JP4268384B2 - Japanese encephalitis virus antigen and method for producing the same - Google Patents

Description

【００９７】
【表２】

【００９８】
Vero細胞を用いたウイルス培養では、ＥＬＩＳＡ価及びＨＡ価は共に培養４日目に最高値を示し、５日目以降はウイルスによる細胞変性効果（cytopathic effect，CPE）が進み、ＥＬＩＳＡ価、ＨＡ価とも低下した。一方、J12#26SF細胞の培養においては、４日目以降もウイルス様粒子を発現し続け、ＥＬＩＳＡ価、ＨＡ価とも上昇した。また、１０日目に培養上清を回収し、新しい培地を添加するとウイルス様粒子の発現は継続した。従って、適当な時期に培地交換することで、１回の細胞培養で大量のウイルス様粒子を発現させることが可能である。上記の結果から明らかなように、J12#26SF細胞の産生する抗原はＨＡ価を示し、図５に見られるように、ＨＡ価とＥＬＩＳＡ価の間にはほぼ直線的な相関関係が見られた。また、表２から明らかなように、J12#26SF細胞は、日本脳炎ウイルスをVero細胞で増殖させた時の培養液に含まれる抗原量の２〜３倍量の抗原を産生することが判明した。本発明の形質転換細胞株であるJ12#26SF細胞は大量且つ持続的に抗原を発現しており、このような大量且つ持続的な抗原の発現は細胞の継代が進んでも（７８代でも）衰えることはなかった。
【００９９】
実施例３
無血清培地からのウイルス様粒子の精製
J12#26SF細胞を、直径１５ｃｍの培養ディッシュを用いて、無血清培地中で７２時間大量培養した。抗原精製用無血清培地としては、VP-SFMに５μｇ／ｍｌのブラストサイジンを添加して用いた。
【０１００】
３，０００ｒｐｍで１０分培養液を遠心し、培養上清を回収した。回収した培養上清をBiomax-100限外濾過膜（MWCO100,000）（米国、Millipore社製）を装着したセントリコンプラス80を用いて３，０００×ｇ、４℃で３０分の遠心を３回行い、容積を１／１１４まで濃縮した。
【０１０１】
濃縮液をSephacryl S-300を充填したHiprep 16/60カラム（１．６×６０ｃｍ）（スウエーデン国、Pharmacia Biotech AB製）にアプライし、ＴＢＳバッファー［50mM Tris-HCl (pH7.5)、150mM NaCl］を用いて流速１ｍｌ／分で分子ふるいクロマトグラフィーを行い、１画分が３．７５ｍｌとなるように分画した。各画分の抗原価をＥＬＩＳＡ法で測定し、ピークフラクションを回収した。目的の抗原は、排除限界域で単一のピークとして溶出された。
【０１０２】
次に、このピーク画分を上部５％（w/w）、底部３０％（w/w）となるように、Gradient Master^TM（model 106）（カナダ国、BIOCOMP Instruments, Inc.製）で調整したショ糖密度勾配液に重層し、SW28ローターおよび超遠心機 L8-70M（いずれも米国、Beckman Instruments, Inc.製）を用いて、４℃、２６，５００ｒｐｍの条件で２時間のショ糖密度勾配遠心分画を行った。遠心後、チューブ底部中央に１９ゲージの穴をあけ、滴下法で１画分が２ｍｌとなるように分画した。各画分の抗原価をＥＬＩＳＡ法で測定し、抗原のピークフラクションを回収した。目的の抗原は単一のピークを形成して分画していた。各フラクションのショ糖濃度はほぼ単調減少直線を描いていた。
【０１０３】
最後に、ショ糖や低分子物質を除くために、このピーク画分をSephadex G-25を充填したHiprep desalting 26/10カラム（２．６×１０ｃｍ）（スウエーデン国、Pharmacia Biotech AB製）にアプライし、ＰＢＳ（−）を用いて流速５ｍｌ／分で分子ふるいクロマトグラフィーを行い、１画分が５ｍｌとなるように分画した。この操作で抗原をショ糖や低分子物質から完全に分離し、同時に溶媒をＰＢＳ（−）に交換した。ショ糖の除去とバッファー交換を行った抗原画分を最終精製物であるウイルス様粒子とした。精製したウイルス様粒子は、J12#26抗原と命名した。なお、高速液体クロマトグラフィーシステムには、AKTA explorer 10S（スウエーデン国、Pharmacia Biotech AB製）を使用した。
【０１０４】
抗原の精製の各段階において、タンパク質量とＥＬＩＳＡ法による抗原量の測定を行った。結果を表３にまとめた。
【０１０５】
【表３】

【０１０６】
表３においては、無血清培地を用いた培養上清中の総抗原量をそれぞれ１００％とし、各ステップにおける収量を基に回収率を算出した。タンパク質の回収率は６．３％であったが、ウイルス様粒子の回収率は４３．５％だった。電気泳動の結果およびウェスタンブロットの結果と併せると、高精製度・高収率で抗原を精製することができた。
【０１０７】
各精製段階のサンプルの抗原量またはタンパク質量を等しくしてそれぞれＳＤＳ−ＰＡＧＥに付した。泳動後、銀染色と同等の感度を持つ蛍光色素のSYPRO-Orangeで泳動分画されたタンパク質を染色した。結果を図６に示した。抗原量を等しく泳動した場合には、精製過程が進むにつれ、同等のシグナル強度を示す分子量５３ｋＤａのタンパク質バンドが等しく観察されたのに対して、その他の混入タンパク質のバンドは減少した。タンパク質量を等しくして泳動した場合には、精製が進むにつれ、上記５３ｋＤａのタンパク質バンドが徐々に強く観察された。更に、５３ｋＤａの主要なタンパク質バンドが日本脳炎ウイルスに特異的な中和抗原のＥタンパク質であるか否かをウェスタンブロットで調べた。結果を図７に示した。日本脳炎ウイルスに対する特異的な抗体によって、５３ｋＤａの主要なタンパク質が認識された。
【０１０８】
J12#26抗原液のタンパク質濃度とＨＡ価を測定した。その結果、タンパク質濃度は４４μｇ／ｍｌであり、ＨＡ価は２５，６００ＨＡだった。従って、J12#26抗原のＨＡ活性は、１μｇ／ｍｌ当たり５８２ＨＡだった。
【０１０９】
実施例４
抗原粒子を構成するタンパク質のＮ末端アミノ酸解析
J12#26抗原がＥタンパク質のみならず、Ｍタンパク質も包含することを確認するために、Ｅタンパク質に相当する５３ｋＤａのバンドの下流に存在するタンパク質のＮ末端のアミノ酸配列を解析した。
【０１１０】
上記で精製したウイルス様粒子に、終濃度が５％になるように５０％トリクロロ酢酸を加えて攪拌し、室温で３０分間静置した。３，０００gで３０分低速遠心後に上清を捨て、残った沈殿を風乾し、１００μlの0.1% NaOHで溶解し、泳動用サンプルとした。泳動用サンプルの抗原量が２０μｇ／レーンとなるようにゲルにアプライしてＳＤＳ−ＰＡＧＥに付した。電気泳動後のゲルに銀染色を施し、タンパク質のバンドを視覚化した。次に、ＳＤＳ−ＰＡＧＥ後のゲルからタンパク質のバンドをＰＶＤＦ膜に転写した。結果を図８に示す。
【０１１１】
図８はＳＤＳ−ＰＡＧＥおよびＰＶＤＦ膜への転写を行った結果であり、左側が銀染色したポリアクリルアミドゲルであり、右側がＰＶＤＦ膜にタンパク質を転写した転写象である。Ｅタンパク質に相当する５３ｋＤａのバンドの下流に存在するバンドのうち、ＪＥＶに由来すると思われる３本のバンド（図８のＪＥＶＬＰ０１〜ＪＥＶＬＰ０３）、即ち、１６．５ｋＤａ、１４．９ｋＤａ及び６．８ｋＤａのバンド、を転写膜から切り出し、メタノール、超純水、メタノールの順番でバンドを洗浄した。各バンドを構成するタンパク質のＮ末端から５残基のアミノ酸配列をシークエンサーを用いて解析した。プロテインシークエンサーにはＧ１０００Ａ（米国、Hewlett Packard Company製）、ＰＴＨアナライザーには１０９０型（米国、Hewlett Packard Company製）、分析プログラムにはRoutine 3.1 PVDFを用いた。結果を表４に示す。
【０１１２】
【表４】

【０１１３】
上記の表４から明らかなように、ＪＥＶＬＰ０１はＭタンパク質（またはその断片）とＥタンパク質断片の混合物であり、ＪＥＶＬＰ０２はＥタンパク質断片であり、ＪＥＶＬＰ０３はＭタンパク質であった。この結果から、J12#26抗原は、Ｅタンパク質と共にＭタンパク質が含まれていることが明らかとなった。
【０１１４】
実施例５
ウイルス様粒子の電子顕微鏡観察
J12#26抗原の形状を電子顕微鏡で観察した。精製したJ12#26抗原を遠心濃縮し、前固定なしでネガティブ染色した。染色したウイルス様粒子を電子顕微鏡下で観察した。電子顕微鏡写真を図９に示す。
【０１１５】
ウイルス様粒子はほぼ均一なサイズの球型粒子状であり、平均直径は２５ｎｍであった。この結果から、精製の完了した抗原が粒子状構造を形成していることが直接的に証明された。また、一般に電子顕微鏡下では、不純物やウイルス様粒子の破壊によって生じた細かな不定形のゴミ様混在物が観察されるが、そのようなものがほとんど見られないことから、精製度はかなり高いものと考えられる。
【０１１６】
実施例６
日本脳炎ウイルス抗原による中和抗体誘導試験
４週齢雌ｄｄＹマウスを１群１０匹に分け、ＰＢＳで希釈した精製J12#26抗原をマウス一匹に付き、総タンパク量で３００ｎｇを腹腔投与免疫した。免疫は１週間おきに２回繰り返した。対照群には５匹のＰＢＳ（−）接種群および現行ワクチン接種群をおいた。２回目の投与から７日目に半数のマウスから、１４日目には残りマウスから無菌的に心臓全採血を行い、血液を３７℃中に１時間放置して凝固させた。血餅を取り除いた後、４℃、５，０００ｒｐｍで１０分遠心し、血清を分離した。血清を回収し、５６℃で３０分血清の非働化を行った。５匹のマウスそれぞれの血清から５０μｌの非働化済み血清をプールして、中和抗体価の測定に用いた。結果は表５に示した。
