JP2023505920A - 重症急性呼吸器症候群ウイルスsars-cov-2に対する発現ベクター - Google Patents
重症急性呼吸器症候群ウイルスsars-cov-2に対する発現ベクター Download PDFInfo
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Abstract
Description
本発明は、バイオテクノロジー、免疫学及びウイルス学に関する。本発明は、重症急性呼吸器症候群ウイルスSARS-CoV-2に対する特異的免疫を誘導するための免疫生物学的薬剤を開発するために製薬産業において使用することができる組換えベクターを包含する。
2019年12月に、湖北省(Hubei)の省都である武漢(Wuhan)において、新型コロナウイルス(SARS-CoV-2)によって引き起こされる疾患が見出された。この疾患によって、迅速な診断法及び患者の臨床管理を含む、公衆衛生の専門家及び医師が対処すべき複雑な作業がもたらされた。SARS-CoV-2ウイルスは急速に世界中に広がり、史上最大規模のパンデミックに発展した。2020年8月19日までに、症例数は22百万を超え、死者数は-791千人であった。
特許請求される発明群の技術的目的は、SARS-CoV-2糖タンパク質に対する持続的な免疫応答を誘導すること、及びSARS-CoV-2糖タンパク質に対する生物学的に有効な防御抗体力価の存在を保証することである。重症急性呼吸器症候群ウイルスSARS-CoV-2に対する特異的免疫を誘導するための免疫生物学的薬剤を作製することが可能になる。
組換えヒトアデノウイルス血清型26のゲノムを含有する発現ベクターを得る方法では、第1段階でヒトアデノウイルス血清型26のゲノムの2つの相同領域を含むプラスミドが構築され、これは次に、制限エンドヌクレアーゼを用いて直線化され、ヒトアデノウイルス血清型26のウイルス粒子から単離されたDNAと混合され、大腸菌(E. coli)細胞において相同組換えが行われる。結果として、E1領域が欠失された組換えヒトアデノウイルス血清型26のゲノムを保有するプラスミドが得られる。次に、遺伝子操作法を用いて、オープンリーディングフレーム6(ORF6)が、ヒトアデノウイルス血清型5のORF6によって置換される。そして、パッケージング能力を拡張するためにE3領域が欠失される。最終的に、発現カセットがベクターに挿入される。
組換えヒトアデノウイルス血清型26のゲノムを含有する発現ベクターの製造
第1段階で、ヒトアデノウイルス血清型26のゲノムと相同の2つの領域(2つの相同性アーム)、及びアンピシリン耐性遺伝子を保有するプラスミド構築物pAd26-Endsを設計した。相同性アームの一方は、ヒトアデノウイルス血清型26のゲノムの開始部分(左側逆位末端反復配列からE1領域まで)、及びpIXタンパク質を含むウイルスゲノムの配列である。他方の相同性アームは、ゲノムの末端を介してORF3 E4領域の後に位置するヌクレオチド配列を含有する。pAd26-Ends構築物の合成は、Moscow企業「Eurogen」ZAOにより実施した。
- 発現カセット配列番号1は、CMVプロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有し、
- 発現カセット配列番号2は、CAGプロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有し、
- 発現カセット配列番号3は、EF1プロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有する。
組換えサルアデノウイルス血清型25のゲノムを含有する発現ベクターの製造
第1段階で、サルアデノウイルス血清型25のゲノムと相同の2つの領域(2つの相同性アーム)を保有するプラスミド構築物pSim25-Endsを設計した。相同性アームの一方は、サルアデノウイルス血清型25のゲノムの開始部分(左側逆位末端反復配列からE1領域まで)、及びE1領域の末端からpIVa2タンパク質までの配列である。他方の相同性アームは、右側逆位末端反復配列を含むアデノウイルスゲノムの末端部分の配列を含有する。pSim25-Ends構築物の合成は、Moscow企業「Eurogen」ZAOにより実施した。
- 発現カセット配列番号4は、CMVプロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有し、
- 発現カセット配列番号2は、CAGプロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有し、
- 発現カセット配列番号3は、EF1プロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有する。
組換えヒトアデノウイルス血清型5のゲノムを含有する発現ベクターの製造
第1段階で、ヒトアデノウイルス血清型5のゲノムと相同の2つの領域(2つの相同性アーム)を保有するプラスミド構築物pAd5-Endsを設計した。相同性アームの一方は、ヒトアデノウイルス血清型5のゲノムの開始部分(左側逆位末端反復配列からE1領域まで)、及びpIXタンパク質を含むウイルスゲノムの配列である。他方の相同性アームは、ゲノムの末端を介してE4領域ORF3の後にヌクレオチド配列を含有する。pAd5-Ends構築物の合成は、Moscow企業「Eurogen」ZAOにより実施した。
- 発現カセット配列番号1は、CMVプロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有し、
