JP2020533966A - Myceliophthora thermophila中でのフルワクチンの産生 - Google Patents
Myceliophthora thermophila中でのフルワクチンの産生 Download PDFInfo
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Abstract
Description
発現構築物
インフルエンザウイルス表面タンパク質
遺伝子操作C1
タンパク質の産生
ワクチン組成
実施例
実施例1−Myceliophthora thermophila(C1)中でのヘマグルチニン(HA)およびノイラミニダーゼ(NA)の発現
−cbh1またはchi1プロモーターを含み、GlaA担体タンパク質を含まないが、ERへの標的のためのcbh1シグナル配列を含む構築物。
−chi1プロモーターおよび担体タンパク質としてGla1(C1グルコアミラーゼ1)を含む構築物。
−担体タンパク質としてEG2(エンドグルカナーゼ2)を含む構築物。
−hex1プロモーター、GlaA担体タンパク質を含み、FLAGタグを含まない構築物。
材料および方法
HAタンパク質標準
C1株
HCは、C1高セルラーゼ株を指し、LCはC1低セルラーゼ株を指す。Prt−はプロテアーゼ欠損、Δalp1は主要な分泌プロテアーゼの遺伝子破壊、Δalp2は高度に発現した液胞プロテアーゼの遺伝子破壊、およびΔchi1はC1中での高度に発現した細胞外C1キチナーゼの遺伝子破壊を指す。すべての株はΔpyr5である。
第1シリーズの構築物の生成
EcoRV−EcoRIで消化したpVJ1(Pcbh1_glaA_kex2_GeneX_Tcbh1)。
EcoRVはkex2部位に位置し、EcoRIはGeneXの停止コドン直後に位置する。
−HA_NC_FLAG−タグxEcoRV−EcoRI(1577bp)
−HA_UR_FLAG−タグxEcoRV−EcoRI(1586bp)
−HA_FL_FLAG−tタグxEcoRV−EcoRI(1655bp)
−HA_NCxEcoRV−EcoRI(1553bp)
EcoRV−EcoRIで消化したpVJ6(Pchi1_glaA_kex2_目的遺伝子_Tcbh1)。
EcoRVはkex2部位に位置し、EcoRIは目的遺伝子の停止コドンの直後に位置する。
(すべての合成断片を、pMKまたはpMSシャトルベクターにGeneArt(登録商標)によってサブクローン化した)。
−HA_NC_FLAG−タグx EcoRV−EcoRI(1577bp)
−HA_UR_FLAG−タグx EcoRV−EcoRI(1586bp)
−HA_FL_FLAG−タグx EcoRV−EcoRI 1655bp)
−HA_NC x EcoRV−EcoRI(1553bp)
*cbh1プロモーターまたはchi1プロモーターを有し、GlaA担体タンパク質を含まないが、ERへの標的化のためのcbh1シグナル配列を含むベクター。このベクターを、次のように生成した:
第3シリーズの構築物の生成
形質転換およびマイクロタイタープレート(MTP)培養
振盪フラスコ培養条件
スポットブロット分析
SDS−PAGEおよびウエスタン分析
2Dゲル電気泳動
赤血球凝集アッセイ
EG2活性アッセイ
BCAアッセイ
分泌されたHAの精製
TMD含有HAの精製
脱グリコシル化実験
In vitro KEX2処理
トリプシン消化
菌糸体抽出物
真菌プロトプラストの調製
実施例2−Myceliophthora thermophila(C1)中で産生されたHAの免疫原性
材料および方法
試験対象組成物
状態:SPF
株:Balb/c ByJマウス
供給業者:Charles River
年齢:D0で9週齢
性別:雌
体重:20〜22g
着色による個体の識別
ケアおよび保守:毎日
ハウジング
場所:動物4匹/すべての群用のケージ/L2バイオセーフティ要件に準拠する空気調整された建物内のケージ
動物/ケージの数:4
食餌:粒状食品(M20、SDS、DIETEX France、St Gratien、France)
水:水道水、自由に、自動給水システムを介する
検疫:N/A
順応:本検査に割り当てられたマウスを、免疫付与の前に5日間、設計されたハウジングに順応させた。
群の定義
群の定義を表4に要約する。
検査スケジュールを、表5に詳述する。
生物学的サンプリング
赤血球凝集抑制(HI)アッセイ
試薬:
−96ウェルV底プレート(NUNC)
−RDE(受容体破壊酵素):10mU/mLのコレラ菌III型由来ノイラミニダーゼ(Sigma)
−Ca++およびMg++非含有PBS(Gibco)
−TPCKトリプシン(Thermo Scientific)
−ニワトリ赤血球(「cRBC」):血清処理用にはPBS中で10%、HIアッセイ用にはPBS中で0.5%(Sanofi Pasteur)