【０１１７】
【表５】

【０１１８】
表５から明らかなように、J12#26抗原を現行ワクチンと同様に接種した群において、現行ワクチンより約３倍高い中和抗体価を誘導した。以上の結果から、本発明の日本脳炎ウイルス抗原は、現行ワクチンと同等の免疫原性を示すことが明らかになった。
【０１１９】
実施例７
日本脳炎ウイルス抗原による感染防御
実施例６と同様に、４週齢雌ｄｄＹマウスを１群１０匹に分け、ＰＢＳで希釈した日本脳炎ウイルス抗原を腹腔投与免疫した。日本脳炎ウイルス抗原には、ＰＢＳで希釈したJ12#26抗原を用い、一匹につき抗原量（ＥＬＩＳＡで求めた量）で３００ｎｇを投与した。対照群にはＰＢＳ（−）接種群および現行ワクチン接種群をおいた。免疫したマウスが６週齢になった時点で、２×１０⁶ＰＦＵの日本脳炎ウイルス［（財）阪大微生物病研究会から分与を受けた日本脳炎ワクチン生産用の種ウイルス株（北京−１株、ＪＷＳ−Ｐ−４）］を腹腔内接種した。腹腔内接種と同時に、マウスに２０μｌのＰＢＳを脳内注射し、血液脳関門を破壊した。接種後２１日間にわたり、マウスの生存または死亡を調査し、生存率（％）を算出した。結果を表６に示した。
【０１２０】
【表６】

【０１２１】
表６から明らかなように、本発明の抗原粒子で免疫したマウスは、現行のワクチンで免疫したマウスと同じように１００％の生存率を示した。従って、本発明の抗原様粒子は日本脳炎ワクチン用抗原として十分な感染防御能を有していた。
【０１２２】
実施例８
日本脳炎ウイルス抗原を用いた日本脳炎の診断
J12#26抗原及び組織培養日本脳炎ウイルス精製抗原（ＢＭ３）をそれぞれタンパク質量が１．０μｇ／ｍｌとなるように、炭酸緩衝液（ｐＨ９．６）で希釈し、固相化抗原とした。抗原をＥＬＩＳＡ用プレートに１００μｌ／ウエルずつ分注し、４℃で一晩放置して抗原を固相化した。ＰＢＳ−Ｔでウエルを洗浄し、１％ＦＢＳを含むＰＢＳを２００μｌ／ウエルずつ分注し、３７℃で２時間ブロッキングした。ＰＢＳ−Ｔでウエルを洗浄し、２００倍に希釈したヒトの血清を各ウエルに１００μｌ加え、３７℃で６０分インキュベートした。ヒト血清には、日本脳炎ウイルス北京株に対する中和抗体価が陰性の血清２検体（Ｓ１とＳ２）と陽性の血清２検体（Ｓ３とＳ４）を用いた。
【０１２３】
ＰＢＳ−Ｔでウエルを洗浄し、ＰＢＳ−Ｔで１０００倍希釈した標識抗体であるHRPO-anti human IgG goat IgG［米国、Rockland Immunochemicals社製（米）］を各ウエルに１００μｌずつ分注し、３７℃で６０分インキュベートした。洗浄後、各ウエルに１００μｌの発色液を加えて２０分間放置して反応させた。発色液は、基質となる３０％過酸化水素２５μｌと発色剤であるＡＢＴＳ [2,2'-Azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) Diammonium Salt]（米国、Sigma社製）２０ｍｇを１００ｍｌのクエン酸緩衝液（ｐＨ４．０）に溶解した溶液を用いた。次に、１．５％のシュウ酸を各ウエルに１００μｌ加えて反応を停止し、４１５ｎｍの吸光度を測定した。吸光度が０．２以上のものを陽性と判定した。結果は表７に示した。
【０１２４】
【表７】

【０１２５】
本発明の抗原を用いてヒト血清中の日本脳炎ウイルスに対する抗体との反応性の有無を試験したところ、中和抗体価及び組織培養日本脳炎ウイルス精製抗原を用いた試験結果の両方と相関した結果が得られた。従って、本発明の抗原は診断剤用の抗原としても利用可能である。
【０１２６】
【発明の効果】
本発明によれば、日本脳炎ウイルス様粒子を包含してなる日本脳炎ウイルス抗原であって、該ウイルス様粒子は、日本脳炎ウイルスのＭタンパク質及びＥタンパク質を包含するが、該粒子中にはＲＮＡを含有していないタンパク質集合体であり、且つ該ウイルス様粒子は赤血球凝集活性を示すことを特徴とする日本脳炎ウイルス抗原が提供される。本発明の抗原を用いることにより、現行の日本脳炎ワクチンに用いられている不活化日本脳炎ウイルスの代わりとなる、マウス脳を使わない、ウイルスを使わない、コスト安の日本脳炎ワクチンおよび診断剤の製造が可能となる。
【０１２７】
【配列表】

【図面の簡単な説明】
【図１】図１は、組換えＤＮＡpCAGJ12bsrの模式図である。
【図２】図２は、１０％ＦＢＳ−ＭＥＭで継代した無血清培地非馴化細胞（Ｊ１２＃２６細胞）の継代数と、培養上清中に発現されたウイルス様粒子量の関係を示すグラフであり、縦軸はＥＬＩＳＡ法で測定したウイルス様粒子量（μｇ／ｍｌ）であり、横軸は継代数である。
【図３】図３は、ＶＰ−ＳＦＭ（無血清培地）で継代した無血清培地馴化細胞（Ｊ１２＃２６ＳＦ細胞）の継代数と、培養上清中に発現されたウイルス様粒子量の関係を示すグラフであり、縦軸はＥＬＩＳＡ法で測定したウイルス様粒子量（μｇ／ｍｌ）であり、横軸は継代数である。
【図４】図４は、日本脳炎ウイルス感染Vero細胞の培養上清のＨＡ価及びＥＬＩＳＡ価の経時変化を示すグラフであり、左の縦軸は精製不活化ウイルス抗原であるTJP**012のＥＬＩＳＡ価を１００とした培養上清のＥＬＩＳＡ価であり、右の縦軸はＨＡ価（ＨＡ活性が認められた最大希釈率）であり、横軸は培養日数であり、○はＥＬＩＳＡ価を表し、◆はＨＡ価を表す。
【図５】図５は、３８代培養したＪ１２＃２６ＳＦ細胞の培養上清のＨＡ価及びＥＬＩＳＡ価の経時変化を示すグラフであり、左の縦軸は精製不活化ウイルス抗原であるTJP**012のＥＬＩＳＡ価を１００とした培養上清のＥＬＩＳＡ価であり、右の縦軸はＨＡ価（ＨＡ活性が認められた最大希釈率）であり、横軸は培養日数であり、○はＥＬＩＳＡ価を表し、◆はＨＡ価を表す。
【図６】図６は、ウイルス様粒子のＳＤＳ−ＰＡＧＥである。図中、レーン２〜６は等しい抗原量をアプライした結果であり、レーン８〜１２は等しいタンパク質量をアプライした結果であり、レーン１と７は分子量マーカー、レーン２と８は限外ろ過で濃縮した培養上澄、レーン３と９はSephacryl S-300クロマトグラフィーのピークフラクション、レーン４と１０はショ糖濃度勾配遠心法で得たピークフラクション、レーン５と１１はSephadex G-25クロマトグラフィーのピークフラクション、そしてレーン６と１２は市販の日本脳炎ワクチンである。
【図７】図７は、図６のＳＤＳ−ＰＡＧＥに対してウサギ抗ＪＥＶ抗血清を用いたウエスタンブロットの結果である。図中、レーン２〜６は等しい抗原量をアプライした結果であり、レーン８〜１２は等しいタンパク質量をアプライした結果であり、レーン１と７は分子量マーカー、レーン２と８は限外ろ過で濃縮した培養上澄、レーン３と９はSephacryl S-300クロマトグラフィーのピークフラクション、レーン４と１０はショ糖濃度勾配遠心法で得たピークフラクション、レーン５と１１はSephadex G-25クロマトグラフィーのピークフラクション、そしてレーン６と１２は市販の日本脳炎ワクチンである。
【図８】図８は、ＴＣＡで処理したウイルス様粒子のＳＤＳ−ＰＡＧＥとＳＤＳ−ＰＡＧＥで分画したタンパク質のバンドをＰＶＤＦ膜に転写した結果であり、右側が銀染色したポリアクリルアミドゲルであり、左側がＰＶＤＦ膜にタンパク質を転写した転写象であり、左端の数字は分子量マーカーであり、２０．９ｋＤａのバンドの下流に３本のバンド（ＪＥＶＬＰ０１〜ＪＥＶＬＰ０３）が存在する。
【図９】図９は、ネガティブ染色したウイルス様粒子を撮影した電子顕微鏡写真であり、スケールを示す横線は１００ｎｍに相当する。[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a Japanese encephalitis virus antigen. More specifically, a Japanese encephalitis virus antigen comprising Japanese encephalitis virus-like particles, wherein the virus-like particles include M protein and E protein of Japanese encephalitis virus, and RNA is contained in the particles. The present invention relates to a Japanese encephalitis virus antigen characterized in that it is a protein aggregate not contained and the virus-like particles exhibit hemagglutination activity. The present invention further relates to a transformed cell expressing Japanese encephalitis virus-like particles, a method for producing a Japanese encephalitis virus antigen, a Japanese encephalitis vaccine, and a diagnostic agent.
