (a) providing a nucleotide sequence encoding an antigen operably linked to a promoter from a gene encoding a costimulatory molecule, wherein the promoter directs expression of the antigen in a subject. And (b) administering the coated core carrier particles to a subject, whereby the antigen is expressed in the subject and in an amount sufficient to elicit an immune response. Administering the nucleotide sequence to the subject.
[0016]
In a further embodiment, the invention is directed to a method for raising an immune response in a vertebrate subject. The method comprises the steps of (a) providing a particle coated with a nucleotide sequence encoding at least one antigen, wherein the nucleotide sequence is operably linked to a promoter from a gene encoding a costimulatory molecule. Wherein the promoter is capable of driving expression of the antigen coding sequence in the subject; and (b) administering the particles to the subject using particle mediated delivery techniques. Whereby the antigen encoded by the nucleotide sequence is expressed in an amount sufficient to elicit an immune response.
[0017]
In the above method, the nucleotide sequence encodes at least one cytokine (eg, a cytokine selected from the group consisting of CD40L, a tumor necrosis factor-related activation-inducing cytokine (TRANCE), and a Flt3 ligand (flt-3L)). And a polynucleotide.
[0018]
These and other embodiments of the present invention will readily occur to those skilled in the art in light of the present disclosure.
[0019]
(Form for implementing the present invention)
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular antigens or antigen-encoding nucleotide sequences. It should also be understood that different applications of the disclosed method may be modified for particular needs in the art. It should also be understood that the terms used herein are for the purpose of describing particular embodiments of the present invention only and are not intended to be limiting.
[0020]
All publications, patents and patent applications (whether or not mentioned above) cited herein are hereby incorporated by reference in their entirety.
[0021]
As used in this specification and the appended claims, the singular forms "a,""an," and "the" refer to plural reference unless the content clearly dictates otherwise. It should be noted that including. Thus, for example, a reference to "antigen" includes a mixture of two or more factors, a reference to "particle" includes a mixture of two or more particles, and a reference to "recipient cells" includes two or more such. And other cells.
[0022]
(Definition)
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The following terms are intended to be defined as set forth below.
[0023]
The term “vaccine composition” contemplates any pharmaceutical composition (eg, a polynucleotide encoding an antigen) that includes an antigen, wherein the composition is used to prevent or treat a disease or condition in a subject. Can be done. Thus, the term covers both subunit vaccines (ie, vaccine compositions containing antigens separated and dispersed from the entire organism to which the antigen is naturally bound, as well as killed, attenuated, or non-killed). A composition comprising activated bacteria, viruses, parasites or other whole microorganisms).
[0024]
The term “transdermal” delivery refers to intradermal (eg, into the dermis or epithelium), transdermal (eg, “percutaneous”) and transmucosal administration (ie, into the skin or mucosal tissue) For example, see, for example, Transdermal Drug Delivery: Developmental Issues and Research Initiatives, Hadgraft and Guy (eds.), McKer, Edk. Controlled Drug Delivery: Fundamentals and Applications, Robinson and Lee (eds.), Marcel Dekk and Inc., (1987); and Transdermal Delivery of Drugs, Vols. 1-3, Kydonieus and Berner (eds.), CRC Press, (1987), and this term is therefore incorporated into US Patent No. 5,630,796. Includes needleless syringe delivery as described, and delivery from particle-mediated delivery of a coated core carrier as described in US Pat. No. 5,865,796.
[0025]
"Core carrier" refers to a guest nucleic acid (eg, DNA) that imparts a defined particle size and provides a sufficiently high density to achieve the moment required for cell membrane transduction, such that the guest molecule A carrier that is coated so that it can be delivered using a particle mediated technique (eg, US Pat. No. 5,100,792). Core carriers typically include materials such as tungsten, gold, platinum, ferrite, polystyrene, and latex. See, for example, Particle Bombardment Technology for Gene Transfer, (1994) Yang, N.M. ed. , Oxford University Press, New York, NY, pages 10-11.
[0026]
By "particle delivery device" or "needleless syringe" is meant an apparatus that delivers a particle composition transdermally without the aid of a conventional needle to pierce the skin. Particle delivery devices for use with the present invention are discussed throughout the specification.
[0027]
"Antigen" refers to a molecule comprising one or more epitopes that stimulates the host immune system to produce a cellular antigen-specific immune response, or a humoral antibody response. Thus, antigens include proteins, polypeptides, antigenic protein fragments, oligosaccharides, polysaccharides, and the like. Further, the antigen can be from any known virus, bacterium, parasite, plant, protist, or fungus, and can be an entire organism. The term also includes tumor antigens. Similarly, oligonucleotides or polynucleotides that express an antigen (eg, in DNA immunization applications) are also included in the definition of antigen. Synthetic antigens (eg, polyepitope, flanking epitopes, and other recombinantly or synthetically derived antigens) are also included (Bergmann et al. (1993) Eur. J. Immunol. 23: 2777-2781; Bergmann et al. (1996) J. Immunol. 157: 3242-3249; Suhrbier, A. (1997) Immunol. And Cell Biol. 75: 402-408; Gardner et al. (1998) 12th World AIDS Conference, Geneva, Switzerland, 28- , 1998).
[0028]
The term “peptide” is used in the broadest sense to refer to a compound of two or more subunit amino acids, amino acid analogs, or other peptidomimetics. These subunits can be linked by peptide bonds or other bonds (eg, esters, ethers, etc.). As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, both glycine and optical isomers of D or L, and amino acid analogs and peptides Includes imitations. When the peptide chain is short, a peptide of three or more amino acids is generally called an oligopeptide. If the peptide difference is long, the peptide is typically called a polypeptide or protein.
[0029]
"T cell epitope" refers to a characteristic of a peptide structure that can generally induce a T cell response. In this regard, it is acceptable in the art that T cell epitopes include a linear peptide determinant that assumes an extended conformation within the peptide bond cleavage of the MHC molecule (Unanue et al. (1987) Science). 236: 551-557). As used herein, a T cell epitope is generally a peptide having at least about 3-5 amino acid residues, and preferably a peptide having at least 5-10 or more amino acid residues. .
[0030]
“Antigen presenting cells” and “APCs” as used herein refer to a histocompatibility complex molecule or portion thereof, or an antigen associated with one or more classical MHC molecules or portion thereof. Intended, any cell that presents on its surface. Examples of suitable APCs are discussed in detail below and include, but are not limited to, whole cells such as Langerhans cells, macrophages, dendritic cells, B cells, hybrid APCs, and Foster antigen presenting cells.
[0031]
Dendritic cells (DC) and Langerhans cells are powerful antigen presenting cells. DCs are a minor component of various immune organs and comprise, for example, about 1% of epidermal cell suspensions (Schuller et al. (1985) J. Exp. Med. 161: 526; and Romani et al. (1989). ) J. Invest. Dermatol. 93: 600). Despite their relative rarity, these cells have been shown to provide all of the signals required for T cell activation and proliferation. The required signals can be classified into two types. The first type, which confers specificity on the immune response, is the T cell receptor / CD3 ("TRC / CD") complex and the major histocompatibility complex ("MHC") class on the surface of the APC. It is mediated through an interaction between I or II proteins. This interaction is necessary, but not sufficient, for T cell activation to occur. In fact, without the second type of signal, the first type of signal can cause T cell allergy (eg, if the T cells are insensitive to additional signals). A second type of signal (eg, called a costimulatory signal) is neither antigen-specific nor MHC-restricted, and in the presence of the first type of signal, the complete proliferative response of T cells and It can lead to induction of T cell effector function. Thus, as noted above, studies accumulated over the past few years have convincingly demonstrated that resting T cells require at least two signals for induction of cytokine gene expression and proliferation. (Schwatrz RH (1990) Science 248: 1349-1356; Jenkins MK (1992) Immunol. Today 13: 69-73). One signal (which gives the property) may be generated by the interaction of the TCR / CD3 complex with the appropriate MHC / peptide complex. The second signal is not antigen specific and is referred to as a "costimulatory" signal.
[0032]
A "co-stimulatory molecule" functions as a receptor ligand pair fr on the surface of antigen presenting cells and T cells. The term encompasses any single molecule or combination of molecules that, when functioning with the peptide / MHC complex bound by the TCR on the surface of the T cell, the Provides a costimulatory effect to achieve activation.
[0033]
Some molecules have been shown to enhance costimulatory activity. These include CD80 (ie, B7-1), CD86 (ie, B7-2 / B70) (Schwatrz RH (1992) Cell 71: 1065-1068), heat stable antigen (HSA) (Liu Y. et al. (1992) J. Exp. Med. 175: 437-445), chondroitin sulfate-modified MHC invariant chain (Naujokas MF, et al. (1993) Cell 74: 257-268), cell adhesion molecule 1 (ICAM- 1) (Van Eventer, GA (1990) J. Immunol. 144: 4579-4586). Each of these molecules appears to achieve costimulation by interacting with their cognate ligand on T cells. Co-stimulatory molecules mediate the signals required to achieve full activation of native T cells under normal physiological conditions. One exemplary receptor-ligand pair is the CD80 and CD86 costimulatory molecules on the surface of APCs and their counterreceptors on T cells, CD28 and CTLA-4 (Ellis et al. (1996) J. Immunol. 56). : 2700-2709; Freeman et al. (1993) Science 262: 909-911; Nabavi et al. (1992) Nature 360: 266-268). Other important costimulatory molecules are CD40 and CD54. Thus, the term refers to CD80, CD86, or other co-stimulatory molecules on an antigen-presenting matrix, such as APCs, as well as cognate ligands when TCRs on the surface of T cells specifically bind to the peptide And fragments of co-stimulatory molecules that result in activation of T cells (only as a complex with another molecule, or as part of a fusion protein). Many of the sequences of the genes encoding costimulatory molecules and their promoter regions are known in the art. Other promoters or fragments thereof can also be determined by the methods known in the art and described herein.
[0034]
As used herein, the term "adjuvant" refers to any substance that enhances the action of a drug, antigen, polynucleotide, vector, and the like. Thus, one example of an adjuvant is a "cytokine". As used herein, the term “cytokine” refers to any one of a number of factors that have various effects on a cell, including, for example, growth, proliferation or maturation. Certain cytokines (eg, TRANCE, flt-3 and CD40L) enhance the immunostimulatory capacity of APCs. Non-limiting examples of cytokines that may be used alone or in combination with the practice of the invention include interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3 (IL-3), interleukin-6 (IL-6), interleukin 12 (IL-12), G-CSF, granulocyte-macrophage colony stimulating factor (GM-CSF), interleukin-1α (IL-1α), interleukin-11 (IL-11 ), MIP-1α, leukemia inhibitory factor (LIF), c-kit ligand, thrombopoietin (TPO), CD40 ligand (CD40L), tumor necrosis factor-related activation-inducing cytokine (TRANCE) and flt3 ligand (flt-3L). No. Cytokines are commercially available from several suppliers, for example, Genzyme (Framingham, MA), Genentech (South San Francisco, CA), Amgen (Thousand Oaks, CA), R & D Systems and Immunex (Seattle, Seattle). The sequences of many of these molecules are also available, for example, from the GenBank database. Although not usually explicitly described, molecules having biological activity similar to wild-type or purified cytokines (eg, recombinantly produced or variants thereof) and nucleic acids encoding these molecules are It is intended to be used within the spirit and scope of the present invention.
[0035]
A composition comprising a selected antigen and an adjuvant, or a vaccine composition co-administered with an adjuvant, elicits an immune response that is greater than the immune response elicited by an equal amount of antigen administered without adjuvant. If capable, it indicates "enhanced immunogenicity." Thus, a vaccine composition may exhibit "enhanced immunogenicity." Either the antigen is more immunogenic or a lower or lower dose of the antigen is required to achieve an immune response in the subject to which the antigen is administered. Such enhanced immunogenicity can be achieved by administering the adjuvant composition and a control of the antigen to the animal and determining antibody titers and / or cell-mediated immunity against the two using standard assays ( For example, it can be determined by comparison using radioimmunoassays, ELISAs, CTL assays, etc., well known in the art.
[0036]
The terms "nucleic acid molecule" and "polynucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length (either deoxyribonucleotides or ribonucleotides), or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, Examples include isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers.
[0037]
Polynucleotides typically consist of specific sequences of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (if the polynucleotide is RNA, uracil ( U) replaces thymine (T)). Thus, the term polynucleotide sequence is the alphabetical representation of a polynucleotide molecule. This alphabetical representation is input into a database in a computer with a central processing unit (CPU) and can be used for bioinformatics applications (eg, functional genomics and homology searches).
[0038]
As used in the context of the present invention, a "gene" is a sequence of nucleotides in a gene nucleic acid (chromosome, plasmid, etc.) to which gene function is related. A gene is a hereditary unit (eg, of an organism) that is a polynucleotide sequence (eg, a mammalian gene) that occupies a particular physical location (“locus” or “gene locus”) in the genome of an organism. DNA sequence). A gene can encode a product (eg, a polypeptide or a polynucleotide (eg, a tRNA)) to be expressed. Alternatively, a gene defines a locus for a particular event / function (eg, binding of a protein and / or nucleic acid (eg, a phage attachment site), where the gene does not encode the product to be expressed). I can do it. Typically, a gene includes coding sequences (eg, sequences encoding a polypeptide) and non-coding sequences (eg, promoter sequences, polyadenylation sequences, transcription regulatory sequences (eg, enhancer sequences)). Many eukaryotic genes have "exons" (coding sequences) interrupted by "introns" (non-coding sequences). In certain cases, a gene may share a sequence with another gene (eg, a duplicated gene).
