JP5894528B2 - 抗原−ノロウイルスpドメインモノマーおよびダイマー、抗原−ノロウイルスp粒子分子、ならびにそれらの製造および使用法 - Google Patents
抗原−ノロウイルスpドメインモノマーおよびダイマー、抗原−ノロウイルスp粒子分子、ならびにそれらの製造および使用法 Download PDFInfo
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Description
本発明は、合衆国政府の支援の下になされ、National Institute of Health(NIH)、National Institute of Allergy and Infectious(認可番号R01 AI37093およびR01 AI055649)およびDepartment of Defense(認可番号PR033018)によってXi Jiangへと表彰された。この研究はUniversity of CincinnatiおよびCincinnati Children's Hospital Medical Center(Center for Clinical and Translational Science and TrainingからのT1助成金)によってMing Tanへも支援された。さらに、Ming TanへのNipert Foundationが出資する感染症助成金がこの研究に寄与した。合衆国政府は本発明についての一定の権利を保有しうる。
本発明は、一般に、ノロウイルス(NOR)カプシドタンパク質のPドメインおよびNOR P粒子、抗原−ノロウイルスPドメインモノマー、抗原−ノロウイルスPドメインダイマーおよび抗原−ノロウイルスP粒子分子の製造、ならびに抗ウイルス剤、薬物送達系およびワクチン開発のためのそれらの使用に関する。
生体材料および生物工学は、現代医学の重要な部分となりつつある急速に成長している分野である。ウイルス性タンパク質ケージは、その万能性およびアレイを形成する傾向のため、提示系を構築するための理想的な基板である。遺伝子操作を通じて、自己集合性ウイルス構造タンパク質は、生体材料合成のための傑出したプラットフォームであり、そして設計されたパターンで外来分子を統合するためのスキャフォールドであり、これはワクチンまたは薬物として使用できる。
本発明は、NOR Pドメインモノマーの遠位部分が外来抗原、特に外来ウイルス性抗原を挿入できるペプチド鎖を含むという発見に関する。得られる抗原−Pドメインモノマーは、12個のダイマーに組織される24個の抗原−Pドメインモノマーからなる、典型的には八面体形態の、抗原−P粒子と称される、ナノ粒子に自然発生的に集合しうる。このP粒子は大腸菌において容易に製造され、極めて安定であり、免疫原性が高い。Pドメインモノマーあたり3個の表面ループが存在し、これは粒子あたり合計72個のループとなり、免疫増進のための外来抗原提示の潜在的部位である。抗原−P粒子は自然発生的に形成され、そしてPドメインモノマーの遠位部分で提示される外来抗原を有する。この抗原−P粒子形成プロセスは、抗原−Pダイマーの濃度に基づいて交換し、または動的に平衡化しうる。抗原−P粒子は、外来ウイルス性抗原に対する抗体を産生し、またかかる外来ウイルス感染のためのワクチンを作製するために有用である。
本明細書において使用するとき、用語「ノロウイルス」、「NOR」、「ノーウォーク様ウイルス」または「NLV」は、ノロウイルスファミリーの任意のウイルスを意味し、限定されないが次のものを含む:ノーウォークウイルス(NV)、MOH、Mexico、VA 207、VA 387、02−1419、C59、VA 115、Hawaii、Snow Mountain、Hillington、Toronto、Leeds、Amsterdam、Idaho Falls、Lordsdale、Grimsby、Southampton、Desert Shield、BirminghamおよびWhite Rivercap。
キメラP粒子の調製。予め製造した[P]タイプ8株のヒトVP8を含むRV VP8−P粒子キメラ(図8、図10のパネルE)を用いる。マウスRVチャレンジ試験のためのマウスVP8を含むキメラワクチンのために、VP8−P粒子キメラのヒトVP8をマウス(EDIM)VP8で置換する。ヒトおよびマウスRVキメラP粒子はいずれも、確立された手法を用いて大腸菌で製造される。GSTアフィニティーカラム(Glutathione Sepharose 4 Fast Flow、HE Healthcare)による部分的精製の後、キメラP粒子はさらにサイズ排除カラムを用いたFPLC系(GE Healthcare)で精製される。得られたキメラP粒子調製物は、高い純度(〜95%)に達し、マウスを免疫する免疫源として使用される。図8に見られるとおり、SDS PAGEのパネルAは、アフィニティーカラム(レーン1)およびさらにP粒子(レーン2)およびPドメインダイマー(レーン3)フラクションでのゲルろ過(パネルD)によって精製された、組換えRV VP8−Pドメインキメラ(53.5kDa)を示す。パネルBおよびCは、VP8(パネルB、レーン4および5)またはNOR VLP(パネルC、レーン6および7)に対する抗体を用いた、VP8−P粒子キメラのウェスタン分析を示す。パネルDにおいて、アフィニティー精製VP8−Pドメインキメラ(レーン1)のゲルろ過は、該タンパク質の大部分がP粒子(〜1300kDa)を形成することを示す。パネルEは、被覆VP8−P粒子キメラ(PP−hVP8C)、組換えVP8(hVP8C)およびP粒子のみ(PP、コントロール)を検出するためにVP8に対する抗体を用いた、酵素免疫アッセイ(EIA)の結果を示す。レーンMは、上から113、92、52、35、29kDaのバンドで、予め染色したタンパク質マーカーを示す。図10は、RV VP8−P粒子キメラの製造を示す。ループ2にクローニングカセットを有するP粒子ベクター(A)は野生型NOR P粒子(B)を製造する。RV VP8コード配列(C)がP粒子ベクター(D)にクローン化されると、それはキメラの最も外側表面でのRV VP8の指標と共に、RV VP8−P粒子キメラ(E)を製造する。