[0002]
[Prior art]
Japanese encephalitis is a serious viral infection caused by Japanese encephalitis virus classified into the genus Flavivirus, and the main symptoms are fever, meningeal symptoms, and cerebral inflammation symptoms (Clinical Virology 13: 166-172, 1985). Although most people end with subclinical infection even after infection, once they develop, the mortality rate is high, and the prognosis is likely to remain sequelae such as intelligence impairment and motor impairment due to CNS injury.
[0003]
Japanese encephalitis virus is a spherical virus with a diameter of about 50 nm, and structural proteins are capsid (C) protein, envelope (E) protein and membrane (M) protein. The (+) strand single-stranded RNA genome of about 11 kb in length is covered with E protein spikes (Annu.Rev.Microbiol44: 649-88, 1990). E protein plays an important role in the protection of infection, such as being involved in adsorption to cells, having a hemagglutination ability, and inducing neutralizing antibodies (Virology 44 (1): 108-24, 1971;J.Virol14 (3): 631-9, 1974;J.Gen.Virol70 (Pt 8): 2037-49, 1989 andActa Virol26 (5): 312-20, 1982).
[0004]
Although it has not shown any signs of an epidemic in Japan in recent years, in the 1960s, thousands of patients developed Japanese encephalitis annually, and about 1,000 people died, which is an intractable disease that has developed into a social problem. Recently, it is one of the diseases that is prevalent mainly in China and Southeast Asia, and WHO is closely monitoring the trend (Clinical Virology 13: 135-143, 1985). Currently, vaccination is the only prevention method for Japanese encephalitis, and there is no effective treatment.
[0005]
The current Japanese encephalitis vaccine is only available from the brain emulsion of virus-infected mice (“Japanese encephalitis vaccine” in Minimum requirements for biological products, Association of Biologicals Manufactures of Japan ed., Pp. 99-101, 1993 ). The current Japanese encephalitis vaccine is a highly safe inactivated vaccine that has been studied several times until now.Appl.Environ.Microbiol39: 54-7, 1980 andVaccine9 (12): 865-7, 1991), because the brain of Japanese encephalitis virus-infected mice is the raw material (BIKEN J11: 25-39, 1968), there is a risk of side reactions due to brain material that may remain or from mouse-invading viruses. In addition, since the virus itself is handled, the manufacturing process involves dangerous work, and therefore the manufacturing cost is high (Vaccine18: 26-32, 2000 andLancet337 (8748): 1044, 1991). As a result, there is a problem that it is difficult to obtain vaccines in developing countries. In view of this situation, WHO has urgently requested the development of a low-cost vaccine that does not use the mouse brain, does not use viruses. In response to this, development research on next-generation Japanese encephalitis vaccines is being promoted by various methods in laboratories around the world (Intervirology44 (2-3): 176-97, 2001).
[0006]
There is a method of expressing an antigen in a recombinant cell into which a structural gene of Japanese encephalitis virus has been introduced by recombinant DNA technology. However, because the E protein of Japanese encephalitis virus is highly toxic to cells, in the animal cell expression system, the expressed cells themselves may be killed by the produced E protein (J.Virol75 (5): 2204-12, 2001). In addition, there is a report that in the yeast expression system, the particle antigen is cleaved and neutralizing antibody cannot be induced (Bull.World Health Organ65 (3): 303-8, 1987). A method of incorporating a structural gene of Japanese encephalitis virus into the genome of vaccinia virus and expressing an antigen in this recombinant vaccinia virus (Japanese Patent Laid-Open Nos. 62-44178 and 64-74082, Japanese Patent Laid-Open Nos. 5-276941 and 6-181792 andJ.Virol64 (6): 2788-95, 1990) and methods of expressing an antigen using a baculovirus expression system (Japanese Patent No. 2511494 and Japanese Patent Laid-Open No. 2000-60567), both of which have been attempted. It has not been successful in expressing antigens that replace inactivated Japanese encephalitis virus.
[0007]
JP 2001-299336 A discloses an animal cell that expresses a virus-like particle obtained by transfecting a cDNA containing a flavivirus precursor membrane (prM) protein gene and an E protein gene into which a mutation has been introduced; Disclosed are virus-like particles expressed by animal cells. In flaviviruses, prM protein is cleaved by furin and matures into a membrane (M) protein, resulting in E protein rearrangement, and hemagglutination and membrane fusion activity appear, indicating cytotoxicity. It has been. In Japanese Patent Application Laid-Open No. 2001-299336, mutations were introduced into the prM protein gene to inhibit prM protein cleavage, thereby reducing cell damage caused by virus-like particles. However, the antigen expression level of the cells disclosed in this publication is very low and is not suitable for practical use.
[0008]
In addition, a DNA vaccine that produces a non-infectious particle antigen using a gene encoding a structural protein of Japanese encephalitis virus is also disclosed (International Publication WO99 / 63095,J.Virol73 (12): 10137-45, 1999,Vaccine 18 (1-2): 68-75, 1999 andJ.Virol73 (7): 5527-34, 1999). Although this method does not use mice or viruses, there are various discussions on immunity induction ability and safety, and international consensus has not yet been obtained (Vaccine 19 (2-3): 155-7, 2000).
[0009]
[Problems to be solved by the invention]
In response to requests from WHO, development of a wide variety of next-generation Japanese encephalitis vaccines is progressing around the world. One of them is a method of producing Japanese encephalitis virus-like particles in genetically modified cells. However, since the virus-like particles themselves have cytotoxicity, the establishment of a high expression system has not been successful.
[0010]
[Means for Solving the Problems]
As a result of intensive studies to solve the above-mentioned problems, the present inventors included a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein prepared in this order from genomic RNA of Japanese encephalitis virus. And constructing a replicable recombinant DNA in which the cDNA can be expressed in a replicable expression vector containing a β-actin promoter, and transforming animal cells with the replicable recombinant DNA. Transformed cells formed by transformation include a Japanese encephalitis virus M protein and E protein, but are a protein aggregate that does not contain RNA in the particle, and a virus-like particle that exhibits hemagglutination activity It was found to express. This virus-like particle had sufficient immunogenicity to be used as an active ingredient such as a vaccine instead of inactivated Japanese encephalitis virus. In addition, the above-mentioned transformed cells were acclimated to a serum-free medium, and expressed in a serum-free medium even after passage of more than 52 passages of a large amount of antigen exceeding 6 μg per ml of culture supernatant. The present invention has been completed based on these findings.
[0011]
Accordingly, an object of the present invention is to provide a Japanese encephalitis virus antigen including Japanese encephalitis virus-like particles, which can replace the inactivated Japanese encephalitis virus used as an active ingredient (antigen) of the current Japanese encephalitis vaccine. It is in.
[0012]
Another object of the present invention is to provide a transformed cell that continuously expresses a large amount of Japanese encephalitis virus-like particles and a method for producing a Japanese encephalitis virus antigen using the same.
[0013]
Another object of the present invention is to provide a low-cost Japanese encephalitis vaccine and diagnostic agent that does not use mouse brain, does not use virus.
[0014]
Next, in order to facilitate understanding of the present invention, basic features and preferred embodiments of the present invention will be listed first.
[0015]
1. A Japanese encephalitis virus antigen comprising Japanese encephalitis virus-like particles, wherein the virus-like particles include M protein and E protein of Japanese encephalitis virus, but the particles do not contain RNA. A Japanese encephalitis virus antigen which is an aggregate and the virus-like particles exhibit hemagglutination activity.
[0016]
2. 2. The Japanese encephalitis virus antigen according to item 1, wherein the virus-like particles are obtained by a method comprising the following steps.
(A) providing a cDNA prepared from the genomic RNA of Japanese encephalitis virus, the cDNA comprising a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein in this order;
(B) constructing a replicable recombinant DNA obtained by incorporating the cDNA into a replicable expression vector containing a β-actin promoter so that the cDNA can be expressed;
(C) transforming animal cells with the replicable recombinant DNA to form transformed cells;
(D) selecting the transformed cells from the parent cells;
(E) culturing the transformed cells in a medium, allowing the transformed cells to express the cDNA and releasing virus-like particles into the medium; and
(F) Isolating the virus-like particles from the medium.
[0017]
3. 3. The Japanese encephalitis virus antigen according to item 2 above, wherein the cDNA is the base sequence of SEQ ID NO: 4 in the sequence listing.
[0018]
4). 4. The Japanese encephalitis virus antigen according to item 2 or 3, wherein the animal cell is an RK13 cell.
[0019]
5). 4. The Japanese encephalitis virus antigen according to any one of items 2 to 3, wherein the medium is a serum-free medium.
[0020]
6). (A) providing a cDNA prepared from the genomic RNA of Japanese encephalitis virus, the cDNA comprising a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein in this order;
(B) constructing a replicable recombinant DNA obtained by incorporating the cDNA into a replicable expression vector containing a β-actin promoter so that the cDNA can be expressed;
(C) transforming animal cells with the replicable recombinant DNA to form transformed cells;
(D) selecting the transformed cells from the parental cells; and
(E) Acclimate the transformed cells to serum-free medium
A transformed cell obtained by a method comprising the above.
[0021]
7. 7. The transformed cell according to item 6 above, wherein the cDNA is the base sequence of SEQ ID NO: 4 in the sequence listing.
[0022]
8). 8. The transformed cell according to item 6 or 7, wherein the animal cell is an RK13 cell.