[0039]
A “coding sequence” or a sequence “encoding” a selected polypeptide is transcribed in vivo (in the case of DNA) and polypeptide when placed under the control of appropriate regulatory sequences (or “control elements”). (In the case of mRNA) is a nucleic acid molecule. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3 'to the coding sequence. Transcription and translation of a coding sequence is typically regulated by "control elements," which include a transcription promoter, a transcription enhancer element, a transcription termination signal, a polyadenylation sequence (3 'of the translation stop codon). ), Sequences that optimize translation initiation (located 5 'of the coding sequence), and translation termination sequences.
[0040]
"Promoter" is a nucleotide sequence that initiates transcription of a polynucleotide encoding a polypeptide. Promoters include inducible promoters (expression of a polynucleotide sequence operably linked to a promoter is driven by an analyte, cofactor, regulatory protein, etc.), repressible promoters (operably linked to a promoter). Expression of the polynucleotide sequence is suppressed by analytes, cofactors, regulatory proteins, etc.), and constitutive promoters. Further, such promoters may also have tissue specificity (eg, the CD80 promoter is inducible only in certain immune cells, and the myoD promoter is inducible only in muscle cells). The term "promoter" or "control element" is intended to include the full-length promoter regions and functional (eg, control transcription or translation) segments of these regions. If the promoter has the same or substantially the same base pair sequence as the region of the promoter region (or its complement) of the costimulatory molecule, or the promoter shows the following sequence identity, the promoter is It is the “origin” of the gene encoding the molecule.
[0041]
A “vector” can transfer a gene sequence to a target cell (eg, a viral vector, a non-viral vector, a microparticle carrier, and a liposome). Typically, “vector construct,” “expression vector,” and “gene transfer vector” refer to any nucleic acid construct that directs the expression of a gene of interest and is capable of transferring a gene sequence to a target cell. Thus, the term includes cloning vehicles, expression vehicles, as well as viral vectors.
[0042]
An "isolated polynucleotide" molecule is an isolated nucleic acid molecule that is distinct from the entire organism in which it is found in nature; a nucleic acid that lacks all or part of the sequence with which it normally associates in nature Is a molecule; or a sequence that is naturally occurring but has a heterologous sequence (defined below) associated within the molecule.
[0043]
"Operably linked" refers to an arrangement of elements wherein the components so described are designed to perform their normal function. Thus, a given promoter operably linked to a coding sequence (eg, the antigen of interest) may result in the expression of the coding sequence, if present, in the presence of regulatory proteins and appropriate enzymes. In some cases, a particular control element need not be contiguous with a coding sequence, as long as the control element functions to direct expression of the coding sequence. For example, an intervening, untranslated but still transcribed sequence may be present between the promoter sequence and the coding sequence, which promoter sequence may still be considered "operably linked" to the coding sequence.
[0044]
As used herein to describe a nucleic acid molecule, "recombinant" means a polynucleotide of genomic, cDNA, semi-synthetic, or synthetic origin, for its origin and manipulation: (1) Does not associate with all or a portion of the naturally associated polynucleotide, and / or (2) ligates to a polynucleotide other than the naturally associated polynucleotide. When used in reference to a protein or polypeptide, the term “recombinant” refers to a polypeptide produced by expression of a recombinant polynucleotide.
[0045]
Techniques for determining nucleic acid “sequence identity” and amino acid “sequence identity” are also known in the art. Typically, such techniques involve determining the nucleotide sequence of the mRNA of a gene and / or determining the amino acid sequence encoded by the mRNA, and comparing these sequences to a second nucleic acid or amino acid sequence. . In general, "identity" refers to an exact nucleotide-to-nucleotide correspondence or an exact amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Two or more sequences (polynucleotides or amino acids) can be compared by determining their "percent identity." The percent identity of the two sequences (whether nucleic acid or amino acid) is the exact match between the two aligned sequences divided by the shorter sequence length and multiplied by 100. Number. Approximate alignments for nucleic acid sequences are provided by the local homology algorithm of Smith and Watermann (Advances in Applied Matrices 2: 482-489 (1981)). This algorithm is described in Dayhoff (Atlas of Protein Sequences and Structure, MO Dayhoff, eds., 5 Supplement 3: 353-358, National Biomedical Research Foundation, Washington, D.C., Washington, D.C. Acids Res. 14 (6): 6745-6763 (1986)), and can be applied to amino acid sequences by using a scoring matrix. An exemplary implementation of this algorithm for determining the percent identity of a sequence is provided by Genetics Computer Group (Madison, WI) in the "BestFit" utility application. Default parameters for this method are described in Wisconsin Sequence Analysis Package Program Manual, Version 8 (1995) (available from Genetics Computer Group, Madison, WI). In the context of the present invention, a preferred method for establishing percent identity is the MPSRCH package of programs copyrighted by the University of Edinburgh (developed by John F. Collins and Shane S. Sturok, and developed by IntelliGenetics, Inc. (Distributed by Mountain View, CA). From this set of packages, the Smith-Waterman algorithm may be used for the scoring table, using default parameters (eg, 12 gap open penalty, 1 gap extension penalty, and 6 gap). From the data generated, the “Match” value reflects “sequence identity”. Other suitable programs for calculating percent identity or similarity between sequences are generally known in the art (eg, another alignment program is BLAST, used with default parameters). For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequence; by = high score; Databases = no duplication, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + Swiss protein + Supdate + PIR. Details of these programs can be found at the following internet address: http: // www. ncbi. nlm. gov / cgi-bin / BLAST.
[0046]
Alternatively, homology is performed by hybridizing the polynucleotide under conditions that form a stable duplex between regions of homology, followed by digestion with a single-strand-specific nuclease, and determining the size of the digested fragment. Can be determined. Two DNA or two polypeptide sequences, as determined using the methods described above, have at least about 80-85%, preferably at least about 90%, and most preferably at least about 95-95%, of the sequence. They are "substantially homologous" to each other if they show sequence identity over a defined length of the molecule. As used herein, substantial homology also refers to sequences that show complete identity to the specified DNA or polypeptide sequence. Substantially homologous DNA sequences can be identified, for example, in Southern hybridization experiments under stringent conditions, as defined for the particular system. For example, stringent hybridization conditions can include 50% formamide, 5 × Denhardt's solution, 5 × SSC, 0.1% SDS, and 100 μg / ml denatured salmon sperm DNA, and the wash conditions are 37 ° C. 2 × SSC, 0.1% SDS at 60 ° C., followed by 1 × SSC, 0.1% SDS at 68 ° C. Defining appropriate hybridization conditions is within the skill of the art. See, eg, Sambrook et al., Supra; DNA Cloning, supra; Nucleic Acid Hybridization, supra.
[0047]
As used herein, the term "treatment" includes any of the following: prevention of infection or re-infection; reduction or elimination of symptoms; and reduction or elimination of pathogens. Treatment may be effected prophylactically (pre-infection) or therapeutically (post-infection). An "effective amount" is an amount sufficient to effect beneficial or desired results. An effective amount can be administered in one or more administrations of a dosage, application. The term “co-administration” or “co-administration” refers to the administration of at least two substances. Simultaneous administration may be achieved by administering the substances at the same time or at different times. Further, co-administration includes delivery using one or more delivery means.
[0048]
With an appropriate immune response, the methods of the present invention can elicit in a immunized subject an immune response characterized by the production of B and / or T lymphocytes specific for viral antigens, wherein The immune response may protect the subject against subsequent infection with a homologous or heterologous virus strain, reduce viral load and / or shedding during infection, or compare the immune response to a non-immunized subject. May result in a faster recovery of the infection, or may prevent or reduce clinical signs of disease symptoms.
[0049]
By “vertebrate subject” is meant any member of the subphylum cordata, especially mammals, including but not limited to humans and other primates. The term does not imply a particular age. Thus, it is intended to include both adult and newborn individuals.
[0050]
(General overview of the present invention)
Before describing the present invention in detail, it is to be understood that this invention is not limited to particular formulations or particular process parameters, which may, of course, vary. It is also understood that the techniques used herein are for the purpose of describing particular embodiments of the present invention only and are not intended to be limiting.
[0051]
DNA-vaccines generally consist of a plasmid encoding the relevant antigen for de novo synthesis by cells present in the target tissue. Viral promoters (eg, promoters from cytomegalovirus (CMV)) are commonly used in DNA-vaccine plasmid constructs to drive expression of the antigen. Delivery of these DNA-vaccine plasmids, both in "naked" form and attached to particles, has been shown to elicit both humoral and cell-mediated immune responses (e.g., Wang et al., (1993) Proc. Natl. Acad. Sci. USA 90: 4156-4160; Tang et al., (1992) Nature 356: 152-154; see Fynan supra).
[0052]
It is known that antigens must be presented to T cells in order to elicit a specific CTL (cytotoxic T cell) response. This presentation is achieved via antigen presenting cells (APC), a class of cells including dendritic cells (DC), Langerhans cells, monocytes, macrophages, and B cells. DCs were first described as morphologically distinct from Langerhans cells in the epidermis of the skin (as reviewed by Bancheraeau et al., (1998) Nature 392: 245-252), and subsequently to native T cells. It was shown to be the most effective APC for cell activation. Lanzaveccia A. ((1993) Science 260: 937-944) and Bancheraeau et al., (1998, supra). The antigen encoded by the injected DNA-vaccine is processed by dendritic cells into peptides and presented to T cells. It has also been shown that DNA-vaccines delivered intraepidermally target Langerhans cells. The targeted Langerhans cells express the DNA-encoded antigen and exit the epidermis, the influx region where the Langerhans cells process the DNA-vaccine encoded antigen and present it to T lymphocytes. Move to lymph nodes. Thus, APC plays an essential role in inducing an effective immune response to DNA-vaccine.
[0053]
APCs exposed to the antigen process the antigen into small fragments (known as epitopes) which are then converted to class I major histocompatibility complex (MHC) for presentation to CD8 + T lymphocytes. And class II MHC for presentation to CD4 + T lymphocytes. However, certain costimulatory molecules, such as CD80 and CD86 (also known as B7-1 and B7-2, respectively), are also required for antigen presentation. Thus, effective activation of T lymphocytes requires two signals at the cell surface at the interface of APC and target T cells. The first activation signal is provided by the binding of the T cell receptor (TCR) to the antigen MHC complex, and the second activation signal comprises a CD80 / CD86 costimulatory molecule on APC and a T lymphocyte. Is known to be provided by binding to the CD28 receptor. After maturation, APCs become susceptible to apoptosis, thus limiting their natural stimulatory capacity.
[0054]
The CD80 / CD86 costimulatory molecules required for successful antigen presentation are not constitutively expressed by ACP. Rather, upon activation (eg, in response to an infectious agent), CD80 / CD86 expression on the APC surface is rapidly upregulated. Signals for inducing CD80 / CD86 expression by APCs can be provided by cytokines released by epithelial and lymphocytes in the site of inflamed tissue infected by the pathogen. In addition, certain cytokines secreted by activated T lymphocytes (eg, IFNγ) can both induce and maintain CD80 / CD86 expression by APCs. To date, two members of the TNF family, CD40 ligand (CD40L or CD154), and a TNF-related activation-inducing cytokine (TANCE), and a factor known as Flt3 ligand (Flt3L), have been implicated in APC maturation. I have. See, for example, Gurunathan et al., (1998) J. Am. Immunol. Pulendran et al., (1998) J. Am. Exp. Med. , 188: 2075-2082, and Wong et al., (1999) J. Am. See Immunol 162: 2251-2258. TRANCE has also been shown to extend the lifespan of mature DCs. (Josien et al., (1999) J. Immun. 162: 2562-2568; Wong et al., (1997) J. Exp. Med., 186: 2075-2080). Co-administration of CD40L and a tumor-specific antigen has been shown to result in the production of IgG1 antibodies that reflect a Th2-type immune response (see Wong et al., (1999) supra).
[0055]
Recently, methods of enhancing a subject's T cell response have been described. These methods require administering a nucleotide encoding an immunizing antigen and a full-length costimulatory molecule under the same transcriptional regulatory element (see, eg, US Pat. No. 5,738,852, and International Publication No. WO 97/32987 (published September 12, 1997)). It is useful to note that these studies illustrate and describe the expression of immune and costimulators under the control of a constitutive promoter (eg, the CMV promoter).
[0056]
In the art, DNA-vaccines that target inducible APCs and methods to effectively enhance the maturation and stimulation lifetime of targeted APCs (eg, by targeting specific cells, or (Due to being active only in specific cells). The invention described herein achieves this goal (eg, by operably linking a polynucleotide encoding an immune factor to a promoter sequence from a gene encoding a costimulatory molecule; and / or Or by co-administering an immune factor and one or more cytokines that enhance the stimulatory life of the APC).
[0057]
More specifically, the present invention provides novel polynucleotides that are particularly useful as vaccines. Typically, the polynucleotide is contained in a vector (eg, a plasmid) that contains the appropriate regulatory elements. In one embodiment, a polynucleotide of the invention comprises a sequence encoding at least one selected antigen. Antigen expression is controlled by transcriptional regulatory elements (eg, promoters) from genes encoding costimulatory molecules (eg, CD80 or CD86). Without being bound by a particular theory, the use of promoter elements from costimulatory molecules may make use of the normal up-regulation of these promoters by APCs during activation and during antigen presentation. It is. Thus, use of the polynucleotides described herein enhances antigen expression, processing and presentation within the APC, as compared to, for example, use of a polynucleotide driven by a constitutive promoter.