- 発現カセット配列番号2は、CAGプロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有し、
- 発現カセット配列番号3は、EF1プロモーター、SARS-CoV-2ウイルスSタンパク質遺伝子、及びポリアデニル化シグナルを含有する。
HEK293細胞における開発発現ベクターによるSARS-CoV-2ウイルスSタンパク質遺伝子の発現の検証
この実験の目的は、構築された組換えアデノウイルスが哺乳類細胞において重症急性呼吸器症候群SARS-CoV-2ウイルスSタンパク質遺伝子を発現する能力を検証することであった。
1)Ad26-CMV-S-CoV2;
2)Ad26-CAG-S-CoV2;
3)Ad26-EF1-S-CoV2;
4)Ad26-null;
5)simAd25-CMV-S-CoV2;
6)simAd25-CAG-S-CoV2;
7)simAd25-EF1-S-CoV2;
8)simAd25-null;
9)Ad5-CMV-S-CoV2;
10)Ad5-CAG-S-CoV2;
11)Ad5-EF1-S-CoV2;
12)Ad5-null;
13)リン酸緩衝食塩水。
開発発現ベクターによる動物免疫化の有効性の評価
免疫化の有効性の主な特徴の1つは抗体力価である。実施例は、免疫化後21日目のSARS-CoV-2糖タンパク質に対する抗体力価の変化に関するデータを提示する。
1)Ad26-CMV-S-CoV2;
2)Ad26-CAG-S-CoV2;
3)Ad26-EF1-S-CoV2;
4)Ad26-null;
5)simAd25-CMV-S-CoV2;
6)simAd25-CAG-S-CoV2;
7)simAd25-EF1-S-CoV2;
8)simAd25-null;
9)Ad5-CMV-S-CoV2;
10)Ad5-CAG-S-CoV2;
11)Ad5-EF1-S-CoV2;
12)Ad5-null;
13)リン酸緩衝食塩水。
1)96ウェルELISAプレートのウェルにタンパク質(S)を+4℃で16時間吸着させた。
2)次に、非特異的な結合を防止するために、1ウェル当たり100μlの量のTPBS中に溶解させた5%の乳によりプレートを「ブロック」した。それを37℃の振とう機において1時間インキュベートした。
3)2倍希釈法を用いて、免疫化マウスからの血清サンプルを希釈した。全体として、各サンプルの12の希釈物を調製した。
4)希釈した血清サンプルのそれぞれの50μlをプレートウェルに添加した。
5)次に、37℃で1時間のインキュベーションを実施した。
6)インキュベーションの後、ウェルをリン酸緩衝液で3回洗浄した。
7)さらに、西洋わさびペルオキシダーゼと結合されたマウス免疫グロブリンに対する二次抗体を添加した。
8)次に、37℃で1時間のインキュベーションを実施した。
9)インキュベーションの後、ウェルをリン酸緩衝液で3回洗浄した。
10)次に、西洋わさびペルオキシダーゼの基質としての役割を果たし、反応によって着色化合物に変換されるテトラメチルベンジジン(TMB)溶液を添加した。硫酸の添加により15分後に反応を停止させた。次に、分光光度計を用いて、各ウェルにおいて450nmの波長で溶液の光学濃度(OD)を測定した。
提供される実施例は全て、発現ベクターの有効性、重症急性呼吸器症候群ウイルスSARS-CoV-2に対する特異的免疫の誘導のための免疫生物学的薬剤を作製するためのその利用可能性、及び産業上の利用可能性を確認する。
Claims (7)
- E1及びE3領域が欠失され、且つORF6-Ad26領域がORF6-Ad5により置換された組換えヒトアデノウイルス血清型26のゲノムを含有し、配列番号1、配列番号2、配列番号3から選択される発現カセットが組み込まれた発現ベクター。
- ヒトアデノウイルス血清型26の親配列として配列番号5が使用されたことを特徴とする、請求項1に記載の発現ベクター。
- E1及びE3領域が欠失された組換えサルアデノウイルス血清型25のゲノムを含有し、配列番号4、配列番号2、配列番号3から選択される発現カセットが組み込まれた発現ベクター。
- サルアデノウイルス血清型25の親配列として配列番号6が使用されたことを特徴とする、請求項3に記載の発現ベクター。
- E1及びE3領域が欠失された組換えヒトアデノウイルス血清型5のゲノムを含有し、配列番号1、配列番号2、配列番号3から選択される発現カセットが組み込まれた発現ベクター。
- ヒトアデノウイルス血清型5の親配列として配列番号7が使用されたことを特徴とする、請求項5に記載の発現ベクター。
- 重症急性呼吸器症候群ウイルスSARS-CoV-2に対する特異的免疫の誘導のための免疫生物学的薬剤を作製するための、請求項1~6のいずれか一項に記載の発現ベクターの利用。
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KR20230088301A (ko) | 2023-06-19 |
CN114845733A (zh) | 2022-08-02 |
ZA202202322B (en) | 2023-12-20 |
CA3152658A1 (en) | 2021-04-22 |
EP4010018A4 (en) | 2022-11-09 |
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IL291022A (en) | 2022-05-01 |
EA037291B1 (ru) | 2021-03-05 |
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MX2022002609A (es) | 2022-06-08 |
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