[0023]
9. (A) culturing the transformed cell according to any one of the preceding items 6 to 8 in a serum-free medium, releasing Japanese encephalitis virus-like particles into the culture solution;
(B) obtaining a culture supernatant from the culture solution;
(C) isolating and purifying the virus-like particles from the culture supernatant,
And a method for producing a Japanese encephalitis virus antigen.
[0024]
10. A Japanese encephalitis vaccine comprising the Japanese encephalitis virus antigen according to any one of the preceding items 1 to 5 as an active ingredient in an immunizing amount.
[0025]
11. A diagnostic agent comprising the Japanese encephalitis virus antigen according to any one of items 1 to 5 as an active ingredient.
[0026]
Hereinafter, the present invention will be specifically described.
As explained in the section of “Prior Art”, the E protein of Japanese encephalitis virus is responsible for receptor binding and membrane fusion of virus particles, has many antigen-protecting epitopes, and forms a particle structure by molecular assembly. It is the first time that it is immunogenic. In mature virus particles, it is known that E protein and M protein exist on the particle surface in a combined form, and E protein having the configuration at this time causes cell damage. On the other hand, in immature virus particles in cells, E protein constitutes a heterodimer with prM protein, which is a glycosylation precursor of M protein. Such immature virus particles have low viral biological activities such as infectivity, hemagglutination (HA) activity, and fusion activity. It is believed that when the prM protein is cleaved by the enzyme furin present in the cell and matures into the M protein, and E protein rearrangement occurs (that is, when the virus particle matures), viral biological activity is exerted. .
[0027]
In JP-A-2001-299336, an attempt was made to establish an E protein antigen-expressing cell line, focusing on the low viral biological activity of immature virus particles in which prM protein is bound to E protein. Specifically, a mutation was introduced into a gene encoding prM protein to inhibit cleavage of prM protein into M protein, and E protein was prevented from adopting a configuration having cytotoxicity. However, since the antigen expressed by this cell line consists of prM protein and E protein, it is an antigen having a clearly different structure from the original virus particle consisting of M protein and E protein, and the expression level of the antigen. Has a very low accumulation of 200 ng / ml after 5 days of culture.
[0028]
Also, Hunt et al.J.Virol.Methods 97: 133-149, 2001), COS-1 cells were transformed with recombinant DNA containing genes encoding prM protein and E protein of Japanese encephalitis virus, and recombinant particulate antigens were expressed in the transformed cells. It was. However, the antigenicity of the antigen expressed by the transformed cells is very low and mice cannot be immunized without an adjuvant.
[0029]
As a result of diligent research to solve such problems, the present inventors have obtained a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein prepared from genomic RNA of Japanese encephalitis virus in this order. Providing a cDNA comprising the construct, constructing a replicable recombinant DNA in which the cDNA can be expressed in a replicable expression vector containing a β-actin promoter, and constructing an animal cell with the replicable recombinant DNA. A transformed cell formed by transformation contains a protein of Japanese encephalitis virus, M protein and E protein, but is a protein aggregate that does not contain RNA in the particle, and shows a hemagglutination activity It has been found that a large amount of such particles are expressed continuously.
[0030]
The virus-like particle contained in the Japanese encephalitis virus antigen of the present invention is a protein aggregate that contains the M protein and E protein of Japanese encephalitis virus, but does not contain RNA in the particle. Since the virus-like particles used in the present invention are protein antigens that do not contain the genomic RNA of Japanese encephalitis virus, there is no fear of infection. It has been confirmed by SDS-PAGE and amino acid sequence analysis that the virus-like particles include E protein and M protein. Specifically, the full length E protein and its fragment and the full length M protein and its fragment were included, and no prM protein was detected. From this, it is considered that the prM protein is not contained at all in the virus-like particle or is contained in a small amount. As described above, in Japanese encephalitis virus, prM protein is cleaved and matured to M protein, so that E protein and M protein exist on the surface of the virus particle. The virus-like particles used in the present invention include E protein and M protein, but are not considered to include prM protein. Therefore, the prM protein is treated in the expression cell in the same manner as in the original Japanese encephalitis virus. It is thought that it matures to M protein.
[0031]
Moreover, the virus-like particle contained in the Japanese encephalitis virus antigen of the present invention exhibits hemagglutination (HA) activity. The HA activity is an activity in which a virus, an antibody or the like causes red blood cells to aggregate. The HA activity is represented by the highest dilution factor at which hemagglutination was observed, and the unit is HAU (HA unit). The Japanese encephalitis virus antigen of the present invention has a HA activity of 150 HAU or more, preferably 500 HAU or more, at a protein content of 1 μg / ml. This HA activity is a value measured at an optimum pH based on the method described in Example 2 of the present application. Specifically, a virus solution diluted with 0.4% ovalbumin-borated caustic soda buffered saline (pH 9.0) was prepared, and an equal amount of 0.33% goose erythrocyte suspension (phosphoric acid at optimum pH) was prepared. And prepared at 37 ° C. for 1 hour, and then the presence or absence of erythrocyte aggregation was determined. In Example 2, the optimum pH of HA activity was 5.8 to 6.0, but the optimum pH differs somewhat depending on the strain of virus-like particle-producing cells and the number of passages thereof. The HAU value is a value measured at an optimum pH.
[0032]
Although the antigen contained in the current Japanese encephalitis vaccine is a virus inactivated with formalin or the like, the inactivated virus contains E protein and M protein, but its surface structure has changed, so it exhibits HA activity. Absent. The virus-like particles contained in the antigen of the present invention include M protein and E protein, and have HA activity, and thus are considered to have a structure and antigenicity similar to the surface structure of the original Japanese encephalitis virus.
[0033]
In order to examine the immunogenicity of the Japanese encephalitis virus antigen of the present invention, the purified antigen was administered to a 4-week-old female ddY mouse twice at weekly intervals, and the neutralizing antibody titer in mouse serum was measured by the plaque reduction method. did. As a result, the neutralizing antibody titer (log) equal to or higher than the current Japanese encephalitis vaccine, specifically 3.15 or higher_Ten) Was observed (see Example 6 herein). As is clear from this result, it was revealed that the antigen of the present invention exhibits immunogenicity equivalent to or higher than that of the current vaccine. Therefore, the antigen of the present invention, which is a protein aggregate including E protein and M protein and includes virus-like particles exhibiting HA activity, has the same immunogenicity as the antigen derived from the original virus. Instead of inactivated Japanese encephalitis virus, it can be used as a vaccine or a diagnosis-like antigen.
[0034]
Furthermore, the virus-like particles contained in the antigen of the present invention are preferably virus-like particles obtained by a method comprising the following steps. (A) providing a cDNA prepared from genomic RNA of Japanese encephalitis virus, the cDNA comprising a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein in this order; (b) a β-actin promoter A replicable recombinant DNA constructed by incorporating the cDNA into a replicable expression vector containing the vector, and (c) transforming an animal cell with the replicable recombinant DNA to form a transformed cell (D) selecting the transformed cells from the parental cells; (e) culturing the transformed cells in a medium, allowing the transformed cells to express the cDNA and releasing virus-like particles into the medium. And (f) isolating the virus-like particles from the medium. Specifically, virus-like particles prepared using the transformed cells of the present invention described later are preferred.
[0035]
The virus-like particles contained in the Japanese encephalitis virus antigen of the present invention are more preferably those isolated from a serum-free medium, and cells adapted to the serum-free medium such as the transformed cells of the present invention are cultured in the serum-free medium. In addition, virus particles isolated from the culture supernatant are more preferable. Normally, when cells are cultured, serum is added to the medium to supplement nutrients necessary for survival, but serum is likely to remain in the purified protein as an impurity. Therefore, virus-like particles isolated from a serum-free medium are virus-like particles having a higher purity than virus-like particles isolated from a normal serum-added medium. Specifically, virus-like particles isolated from serum-free media are unlikely to contain not only serum-derived proteins but also potentially dangerous viruses and abnormal prions contained in serum. When used as an agent, purification and quality control are easy, and it can be produced at low cost.
[0036]
As a serum-free medium used for isolation of virus-like particles, a serum-free medium described later may be used as a medium for transformed cells adapted to the serum-free medium. The method for isolating virus-like particles from the medium is not particularly limited, and may be performed according to a commonly used method for purifying protein antigens. A specific method will be described together with a method for producing the Japanese encephalitis virus antigen of the present invention described later.
[0037]
The present invention further provides (a) a cDNA prepared from the genomic RNA of Japanese encephalitis virus, the cDNA comprising a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein in this order; ) Constructing a replicable recombinant DNA in which the cDNA can be expressed in a replicable expression vector containing a β-actin promoter, and (c) transforming an animal cell with the replicable recombinant DNA. Providing a transformed cell obtained by a method comprising: forming a transformed cell; (d) selecting the transformed cell from a parent cell; and (e) acclimating the transformed cell to a serum-free medium. .
[0038]
As described above, when prM protein is cleaved and matured to M protein and E protein rearrangement occurs, HA activity and membrane fusion activity appear and cause cell damage. Because of this phenomenon, it has been considered difficult to express in animal cells virus-like particles having a structure close to the original surface structure of Japanese encephalitis virus, including E protein and M protein. In addition, as in the study by Hunt et al., Even if the expression of the particulate antigen including the M protein and the E protein of Japanese encephalitis virus by animal cells is successful, the particles have such an antigen value as that of PBS without adjuvant. The antigenicity of the dendritic antigen was low. However, in the present invention, when animal cells are transformed to express the Japanese encephalitis virus prM protein and E protein cDNA under the control of the β-actin promoter, surprisingly, the Japanese encephalitis virus A virus-like particle that includes M protein and E protein, but does not contain RNA in the particle and exhibits hemagglutination activity, ie, the original Japanese encephalitis virus We succeeded in expressing a large number of virus-like particles with a structure and antigenicity similar to the surface structure. As is apparent from FIG. 3, the transformed cell of the present invention produces virus-like particles exceeding 6 μg (at the maximum exceeding 12 μg) per 1 ml of the culture supernatant. There was no decline even after. For example, since the antigen expression level in Japanese Patent Application Laid-Open No. 2001-29936 is 200 ng / ml, the present invention has achieved an antigen expression level that is more than 30 times that amount.