[0058]
The invention also includes compositions wherein the expression vector comprising a costimulatory molecule promoter and a sequence encoding the antigen further comprises an adjuvant (eg, a cytokine). Preferably, the cytokine is, for example, by enhancing the immunostimulatory capacity of the APC, increasing the expression of costimulatory ligand on the APC surface, stabilizing the antigen / MHC complex, and / or Enhances the immune response by inhibiting apoptosis of Also included are methods of co-administering a polynucleotide bearing an antigen operably linked to a costimulatory molecule promoter with an adjuvant. The selected adjuvant can be provided in the form of a polynucleotide under appropriate regulatory control, or can be provided as a polypeptide (eg, a recombinantly produced polypeptide). When administered as nucleotides, the sequence encoding the cytokine and the sequence encoding the antigen may be maintained on the same vector or on different vectors. Thus, the sequence encoding the cytokine may be under the control of a promoter from the costimulatory molecule or under the control of a different promoter (eg, a constitutive promoter). Further, the sequence encoding the cytokine may be located either 3 'or 5' to the sequence encoding the antigen.
[0059]
The invention further includes a vaccine composition comprising an antigen in combination with at least one cytokine that enhances the stimulation or viability of dendritic cells. As noted above, such cytokines (eg, TRANCE, flt3, CD40L) can increase the expression of costimulatory molecules on the surface of APCs, stabilize antigen / MHC complexes, or reduce their apoptosis. May be prevented, thereby increasing the stimulation life of the APC. In particular, compositions comprising a polynucleotide encoding at least one antigen and a peptide cytokine, particularly a cytokine such as TRANCE, flt-3 or CD40L, are described. Another composition includes a composition comprising at least one antigen (eg, a peptide) and at least one cytokine (eg, TRANCE, flt-3 and / or CD40L). Still another composition comprises a polynucleotide encoding at least one antigen (eg, a peptide) and at least one cytokine (eg, TRANCE, flt-3 and / or CD40L). It is understood that more than one antigen can be used in combination with one or more cytokines.
[0060]
A polynucleotide of the invention can be introduced into a cell in vitro or in vivo, for example, by transfection or by coating the polynucleotide on the particle and administering the coated particle to the cell. Alternatively, the polynucleotides and / or peptides may be provided in the form of microparticles (eg, powders), as described below and in the disclosures of WO 97/48485 and WO 98/10750, which are hereby incorporated by reference. Considered enough.
[0061]
Accordingly, the invention includes a method of raising an immune response, preferably a CTL response, in a vertebrate subject by administering a polynucleotide encoding at least one selected antigen, wherein the method comprises the steps of: The sequence encoding the antigen is operably linked to the regulatory elements of the costimulatory molecule. Also provided is a method of eliciting an immune response in a vertebrate subject by co-administering the selected antigen with a cytokine such as TRANCE, flt-3L or CD40L.
[0062]
(antigen)
The compositions and methods described herein include, but are not limited to, a number of conditions, including cancer, allergies, toxicity and infection by pathogens such as viruses, bacteria, fungi, and other pathogenic organisms. No) is useful in eliciting an immune response to a wide variety of antigens for treatment and / or prevention.
[0063]
Suitable viral antigens for use in the present compositions and methods include, but are not limited to: hepatitis family viruses (hepatitis A virus (HAV), hepatitis B virus (HBV), C Antigens obtained or derived from hepatitis hepatitis virus (HCV), hepatitis delta virus (HDV), hepatitis E virus (HEV) and hepatitis G virus (HGV). See, for example, International Publication Nos. WO 89/04669; WO 90/11089; and WO 90/14436. The HCV genome encodes several viral proteins, including E1 and E2. See, for example, Houghton et al. (1991) Hepatology 14: 381-388. Nucleic acid molecules (including sequences encoding these proteins) and antigenic fragments thereof find use in the present methods. Similarly, the coding sequence for the δ antigen from HDV is known (see, eg, US Pat. No. 5,378,814).
[0064]
Similarly, a wide variety of proteins from the herpesvirus family can also be used as antigens in the present invention, including proteins from herpes simplex virus (HSV) types 1 and 2, such as HSV-1 and HSV-2 glycoproteins gB, gD and gH); antigens from varicella-zoster virus (VZV), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) (including CMV gB and CMV gH); and others (Eg, Chee et al. (1990) Cytomegaroviruses (ed. JK McDougall, Ed. Springer-Verlag, 125-169; McGeoch et al., (1)), which are derived from human herpesviruses (e.g., HHV6 and HHV7). 88) J. Gen. Virol. 69: 1531-1574; U.S. Patent No. 5,171,568; Baer et al. (1984) Nature 310: 207-211; and Davison et al. (1986) J. Gen. Virol. 1759-1816).
[0065]
Human immunodeficiency virus (HIV) antigens, such as the gp120 molecule for a number of HIV-1 and HIV-2 isolates, including members of various genetic subtypes of HIV, are known and reported. (Eg, Myers et al., Los Alamos Database, Los Alamos National Laboratory, Los Alamos, New Mexico (1992); and Modrow et al. (1987) J. Virol. 61: 570-578 alone). Antigens from the isolate or comprising the obtained nucleic acid sequence find use in the methods of the invention. In addition, any of a variety of HIV isolates (eg, one or more of various envelope proteins (eg, gp160 and gp41), gag antigens (eg, p24gag and p55gag), and HIV pol, env, tat, vif, rev, Other immunogenic portions (including proteins from the nef, vpr, vpu and LTR regions) find use herein.
[0066]
Antigens from or derived from other viruses also find use herein and include, but are not limited to, the Picornaviridae family (eg, poliovirus, rhinovirus, etc.). Caliciviridae; Togaviridae (eg, rubella virus, dengue virus, etc.); Flaviviridae; Coronaviridae; Reoviridae (eg, rotavirus); Orthomyxoviridae (eg, influenza virus A, Virnaviridae; Rhabodoviridae (eg, rabies virus); Filoviridae; Paramyxoviridae (eg, mumps virus, measles virus, RS) Virus, parainfluenza virus Bunyaviridae; arenaviridae; retroviridae (e.g., HTLV-1; HTLV-II; HIV-1 (also known as HTLV-III, LAV, ARV, hTLR, etc.)), which is isolated Goods HIV _IIIb , HIV _SF2 , HIV _LAV , HIV _LA1 , HIV _MN HIV-1 _CM235 , HIV-1 _US4 HIV-2; especially simian immunodeficiency virus (SIV); papillomavirus, tick-borne encephalitis virus and the like. For a description of these and other viruses, see, eg, Virology, 3rd Edition (WK Joklik, 1988); Fundamental Virology, 2nd Edition (BN Fields and DM Knipe, 1991). checking ...
[0067]
In some situations, the selected antigen is obtained or derived from a viral pathogen that typically enters the body through mucosal surfaces, and is subject to human disease (eg, HIV (AIDS), influenza virus (Flu ), Herpes simplex virus (genital infection, herpes simplex, STD), rotavirus (diarrhea), parainfluenza virus (respiratory infection), poliovirus (polio), respiratory syncytial virus (respiratory infection), measles virus and epidemic parotid gland Is known or associated with, but not limited to, the inflammation viruses (measles, mumps), rubella virus (rubella), and rhinovirus (common cold) May be desirable.
[0068]
Suitable bacterial and parasite antigens may be obtained or derived from known causative agents that cause the following diseases: Diphtheria, pertussis, tetanus, tuberculosis, bacterial pneumonia or fungal Pneumonia, otitis media, gonorrhea, cholera, typhoid fever, meningitis, mononucleosis plague, bacillary dysentery or salmonellosis, Legionaire's Disease, Lyme disease, leprosy, malaria, hookworm, onchocerciasis, Schistosomiasis, trypanosomiasis, Leshmaniasis, Giardia, amoebiasis, filariasis, Borrelia, and trypanosomiasis. Still further antigens include non-conventional pathogens (eg, Kourou, Creutzfeldt-Jakob disease (CJD), scrapie, causative agents of infectious encephalopathy of mink and chronic wasting disease), or prions such as mad cow disease. Can be obtained from or derived from natural proteinaceous infectious particles.
[0069]
Particular pathogens from which the antigen may be derived include: tuberculosis, Chlamydia, N.M. gonorrhoeae, Shigella, Salmonella, Vibrio Cholera, Treponema pallidua, Pseudomonas, Bordetella pertussis, Brucella, Franciscella tulorensis, Helicobacter pylori, Leptospria interrogaus, Legionella pneumophila, Yersinia pestis, Streptococcus (A and B type), Pneumococcus, Meningococcus, Hemophilus influenza ( b-type), Toxoplasma gondic, Compylobacteriosis, Moraxella cat rrhalis, Donovanosis, and Actinomycosis; as fungal pathogens, include Candidiasis and aspergillosis; Taenia as parasites pathogens, Flukes, Roundworms, Amebiasis, Giardiasis, Cryptosporidium, Schistosoma, Pneumocystis carinii, include Trichomoniasis and Trichinosis. Thus, the present invention also provides neuro-veterinary diseases (eg, foot and mouth diseases, coronavirus, Pasteurella multitocida, Helicobacter, Strongylus vulgaris, Actinobacillus pleuropneumonia, bovine viral diarrhea virus (BVeVela, Bovine Viral Diarrhea). Bordetella pertussis, Bordetella parapertussis and brochiseptica) can be used to provide an appropriate immune response.
[0070]
Typically, nucleotide sequences corresponding to one or more of the above-listed antigens are used in the production of polynucleotides, as shown below.
[0071]
(Isolation of Gene and Polynucleotide Constructs)
The invention provides a polynucleotide encoding at least one antigen (eg, an antigen from and / or expressed from a virus, bacterium, fungus, worm, toxin, allergic or cancer cell). The antigen is operably linked to a non-virus-specific, cell-specific or tissue-specific promoter (eg, a promoter from a regulatory element that controls transcription of a sequence encoding a costimulatory molecule). These polypeptides are useful in eliciting an immune response to the antigen, particularly in activating T lymphocytes.
[0072]
The nucleotide sequence selected for use in the present invention can be from a known source (eg, by isolating the same from a cell containing the desired gene or nucleotide sequence using standard techniques). . Similarly, nucleotide sequences can be produced synthetically using standard modes of polynucleotide synthesis well known in the art. See, eg, Edge et al. (1981) Nature 292: 756-762; Nambair et al. (1994) Science 223: 1299-1301; Jay et al. Biol. Chem. 259: 6311-6317. In general, synthetic oligonucleotides can be prepared using the phosphotriester method (described in Edge et al., Supra, and Duckworth et al. (1981) Nucleic Acids Res. 9: 1691-1706) or the phosphoramidite method (Beaucage et al. (1981) Tet. Letts. 22: 1859, and described in Matteucci et al. (1981) J. Am. Chem. Soc. 103: 3185). Synthetic oligonucleotides can also be prepared using commercially available automated oligonucleotide synthesizers. Thus, nucleotide sequences can be designed with the appropriate codons for a particular amino acid sequence. In general, one will select preferred codons for expression in the intended cell. The complete sequence is assembled from overlapping oligonucleotides by standard methods and assembled into the complete coding sequence. See, eg, Edge et al., Supra; Nambair et al., Supra, and Jay et al., Supra.
[0073]
Another way of obtaining a nucleic acid sequence for use herein is by recombinant means. Thus, the desired nucleotide sequence can be excised from a plasmid carrying the same using standard restriction enzymes and procedures. Site-specific DNA cleavage is performed by treatment with an appropriate restriction enzyme under conditions generally understood in the art, and details are specified by the manufacturer that markets the restriction enzyme. If desired, size separation of the cleaved fragments can be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques.
[0074]
Restriction cleavage fragments were prepared using standard techniques in the presence of four deoxynucleotide triphosphates (dNTPs) using standard techniques. The DNA can be blunt-ended by treating with a large fragment of E. coli DNA polymerase I (Klenow). The Klenow fragment is packed with a 5 'single stranded overhang, but digests the protruding 3' single stranded, even in the presence of four dNTPs. If desired, selective repair can be performed by supplying only one or several selected dNTPs within the limits dictated by the nature of the overhang. After Klenow treatment, the mixture could be extracted using, for example, phenol / chloroform and ethanol precipitated. Treatment under appropriate conditions with S1 nuclease or BAL-31 results in hydrolysis of any single-stranded portion.
[0075]
Yet another suitable method of isolating a specific nucleic acid molecule is by the polymerase chain reaction (PCR). Mullis et al. (1987) Methods Enzymol. 155: 335-350. This technique uses a DNA polymerase (a normal thermostable DNA polymerase) to replicate a region of the desired DNA. The region of this DNA to be replicated is identified by a specific sequence of oligonucleotides complementary to the opposite end and the opposite strand of the desired DNA that primes the replication reaction. The product of the first round of replication is its own template for subsequent replication, and thus repeated successive cycles of replication are performed by incorporating these added sequences into the oligonucleotide primers, Amplification occurs (see, eg, PCR Protocols, A Guide to Methods and Applications, Innis et al. (Eds.) Harcourt Brace Jovanovich Publishers, NY (1994)). The PCR conditions used for each amplification reaction are empirically determined. Many parameters affect the success of the reaction. Among them are annealing temperature and time, extension time, Mg2 + and ATP concentrations, pH, and relative concentrations of primers, templates, and deoxyribonucleotides. One example of suitable PCR conditions is found below in the Examples. Once the coding sequence for the desired protein has been prepared or isolated, such a sequence can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Ligations to other sequences are performed using standard procedures known in the art.