−インフルエンザ株A/New Caledonia/20/99、透明尿膜腔液1900赤血球凝集単位(HAU)/50μL
観察および逸脱
結果
臨床モニタリング
体液性応答
実施例3−HAの撹拌タンク発酵
Claims (24)
- 少なくとも1つのC1調節配列に作動可能に連結されるインフルエンザウイルス表面タンパク質をコードする核酸配列を含む発現構築物を含む、前記インフルエンザウイルス表面タンパク質を産生するために遺伝子改変されたMyceliophthora thermophila C1であって、前記インフルエンザウイルス表面タンパク質が、その外部ドメインおよび膜貫通ドメインを含みかつ前記C1中で膜結合タンパク質として発現される、C1。
- 前記インフルエンザウイルス表面タンパク質が、ヘマグルチニン(HA)である、請求項1に記載のC1。
- 前記発現構築物が、前記HAをコードする前記核酸配列にインフレームで連結されるC1シグナルペプチドをコードする核酸配列をさらに含む、請求項2に記載のC1。
- 前記HA亜型が、インフルエンザA−H1、H2、H3、H4、H5、H6、H7、H8、H9、H10、H11、H12、H13、H14、H15およびH16、ならびにインフルエンザB亜型からなる群から選択される、請求項2に記載のC1。
- 前記HA亜型が、インフルエンザA亜型H1、H2、およびH3、ならびにインフルエンザB亜型から選択されるヒトに感染する亜型である、請求項2に記載のC1。
- 前記HAが、A/New Caledonia/20/99(H1N1)、A/California/04/2009(H1N1)、A/Uruguay/716/07(H3N2)(A/Brisbane/10/07−様)、B/Florida/04/2006:B Yamagata lineage、A/Puerto Rico/08/1934(H1N1)、およびA/Texas/50/2012(H3N2)からなる群から選択されるインフルエンザウイルス株に由来する、請求項2に記載のC1。
- 前記少なくとも1つのC1調節配列が、C1プロモーターを含む、請求項1に記載のC1。
- 前記C1プロモーターが、hex1(ウォロニンボディ)、cbh1(セロビオヒドロラーゼ1)およびchi1(キチナーゼ1)プロモーターからなる群から選択される、請求項7に記載のC1。
- 前記C1プロモーターが、hex1プロモーターである、請求項7に記載のC1。
- 前記C1シグナルペプチドが、Gla1(グルコアミラーゼ1、C1)、GlaA(グルコアミラーゼA、アスペルギルス)およびCbh1(セロビオヒドロラーゼ1、C1)からなる群から選択されるタンパク質に由来するシグナルペプチドである、請求項3に記載のC1。
- 前記C1シグナルペプチドが、Cbh1に由来する、請求項10に記載のC1。
- 前記発現構築物が、HAに融合されたCbh1シグナルペプチドをコードする核酸配列に作動可能に連結されるhex1プロモーターを含む、請求項1に記載のC1。
- 前記HAが、A/New Caledonia/20/99(H1N1)、A/California/04/2009(H1N1)、B/Florida/04/2006:B Yamagata lineage、A/Puerto Rico/08/1934(H1N1)、およびA/Texas/50/2012(H3N2)からなる群から選択されるインフルエンザウイルス株に由来する、請求項12に記載のC1。
- 前記インフルエンザウイルス表面タンパク質が、ノイラミニダーゼ(NA)である、請求項1に記載のC1。
- 前記NA亜型が、インフルエンザA−N1、N2、N3、N4、N5、N6、N7、N8およびN9ならびにインフルエンザB亜型からなる群から選択される、請求項14に記載のC1。
- 前記NA亜型が、インフルエンザA亜型N1およびN2ならびにインフルエンザB亜型から選択されるヒトに感染する亜型である、請求項14に記載のC1。
- 前記発現構築物が、NAをコードする核酸配列に作動可能に連結されるhex1プロモーターを含む、請求項14に記載のC1。
- 前記NAが、前記インフルエンザウイルス株A/New Caledonia/20/99(H1N1)由来である、請求項17に記載のC1。
- 前記C1株が、W1L#100I(prt−Δalp1Δchi1Δalp2Δpyr5)寄託番号CBS141153、UV18−100f(prt−Δalp1、Δpyr5)寄託番号CBS141147、W1L#100I(prt−Δalp1Δchi1Δpyr5)寄託番号CBS141149、およびUV18−100f(prt−Δalp1Δpep4Δalp2、Δprt1Δpyr5)寄託番号CBS141143からなる群から選択される、請求項1に記載のC1。