[0039]
In the present invention, in order to obtain virus-like particles having the same structure as the surface structure of Japanese encephalitis virus, a cDNA prepared from genomic RNA of Japanese encephalitis virus, a cDNA fragment encoding prM protein and E protein A cDNA comprising a cDNA fragment encoding in this order is used. The cDNA used in the present invention includes a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein in the direction from the 5 'end to the 3' end. The base sequence of the cDNA is not particularly limited, but preferably includes a base sequence encoding a signal peptide upstream (5 'side) of the cDNA fragment encoding the prM protein. The base sequence of the DNA may be the base sequence of any virus strain identified as the Japanese encephalitis virus, but considering the safety and effectiveness of the antigen, the Japanese encephalitis virus used in the current Japanese encephalitis vaccine It is preferable to use a base sequence of a strain, such as a Beijing strain. For example, a cDNA sequence encoding the signal peptide to the E protein of the Japanese encephalitis virus Beijing strain described in SEQ ID NO: 4 in the sequence listing can be used. In the base sequence of SEQ ID NO: 4, the 10th to 78th bases encode a signal peptide (SEQ ID NO: 5) consisting of 23 amino acid residues, and the 79th to 579th bases consist of a prM protein consisting of 167 amino acid residues (sequence) No. 6), of which the 355th to 579th bases encode the M protein (SEQ ID NO: 7) consisting of 75 amino acid residues generated by cleavage of the prM protein, and the 580th to 2079th bases are 500 amino acid residues The E protein consisting of (SEQ ID NO: 8) is encoded.
[0040]
Next, a replicable recombinant DNA is constructed by incorporating the above-described cDNA into a replicable expression vector containing a β-actin promoter so as to allow expression. The recombinant DNA is not particularly limited as long as the Japanese encephalitis virus cDNA is expressed under the control of the β-actin promoter. The Japanese encephalitis virus cDNA can be inserted into an expression vector containing the β-actin promoter, It can be prepared by replacing the control region with an expression cassette containing a β-actin promoter and Japanese encephalitis virus DNA. As such a recombinant DNA, the recombinant plasmid pCAGJ12bsr of FIG. 1 including the base sequence described in SEQ ID NO: 4 of the Sequence Listing can be mentioned. This recombinant plasmid also contains a blasticidin resistance gene in order to select transformed cells using blasticidin resistance as an index.
[0041]
Next, animal cells are transformed with the above-described replicable recombinant DNA. The animal cell to be transformed in the present invention is not particularly limited as long as it can be cultured in a culture solution having a low serum concentration, is low in sensitivity to Japanese encephalitis virus infection, and has a high protein secretion ability. Examples of such animal cells include RK13 cells. RK13 cells are a cell line derived from rabbit normal kidney cells and are available from the American Type Culture Collection (ATCC) (ATCC No. CCL-37). RK13 cells are a cell line that can be cultured in a culture solution having a low serum concentration and can be maintained for a long period of time. In addition, it is considered to be resistant to the cytotoxicity caused by E protein because of its low susceptibility to Japanese encephalitis virus infection. In addition, it is known that the Golgi apparatus is developed in normal kidney cells of rats and the protein secretion ability is high (The Journal of Cell Biology, 144, 1135-1149, 1999). RK13 cells are also considered to have high protein secretion ability. In Example 1 of the present application, Japanese encephalitis virus cDNA was expressed in RK13 cells having such characteristics under the control of the β-actin promoter. The resulting transformed cells expressed a large amount of Japanese encephalitis virus particles having a structure and antigenicity similar to the surface structure of the original Japanese encephalitis virus without being damaged by E protein. .
[0042]
Selection of transformed cells from parent cells may be performed based on a selection factor contained in the recombinant DNA. In Example 1 of the present application, blasticidin resistance was used as a selection factor. However, a recombinant DNA was constructed so as to confer an appropriate selection factor, for example, drug resistance or auxotrophy, and transformed cells were used based on that. Can be sorted.
[0043]
The medium for maintaining and culturing the transformed cells of the present invention is not particularly limited as long as the transformed cells can grow, and a medium usually used for culturing RK13 cells should be used. Can do. For example, an MEM (Eagle's Minimal Essential Medium) supplemented with 10% FBS can be used as the medium. The cell culture temperature is 35 to 38 ° C, preferably 36 to 37 ° C.
[0044]
Furthermore, the transformed cells are acclimated to a serum-free medium. Usually, when culturing cells, a medium supplemented with serum is used. However, if serum is not added to the medium, the adhesiveness of the cells becomes weak, and the pH buffering capacity of the medium, CO₂Since the retention ability and the ability to retain cells from toxic substances are reduced, problems such as the cells failing to divide or produce antigens occur. If the antigen itself is cytotoxic, such as Japanese encephalitis virus antigen, and even its expression is difficult, culturing in a serum-free medium that gives great stress to the transformed cells could not be considered. For example, in Japanese Patent Application Laid-Open No. 2001-299336, when 10% FBS contained in the culture medium of a Japanese encephalitis virus antigen-producing cell line was changed to 0.1% BSA, the antigen expression level decreased to about 1/3. .
[0045]
In the present invention, in order to obtain transformed cells acclimatized to a serum-free medium without causing the above-mentioned problems, the serum concentration of a medium containing 5 μg / ml blasticidin is sequentially decreased by ½. The transformed cells were subcultured to the medium, and finally the medium was completely switched to a serum-free medium. Specifically, the transformed cells cultured in 10% FBS-MEM are cultured in 1st to 8th generations in 5% FBS-MEM, and in the 9th to 16th generations in 2.5% FBS-MEM, The 17th to 18th generations were cultured with 1.25% FBS-MEM, and the 19th to 20th generations were cultured in 1.25% FBS-VP-SFM [VP-SFM (Gibco BRL, USA) with 1.25% FBS. To which was added]. Next, the transformed cells were subcultured with VP-SFM containing no serum to obtain serum-free medium-conditioned antigen-expressing cells. The serum-free medium-conditioned cells thus obtained produced virus-like particles in a larger amount than conventional Japanese encephalitis virus antigen-producing cells, although the production of virus-like particles was somewhat inferior to non-conditioned cells. Moreover, its ability to produce virus-like particles is not lost even after passage (see FIGS. 2 and 3 of the present specification). In the case of a serum-added medium, it is difficult to supply a large amount of a uniform medium because there is a difference between serum lots, but in the case of a serum-free medium, there is no such problem.
[0046]
The serum-free medium for conditioning the transformed cells is not particularly limited as long as the transformed cells of the present invention can grow and express virus-like particles. For example, it can be subcultured with a medium in which Eagle MEM is used as the basal medium and the concentration of the added serum is reduced. As serum-free medium for culturing serum-free medium-conditioned cells, Eagle MEM, hormones such as insulin and hydrocortisone, epidermal growth factor (EGF), platelet-derived growth factor (platelet) Derived growth factor (PDGF), growth factor such as fibroblast growth factor (FGF), cell adhesion and growth factor such as fibronectin, low density lipoprotein (LDL), transferrin, phospholipid, etc. A culture medium can be used. These additives are preferably not derived from humans or animals. A synthetic medium such as VP-SFM (manufactured by GIBCO BRL, USA), EX-CELL 525 (manufactured by JRH Bioscience, USA) can also be used.
[0047]
In addition, in order to limit the growth of cells in which the recombinant DNA has been detached and no longer produce antigens, when culturing transformed cells, the cells are always selectively selected with the selection factor introduced into the recombinant DNA. It is necessary to culture. Since the transformed cells prepared in Example 1 of the present application are transformed with a recombinant DNA introduced with a blasticidin resistance gene as a marker, in order to maintain antigen-producing cells, blasticidin is added to S The transformed cells were cultured in a medium supplemented with 5 μg / ml of hydrochloride.
[0048]
The present invention further comprises (a) culturing the transformed cell of the present invention in a serum-free medium, releasing Japanese encephalitis virus-like particles into the culture solution, (b) obtaining a culture supernatant from the culture solution, c) A method for producing a Japanese encephalitis virus antigen, comprising isolating and purifying the virus-like particles from the culture supernatant. First, transformed cells adapted to a serum-free medium are cultured in a serum-free medium. The serum-free medium for culturing the transformed cells of the present invention is not particularly limited as long as the transformed cells can proliferate and express virus-like particles, but are used when acclimating the transformed cells to the serum-free medium. A fresh medium may be used. Specifically, Eagle's MEM to which an additive in place of serum (growth factor, cell adhesion / proliferation factor, etc.) or a synthetic medium can be used. The culture of the transformed cells is not particularly limited as long as it is a method for culturing animal cells, but the culture temperature is 35 to 38 ° C, preferably 36 to 37 ° C.
[0049]
Next, cells are removed from the culture solution to obtain a culture supernatant. The method for obtaining the culture supernatant is not particularly limited, and a commonly used method such as centrifugation or filtration can be used.