[0076]
As described in detail below, the selected nucleotide sequence can be placed under the control of regulatory sequences, such as a promoter, so that the sequence encoding the desired protein contains a vector comprising the expression construct. Transcribed into RNA in the transformed host tissue.
[0077]
(promoter)
The choice of promoter will be central to the construction of the particular polynucleotide described herein. Thus, the invention provides for the expression of a selected antigen driven by a non-viral, preferably mammalian cell (or tissue) -specific promoter. In a preferred embodiment, the promoter is from a regulatory sequence that controls transcription of the costimulatory molecule (eg, from the CD80 (also known as B7-1), CD86 (B7-2 and also known), CD40 or CD54 genes. promoter). Other suitable promoters can be readily determined using the teachings herein.
[0078]
Genomic constructs (including promoter mapping) of suitable costimulators (eg, CD80 and CD86) are described, for example, in Zhang et al. (1996) Gene 183: 1-6; Selvakumar et al. (1993) Immunogenetics 38: 292-295 and Fong et al. (1996) J. Amer. Immunol. 157: 4442-4450. These studies show that the CD80 (B7-1) gene promoter has three positive regulatory regions: a distal region from -2597 to -1555 containing a putative transcription factor binding site; a region near -130 to -110 containing tandem repeats. And a downstream region from +269 to +25 (Zhang et al., Supra). Cleavage at nucleotide positions -906 to -84 has been shown to result in increased transcriptional activity in CD80-expressing Raji cells, and regulatory elements around -41 are identified (Fong et al., Supra). In addition, mapping of the CD80 promoter or other costimulatory molecule promoter can be performed using methods known in the art in light of these references and the teachings herein.
[0079]
The invention also provides a method of eliciting an immune response using an immunizing agent in combination with at least one cytokine that enhances the stimulatory lifespan of APCs (eg, dendritic cells). In these embodiments, either the immunizing agent or the cytokine can be delivered to the subject as a polynucleotide encoding the polypeptide of interest. These polynucleotides include a wide variety of promoters (eg, constitutive promoters (eg, CMV, SV-40, housekeeping gene promoters, etc.), inducible promoters (eg, metallothionein), heat shock, cytochromes, protein tyrosines. Kinases, carbon monoxide synthesis promoters, etc.), and tissue or cell specific promoters (including, for example, CD80 / 86, muscle creatine kinase, etc.).
[0080]
In addition to the promoter, it may be desirable to add other regulatory sequences that can regulate the expression of the protein sequence encoded by the delivered nucleotide sequence. Suitable additional regulatory sequences are known to those of skill in the art and, by way of example, cause the expression of a coding sequence that is represented or not represented in response to a chemical or physical stimulus, including the presence of a regulatory compound. Sequences. Other types of regulatory elements may also be present in the vector, for example, an enhancer sequence.
[0081]
The expression vector is constructed so that the particular coding sequence is localized in the vector with the appropriate regulatory sequences, such that the positioning and orientation of the coding sequence with respect to the control sequence is based on the control sequence of the coding sequence. It allows transcription under "control" (ie, RNA polymerase binding to a DNA molecule at a control sequence transcribes the coding sequence). Modification of the sequence encoding the particular protein of interest may be desired to achieve this termination. For example, in some cases, it may be necessary to modify the sequence so that it is joined to the control sequence in the proper orientation (ie, to maintain the reading frame). Control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into the vector. Alternatively, the coding sequence can be cloned directly into an expression vector that already contains the control sequences and appropriate restriction sites.
[0082]
Generally, the nucleic acid molecules used in the subject methods include a coding region with appropriate control sequences and, optionally, ancillary nucleotide sequences encoding cytokines or other immune enhancing polypeptides. Nucleic acid molecules generally contain the necessary elements and are prepared in the form of a vector that directs transcription and translation in the recipient cell.
[0083]
(Adjuvant)
To enhance the immune response in a subject, the compositions and methods described herein may further include auxiliary substances (eg, adjuvants) (eg, pharmacological agents, cytokines, etc.). Suitable adjuvants include any substance that enhances a subject's immune response to a polynucleotide of the present invention. Non-limiting examples include cytokines (eg, Flt3 ligand, CD40L and TRANCE). As noted above, these cytokines may affect any number of pathways (eg, stabilizing the antigen / MHC complex, thereby making the antigen / MHC complex more present on the cell surface). Can enhance the immune response by increasing APC maturation or by prolonging APC survival (eg, inhibiting apoptosis). For example, recent studies suggest that CD40L and Flt3 ligand may be supplied adjuvanted in a mouse model (Gurunathan et al. (1998) J. Immunol. 161.4563 and Pulendran et al. (1998), J Immunol. 188. : 2075). Wong et al. (1997) J. Am. Exp. Med. 186: 2075 reports that TRANCE can promote the lifespan of mature dendritic cells. As noted above, these cytokines, delivered either as peptides or as polynucleotides encoding functional proteins, are also useful in eliciting an immune response.
[0084]
Ancillary substances can be administered simultaneously with, prior to, or subsequent to administration of a costimulatory molecule promoter-driven polynucleotide, as a protein or other macromolecule, a peptide antigen or a polynucleotide encoding the antigen of interest. Cytokines can be obtained from a variety of sources, such as Immunex (Seattle, WA), Genentech (South San Francisco, CA) and Amgen (Thousand Oaks, CA). Alternatively, cytokines can be produced using various methods known to those of skill in the art in light of the teachings herein. In particular, cytokines can be isolated directly from natural sources using standard purification techniques. Alternatively, cytokines can be produced recombinantly using the expression systems described above and purified using known techniques. The cytokine can also be synthesized via chemical polymer synthesis (eg, solid phase peptide synthesis) based on known amino acid sequences or amino acid sequences derived from the DNA sequence of the molecule of interest. Such methods are described, for example, in J.P. M. Stewart and J.M. D. Young, Solid Phase PeptideSynthesis, 2nd edition, Pierce Chemical Co. Rockford IL (1984); Barany and R.M. B. Merrifield, The Peptides: Analysis, Synthesis, Biology, author E.M. Gross and J.M. Meinenhofer, vol. 2, Academic Press, New York, (1980), pp. 254.
[0085]
Alternatively, an auxiliary nucleic acid sequence encoding a peptide (eg, a cytokine) known to stimulate, modify, or regulate the immune response of the host is co-administered as a polynucleotide with the polynucleotide or peptide antigen encoding the antigen described above. obtain. The gene sequences for many of these cytokines are known (eg, GenBank and other publicly available databases; Wong et al. (1997) J. Biol Chem 272 (40): 25190-4 (TRANCE); Lymanet et al. (1995) Oncogene 11 (6): 1165-72 (flt3); Spriggs et al. (1992) J. Exp. Med. 176.1543-1550 and Armitage et al. (1992) Nature 357: 80-82 (CD40L); Spriggs ( (1992) Immunol Ser. 56: 3-34 (TNF-α); Morgan et al. (1976) Science 193: 1007-1008 (IL-2); U.S. Patent No. 5,187,077 (LIF); hoff et al (1987) Immunol.139: 4116-4121 (IL-6); in that the reference, etc.).
[0086]
Thus, a suitable cytokine may be provided by administration of a polynucleotide encoding the cytokine (or encoding an active fragment thereof). These cytokine-encoding nucleotides can be administered either on the same vector carrying the antigen-encoding sequence, or on a different vector. In some cases, it may be desirable to design the polynucleotide under the control of both the antigen coding sequence and the cytokine coding sequence under the same promoter (eg, from a costimulatory molecule). In other cases, it may be desirable to use a cytokine coding sequence under the control of a different promoter (eg, a constitutive promoter).
[0087]
(Administration of polynucleotide and adjuvant)
The polynucleotides and auxiliary substances described herein can be administered by any suitable method. In a preferred embodiment, the polynucleotides are administered by coating them on particles and then administering the particles to a subject or cell, as described below. However, the polynucleotide may also be delivered using a viral vector known in the art, or using a non-viral vector system, for example, as described in US Pat. No. 5,589,466. Can be done.
[0088]
(Virus vector)
Numerous virus-based systems have been used for gene delivery. For example, retroviral systems are known and packaging systems with integrated defective proviruses ("helpers") that express all genes of the virus are commonly used, but the package known as the psi sequence is used. Cannot package its own genome due to the deletion of the packaging signal. Thus, this cell line produces an empty viral coat. The producer strain may be derived from a packaging strain that includes, in addition to helpers, a viral vector that contains the necessary sequences in cis for replication and packaging of the virus, known as long terminal repeats (LTRs). The gene of interest can be inserted into a vector and packaged into a viral envelope synthesized by a retroviral helper. The recombinant virus can then be isolated and delivered to a subject (see, for example, US Pat. No. 5,219,740). Representative retroviral vectors include, for example, LHL, N2, LNSAL, described in US Pat. No. 5,219,740, which is hereby incorporated by reference in its entirety. Vectors such as, but not limited to, the LSHL and LHL2 vectors, and derivatives of these vectors (eg, the modified N2 vectors described herein). Retroviral vectors can be constructed using techniques well known in the art. See, for example, U.S. Patent No. 5,219,740; Mann et al. (1983) Cell 33: 153-159.
[0089]
Adenovirus-based systems have been developed for gene delivery and are suitable for delivering the polynucleotides described herein. Human adenoviruses are double-stranded DNA viruses that enter cells by receptor-mediated endocytosis. These viruses are particularly suitable for gene transfer. Because they are easy to grow and manipulate, and they exhibit a wide host range in vivo and in vitro. For example, adenoviruses can infect human cells of hematopoietic, lymphoid, and bone marrow origin. In addition, adenovirus infects resting and replicating target cells. Unlike retroviruses, which integrate into the host genome, adenoviruses persist extrachromosomally, thus minimizing the risks associated with insertional mutagenesis. This virus is easily produced at high titers and is stable so that it can be purified and stored. Even in replication competent form, adenovirus causes only low levels of morbidity and is not associated with human malignancies. Therefore, adenovirus vectors that take advantage of these advantages have been developed. For a description of adenovirus vectors and their uses, see, eg, Haj-Ahmad and Graham (1986) J. Am. Virol. 57: 267-274; Bett et al. (1993) J. Am. Virol. 67: 5911-5921; Mittereder et al. (1994) Human Gene Therapy 5: 717-729; Seth et al. (1994) J. Am. Virol. 68: 933-940; Barr et al. (1994) Gene Therapy 1: 51-58; Berkner, K. et al. L. (1988) BioTechniques 6: 616-629; Rich et al. (1993) Human Gene Therapy 4: 461-476.
[0090]
Adeno-associated virus vectors (AAV) can also be used to administer the polynucleotides described herein. AAV vectors can be derived from any AAV serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, and the like. An AAV vector may have one or more AAV wild-type genes, preferably rep and / or cap genes, deleted in whole or in part, but with one or more functional adjacent inverted terminal repeats (ITRs). Keep the array. Functional ITR sequences are required for AAV virion rescue, replication and packaging. Thus, AAV vectors include at least those sequences required in cis for viral replication and packaging (eg, functional ITRs). The ITR sequence need not be a wild-type nucleotide sequence and can be altered, for example, by nucleotide insertion, deletion or substitution, as long as the sequence provides for functional rescue, replication and packaging.
[0091]
The AAV expression vector is constructed using known techniques, and provides at least control elements including a transcription initiation region, a target DNA, and a transcription terminal region as components operably linked in the transcription direction. A control element that is functional in a mammalian cell is selected. The resulting construct, comprising the operably linked components, is linked to a functional AAV ITR sequence (5 'and 3'). Suitable AAV constructs can be designed using techniques well known in the art. For example, U.S. Patent Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published January 23, 1992) and WO 93/03769 (published March 4, 1993); Lebkowski. (1988) Molec. Cell. Biol. 8: 3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B .; J. (1992) Current Opinion in Biotechnology 3: 533-539; Muzyczka, N.M. (1992) Current Topics in Microbiol. and Immunol. 158: 97-129; Kotin, R .; M. (1994) Human Gene Therapy 5: 793-801; Shelling and Smith (1994) Gene Therapy 1: 165-169; and Zhou et al. (1994) J. Am. Exp. Med. 179: 1867-1875.
[0092]
(Conventional pharmaceutical preparation)
Formulation of a preparation containing a polynucleotide of the present invention, with or without the addition of an adjuvant composition, can be performed using standard pharmaceutical formulation chemistry and methodology. All of these are readily available to those skilled in the art. For example, a composition comprising one or more nucleic acid molecules (eg, present in a plasmid or viral vector) is combined with one or more pharmaceutically acceptable excipients or vehicles to provide a liquid preparation. I can do it.
[0093]
Auxiliary substances such as wetting or emulsifying agents, pH interfering substances and the like can be present in excipients or vehicles. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not elicit an immune response in the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as, for example, water, saline, polyethylene glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts may also be included therein: mineral salts (eg, hydrochloride, hydrobromide, phosphate, sulfate, etc.); and salts of organic acids (eg, acetate, Propionate, malonic acid salt, benzoate, etc.). Although not required, if a peptide, protein or other molecule is to be included in the vaccine composition, it is also preferred that the preparation include pharmaceutically acceptable excipients, which in particular act as stabilizers for them. Examples of suitable carriers that also act as stabilizers for the peptide include, but are not limited to, the following pharmaceutical grades: glucose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran and the like. Other suitable carriers include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycol (PEG), and combinations thereof. . Pharmaceutically acceptable excipients, vehicles and auxiliary substances are available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ 1991), which is hereby incorporated by reference. .