- インフルエンザウイルス表面タンパク質を産生するための方法であって、前記インフルエンザウイルス表面タンパク質を発現するのに好適な条件下で請求項1に記載のMyceliophthora thermophila C1を培養すること、および前記インフルエンザウイルス表面タンパク質を回収することを含む、方法。
- 前記インフルエンザウイルス表面タンパク質を回収することが、菌糸体からの抽出を含む、請求項20に記載の方法。
- Myceliophthora thermophila C1中で膜結合インフルエンザウイルス表面タンパク質を発現するための発現構築物であって、インフルエンザウイルス表面タンパク質をコードする核酸配列に作動可能に連結される少なくとも1つのC1調節配列を含み、前記インフルエンザウイルス表面タンパク質が、その外部ドメインおよび膜貫通ドメインを含む、発現構築物。
- 請求項1に記載の改変Myceliophthora thermophila C1によって産生される実質的に純粋な、組換えインフルエンザウイルス表面タンパク質であって、純度95%以上に精製されならびに活性でかつ免疫原性であり、かつワクチンとして使用される場合に防御免疫応答を誘導する、インフルエンザウイルス表面タンパク質。
- 請求項1に記載の改変Myceliophthora thermophila C1に産生されるインフルエンザウイルス表面タンパク質を含むインフルエンザワクチン組成物。
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US201762547885P | 2017-08-21 | 2017-08-21 | |
US62/547,885 | 2017-08-21 | ||
PCT/IB2018/056003 WO2019038623A1 (en) | 2017-08-21 | 2018-08-09 | PRODUCTION OF AN INFLUENZA VACCINE IN MYCELIOPHTHORA THERMOPHILA |
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EP (1) | EP3672630A4 (ja) |
JP (1) | JP2020533966A (ja) |
KR (1) | KR20200090143A (ja) |
CN (1) | CN111315408A (ja) |
AU (1) | AU2018319501A1 (ja) |
CA (1) | CA3073646A1 (ja) |
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KR20230154261A (ko) * | 2021-03-11 | 2023-11-07 | 다이아딕 인터내셔널 (유에스에이), 인크. | N-연결된 글리코실화가 없거나 감소된 외인성 단백질의 생산을 위한 유전적으로 변형된 사상 진균 |
WO2023225646A1 (en) | 2022-05-20 | 2023-11-23 | Phibro Animal Health Corporation | Using fungal constructs to produce viral protein antigens |
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WO2016097369A1 (en) * | 2014-12-19 | 2016-06-23 | Sanofi Pasteur | Multimerization of recombinant protein by fusion to a sequence from lamprey |
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KR20200090143A (ko) | 2020-07-28 |
US20200215183A1 (en) | 2020-07-09 |
CA3073646A1 (en) | 2019-02-28 |
US20230346913A1 (en) | 2023-11-02 |
EP3672630A1 (en) | 2020-07-01 |
US11738080B2 (en) | 2023-08-29 |
EP3672630A4 (en) | 2021-06-02 |
IL272568A (en) | 2020-03-31 |
CN111315408A (zh) | 2020-06-19 |
AU2018319501A1 (en) | 2020-04-02 |
WO2019038623A1 (en) | 2019-02-28 |
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