[0050]
Virus-like particles are purified from the obtained culture supernatant. The method for purifying virus-like particles is not particularly limited, and various methods used for purifying proteins such as antigens may be combined. The Japanese encephalitis virus E protein is known to exhibit immunogenicity only when it is assembled into a particle structure, and the virus-like particle contained in the antigen of the present invention is also similar to the Japanese encephalitis virus particle. In addition, it is unstable to solubilizers, EDTA, organic solvents, etc. under extreme pH conditions such as centrifugal force and osmotic pressure, acid and alkali, and loses its particle structure and loses its antigenicity. there is a possibility. Therefore, it is desirable that virus-like particles can be isolated and purified in a simple manner and in a high yield under mild purification conditions. Specific purification methods include physical low-speed centrifugation, ultracentrifugation, filtration, molecular sieving, membrane concentration, etc., chemical precipitation agents, solubilizers, desorbents, dispersants, etc. Induction, column chromatography, dialysis, salting out, and the like can be used in combination. For example, the culture supernatant can be concentrated by centrifugation through an ultrafiltration membrane, and gel chromatography and sucrose density gradient centrifugation can be combined. In applying these means, physicochemical conditions such as temperature, pressure, pH, and ionic strength can be set as appropriate.
[0051]
For example, in Example 3 of the present specification, the culture supernatant is centrifuged and concentrated with an ultrafiltration membrane, and the virus-like particles are purified by a combination of gel chromatography and sucrose density gradient centrifugation. A high antigen recovery rate of over% was achieved. Purity assay experiments by protein fluorescence staining and Western blot after SDS-PAGE revealed that the produced antigen was highly purified, and the purified antigen was observed with an electron microscope. The particles were found to be in the form of small spherical particles. Also from this, it was possible to purify virus-like particles easily in high yield without losing the particle structure of virus-like particles.
[0052]
Furthermore, the present invention provides a Japanese encephalitis vaccine comprising the Japanese encephalitis virus antigen of the present invention in an effective amount as an active ingredient. The Japanese encephalitis virus antigen of the present invention induces antibodies in mice as in the case of the current Japanese encephalitis vaccine, as is clear from the results of the neutralizing antibody test of Example 6 and the protection test of Example 7 of the present specification. And can prevent infection by Japanese encephalitis virus. Therefore, the vaccine containing the Japanese encephalitis virus antigen of the present invention as an active ingredient is a vaccine that replaces the current Japanese encephalitis vaccine containing inactivated Japanese encephalitis virus as an active ingredient.
[0053]
Japanese encephalitis vaccine can be produced as follows.
The purified antigen of the present invention is suspended in a solvent such as an isotonic saline solution, a buffer solution, or a tissue culture solution, for example, PBS (phosphate buffer saline) to prepare a vaccine stock solution. If necessary, the steric structure can be immobilized by immobilizing the vaccine antigen with a conventional immobilizing agent. Examples of the immobilizing agent include formalin, phenol, glutardialdehyde, β-propiolactone and the like. The immobilizing agent can be added to the antigen before preparing the vaccine stock solution or added to the vaccine stock solution.
[0054]
Next, the vaccine stock solution is diluted to prepare a vaccine solution. For example, PBS is used as the vaccine stock solution, and the amount of antigen in the vaccine is an amount necessary for inducing antibody production and producing immunity, for example, a protein content of 1 to 20 μg / ml, preferably 10 μg / ml. Dilute as follows. For example, the HA value when the protein content is adjusted to 10 μg / ml is 1,500 to 24,000 HAU, preferably 5,000 to 10,000 HAU. When preparing a vaccine solution, a stabilizer for enhancing the heat resistance of the vaccine or an adjuvant as an adjuvant for enhancing the immunogenicity may be added and mixed. Examples of the stabilizer include saccharides and amino acids, and examples of the adjuvant include mineral oil, vegetable oil, alum, aluminum compound, bentonite, silica, muramyl dipeptide derivative, thymosin, and interleukin.
[0055]
The vaccine solution is dispensed into a container of an appropriate volume, for example, a vial of about 0.5 to 20 ml, sealed and sealed, and then used as a vaccine. Such a vaccine can be used not only in liquid form but also as a dry preparation by lyophilization after dispensing.
[0056]
The vaccine of the present invention may be inoculated to the recipient similarly to the current Japanese encephalitis vaccine. For example, a dose of about 0.2 to 0.5 ml of vaccine may be inoculated subcutaneously 1 to 3 times at intervals of about 1 to 4 weeks. In addition, the dried preparation is used after returning to its original volume after dissolution with sterile distilled water or the like before inoculation.
[0057]
Furthermore, the antigen of the present invention can be used in place of a conventionally used antigen as a diagnostic antigen. As shown in Example 8 of the present specification, the antigen of the present invention can be used for diagnosis of Japanese encephalitis virus infection carried out by ELISA or the like. Specifically, the antigen of the present invention can be used for ELISA, hemagglutination inhibition (HI) test, and the like.
[0058]
The concentration of the diagnostic antigen is not particularly limited, but a solution having a protein content of 1 μg / ml is suitable. For example, the HA value when the protein content is adjusted to 1 μg / ml is 150 to 2,400 HAU, preferably 500 to 1,000 HAU.
[0059]
The antigen for vaccine and diagnostic antigen of the present invention is preferably an antigen including virus-like particles isolated from a serum-free medium. As described above, virus-like particles isolated from serum-free medium are not only proteins derived from serum, but are less likely to be contaminated with viruses or abnormal prions that may be contained in serum. Therefore, purification and quality control are easy when used as a vaccine or diagnostic agent.
[0060]
DETAILED DESCRIPTION OF THE INVENTION
EXAMPLES Hereinafter, although an Example is given and this invention is further demonstrated, this invention is not limited to these at all.
[0061]
The following describes the ELISA method, the total protein quantification method, SDS-PAGE, Western blot, plaque method and neutralizing antibody titer measurement method in the present application.
[0062]
ELISA ( Enzyme linked immunosorbent assay ) Law
96-well immunoplate (Coaster, USA)^ROn the other hand) and anti-E protein monoclonal antibody (Group-8, Clone 503, IgG) with high neutralizing activity against Japanese encephalitis virus (J.Immunol141 (10): 3606-10, 1988; dispensed by Dr. Kotaro Yasui of the Tokyo Metropolitan Institute for Neurosciences (hereinafter often referred to as “503 monoclonal antibody”), 50 μl each in a well. The antibody was immobilized by incubating overnight at 4 ° C. Plates were washed with PBS-T (8.0 g NaCl, 0.2 g KCl, 2.88 g Na₂HPO_Four/ 12H₂O, 0.2 g KH₂PO_Four, 0.5 ml of Tween20 was dissolved in distilled water, and the resulting solution was washed 4 times with a final volume of 1 liter solution), and each washing was performed for about 8 seconds. After washing, 50 μl / well of diluted antigen was applied and incubated at 37 ° C. for 1 hour. As a diluted antigen, a purified inactivated virus antigen (TJP ** 012), which is also used as a standard antigen and received from the Osaka University Society for Microbial Diseases, was serially diluted twice with PBS-T. Using. As the dilution factor, a factor corresponding to the linear region of the calibration curve prepared with the standard antigen diluent was used.
[0063]
After washing away unbound proteins, 50 μl of 503 monoclonal antibody labeled with horse radish peroxidase was applied to each well and incubated at 37 ° C. for 1 hour. After removing the labeled antibody by washing, TMB as a chromogenic substrate⁺(DAKO, Denmark) 50 μl was added to each well and left at room temperature for 15 minutes to carry out the enzyme reaction. The enzyme reaction was stopped by adding 50 μl of 1 mol / liter sulfuric acid to each well. Absorbance was measured at a wavelength of 450/690 nm using a plate reader (Multiskan MS) (Thermo Labsystems Inc., USA).
[0064]
As the standard antigen, purified inactivated virus antigen (TJP ** 012) was used. An ELISA of this antigen diluted 2-fold was performed in the same manner as described above to obtain a calibration curve. The amount of antigen (ELISA value) of the sample was calculated from the calibration curve.
[0065]
Total protein quantification
An equal amount of 10% (w / v) TCA solution was added to the sample, mixed, and then incubated on ice for 10 minutes. Centrifugation was performed at 4 ° C. and 12,000 rpm for 20 minutes, and the supernatant was removed. The remaining precipitate was washed by adding a 5% (w / v) TCA solution, and centrifuged at 4 ° C. and 12,000 rpm for 10 minutes to collect the precipitate. This precipitate was solubilized with 0.1N NaOH and used for protein quantification. For quantification, a color development system of BCA Protein Assay (manufactured by Pierce Chemical Company, USA) was used. 12.5 μl of sample and 100 μl of diluted reagent solution were mixed in a microplate and incubated at 37 ° C. for 30 minutes. Absorbance was measured at a wavelength of 570 nm using a plate reader.
[0066]
A calibration curve was prepared using bovine serum albumin (2 mg / ml) (Pierce Chemical Company, USA) as a standard protein, and the protein concentration of the sample was calculated from the calibration curve.
[0067]
SDS-polyacrylamide gel electrophoresis (SDS-PAGE)
Sample electrophoresis was performed on 12% SDS-polyacrylamide gel. Each sample was mixed with an equal volume of sample buffer [solution containing 0.5M Tris-HCl (pH 6.8), 4% SDS, 14 mM 2-mercaptoethanol], heated at 100 ° C. for 10 minutes, and protein in the sample. Was denatured to obtain a sample for electrophoresis. The obtained specimen was applied to the gel at 10 μl / lane. Electrophoresis was performed using electrophoresis buffer [25 mM Tris-HCl (pH 8.8), 192 mM glycine, 0.1% SDS] at a constant current of 10 mA in the concentrated gel and 20 mA in the separation gel. Inactivated purified antigen used in the current Japanese encephalitis vaccine was used as the standard antigen, and Prestained SDS-PAGE Standards, low range (BIO-RAD Laboratories, USA) was used as the molecular weight marker. Protein is SYPRO^R Fluorescent staining was performed using Orange Protein gel stain (Molecular Probes, USA). Specifically, SYPRO^R The gel was immersed in a staining solution obtained by diluting Orange Protein gel stain with a 7.5% acetic acid solution 5000 times and incubated at room temperature for 40 minutes. An image analyzer LAS-1000 (manufactured by Fuji Film Co., Ltd., Japan) was used for fluorescence detection.