[0094]
Certain facilitators of nucleic acid uptake and / or expression ("transfection facilitators") can also be included in the compositions (e.g., non-viral vector compositions (e.g., bupivacaine, cardiac toxin) enhancers such as cardiotoxin) and sucrose) and transfection-enhancing vehicles (eg, liposomal preparations conventionally used to deliver nucleic acid molecules). Anionic and neutral liposomes are widely available and are well known to deliver nucleic acid molecules (eg, Loposomes: A Practical Approach (1990) RPC New Edition, IRL Press). Cationic lipid lipid preparations are also well-known vehicles for use in delivering nucleic acid molecules. Suitable preparations include DOTMA (N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride), (trade name Lipofectin) ^TM And DOTAP (1,2-bis (oleyloxy) -3- (trimethylamino) propane) (see, for example, Felgner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7416). (1989) Proc. Natl. Acad. Sci. USA 86: 6077-6081; U.S. Patent Nos. 5,283,185 and 5,527,928, and International Publication Numbers WO90 / 11092, WO91 / 15001. And WO 95/26356). These cationic lipids can preferably be used in association with neutral lipids, such as DOPE (dioleyl phosphatidylethanolamine). Still further transfection-enhancing compositions that can be added to the lipid or liposome preparations include spermine derivatives (eg, WO 93/18759) and membrane osmosis such as GALA, gramicidin S and cationic bile salts. Compounds (for example, International Publication No. WO93 / 19768).
[0095]
Alternatively, the nucleic acid molecules of the invention can be encapsulated, adsorbed to, or bound to a particulate carrier. Suitable particulate carriers include polymethyl methacrylate polymers and particulate carriers derived from poly (lactide) and PLG microparticles derived from poly (lactide-co-glycolide). See, for example, Jeffery et al. (1993) Pharm. Res. 10: 362-368. Other particle systems and polymers may also be used (eg, polymers: for example, polylysine, polyarginine, polyornithine, spermine, spermidine, and conjugates of these molecules).
[0096]
Accordingly, formulated vaccine compositions typically include a polynucleotide (eg, a plasmid) containing a sequence encoding the antigen of interest in an amount sufficient to mount an immunological response. An appropriate effective amount can be readily determined by one skilled in the art. Such amounts fall into a relatively broad range that can be determined through routine testing. For example, an immune response can be obtained using as little as 1 μg of DNA, while in other administrations, up to 2 mg of DNA is used. Effective doses of polynucleotides comprising genomic fragments are generally expected to fall in the range of about 10 μg to 1000 μg, but doses above or below this range are also effective. Has been found. Thus, the composition may comprise from about 0.1% to about 99.9% of the polynucleotide molecule.
[0097]
(Administration of conventional pharmaceutical preparations)
Administration of the above pharmaceutical preparations can be effected continuously or intermittently in a single dose during the course of treatment. Delivery is most typically done via conventional needles and syringes for liquid compositions and for liquid suspensions containing particulate compositions. Additionally, various liquid ink injectors are known in the art and can be used to administer the compositions of the present invention. The most effective means and methods for determining the dosage of administration are well known to those of skill in the art, and will vary with the delivery vehicle, therapeutic composition, target cells, and the subject being treated. Single and multiple administrations can be carried out using dosage levels and patterns selected by the physician. It should be understood that one or more antigen sequences can be performed by the polynucleotide construct. Alternatively, separate vectors (eg, plasmid or viral vectors), each containing a sequence that expresses one or more antigens, can also be delivered to the subject as described herein.
[0098]
Further, it is also contemplated that polynucleotides that can be delivered by the methods of the invention can be combined with other suitable compositions and therapies. For example, to increase the immune response in a subject, the compositions and methods described herein may further include auxiliary substances such as pharmacological agents, cytokines, etc. (eg, adjuvants). Auxiliaries can be administered, for example, as a protein or other macromolecule, simultaneously with, prior to, or subsequent to administration of a polynucleotide described herein. The nucleic acid molecule composition can be administered directly to the subject, or can be administered ex vivo to cells from the subject using methods known to those of skill in the art.
[0099]
(Coated particles)
In one embodiment, the polynucleotide (eg, a DNA vaccine) and / or adjuvant is delivered using carrier particles (eg, a core carrier). Particle-mediated methods for delivering such nucleic acid preparations are known in the art. Thus, once prepared and appropriately purified, the nucleic acid molecules and / or adjuvants described above can be coated onto carrier particles (eg, a core carrier) using various techniques known in the art. The carrier particles are selected from materials having a suitable density in a range of particle sizes typically used for intracellular delivery from a suitable particle-mediated delivery device. The optimal carrier particle size will, of course, depend on the diameter of the target cell. Alternatively, colloidal gold particles can be used, where the coated colloidal gold is administered (eg, injected) to a tissue (eg, skin or muscle) and subsequently taken up by immunocompetent cells It is.
[0100]
For the purposes of the present invention, tungsten, gold, platinum and iridium carrier particles may be used. Tungsten and gold particles are preferred. Tungsten particles are readily available with an average size of 0.5-2.0 μm in diameter. While such particles have optimal densities for use in particle accelerated delivery methods and allow for highly efficient coatings using DNA, tungsten is potentially not available for certain cell types. Can be toxic. Gold particles or microcrystalline gold (eg, gold powder A1570 (available from Engelhard Corp., East Newark, NJ)) also find use in the present invention. Gold particles provide uniform size (available in particle sizes of 1-3 μm from Alpha Chemicals or in a range of particle sizes including 0.95 μm from Degussa, South Plainfield, NJ) and reduced toxicity. .
[0101]
Numerous methods are known and described for coating or precipitating DNA or RNA on gold or tungsten particles. The majority of such methods generally involve the transfer of a predetermined amount of gold or tungsten to plasmid DNA, CaCl ₂ And combined with spermidine. The resulting solution is vortexed continuously during the coating procedure to ensure uniformity of the reaction mixture. After precipitation of the nucleic acids, the coated particles can be transferred to a suitable membrane and dried prior to use, coated on the surface of a sample module or cassette, or a delivery cassette for use in a suitable particle delivery device Can be loaded.
[0102]
Peptide adjuvants (eg, cytokines) can be coated on suitable carrier particles (eg, gold or tungsten). For example, by simply mixing the two components in empirically determined ratios, by ammonium sulfate precipitation or other solvent precipitation methods known to those skilled in the art, or by chemically coupling the peptide to carrier particles. By means of the peptide can be attached to the carrier particles. The coupling of L-cysteine residues to gold has been described previously (Brown et al., Chemical Society Reviews: 271-311 (1980)). Other methods include, for example, dissolving the peptide antigen in absolute ethanol, water, or an alcohol / water mixture, adding this solution to the quantified carrier particles, and then flowing air or nitrogen while vortexing. Drying the mixture underneath. Alternatively, the peptide antigen can be dried on the carrier particles by centrifugation under reduced pressure. Once dried, the coated particles can be suspended in a suitable solvent (eg, ethyl acetate or acetone) and milled (eg, by sonication) to provide a substantially uniform suspension. I will provide a.
[0103]
(Administration of coated particles)
According to these formations, carrier particles coated with either a nucleic acid preparation or a peptide or protein adjuvant preparation are delivered transdermally to a subject using, for example, particle-mediated delivery techniques.
[0104]
A variety of particle delivery devices suitable for particle mediated delivery technology are known in the art and are all suitable for use in the practice of the present invention. Current device designs use explosive, electrical or gaseous firing to propel the coated carrier particles toward target cells. The coated carrier particles themselves can be releasably attached to a movable carrier sheet, or can be removably attached to a surface through which an air stream passes, lifting the particles off this surface and directing them to a target. Accelerate. An example of a gas firing device is described in U.S. Patent No. 5,204,253. Explosive devices are described in U.S. Pat. No. 4,949,050. One example of an electrically launched particle accelerator is described in US Pat. No. 5,120,657. Another electrical launcher suitable for use herein is described in U.S. Patent No. 5,149,655. The disclosures of all of these patents are incorporated herein by reference in their entirety.
[0105]
The coated particles are administered to the subject to be treated in a manner compatible with the dosage formulation, and in an amount effective to produce the desired immune response. Generally, when the nucleic acid molecule is in the range of 0.001 to 100.1 μg, more typically 0.01 to 10.0 μg, nucleic acid molecule per single dose, and the peptide or protein molecule is When between 1 μg and 5 mg, more typically between 1 and 50 μg, the amount of composition delivered depends on the subject being treated. The exact amount required will vary depending on the age and general condition of the individual to be immunized, and the particular nucleotide sequence or peptide chosen, as well as other factors. An appropriate effective amount can be readily determined by one of ordinary skill in the art in reading details instantly.
[0106]
Thus, an effective amount of an antigen, or nucleic acid code therefor, described herein is sufficient to produce an appropriate immune response in an immunized subject, and is determined through routine testing. Fall into a relatively wide range that can be done. Preferably, the coated particles are delivered to appropriate recipient cells to produce an immune response (eg, T cell activation) in the treated subject.
[0107]
(Particulate composition)
Alternatively, the antigen of interest (as well as one or more selected adjuvants) can be formulated as a particulate composition. More specifically, the formulation of particles containing the antigen and / or adjuvant of interest can be performed using standard pharmaceutical formulation chemistry, and all those methodologies will be readily apparent to those skilled in the art. Available. For example, one or more antigens and / or adjuvants can be combined with one or more pharmaceutically acceptable excipients or vehicles to provide an antigen, adjuvant, or vaccine composition. Auxiliary substances such as wetting or emulsifying agents, pH interfering substances and the like can be present in excipients or vehicles. These excipients, vehicles and auxiliary substances are generally pharmaceutical agents that do not elicit an immune response in the individual receiving the composition and can be administered without undue toxicity. Pharmaceutically acceptable excipients include, but are not limited to, liquids such as, for example, water, saline, polyethylene glycol, hyaluronic acid, glycerol and ethanol. Pharmaceutically acceptable salts may also be included therein: mineral salts (eg, hydrochloride, hydrobromide, phosphate, sulfate, etc.); and salts of organic acids (eg, acetate, Propionate, malonic acid salt, benzoate, etc.). Although not required, it is also preferred to include a pharmaceutically acceptable antigen composition that acts as a stabilizer, especially for peptides, proteins or other antigens. Examples of suitable carriers that also act as stabilizers for the peptide include, but are not limited to, the following pharmaceutical grades: glucose, sucrose, lactose, trehalose, mannitol, sorbitol, inositol, dextran and the like. Other suitable carriers include, but are not limited to, starch, cellulose, sodium or calcium phosphate, citric acid, tartaric acid, glycine, high molecular weight polyethylene glycol (PEG), and combinations thereof. . A thorough discussion of pharmaceutically acceptable excipients, carriers, stabilizers and auxiliary substances is incorporated herein by reference for REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., NJ. 1991).
[0108]
The formulated composition will contain an antigen of interest in an amount sufficient to mount an immunological response, as defined above. An appropriate effective amount can be readily determined by one skilled in the art. Such amounts fall within a relatively broad range, generally from 0.1 μg to 25 mg or more of the antigen of interest, and particular suitable amounts may be determined by routine testing. The composition comprises from about 0.1% to about 99.9% of the antigen. If an adjuvant is included in the composition, or if the method is tried to provide a particulate adjuvant composition, the adjuvant is present in an appropriate amount as described above. The composition can then be purified using standard techniques (eg, simple evaporation (air drying), vacuum drying, spray drying or lyophilization, spray-lyophilization, spray cooling, precipitation, suspension). Prepared as particles (eg, by liquid particle formation). If desired, the residual particles can be densified using the techniques described in International Application WO 97/48485, which is incorporated herein by reference.
[0109]
Using these methods, sizes ranging from about 0.1 to about 250 μm, preferably about 10 to about 150 μm, and most preferably about 20 to about 60 μm; and about 0.1 to about 25 g / cm ³ Particle density in the range of about 0.5 to about 3.0 g / cm ³ Nucleic acid particles having the above bulk density can be obtained.
[0110]
Similarly, a size in the range of about 0.1 to about 250 μm, preferably about 0.1 to about 150 μm, and most preferably about 20 to about 60 μm; about 0.1 to about 25 g / cm ³ Particle density in the range of about 0.5 to about 3.0 g / cm ³ And most preferably from about 0.8 to about 1.5 g / cm ³ Particles of the selected adjuvant having a bulk density can be obtained.
[0111]
Single or multi-dose containers, the particles of which can be packaged prior to use, contain a suitable amount of particles containing the antigen of interest and / or a selected adjuvant (eg, a vaccine composition). It may include a sealed container for inclusion. The particle composition can be packaged as a sterile formulation, and the sealed container can be designed to maintain sterility of the formulation until use in the methods of the present invention. If desired, the container can be adapted for direct use in a needleless syringe system, and can take the form of a capsule, foil pouch, sachet, cassette, and the like.
[0112]
The container in which the particles are packaged is further labeled to identify the composition, and provides clear dosage information. Further, the container may be affixed with a precautionary statement in the form prescribed by a government agency (eg, Food and Drug Administration), which may include an antigen, adjuvant (or vaccine) included for administration to humans. Composition) is approved by federal law.