Hisタグを含むキメラP粒子の製造。我々の研究は、P粒子のループ2に小さなエピトープ、ポリヒスチジン(His)タグを挿入することで開始した(図3および14)。GST遺伝子融合系を用いた大腸菌におけるこのPタンパク質の発現および精製は、高収率(>10mg/リットル培養)で予測したサイズ(〜35kDa)を有するタンパク質が得られ、次いでトロンビンでGSTタグを除去するために(〜27kDa)タンパク質を消化した(図14C)。ゲルろ過クロマトグラフィー、次いでSDS PAGEおよびウェスタンブロット分析によってキメラPタンパク質のP粒子形成が示され、ここでキメラP粒子は〜840kDaの主要なピークを形成した(図14DおよびE、データは示さず)。キメラP粒子上での挿入したHisタグの露出は、Talon樹脂へのその特異的結合、続く250mMのイミダゾールによる溶出によって示され(図7A、溶出1および2)、これは高純度のキメラP粒子をもたらした。したがって、P粒子−Hisタグキメラはまた、アフィニティーTalon樹脂を用いて大腸菌からも精製できる(データは示さず)。
Claims (18)
- 挿入された外来タンパク質またはポリペプチド抗原を含む少なくとも1個の表面ループを有するノロウイルス(NOR)Pドメインモノマーを含む、組換え抗原−Pドメインモノマーであって、該少なくとも1個の表面ループがループ2(T371−D374)を含む第一のループを含む、組換え抗原−Pドメインモノマー。
- NOR Pドメインモノマーがさらに第二のループおよび第三のループを含み、かつ、該少なくとも1個の表面ループがさらに該第二のループおよび該第三のループの1個以上を含む、請求項1の組換え抗原−Pドメインモノマー。
- NOR PドメインモノマーがNOR株VA387のPドメインであり、該第二のループがループ1(I293−H297)であり、該第一のループがループ2(T371−D374)であり、該第三のループがループ3(D391−N394)である、請求項1または2の組換え抗原−Pドメインモノマー。
- 挿入された外来タンパク質またはポリペプチド抗原がロタウイルス(RV)VP8抗原である、請求項1または2の組換え抗原−Pドメインモノマー。
- 挿入された外来タンパク質またはポリペプチド抗原が少なくとも2個の類似のまたは異なる抗原を含む、請求項1または2の組換え抗原−Pドメインモノマー。
- 該少なくとも2個の抗原が異なるものである、請求項5の組換え抗原−Pドメインモノマー。
- 24個のPドメインモノマーからなる抗原−P粒子であって、該24個のPドメインモノマーが、挿入された外来タンパク質またはポリペプチド抗原を含む少なくとも1個の表面ループを有するノロウイルス(NOR)Pドメインモノマーである少なくとも1個の組換え抗原−Pドメインモノマーを含み、ここで、該抗原−P粒子は抗原単独の免疫原性と比較して抗原の免疫原性を向上させうるものであり、該少なくとも1個の表面ループがループ2(T371−D374)を含む第一のループを含むものである、抗原−P粒子。
- 該24個のPドメインモノマーが、1個以上の野生型NOR Pドメインモノマーを含む、請求項7の抗原−P粒子。
- 異なる修飾をされた2個以上の組換え抗原−Pドメインモノマーを含む、請求項7または8の抗原−P粒子。
- 少なくとも1個の表面ループが3個のループであり、該3個のループの各々が、挿入された複数の異なる外来抗原を含む、請求項1の組換え抗原−Pドメインモノマー。
- 少なくとも1個の表面ループ中に、挿入された外来リガンドまたはシグナルペプチドをさらに含む、請求項1の組換え抗原−Pドメインモノマー。
- 挿入された外来リガンドまたはシグナルペプチドが、特定の臓器または組織における対応するレセプターを標的とすることができるものである、請求項11の組換え抗原−Pドメインモノマー。
- 挿入された外来シグナルペプチドが、5アミノ酸CNGRCである、請求項12の組換え抗原−Pドメインモノマー。
- 化学反応によって、表面露出リシンおよびシステインを介してP粒子のループに挿入されている薬物のコンジュゲートをさらに含む、請求項1の組換え抗原−Pドメインモノマー。
- 表面ループの少なくとも1個に挿入されたリガンドまたはシグナルペプチド、ならびに表面ループの少なくとも1個に挿入された薬物のコンジュゲートをさらに含み、それによって疾患を有する特定の組織または臓器に薬物を標的化する薬物送達系を提供する、請求項1の組換え抗原−Pドメインモノマー。
- NOR Pドメインモノマーの表面ループをコードする配列の中に少なくとも1対の制限部位を含む、組換えPドメインモノマーベクターであって、少なくとも1個の表面ループがループ2(T371−D374)を含む、組換えPドメインモノマーベクター。
- コードされるNOR Pドメインモノマーの表面ループが第一のループ、第二のループおよび第三のループを含み、かつ、該少なくとも1対の制限部位が、該第一のループ、該第二のループおよび該第三のループの少なくとも1つをコードする配列の中にある、請求項16の組換えPドメインモノマーベクター。
- 該配列にコードされる表面ループが、該少なくとも1個の表面ループの長さおよび露出を伸長させるスペーサーを含む、請求項16の組換えPドメインモノマーベクター。
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