[0068]
Western blot
The protein separated from the gel subjected to SDS-PAGE as described above was transferred to a polyvinylidene difluoride (PVDF) membrane. Transcription was performed by applying a constant current of 180 mA for 1 hour under ice cooling in a buffer containing 25 mM Tris-HCl (pH 8.8) and 192 mM glycine-20% methanol. PVDF membrane is Immobilon pretreated with methanol for 10 minutes at room temperature^TM(Millipore, USA) was used. The transferred membrane was blocked with 2% skim milk-PBS, then immersed in a solution obtained by diluting a rabbit anti-Japanese encephalitis virus (Beijing strain) polyclonal antibody used as a primary antibody 100-fold with PBS-T, and shaken at room temperature for 1 hour. did. After washing and removing the non-specifically adsorbed antibody with PBS-0.05% Tween 20, Alkaline phosphatase-labeled anti-rabbit IgG antibody (ALI 3405) (manufactured by BioSource Interna-tional, USA) was 5,000 times to 10 times with PBS-T. It was immersed in a solution diluted 000 times and further shaken at room temperature for 1 hour. BCIP / NBT Phosphatase Substrate (3-Component system) (manufactured by Kirkegaard & Perry Laboratories, Inc., USA) was used as the chromogenic substrate system.
[0069]
Plaque method
The cultured Vero cells (cultured with 10% FBS-MEM) are inoculated with a virus sample, and CO₂Incubated for 90 minutes in incubator. The plate was shaken every 15 minutes to allow the cells to adsorb the virus evenly. The cells from which the sample solution has been removed are overlaid with MEM containing 2% methylcellulose and 2% FBS, and CO.₂The cells were cultured for 3 days in an incubator. After confirming the presence or absence of plaque formation under an inverted microscope, the cells were treated with a 10% formalin solution for 1 hour on the 4th day of culture to inactivate the virus and fix the cells. Formalin and methylcellulose were washed away in cold water and stained with 0.038% methylene blue. A cell group denatured or necrotized by infection with a virus is not stained, and thus is measured as a plaque.
[0070]
Measurement of neutralizing antibody titer
Serum was serially diluted and mixed with an equal volume of Japanese encephalitis virus specimen. After incubation at 37 ° C. for 90 minutes, the virus in the Japanese encephalitis virus specimen was treated with the antibody (ie, neutralization was performed). The number of infectious viruses remaining in the Japanese encephalitis virus specimen after the neutralization reaction was measured by the plaque method. The neutralizing antibody titer was defined as the maximum serum dilution ratio required to reduce the number of plaques to 50% with the average number of plaques in each well being 100% in a plate infected with antibody-untreated Japanese encephalitis virus. .
[0071]
Example 1
Establishment of transformed cells that continuously express the Japanese encephalitis virus antigen
(1) Preparation of Japanese encephalitis virus RNA
As a Japanese encephalitis virus (hereinafter abbreviated as “JEV”), a purified JEV produced as a Japanese encephalitis virus vaccine at the Osaka University Microbial Disease Research Society was used.
[0072]
Take 100 μl of purified JEV in a 1.5 ml sampling tube and add 400 μl of Chaos Buffer consisting of Chaos / 2ME Buffer (4.2 M guanidine thiocyanate, 0.5% sarkosyl and 25 mM Tris-HCl (pH 8.0)). 25 μl of 2-mercaptoethanol was added to 400 μl) and mixed by pipetting to obtain a virus sample.
[0073]
RNA extraction of the obtained virus sample was performed as follows. 400 μl TE saturated phenol [phenol saturated with TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0))] and 50 μl phenol extraction buffer [100 mM Tris-HCl (pH 8.0) , 10 mM EDTA and 1% SDS] and mixed with Vortex. Then, it centrifuged at 15,000 rpm for 10 minutes, and left still at 65 degreeC for 30 minutes. Furthermore, 200 μl of chloroform was added, mixed with Vortex, centrifuged at 15,000 rpm for 10 minutes, and separated into an upper layer (RNA-containing TE layer) and a lower layer (phenol-chloroform layer). The upper layer was transferred to another sampling tube. 100 μl of TE buffer was added to the remaining phenol / chloroform layer to obtain a mixture. RNA was re-extracted from the obtained mixture in the same manner as described above to obtain an RNA-containing TE layer. A crude RNA sample was prepared together with the RNA-containing TE layer obtained in the first round.
[0074]
The crude RNA sample was extracted twice with phenol / chloroform and once with chloroform. Phenol / chloroform extraction was performed by adding an equal volume of 1 mM Tris-HCl (pH 8.0) saturated phenol / chloroform / isoamyl alcohol (25: 24: 1) to the sample, vortexing, and centrifuging at 12,000 rpm for 5 minutes. The upper layer was transferred to another sampling tube. Chloroform extraction was performed by vortexing the same amount of chloroform as the sample, centrifuging at 12,000 rpm for 5 minutes, and then transferring the upper layer to another sampling tube. Finally, 1/10 amount of 3M sodium acetate and 2.5 times amount of ethanol were added to the upper layer, vortexed, centrifuged at 12,000 rpm for 15 minutes, and the supernatant was discarded. The remaining precipitate was washed with 80% ethanol, centrifuged at 12,000 rpm for 5 minutes, and then the supernatant was discarded. The precipitated RNA was air-dried and dissolved in purified water to obtain JEV RNA.
[0075]
(2) cDNA production, PCR and cloning
The above method (1) was repeated, and RNA was recovered from a total of 900 μl of purified JEV and used for cDNA synthesis.
[0076]
Synthesis of cDNA was performed by PCR using a cDNA synthesis kit (superscript choice system for cDNA synthesis) manufactured by Gibco BRL, USA. For the synthesis of the first strand, together with the random primer attached to the kit,Virology 161: 497-510, 1987 or a synthetic primer prepared every about 500 bases based on the total RNA sequence of Japanese encephalitis virus genome published in GenBank Accession No. NC001437. PCR was performed with 10 μl of cDNA (1/20 of that obtained in one cDNA synthesis reaction), 1 μl of primer concentration, and the reaction conditions were 94 ° C. for 1 minute, 55 ° C. for 45 seconds, and 72 ° C. for 2 minutes. 35 cycles were performed.
[0077]
Molecular Cloning, Cold Spring Harbor Lab. According to the method described in 2001, 9.66 to 9.75 or using Megalabel (TaKaRa, Japan), the 5 ′ end of the synthesized JEV cDNA was phosphorylated, and it was converted to the SmaI site of pUC18 (TaKaRa, Japan). Alternatively, a recombinant plasmid was obtained by ligation to the SrfI site of pBlueScript SK (+) (manufactured by STRATAGEN, USA). E. coli competent cells are transformed with the obtained recombinant plasmid to obtain a transformed bacterium, and the obtained transformed bacterium is grown on an agar plate.Virology, 161: 497-510, 1987. The following three oligonucleotides (SEQ ID NOs: 1 to 3) prepared based on the base sequence of Japanese encephalitis virus reported in:
5'-GGGAGCCCTCTCAAAGCTTCTGCC-3 '(SEQ ID NO: 1)
5'-CCCCGCTCTTTGGAGGCACATTGC-3 '(SEQ ID NO: 2)
5'-CCCATCTTCCCTATACCCTTTCGC-3 '(SEQ ID NO: 3)
Screening was carried out by colony hybridization using as a probe. E. coli of a positive colony was cultured, and plasmid DNA was extracted. The extracted plasmid DNA was treated with an appropriate restriction enzyme, and the size of the DNA incorporated in the plasmid was examined by agarose electrophoresis. The base sequence of the cloned DNA of the desired size was confirmed by Southern hybridization using the oligonucleotides of SEQ ID NOs: 1 to 3 as probes.
[0078]
To confirm the base sequence, plasmid DNA is used as a template.³²Sanger method using P (Sequenase V.2) (Amersham Bioscience, USA) or PCR-fluorescence sequencer method was used. Regarding JEV cDNA, the sequences of a plurality of clones (one region of 3 clones or more) that were independently cloned were examined. As the sequencing primer, the same synthetic primer as that used in the above PCR was used.
[0079]
The target Japanese encephalitis virus cDNA, ie, the cDNA fragment encoding the signal peptide, the cDNA fragment encoding the prM protein, and the cDNA fragment encoding the E protein are linked in this order, and the signal peptide of the Japanese encephalitis virus Beijing strain, prM protein And J12 cDNA encoding E protein was obtained.
[0080]
(3) Construction of replicable recombinant DNA
The J12 cDNA obtained above was transformed into the pCAGGS expression vector (Gene 108: 193-199 (1991) to obtain a recombinant plasmid pCAGJ12. A fragment containing CMV-IE, β-actin promoter, intron and J12 cDNA was recovered from the obtained pCAGJ12, and pEFBOSbsr [pSV2bsr (made by Kaken Pharmaceutical, Japan) was inserted into the plasmid containing the FE-BOS promoter. Inserted from the control region of M. Tatsumi] to obtain recombinant DNApCAGJ12bsr having blasticidin resistance. The obtained pCAGJ12bsr is shown in FIG.