[0113]
(Administration of particulate composition)
Following their formation, the particulate composition (eg, a powder) can be delivered transdermally to a vertebrate using a suitable transdermal delivery technique. Various particle delivery techniques suitable for administering the substance of interest are known in the art and find use in the practice of the present invention. A particularly preferred transdermal particle delivery system uses a needleless syringe to launch a controlled dose of solid particles into and through intact skin and tissue. See, for example, U.S. Patent No. 5,630,796 to Bellhouse et al., Which describes a needleless syringe (also known as a "PowderJect (R) particle delivery device"). Other needleless syringe configurations are known in the art and are described herein.
[0114]
The particulate composition (including the antibody of interest and, optionally, an adjuvant) is then delivered via a transdermal delivery technique. Preferably, the particulate composition is delivered via a powder injection method (e.g., a needleless syringe (WO 94/24263, WO 96/04947, WO 96/12513 and WO 96/20022, all of which are incorporated by reference). Is delivered from a needleless syringe) as described in)), incorporated herein by reference)). Delivery of particles from such particulate delivery devices is generally performed with particles having a size in the range of 0.1-250 μm, preferably in the range of about 10-70 μm. Particles larger than about 250 μm can also be delivered from the device, with the upper limit being that the size of the particles causes undesired damage to skin cells. The actual distance that the delivered particles penetrate the target surface was determined by the particle size (eg, nominal particle diameter considered as a nearly spherical particle profile), particle density, initial velocity at which the particles hit the surface, and targeted Depends on skin tissue density and kinematic viscosity. In this regard, the optimal particle density for use in needleless injection is generally about 0.1 g / cm ³ And 25g / cm ³ , Preferably about 0.9 g / cm ³ And 1.5g / cm ³ And injection speeds generally range between about 100 m / s and 3,000 m / s. With appropriate gas pressure, particles having an average diameter between 10 μm and 70 μm can be accelerated through the nozzle at a speed approaching the ultrasonic velocity of the driving gas stream.
[0115]
If desired, these particle delivery devices (eg, needle-free syringes) can be provided in a pre-filled form containing an appropriate dose of particles containing the antigen of interest and / or the selected adjuvant. . The filled syringe may be packaged in a hermetically sealed container, which may be further labeled as described above.
[0116]
A composition comprising a therapeutically effective amount of powder particles, as described herein, can be delivered to any suitable target tissue via the particle delivery device described above. For example, the composition comprises muscle, skin, brain, lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, cartilage, pancreas, kidney, gallbladder, intestine, ovary, uterus, rectum, nervous system, eye, It can be delivered to glands, and connective tissue. With respect to nucleic acid molecules, it is preferably delivered to differentiated cells for a period of time, and the molecule is expressed there; however, the molecule may be undifferentiated or partially differentiated cells (eg, blood cells). Stem cells and skin fibroblasts).
[0117]
The powdered composition is administered to the subject so as to be treated in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and / or therapeutically effective. The amount of composition to be delivered (generally, 0.5 μg / kg to 100 μg / kg of nucleic acid molecule per dose) depends on the subject to be treated. The dose can be as low as 0.5 μg for a 50 kg subject (ie, about 0.01 μg / kg). Administration of other drugs (eg, physiologically active peptides and proteins) will generally range from about 0.1 μg to about 20 mg, preferably 10 μg to about 3 mg. The exact amount required will vary depending on the age and general condition of the individual to be treated, the severity of the condition to be treated, the particular preparation delivered, the site of administration, and other factors. I do. An appropriate effective amount can be readily determined by one skilled in the art.
[0118]
Accordingly, a "therapeutically effective amount" of the particulate composition of the present invention is sufficient to achieve treatment or prevention of the disease or condition, and is a relatively broad range that can be determined through trials.
[0119]
(Vaccination method)
As will be apparent to one of skill in the art in light of the present specification, vaccines comprising the above-described polynucleotides (DNA vaccines) may be effective in a single dose, either continuously or intermittently throughout the course of this treatment. The most effective means and methods for determining the dosage of administration are well known to those of skill in the art, and will vary with the delivery vector, the nature of the composition, the particular therapy sought, the target cells, and the subject being treated. I do. Single and multiple administrations can be carried out with the dose level and pattern being selected by the appropriate physician. It should be understood that one or more antigens can be expressed by the delivered polynucleotide. Alternatively, individual vectors, each expressing one or more different antigens under the control of a costimulatory molecule promoter, can be delivered to a subject as described herein.
[0120]
It is further contemplated that the immunizing antigen (eg, polynucleotide or peptide) delivered by the methods of the invention will be combined with other suitable compositions and therapies. For example, a T cell response can be enhanced by delivering a polynucleotide or peptide described herein with one or more additional agents (eg, cytokines) before, after, or simultaneously with (1 A) a polynucleotide having a costimulatory molecule promoter that drives antigen expression; (2) a polynucleotide encoding at least one antigen or (3) a peptide antigen. These additional agents can be provided in various forms (eg, purified molecules) or by vectors encoding full-length or fractional fragments of the polypeptide. The vector can be different from the vector carrying the sequence encoding the antigen, or can be carried on the same vector. Whether the cytokine coding sequence is the same or a different vector, this expression can be driven by the same promoter or a different promoter that drives antigen expression. In this method, APC mature cytokines (eg, CD40 ligand (CD40L or CD154), tumor necrosis factor-related activity-inducing cytokine (TRANCE), and Flt3 ligand (flt-3L)) express costimulatory ligand on APCs. , By stimulating the antigen / MHC complex, or by inhibiting APC apoptosis, can obviously be provided to enhance the immune response.
[0121]
The following are examples of specific embodiments for carrying out the present invention. These examples are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.
[0122]
(Example)
Efforts have been made to ensure accuracy with respect to members used (eg, amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.
[0123]
Example 1: Nucleic acid immunization using CD80 promoter driven plasmid
The following studies were performed to identify and evaluate efficacy of nucleic acid immunity using DNA vaccine plasmids containing the CD80 or CD86 promoter.
[0124]
(A. Preparation of plasmid)
Mouse and human CD80 gene promoter DNA sequences were obtained from GenBank Database. DNA primers for synthesizing the mouse CD80 promoter by PCR were obtained from Life Technologies, Gibco BRL and had the following sequences:
(1) 5'-ACG CGT CGA CTC TAG AAG GAG ACA TTC AGC TG-3 '(SEQ ID NO: 1)
(2) 5′-ACG CGT CGA CAG CTT TCA TGG CCT AGC TGC TA-3 ′ (SEQ ID NO: 2)
(3) 5'-ATT CGG CCG CGG TCT AGA GCC AAT GGA GCT TAG G-3 '(SEQ ID NO: 3)
(4) 5′-ATT CGG CCG CGG AGA GTT CTG AAT CAG GGT GT-3 ′ (SEQ ID NO: 4).
[0125]
Similarly, DNA primers for synthesizing the human CD80 promoter by PCR were obtained from Life Technologies, Gibco BRL and had the following sequences:
(5) 5'-ACG CGT CGA CAG TCT TCC TCA TCC CAC CA-3 '(SEQ ID NO: 5)
(6) 5′-ACG CGT CGA CCA TCA CAC AGC AAG GCT AG-3 ′ (SEQ ID NO: 6)
(7) 5′-ACG CGT CGA CGT TTG TTA GTC CAT GCA CG-3 ′ (SEQ ID NO: 7)
(8) 5'-TCC CCG CGG AGA GAG GCG ACA TTT C-3 '(SEQ ID NO: 8).
[0126]
Fragments of the CD80 promoter (human and mouse) were combined with 200 μg of human or mouse gene DNA, 1 × Trbo ^TM Pfu buffer (Stratagene, La Jolla, CA, 20 mM Tris-Cl, pH 8.8, 2 mM MgSO ₄ , 10 mM KCl, 10 mM (NH ₄ ) ₂ SO ₄ , 0.1% Triton, 0.1 mg / ml nuclease-free BSA), 20 pm 5 ′ primer, 20 pm 3 ′ primer, 200 μm dNTP, 1U Perfect Match ^Tm PCR enhancer (StrataGene, La Jolla, CA) and 2.5U Pfu Turbo ^TM (Stratagene) was amplified by PCR by reacting 35 cycles of 96 ° C. (45 seconds); 55 ° C. (45 seconds) and 72 ° C. (45 seconds). After completion of 35 cycles, the PCR product was kept at 4 ° C. This PCR product is used for QLAquick ^Tm Purified by PCR purification kit (Qiagen Corporation) according to manufacturer's instructions.
[0127]
The purified PCR product was added to buffer # 3 (100 mM NaCl, 50 mM Tris-Cl, 10 mM MgCl ₂ Double digestion with Sal I (New England BioLabs) and Sac II (New England BioLabs) in 1 mM DTT, pH 7.9) at 37 ° C. overnight. The digested PCR product was run on a 1% agarose gel and the correct band was excised from the gel. The DNA in this gel slice was ^Tm Purification was performed using an ethidium bromide spin column (Supelco).
[0128]
A plasmid was constructed by removing the CMV promoter from plasmid pWRG7128 (Tacket et al. (1999) Vaccine 17: 2826) and replacing it with the amplified CD80 promoter segment. This plasmid, pWRG7128, contains, in addition to appropriate control elements, a sequence encoding the hepatitis B surface antigen (HBsAg), which is under the transcriptional control of the cytomegalovirus (CMV) promoter, and is transfected into most cell types. It was shown to produce HbsAg particles when transfected. This pWRG7128 plasmid was constructed as follows. The cloning vector pWRG7077 (Schmaljohn et al. (1997) J Virol. 71: 9563-9569) was digested with the HBsAg code by digesting the vector to completion with BamH1 followed by partial digestion with Hind3. Prepared to accept the sequence. After blunting the 5 'overhang by treatment with Klenow fragment and deoxyribonucleotides, this 4.3 kB vector fragment was isolated. The 1.35 kB HbsAg insert fragment (containing the untranslated pre-S2 sequence, the 226 amino acid HbsAg coding sequence of the adw strain, and the HBV enhancer element) was digested with BamH1 to give the plasmid pAM6 (ATCC, Rockford, MD). After blunting the ends by treatment with the Klenow fragment and deoxyribonucleotides, the fragment was isolated and ligated to the 4.3 kB vector fragment as described above. The resulting recombinant was screened for the proper origin of the insert, and the correct isolate was identified and named intermediate plasmid (pWRG7074). To remove the start of the codon of the X protein (located at the 3 'end of the pAM6 1.35 kB insert), the 4.86 kB vector fragment was digested with Bgl2 and the ends were blunted using Klenow fragment and deoxyribonucleotides. The pWRG7074 plasmid is isolated from the pWRG7074 plasmid by digestion with BstX1, the 754 bp insert fragment is digested with Nco1, treated with mung bean nuclease, and digested with BstX1 to obtain a single fragment from the pWRG7074 construct. Released. The resulting vector and insert fragment are then ligated together to form the analytical plasmid pWRG7128. This plasmid construct is shown in Table 1 and FIGS.
[0129]
(Table 1: Representative plasmids)
[0130]
[Table 1]

pWGR7128 was digested with Sal I and Sac II in buffer # 3. The digested vector is run on a 1% agarose gel, the correct vector band is excised from the gel, and GenElute ^Tm Purified using an ethidium bromide spin column.
[0131]
The promoter was ligated into the prepared vector, and 20 ng of the vector and 100 ng of the insert promoter were ligated to a 1 × T4 DNA ligase buffer (50 mM Tris-Cl, 10 mM MgCl 2) with 6 Wise unit T4 DNA ligand (New England Biolabs). ₂ , 10 mM DTT, 1 mM ATP, 25 μg / ml BSA, pH 7.8) at 15 ° C. overnight.
[0132]
The ligation mixture was diluted 1: 3 in saline for appropriately ligated fragments of the construct. 5 μl of the diluted mixture was added to 50 μl of MAX Efficiency DH5α Competent Cells ^TM (Gibco-BRL) and incubated on ice for 30 minutes. The transformed mixture was heat shocked at 42 ° C. for 45 minutes and quickly returned to a 2 minute incubation. One ml of SOC medium was added to the transformed cells and shaken at 37 ° C for 1 hour. After incubation, the cells were briefly centrifuged and plated on kanamycin-LB plates. The plate was incubated overnight at 37 ° C.
[0133]
To confirm ligation successfully, a single clone was picked and incubated in 3.0 ml of LB / Kan medium and cultured at 37 ° C. with shaking overnight. Plasmids were isolated from the medium, digested with Sal I and Sac II, and visualized on a 1% agarose gel stained with ethidium bromide.
[0134]
(B. Expression of antigen)
2 ml of B16 cells were added to 2 × 10 ⁵ Cells were seeded in 6-well plates per ml. The cells are reduced to 5% CO ₂ And incubated overnight at 37 ° C. to reach 60-80% confluency. Endotoxin-free plasmid DNA reduced in 2 μg serum medium (Gibco-BRL) (in 100 μl Opti-MEM®1) and lipofectin reduced in 10 μl serum medium (in 90 μl Opti-MEM® 1). And incubated separately for 45 minutes at room temperature. The two solutions were then mixed and incubated for 15 minutes at room temperature. During this incubation, B16 cells were washed three times in serum free medium. For each transfection, 0.8 ml of Opti-MEM® 1 with reduced serum medium was added to the mixed solution and the complex was plated on B16 cells. The cells were then incubated at 37 ° C. for 5 hours. Subsequently, 1.0 ml of 20% FBS medium was added. The medium was collected after 48 hours for antigen expression analysis.
[0135]
The expression of HBsAg in transfected B16 cells is shown in Table 2. The cells were treated in one of three ways: (1) transfection with a positive control (ie, pWRG7128) expressing the antigen (ie, HBsAg) in B16 cells; (2) Transfection with plasmids p5020 and p5021 and (3) no transfection (negative control).
