JP4578973B2 - 免疫刺激のためのihnvg蛋白質 - Google Patents
免疫刺激のためのihnvg蛋白質 Download PDFInfo
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- JP4578973B2 JP4578973B2 JP2004537104A JP2004537104A JP4578973B2 JP 4578973 B2 JP4578973 B2 JP 4578973B2 JP 2004537104 A JP2004537104 A JP 2004537104A JP 2004537104 A JP2004537104 A JP 2004537104A JP 4578973 B2 JP4578973 B2 JP 4578973B2
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Description
第一側面において、本発明は、IHNV G蛋白質のコード配列の部分を含みおよびIHNV以外の病原性生物に由来する第二蛋白質のコード配列の部分をさらに含み、但し、第二蛋白質のコード配列はVHSVのM遺伝子の配列のすべてでなく部分でもない、発現ベクターを提供する。IHNV G蛋白質の部分は、IHNV G蛋白質の単離されたリーダー配列でもよい。第二蛋白質が魚類の病原体であることが好ましく、場合によりウイルスに由来する抗原であることが好ましい。IHNV G蛋白質のコード配列の部分は、第二蛋白質のコード配列と融合していてもよい。
本発明は、核酸ワクチンから発現されるIHNV G蛋白質またはそのリーダー配列(シグナル配列とも称される)が第二抗原と共に存在することによって免疫応答の促進が可能となり、その結果、第二抗原が由来するところの病原体に対する防護が強化されるという観察結果に基づく。この効果についての説明は未だに明らかではないが、G蛋白質上に免疫調節モチーフが存在することに関係があり得る。抗原蛋白質がG蛋白質またはそのリーダー配列に融合した結果、抗原が細胞表面に移行し、その結果、宿主の免疫系へのペプチドの露出が促進されるということも、もっともらしい。あるいは、防護の強化は、これらの効果の組み合わせを介した相乗効果によるものであり得る。
タイセイヨウサケの稚魚(体重は9−26g)を、8℃の新鮮な水が入った直径が1メートルの円形の二つのタンクに入れる。そしてワクチン接種の24時間前から、空腹にさせる。ワクチン接種のために、約0.5g/リットルの濃度の3−アミノ安息香酸エチルエステル(MS222、Sigma、プール、英国)を用いて魚に麻酔をかける。核酸ワクチン(10μgのDNA/50μLの投与量)を、左側の背部の側面の、背びれの少し下側の領域に、筋肉注射によって投与する。オイルアジュバント死滅IPNVワクチン、PBSのコントロールを、腹腔内注射(100μL)によって投与する。それぞれの処理群には40匹の魚があり、6種のワクチンのそれぞれについて、ワクチンあたり2回反復する。
(1)pUKベクター:カナマイシン耐性遺伝子、CMV最初期プロモーター、マルチクローニング部位、およびウシ成長ホルモンのポリアデニル化シグナル(BGHポリA)を保持するクローニングベクター。
(2)pUK+VP2:IPNV VP2の完全なコード配列がそのベクターのマルチクローニング部位内に組み込まれているpUKベクター。
(3)pUK+IHNG:IHNV G蛋白質の完全なコード配列がそのベクターのマルチクローニング部位内に組み込まれているpUKベクター。
(4)pUK+IHNG+VP2:pUKベクター。IHNウイルスの糖蛋白質(G)とインフレームで融合したIPNV VP2蛋白質の完全なコード配列が、G蛋白質がVP2の5’となるようにマルチクローニング部位内に組み込まれているpUKベクター。
(5)IPNV+オイル:オイルでアジュバント化されたIPNウイルスの非活性化調製品。
(6)PBS(ネガティブコントロール)。
ワクチン接種から807度日後に、IPNウイルスの「Cole−Deep」株を2回凍結させて通過させた上清を、滅菌したPBSで五倍に希釈して最終濃度を1×107TCID50/mLとする。MS222で魚に麻酔をかけ、そしてそれぞれの魚の投与量が106TCID50となるように、100μLのウイルス懸濁液を腹腔内に注射することによって、群れごとに投与する。投与時の海水の温度は約11℃であり、それぞれのタンク内での水の流速は約5リットル/分である。
平均体重が10g(6月齢未満)のタイセイヨウサケの稚魚を、12±1℃で流速が2.5L/分の水に少なくとも7日間順応させる。この魚に、一日当たり体重の1.5%の市販のペレット餌を与える。