[0136]
(Table 2. In vitro expression of HBsAg by transfecting cells with virus)
(Plasmid)
[0137]
[Table 2]

* Data are shown as average OD values (492.6 wavelength) for supernatants from two separate media and use the HBV Surface Antigen Kit (Abbott Laboratories Diagnostic Division (Auszyme Monoclonal, List No. 1980-24)). And analyzed.
[0138]
As shown in Table 2, unlike the positive control plasmid pWRG7128, no HBV surface antigen expression was detected in cultured B16 cells transfected with plasmids p5020 and p5021. Thus, the CD80 promoter does not drive expression of antigen in non-antigen presenting cells.
[0139]
(C. Preparation of coated microparticles)
Plasmid DNA was placed on 1-3 μm gold particles (Degussa Corp., South Plainfield, NJ) on Eisenbraun et al. (1993) DNA Cell Biol. 12: 791-797. Briefly, gold particles are suspended in 50 mM spermidine and mixed with an equal amount of plasmid in water. The solution is mixed on a vortex and half the volume of 1 M CaCl 2 of the gold / DNA mixture. ₂ Was dropped. The mixture was incubated at room temperature for 10 minutes and centrifuged to pellet the particles. The particles were washed three times with 100% ethanol and resuspended in ethanol containing 0.05-0.5% polyvinylpyrrolidone (PVP). The DNA-coated gold particles are then loaded into Tefzel® tubes as described in U.S. Pat. No. 5,584,807 to McCabe, which tubes are cut to 1.27 cm length and placed in PowderJect® (TM) XR-1 particle delivery device (PowerJect Vaccines, Inc. Madison, WI). Helium pulsed XR-1 particle delivery devices have been described previously (see, for example, US Pat. Nos. 5,584,807 and 5,865,796). At vaccination, each 1.27 cm cartridge contained 0.5 mg of gold particles coated with 2 μg of plasmid DNA.
[0140]
(D. Antibody response)
Based on the positive results found in the above in vitro transfection studies, vaccination studies were initiated using an in vivo particle mediated delivery method. Animal subjects receiving nucleic acid immunization in this study were: (1) a first experimental group of four mice vaccinated by particle-mediated delivery to the epidermis using the pWGR7128 positive control; and (2) the CD80 promoter A second experimental group of six mice vaccinated by particle-mediated delivery to the epidermis using the driven HBsAg plasmids p5020 and p5021 was included. Blood samples were collected from each animal before immunization (naive mice).
[0141]
Mice were immunized (primed) with plasmids p5020 and p5021 or control pWGR7128 by particle-mediated delivery (0.5 mg gold / shot coated with 2 μg DNA / mg gold) on gold particles. For immunity, the mice were shaved and the particles were delivered to the abdominal skin using an XR-1 device in a 500 psi helium-operated study barrel. Two shots were given to each mouse per immunization. Four weeks later, the mice were boosted according to the same immunization protocol used for prime immunization.
[0142]
Serum was collected from the animals before immunization, two and four weeks after priming, and two weeks after boost. Serum antibody levels were analyzed using the AUSAB EIA kit (Abbott Laboratories Diagnostics Division) according to the instructions provided by the manufacturer. Antibody reactivity to the hepatitis B virus (HBV) surface antigen was not detected in serum samples taken from animals immunized with plasmids p5020 and p5021, but two weeks after boost, serum antibody titers were It was> 3000 mlU / ml in mice immunized with pWGR7128 (Table 3).
[0143]
(Table 3. HBsAg-specific serum antibody titers in mice immunized with the CD80 plasmid)
[0144]
[Table 3]

^* Data are shown as mean HBV surface antigen antibody titers (mlU / ml) for the indicated groups of mice. Serum antibody titers were determined using an assay kit from Abbott Laboratories Diagnostics Division (AUSAB EIA, List 9006-24).
[0145]
Thus, the CD80 promoter driven plasmid does not cause an increase in the average antibody titer in serum.
[0146]
(E. Cell-mediated immune response)
Based on the positive results found in the serum antibody titer analysis described above, a study of cytotoxic T cell activity in immunized mice was performed. Cytotoxic T cell (CTL) activity was analyzed using splenocytes from immunized mice sacrificed two weeks after boost. As shown in FIG. 8, the CTL response elicited by immunization with plasmids p5020 and p5021 was more similar to the response elicited by plasmid pWGR7128. These responses were much greater than those found in splenocytes collected from naive mice.
[0147]
As a result of the above studies, it can be found that nucleic acid immunization provides a T cell-specific immune response when antigen expression is driven by a promoter derived from a costimulatory molecule. Furthermore, the T cell response is comparable to that found using the positive control.
[0148]
(Example 2: SIV immune response)
To determine whether the expression vector encoding the human CD80 promoter-driven HBsAg (hCD80-HBsAg) is expressed in monkey dendritic cells, the following study is performed. Monkey dendritic cells (DCs) were essentially purified by van der Meide et al. Med. Primatol. 24: 271-281, isolated from PBMC. The isolated DCs are then transfected with a plasmid encoding a human CD80 promoter-driven HBsAg essentially as described in Example 1 for B16 cells. Non-APC lines (eg, monkey COS cells) are similarly transfected. Expression of HBsAg in the supernatant and / or cells is performed by immunohistochemical staining.
[0149]
Studies are performed to determine the extent of HBsAg expression in APCs, taking into account cell-specific expression. The plasmid hCD80-HBsAg is delivered to the epidermis of monkey skin using a PowderJect® XR particle delivery device. Various additional epidermal sites will also be studied. Gold is used as a negative control, while CMV-HBsAg is used as a positive control. GM-CSF is administered to test dendritic cell recruitment. Biopsies are performed at the site of administration 24 or 48 hours later. Tissues are fragmented and HBsAg expression in dendritic cells is assessed by immunohistochemical staining using specific antibodies for HBsAg and dendritic cell surface markers. HBsAg expression in dendritic cells and Langerhans cells is assessed.
[0150]
An hCD80-SIV expression vector is constructed, for example, by replacing the sequence encoding HBsAg with the appropriate SIV coding sequence. Monkeys are immunized with the hCD80-SIV construct and compared to monkeys immunized with the CMV-SIV plasmid (positive control). Delivery of the plasmid is performed using a particle delivery device. Antigen expression, antibody response, and CTL activation are assessed essentially as described in Example 1 above. The monkeys are then challenged with pathogenic SIV and monitored for clinical expression of SIV.
[0151]
(Example 3: Use of cytokine adjuvant)
The following studies were performed to assess the ability of nucleic acids encoding TNF-related activation-inducing kinase (TRANCE) to enhance the immune response to co-administered antigen sequences.
[0152]
(A. Plasmid preparation)
The mouse TRANCE cDNA coding sequence was derived from the mRNA sequence (GenBank number AF0113170) and cloned into the insertion site of the pFLAG-CMV2 expression vector (Sigma, catalog number E4026), and the TRANCE coding sequence was under transcriptional control of the CMV2 promoter. The resulting expression construct was obtained. This plasmid construct was named pTRANCE.
[0153]
Plasmids containing sequences encoding hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) were constructed as follows. Both sequences encoding HBcAg and BHBsAg were obtained from the HBV clone pAM6 (ATCC Accession No. 45020). To create the HBsAg coding region, the pAM6 construct was cut with NcoI and treated with mung bean nuclease to remove the X antigen start codon. The resulting DNA is then cut with BamHI and treated with T4 DNA polymerase to make the DNA blunt-ended, creating an HBsAg expression cassette. The HBsAg expression cassette is present in a 1.2 kB fragment. The plasmid construct pPJV7077 (Schmaljohn et al. (1997) J. Virol. 71: 9563-9569) containing the full-length human CMV (Towne species) immediate early promoter (along with enhancers) was cut with HindIII and BglII, followed by T4 DNA polymerase and Treatment with bovine alkaline phosphatase generated blunt-ended DNA, and the HBsAg expression cassette was ligated into the plasmid to yield the pWRG7128 construct.
[0154]
To create the HBcAg coding region, the pAM6 construct was cut to create an HBcAg expression cassette, after which the HBcAg sequence was truncated by site-directed mutagenesis and the C-terminal arginine-rich region was removed from the core antigen particles ( This deletion does not interfere with particle formation). This truncated HBcAg sequence was then cloned into a plasmid construct containing the human elongation factor promoter ("hELF", Mizushima et al. (1990) Nucl. Acids Res. 18: 5322) to obtain an HBcAg vector construct.
[0155]
(A) CMV promoter / enhancer, intron A-5 'untranslated region, and human tissue plasminogen activator (hTPA) signal peptide ("CMV-IA-TPA"); or (b) bovine growth hormone polyA sequence Expression cassettes containing (bGHpA) were each obtained from the JW4303 vector construct (provided by Dr. Harriet Robinson of the University of Massachusetts) and inserted into the plasmid backbone. The resulting construct was cut with NheI, filled in with polymerase, and then cut with BamHI to create a vector fragment containing the pUC19 origin of replication, the ampicillin resistance gene and the bGHpA sequence. This plasmid backbone was cut a second time with SalI, filled with polymerase, and cut with BamHI to release vector fragments, including the CMV-IA-TPA vector fragment. The two vector fragments were ligated together to obtain a construct named pWRG7054.
[0156]
The pWRG7054 construct was cut with Nhel, filled in with polymerase, and cut with BamHI to create a vector fragment. The HBcAg vector construct was cut with NcoI, filled in with polymerase, and cut with BamHI to create an insert fragment. The two fragments were then ligated together to give a construct named pWRG7063.
[0157]
PEL-Bos was cut with EcoRI and dephosphorylated with bovine intestinal phosphatase to create a vector fragment. The pWRG7063 plasmid was cut with HindIII, filled in with polymerase, and cut with EcoRI to create an insert fragment containing the hTPA signal peptide, the HBcAg antigen sequence and the bGHpA region. These two fragments were ligated together to give a construct named pWRG7145.
[0158]
The pWRG7128 construct was cut with EcoRI and dephosphorylated with bovine intestinal phosphatase to create a vector fragment containing the HBsAg coding region under the transcriptional control of the hCMV promoter. This pWRG7145 construct was cut with MfeI and EcoRI to create an insert fragment containing the hELF promoter / intron, hTPA signal peptide sequence, HBcA antigen sequence and bGHpA region. These two fragments were then ligated together to yield the pPJV7193 plasmid construct containing the HBcAg and HBsAg coding sequences.
[0159]
(B. Vaccine preparation and immunization)
A panel of DNA vaccine compositions was organized using various combinations of the following DNA plasmids: pPJV7193 construct (encoding hepatitis B surface antigen and hepatitis B core antigen); pPJV7046 construct (empty DNA plasmid); And pTRANS constructs (encoding TRANCE cytokines). The final concentration of DNA in each vaccine composition was 2.0 μg / mg gold and the concentration of the antigen construct (pPJV7193) was kept constant in all compositions, while changing the concentration of the TRANCE construct I let it. The actual concentration of each construct present in the panel of DNA vaccine compositions is reported in Table 4 below.
[0160]
(Table 4. TRANCE DNA vaccine composition)
[0161]
[Table 4]

Plasmid DNAs (pPJV7193, pPJV7046 and pTANCE) were combined at the ratios reported in Table 4 above, and the plasmid mixture was then coated on 1-3 μm gold particles using the technique described in Example 1 above. A final concentration of 2 μg DNA / mg gold was obtained. The DNA-coated gold particles are then loaded into Tefzel® tubes as in Example 1 above and cut into lengths provided as cartridges for the PowderJect® XR particle delivery device. Each cartridge contained 0.5 mg of gold particles.
[0162]
Six experimental groups of four Balb / c mice were each assembled and immunized (prime) by particle-mediated delivery of plasmid DNA coated on gold particles using the DNA vaccine compositions listed in Table 4 above. did. For immunization, the mice were shaved and the particles were delivered to the abdominal skin in a single shot using a 500 psi helium-operated particle delivery device. The mice were sacrificed two weeks after immunization and the spleens were removed for ELISPOT analysis.
[0163]
(C. ELISPOT analysis)
ELISPOT filter plates were coated with rat anti-mouse IL-4 antibody at a concentration of 0.75 μg / well in 0.1 M carbonate buffer. The plate was incubated overnight at 4 ° C. After two washes with phosphate buffered saline (PBS), the plates were blocked with 100 μl of RPMI medium supplemented with 10% fetal bovine serum (FBS) for 1 hour at 37 ° C. This medium was discarded after blocking. 1 × 10 5 dissolved and supplemented with RPMI (supplemented with 10% FBS, sodium pyruvate, and non-essential amino acids) ⁷ Splenocytes with erythrocytes resuspended in cells / ml at 1 × 10 5 per well ⁶ 0.5 x 10 cells / well ⁶ Added at cell concentration. 100 μl of whole hepatitis B virus core antigen (BioDesign), diluted to 20 μg / ml in RPMI supplemented with 10% FBS, was added to the wells and the plates were incubated at 37 ° C. for 48 hours. After the incubation, the cells and medium were discarded and the plate was washed twice with PBS, followed by washing with deionized water to lyse any remaining cells, and then twice more with PBS. The detection antibody (biotinylated rat anti-mouse IL-4) was diluted to 1 μg / ml in PBS, and 50 μl was added to each well and incubated for 1 hour at room temperature. The plate was then washed 5 times with PBS and 50 μl of streptavidin alkaline phosphatase conjugate (1: 1000 dilution in PBS) was added to each well. After 1 hour incubation at room temperature, the plates were washed 5 times with PBS and 50 μl of chromagenic alkaline phosphatase substrate was added to each well. As soon as spots appeared, the reaction was stopped by rinsing the plate with tap water. Spots were counted and the results were graphed in FIG. 9 as specific spots per million splenocytes. As can be found by reviewing the data shown in FIG. 9, HBcAg-specific IL-4 production is increased compared to controls (compositions containing no pTRANCE) and dose-related to specific spots. Decreased.