(1)pUKベクター:カナマイシン耐性遺伝子、CMV最初期プロモーター、マルチクローニング部位、およびウシ成長ホルモンのポリアデニル化シグナル(BGHポリA)を保持するクローニングベクター。
(2)pUK−HA:ISAV血球凝集素蛋白質の完全なコード配列がマルチクローニング部位内に組み込まれているpUKベクター。
(3)pUK−IHNg:IHNV G蛋白質の完全な配列がマルチクローニング部位内に組み込まれているpUKベクター。
(4)pUK−HA−IHNg:ISAV血球凝集素蛋白質の完全なコード配列がマルチクローニング部位内に組み込まれ、およびその5’末端がIHNV G蛋白質のリーダー配列に融合したpUKベクター。
(5)ISAV+オイル:オイルでアジュバント化されたISAウイルスの非活性化調製品。
RPS=1−(ワクチンでの死亡率%/pUKコントロールでの死亡率%)×100
結果:
ネガティブコントロール群(pUKプラスミド)についての累積死亡率は94%である。最も優れた防護は、ポジティブコントロールの一価の非活性化ISAVワクチンの注射によって誘導される(94%のRPS)。ネガティブコントロールとしてのpUK−IHNgプラスミドの注射によって、いくらかの非特異的な防護が誘導される(26%のRPS)。pUK−HAによって、高いレベルの防護が付与される(49%のRPS)。3’末端におけるIHNV G蛋白質のリーダー配列の追加(pUK−HA−IHNg)によって、この防護は有意に強化される(60%のRPS)。これらの結果から、核酸ワクチンにIHNV G蛋白質配列(または少なくともそのリーダー配列)を含めてIHNV以外の感染症に対する免疫性を促進するという有意性についてのさらなる支持が提供される。G蛋白質のリーダー配列は、魚の内側の細胞表面に異種抗原を局在化させている可能性がある。または、魚類の免疫系を非特異的に刺激する蛋白質の範囲内の特別のモチーフが存在することもあり得る。
Claims (15)
- IHNV G蛋白質のコード配列の部分を含みおよびIHNV以外の病原性生物に由来する第二蛋白質のコード配列の部分をさらに含み、前記IHNV G蛋白質のコード配列の部分と第二蛋白質のコード配列の部分は同一のDNA発現ベクター上にてインフレームで融合しており、前記IHNV G蛋白質のコード配列の部分はIHNV G蛋白質のリーダー配列をコードする配列を含む、DNA発現ベクター。
- 第二蛋白質のコード配列が、魚類の病原体に由来する、請求項1に記載のDNA発現ベクター。
- IHNV G蛋白質のリーダー配列が配列番号:1である、請求項1または2に記載のDNA発現ベクター。
- IHNV G蛋白質の完全なコード配列を含む、請求項1から3のいずれか1項に記載のDNA発現ベクター。
- 第二蛋白質が、ISAV、IPNV、VHSV、NNV、イリドウイルス、SVCV、SPDV、レニバクテリウム・サルモニナルム(Renibacterium salmoninarum)、ピシリケッチア・サルモニス(Piscirickettsia salmonis)、ビブリオ・スピーシーズ(Vibrio spp.)、アエロモナス・スピーシーズ(Aeromonas spp.)、エルジニア・ルケリ(Yersinia ruckerii)、シュードモナス・スピーシーズ(Pseudomonas spp.)、ノカルジア・スピーシーズ(Nocardia spp.)およびフォトバクテリウム・ダムセラ・サブスピーシーズ・ピシシダ(Photobacterium damselae subsp. piscicida)から選択される病原体に由来する、請求項1から4に記載のDNA発現ベクター。
- 第二蛋白質が、IPNVに由来するVP2蛋白質かまたはISAVに由来する血球凝集素蛋白質である、請求項5に記載のDNA発現ベクター。
- 医薬適合性の担体と共に、請求項1から6のいずれか1項に記載のDNA発現ベクターを含有する核酸ワクチン。
- IHNV G蛋白質の部分および魚類のIHNV以外の病原性生物に由来する第二蛋白質の部分を含み、前記IHNV G蛋白質の部分はIHNV G蛋白質のリーダー配列を含む、融合蛋白質。
- 第二蛋白質が、ISAV、IPNV、VHSV、NNV、イリドウイルス、SVCV、SPDV、レニバクテリウム・サルモニナルム、ピシリケッチア・サルモニス、ビブリオ・スピーシーズ、アエロモナス・スピーシーズ、エルジニア・ルケリ、シュードモナス・スピーシーズ、ノカルジア・スピーシーズおよびフォトバクテリウム・ダムセラ・サブスピーシーズ・ピシシダから選択される病原体に由来する、請求項8に記載の融合蛋白質。
- 第二蛋白質が、IPNVに由来するVP2蛋白質である、請求項9に記載の融合蛋白質。
- 第二蛋白質が、ISAVに由来する血球凝集素蛋白質である、請求項9に記載の融合蛋白質。