[0164]
(D. Antibody analysis (ELISA))
The above study was repeated using a coated DNA particle cartridge prepared as described above, but with the following modifications: pPJV7193 construct (encoding hepatitis B surface antigen and hepatitis B core antigen) and pTRANCE. The construct (encoding the TRANCE cytokine) was coated on another batch of gold particles (pTRANCE and pPJV7046 plasmids were matched as described above and this combination was coated on gold particles, while pTRANCE plasmid was Coated on a batch of gold particles). The two batches of coated gold particles were then mixed together before encoding the Tefzel® tube. The same concentration of DNA as reported in Table 4 above was used with one exception, except that a 625: 1 ratio was not included in this second study. Each experimental group of four Balb / c mice was immunized as described above, and the mice were sacrificed two weeks after immunization, and sera were collected for antibody analysis by ELISA.
[0165]
For antibody analysis, ELISA plates were coated with 100 μl of whole hepatitis B virus core antigen (BioDesign), diluted to 0.1 μg / ml in PBS and incubated at 4 ° C. overnight. The plate was washed once with PBS with 0.05% Tween 20 (PBS-T) and then blocked with 300 μl of blocking solution (PBS and 5% dry milk) for 1 hour at room temperature. The blocking solution was discarded and 100 μl of serially diluted serum was added. The plate was incubated for 1 hour at room temperature, followed by three washes with PBS-T. The conjugated antibody (goat anti-mouse IgG-horseradish peroxidase) was diluted 1: 5000 in PBS and 2% dry milk. 100 μl of the conjugated antibody was added to each well and the plate was incubated for 1 hour at room temperature. The plate was then washed 5 times with PBS-T and 100 μl of TMB substrate was added to each well. After 15 minutes, the reaction was quenched with ₂ SO ₄ Stopped at The absorbance was read at 450 nm. The geometric mean of each experimental group was then calculated and plotted in FIG. 10 against dilution. As can be found by reviewing the data shown in FIG. 10, anti-HBcAg antibody response was increased in all groups receiving the pTANCE adjuvant composition, except for the composition containing a 25: 1 ratio.
[0166]
Thus, the addition of the pTRANCE cytokine encoding the plasmid construct in the hepatitis DNA vaccine composition enhanced both cellular and humoral immune responses to the antigen of interest.
[0167]
Accordingly, novel compositions that elicit immunity are described. Methods of using these compositions are also described. While the preferred embodiments of the invention will be described in some detail, it will be understood that obvious modifications can be made as defined by the appended claims without departing from the spirit and scope of the invention. .
[Brief description of the drawings]
FIG.
FIG. 1 is a schematic diagram of a CD80 promoter-driven expression vector p5020. This plasmid vector was constructed from pWRG7128 (mammalian expression vector) based on the pUC19 backbone. The CMV promoter was removed and replaced with a 254 bp PCR fragment obtained by amplification of the mouse CD80 promoter (obtained from Life Technologies, Gibco BRL). This plasmid also contains regulatory elements and the bovine growth hormone poly-A signal and is operably linked to the full-length cDNA encoding hepatitis B surface antigen.
FIG. 2
FIG. 2 is a schematic diagram of the CD80 promoter-driven expression vector p5021. This plasmid vector was constructed from pWRG7128 (mammalian expression vector) based on the pUC19 backbone. The CMV promoter was removed and replaced with a 489 base pair PCR fragment obtained by amplification of the mouse CD80 promoter (obtained from Life Technologies, Gibco BRL). This plasmid also contains regulatory elements and the bovine growth hormone poly-A signal and is operably linked to the full-length cDNA encoding hepatitis B surface antigen.
FIG. 3
FIG. 3 is a schematic diagram of the CD80 promoter-driven expression vector p5022. This plasmid vector was constructed from pWRG7128 (mammalian expression vector) based on the pUC19 backbone. The CMV promoter was removed and replaced with a 3123 base pair PCR fragment obtained by amplification of the mouse CD80 promoter (obtained from Life Technologies, Gibco BRL). This plasmid also contains regulatory elements and the bovine growth hormone poly-A signal and is operably linked to the full-length cDNA encoding hepatitis B surface antigen.
FIG. 4
FIG. 4 is a schematic diagram of the CD80 promoter-driven expression vector p5023. This plasmid vector was constructed from pWRG7128 (mammalian expression vector) based on the pUC19 backbone. The CMV promoter was removed and replaced with a 3357 base pair PCR fragment obtained by amplification of the mouse CD80 promoter (obtained from Life Technologies, Gibco BRL). This plasmid also contains regulatory elements and the bovine growth hormone poly-A signal and is operably linked to the full-length cDNA encoding hepatitis B surface antigen.
FIG. 5
FIG. 5 is a schematic diagram of the CD80 promoter-driven expression vector p5024. This plasmid vector was constructed from pWRG7128 (mammalian expression vector) based on the pUC19 backbone. The CMV promoter was removed and replaced with a 578 base pair PCR fragment obtained by amplification of the mouse CD80 promoter (obtained from Life Technologies, Gibco BRL). This plasmid also contains regulatory elements and the bovine growth hormone poly-A signal and is operably linked to the full-length cDNA encoding hepatitis B surface antigen.
FIG. 6
FIG. 6 is a schematic diagram of the CD80 promoter-driven expression vector p5025. This plasmid vector was constructed from pWRG7128 (mammalian expression vector) based on the pUC19 backbone. The CMV promoter was removed and replaced with a 202 base pair PCR fragment obtained by amplification of the mouse CD80 promoter (obtained from Life Technologies, Gibco BRL). This plasmid also contains regulatory elements and the bovine growth hormone poly-A signal and is operably linked to the full-length cDNA encoding hepatitis B surface antigen.
FIG. 7
FIG. 7 is a schematic diagram of the CD80 promoter-driven expression vector p5026. This plasmid vector was constructed from pWRG7128 (mammalian expression vector) based on the pUC19 backbone. The CMV promoter was removed and replaced with a 294 base pair PCR fragment obtained by amplification of the mouse CD80 promoter (obtained from Life Technologies, Gibco BRL). This plasmid also contains regulatory elements and the bovine growth hormone poly-A signal and is operably linked to the full-length cDNA encoding hepatitis B surface antigen.
FIG. 8
FIG. 8 is a graph showing the cytotoxic T cell (CTL) response elicited in mice immunized with a plasmid encoding hepatitis B antigen (HBsAg) under the control of the CMV promoter or the CD80 promoter.
FIG. 9
FIG. 9 is a histogram showing anti-hepatitis B core antigen-specific IL-4 production in splenocytes from mice immunized with a plasmid encoding hepatitis B core / surface antigen and a TRANCE cytokine adjuvant.
FIG. 10
FIG. 10 is a graph showing anti-hepatitis B core antigen-specific antibody production in mice immunized with the plasmid encoding hepatitis B core / surface antigen and the TRANCE cytokine adjuvant.

Claims (38)

A polynucleotide comprising a first promoter from a gene encoding a costimulatory molecule and a first sequence encoding at least one antigen, wherein the first sequence activates the first promoter. A polynucleotide, operably linked. The polynucleotide according to claim 1, wherein the promoter is derived from a CD80 (B7-1) gene. The polynucleotide according to claim 1, wherein the promoter is derived from a CD86 (B7-2) gene. The polynucleotide of any one of claims 1 to 3, further comprising a second sequence encoding at least one cytokine operably linked to the first promoter. 4. The polynucleotide of any one of claims 1-3, further comprising a second sequence encoding at least one cytokine and a second promoter, wherein the second sequence is A polynucleotide operably linked to a second promoter. The polynucleotide of claim 5, wherein said second promoter is a constitutive promoter. The polynucleotide according to any one of claims 4 to 6, wherein the cytokine is from the group consisting of CD40 ligand (CD40L), tumor necrosis factor-related activation-inducing cytokine (TRANCE), and Flt3 ligand. The polynucleotide to be selected. A core carrier coated with the polynucleotide according to claim 1. 9. The core carrier of claim 8, wherein said carrier is comprised of gold. A dosing container suitable for use in a particle delivery device, said dosing container comprising a coated carrier according to claim 8 or 9. A particle delivery device filled with a coated carrier according to claim 8 or 9. A pharmaceutical composition comprising the polynucleotide of any one of claims 1 to 7 and a pharmaceutically acceptable excipient. A pharmaceutical composition comprising the polynucleotide according to any one of claims 1 to 3, a pharmaceutically acceptable excipient and a cytokine. 14. The pharmaceutical composition according to claim 13, wherein the cytokine is selected from the group consisting of CD40L, tumor necrosis factor-related activation-inducing cytokine (TRANCE) and Flt3 ligand. Use of a nucleotide sequence encoding an antigen operably linked to a promoter from a gene encoding a costimulatory molecule, in the manufacture of a medicament for raising an immune response in a vertebrate subject, wherein the nucleotide sequence comprises Use wherein the promoter is capable of directing the expression of the antigen in the subject. 16. Use according to claim 15, wherein the costimulatory molecule is CD80 or CD86. 17. Use according to claim 15 or 16, wherein the nucleotide sequence further encodes at least one cytokine for the subject. 17. Use according to claim 15 or 16, wherein said nucleotide sequence and at least one cytokine are co-administered to said subject. 19. The use according to claim 17 or 18, wherein the cytokine is selected from the group consisting of CD40L, tumor necrosis factor-related activation-inducing cytokine (TRANCE) and Flt3 ligand (flt-3L). 20. Use according to any one of claims 15 to 19, wherein the nucleotide sequence is coated on a core carrier particle suitable for administration to the subject using a particle mediated transdermal delivery technique. 21. Use according to claim 20, wherein the core carrier particles consist of gold. 22. The use according to any one of claims 15 to 21, wherein the administration of the coated carrier particles is repeated to provide a prime administration and a booster administration. A vaccine composition, comprising:
(A) an expression vector comprising a polynucleotide encoding at least one antigen; and (b) a CD40 ligand (CD40L), a tumor necrosis factor-related activation-inducing cytokine (TRANCE), and a Flt3 ligand (flt-3L). At least one cytokine,
A vaccine composition comprising: A vaccine composition, comprising:
(A) at least one peptide antigen; and (b) poly-encoding at least one cytokine selected from CD40 ligand (CD40L), tumor necrosis factor-related activation-inducing cytokine (TRANCE) and Flt3 ligand (flt-3L). An expression vector comprising a nucleotide,
A vaccine composition comprising: A vaccine composition, comprising:
(A) at least one peptide antigen; and (b) at least one cytokine selected from CD40 ligand (CD40L), a tumor necrosis factor-related activation-inducing cytokine (TRANCE) and Flt3 ligand (flt-3L);
A vaccine composition comprising: 24. The vaccine composition according to claim 23, wherein said expression vector and / or said at least one cytokine are coated on a core carrier. 25. The vaccine composition according to claim 24, wherein the expression vector and / or the at least one peptide antigen is coated on a core carrier. 26. The vaccine composition according to claim 25, wherein said at least one peptide antigen and / or said at least one cytokine are coated on a core carrier. 29. A dosing container suitable for use in a needleless syringe, wherein the dosing container comprises a coated core carrier according to any one of claims 26-28. 29. A particle delivery device filled with the coated core carrier of any one of claims 26-28. 26. Use of a vaccine composition according to any one of claims 23 to 25 in the manufacture of a medicament for raising an immune response in a vertebrate subject. 32. The use of claim 31, wherein the vaccine composition is coated on core carrier particles suitable for administration to the subject using a particle mediated transdermal delivery technique. 33. Use according to claim 32, wherein the core carrier particles are comprised of gold. 34. Use according to any one of claims 32 or 33, wherein the administration of the coated particles is repeated to provide a prime administration and a booster administration. A method for raising an immune response in a vertebrate subject, the method comprising:
(A) providing a nucleotide sequence encoding an antigen operably linked to a promoter from a gene encoding a costimulatory molecule, wherein the promoter can direct expression of the antigen in the subject; And (b) administering the nucleotide sequence to the patient, whereby the antigen is expressed in an amount sufficient to elicit an immune response.
A method comprising: A method for raising an immune response in a vertebrate subject, the method comprising:
(A) providing a core carrier particle coated with a nucleotide sequence encoding at least one antigen, wherein the nucleotide sequence is operably linked to a promoter from a gene encoding a costimulatory molecule; Wherein said promoter is capable of driving expression of an antigen coding sequence in said subject; and (b) administering said coated core carrier particles to said subject using particle mediated delivery techniques. Wherein the antigen is expressed in an amount sufficient to elicit an immune response,
A method comprising: A method for raising an immune response in a vertebrate subject, the method comprising:
(A) providing a vaccine composition according to any one of claims 23 to 25; and (b) administering the composition to the subject in an amount sufficient to elicit an immune response. ,
A method comprising: A method for raising an immune response in a vertebrate subject, the method comprising:
(A) providing a vaccine composition according to any one of claims 26 to 28; and (b) administering the composition of step (a) to the patient using particle mediated delivery technology. Process,
A method comprising:

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