- 医薬適合性の担体と共に、請求項9から11のいずれか1項に記載の融合蛋白質をコードする配列を含む、核酸ワクチン。
- 魚類における病原体の感染を予防または治療するための薬剤の調製における請求項1から6のいずれか1項に記載のDNA発現ベクター、または、請求項8から11のいずれか1項に記載の融合蛋白質の使用。
- 魚類における細菌、ウイルス、真菌または原虫の感染を予防または治療するための薬剤の調製における、請求項13に記載の使用。
- 免疫応答を促進させ、その結果、第二蛋白質が由来する病原体に対する防護を強化するための薬剤の調製における、請求項13または14に記載の使用。
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GB0221552A GB0221552D0 (en) | 2002-09-17 | 2002-09-17 | Organic compounds |
GB0221553A GB0221553D0 (en) | 2002-09-17 | 2002-09-17 | Organic compounds |
PCT/EP2003/010305 WO2004026338A1 (en) | 2002-09-17 | 2003-09-16 | Ihnv g protein for immune stimulation |
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US8163893B2 (en) | 2006-02-15 | 2012-04-24 | The Regents Of The University Of Caifornia | Pseudotyped retroviral vectors and methods of use thereof |
US8343505B2 (en) * | 2007-12-04 | 2013-01-01 | Schweitzer Co., Ltd. | Subunit vaccine for aquaculture |
CA2884918C (en) * | 2012-09-17 | 2019-01-08 | Novartis Tiergesundheit Ag | Salmonid alphavirus and uses thereof |
FR2996856B1 (fr) * | 2012-10-15 | 2015-06-05 | Agronomique Inst Nat Rech | Novirhabdovirus recombinant utilisable comme vecteur d'antigenes |
CN109136200B (zh) * | 2018-09-20 | 2022-02-18 | 中国水产科学研究院黑龙江水产研究所 | 一种重组传染性造血器官坏死病毒及其构建方法与应用 |
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AU2003277863B2 (en) | 2007-02-22 |
WO2004026338A1 (en) | 2004-04-01 |
DE60315858T2 (de) | 2008-05-21 |
CA2498896A1 (en) | 2004-04-01 |
EP1553979B1 (en) | 2007-08-22 |
DK1553979T3 (da) | 2007-12-27 |
ATE370746T1 (de) | 2007-09-15 |
HK1082666A1 (en) | 2006-06-16 |
CY1107784T1 (el) | 2013-06-19 |
EP1553979A1 (en) | 2005-07-20 |
ES2288627T3 (es) | 2008-01-16 |
AU2003277863A1 (en) | 2004-04-08 |
HK1080712A1 (en) | 2006-05-04 |
JP2006504414A (ja) | 2006-02-09 |
CA2498896C (en) | 2014-10-21 |
CN100339131C (zh) | 2007-09-26 |
CN1681530A (zh) | 2005-10-12 |
NO20051840L (no) | 2005-06-16 |
NO337372B1 (no) | 2016-03-29 |
DE60315858D1 (de) | 2007-10-04 |
PT1553979E (pt) | 2007-11-27 |
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