[0081]
(4) Preparation of Japanese encephalitis virus-like particle persistently expressing cells (J12 # 26 cells)
RK13 cells (ATCC No: CCL-37) were cultured at 36-37 ° C. in 10% fetal bovine serum (FBS) -added Eagle's minimal medium (MEM) until they became monolayers, and FuGENE6 (Roche Diagnostic Co., France) was used to transform with pCAGJ12bsr. The cells were cultured for 2 days (the medium was changed once in the middle), then the culture solution was removed, and PBS (-) containing 0.2% trypsin was added to the culture solution to add about 1/10 volume. Next, the cells are detached and dispersed by pipetting, suspended, diluted to 6 times the original culture volume with a medium containing 10 μg / ml blasticidin (manufactured by Kaken Pharmaceutical, Japan), and φ10 A blastcidin-resistant strain was selected by culturing for 10 days on the plate. The medium was changed on the 4th and 7th days. On day 10, 23 blasticidin resistant strains were selected, transferred to a 24-well plate containing 0.5 ml of medium and cultured. As the medium, the same blasticidin-containing MEM as described above was used. On the 4th day from the start of culture in a 24-well plate, 0.5 ml of a new medium was added, and further cultured for 2 to 5 days. About each cell line, a part of culture solution was sampled and it was confirmed by ELISA method whether the culture medium contained an antigen.
[0082]
In addition, cells grown in a 24-well plate are sequentially transferred to a φ6 or φ10 plate, and the culture scale is expanded and cultured for 3 to 6 days.²The flask was transferred to the flask and further cultured for 3 to 6 days. The culture scale was expanded by the following method. When the cells became monolayers, the culture solution was removed, and PBS (-) containing 0.2% trypsin was added to the culture solution to add about 1/10 volume. Next, the cells were detached and dispersed by pipetting, suspended, diluted to 6 times the original culture volume with a medium, and transferred to a new container.
[0083]
The cultured cells were stored frozen at -80 ° C. A portion of this stored cell is 25 cm²Further, the culture was subjected to ultracentrifugation to obtain a culture supernatant. The Japanese encephalitis virus antigen contained in the obtained culture supernatant was confirmed by Western blot. Cell lysates were also prepared for cultured cells, and the presence of Japanese encephalitis virus antigen was confirmed by Western blot.
[0084]
According to the result of ELISA and the result of Western blot, # 10 and # 20 among 23 transformants (# 1 to # 23) expressed a large amount of antigen.
[0085]
(5) Cloning of transformants
Using a 96-well microplate, cell lines # 10 and # 20 were diluted with a medium so that each would be 0.2 cells / 100 μl, and 100 μl was dispensed into each well and cultured. On the 4th day from the start of the culture, 100 μl of a new medium was added, and on the 8th, 12th and 15th days from the start of the culture, half of the medium was replaced with a new medium. From the 12th to the 20th day from the start of the culture, the grown cells were sequentially transferred to a 24-well plate to expand the culture scale, and 20 clones for the cell line # 10 and 27 clones for the cell line # 20 (J12 # 20.01 to J12 # 20.27). These clones were cultivated from 17 to 29 days from the start of culture with a φ6 plate and then 75 cm.²The culture scale was expanded to the flask. The culture scale was expanded by the method described above, and the cultured cells were stored frozen at -80 ° C. On the way, the antigen titer of the culture broth was confirmed by ELISA. Since the clone of cell line # 20, J12 # 20.14, showed the highest ELISA titer, this clone was subjected to the second cloning.
[0086]
Cell line J12 # 20.14 was cultured in 0.2 cells / 100 μl medium using a 96-well microplate. On the 8th day from the start of the culture, 100 μl of a new medium was added, and on the 14th, 19th and 22nd days from the start of the culture, half of the medium was replaced with a new medium. On the 18th to 27th days from the start of the culture, the grown cells were sequentially transferred to a 24-well plate to expand the culture scale, and 51 clones were obtained. Furthermore, from the beginning of culture to the 23rd to 34th, 75cm from the plate of φ6 and then φ10²The culture scale was expanded to the flask of (2), and the cultured cells were stored frozen at -80 ° C. On the 19th day from the start of the culture, 50 μl of the medium was sampled and the antigen titer was confirmed by ELISA. In addition, the growth state and cell morphology of cells during the culture were observed with the naked eye. As a result, out of 51 clones of J12 # 20.14, clone 26 was selected as an antigen-expressing cell and designated as J12 # 26 cell.
[0087]
(6) Acclimatization of J12 # 26 cells to serum-free medium
J12 # 26 cells subcultured to 10 generations with 10% FBS-MEM and cryopreserved were used in the following experiments. The cryopreserved cells were thawed and cultured in 10% FBS-MEM at 36-37 ° C. Hereinafter, 5 μg / ml blasticidin was added to the medium used for acclimation. J12 # 26 cells were acclimated to serum-free medium by subculturing the cells using a medium in which the added serum concentration was successively decreased by 1/2, 5%, 2.5%, and 1.25%. Specifically, 1st to 8th generations are cultured with 5% FBS-MEM, 9th to 16th generations are cultured with 2.5% FBS-MEM, and 17th to 18th generations are cultured with 1.25% FBS-MEM. The 19th to 20th generations were cultured in 1.25% FBS-VP-SFM [VP-SFM (Gibco BRL, USA) with 1.25% FBS added]. Next, J12 # 26 cells were passaged with VP-SFM containing no serum to obtain J12 # 26SF cells, a serum-free medium-conditioned antigen expression strain.
[0088]
(7) Confirmation of antigen production by ELISA
In order to confirm the antigen production amount of J12 # 26SF cells, the cells were passaged with VP-SFM, and the antigen titer of the culture supernatant was measured by ELISA. As a control, the antigen titer of the culture supernatant of non-acclimated cells (J12 # 26 cells passaged with 10% FBS-MEM) was also measured in the same manner. The results are shown in FIGS.
[0089]
As is clear from FIGS. 2 and 3, J12 # 26SF cells conditioned to serum-free medium (FIG. 3) are compared to non-conditioned cells J12 # 26 cells (FIG. 2) passaged with 10% FBS-MEM. Although inferior, it produced a sufficient amount of antigen. It was also revealed that the amount of antigen production did not decay even after passage.
[0090]
Example 2
J12 # 26SF Changes in HA value and ELISA value of virus-like particles expressed by cells over time
In order to confirm that the antigen expressed by J12 # 26SF cells showed HA activity, the HA value and ELISA value were measured, and the change with time was examined.
[0091]
For measurement of HA titer and ELISA titer, J12 # 26SF cells passaged for 38 passages with VP-SFM containing 5 μg / ml blasticidin were used, and as a control, Vero cells on the 4th day after culture were treated with Japanese encephalitis virus. What was infected with (Beijing strain) (JEV infected Vero cells) was used. J12 # 26SF cells were cultured in VP-SFM containing 5 μg / ml of blasticidin and were cultured for 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 10 days, 14 days and 17 days from the start of the culture. The culture supernatant was sampled on the eye. On the 10th day, the medium was changed. JEV-infected Vero cells were cultured in MEM containing 2% bovine serum, and the culture supernatant was sampled every day from day 1 to day 7 after the start of culture.
[0092]
(1) Measurement of HA value
Goose erythrocytes were used for the measurement of HA value. ACD was added to goose blood so that the ratio of blood to ACD (acid-citrate-dextrose) was 10: 1.5. Thereto, 2.5 volumes of DGV (dextrose-gelatin-veronal) was added and mixed, and the resulting mixture was centrifuged at 1,000 rpm for 15 minutes. The supernatant was discarded, and the same amount of DGV was added to the remaining sediment to suspend it, and then centrifuged again. This operation was repeated a total of 4 times to obtain a sediment. The obtained sediment was suspended in physiological saline to obtain an 8% erythrocyte suspension.
[0093]
50 μl of 0.4% egg albumin borated sodium hydroxide buffered saline (pH 9.0) was dispensed into each well of a U-shaped microplate. Place 100 μl of the sampled culture supernatant in the next well of the dilution series, and use a multichannel autopipette to dilute by repeatedly moving and pipetting 50 μl from the undiluted sample toward the next hole in order, and then dilute it twice. A measurement sample was obtained. 50 μl of 0.33% erythrocyte suspension prepared in phosphate buffer at an optimum pH (pH 6.0 for culture supernatant of J12 # 26SF cells, pH 6.2 for JEV-infected Vero cells) They were added one by one and immediately mixed with a micromixer. After standing at 37 ° C. for 1 hour, the aggregation reaction was determined. Red blood cells settled in the center of the bottom of the well, and those that looked like red dot clusters were negative. Red blood cells spread and settled across the bottom, and those that did not flow out even when tilted were positive. The HA value was expressed as the highest dilution factor of the virus solution that is positive for hemagglutination.
[0094]
(2) Measurement of ELISA value
The culture supernatant of J12 # 26SF cells (passage 38) and the culture supernatant of JEV-infected Vero cells were serially diluted 2-fold with PBS-T, and each was used as a sample. The ELISA titer was measured using a 503 monoclonal antibody. As a standard for the ELISA titer, TJP ** 012 which is a purified inactivated virus antigen was measured in the same manner, and the ELISA titer of the sample was converted with the ELISA titer of TJP ** 012 as 100. In addition, the ELISA titer was also measured in the culture supernatant of J12 # 26SF cells passaged 78 times. The HA titer and ELISA titer of JEV-infected Vero cells are shown in Table 1 and FIG. 4, and the HA titer and ELISA titer of virus particles of J12 # 26SF cells are shown in Table 2 and FIG.
[0095]
For the culture supernatant of JEV-infected Vero cells, the infectivity titer was measured by the plaque method using a sample diluted 10-fold, and the results are shown in Table 1.
[0096]
[Table 1]

[0097]
[Table 2]

[0098]
In virus culture using Vero cells, both ELISA and HA values reached their highest values on the 4th day of culture, and the cytopathic effect (CPE) caused by the virus progressed from the 5th day onwards. Both declined. On the other hand, in the culture of J12 # 26SF cells, virus-like particles continued to be expressed after the 4th day, and both the ELISA value and the HA value increased. On the 10th day, when the culture supernatant was collected and a new medium was added, the expression of virus-like particles continued. Therefore, a large amount of virus-like particles can be expressed in one cell culture by changing the medium at an appropriate time. As is clear from the above results, the antigen produced by J12 # 26SF cells showed HA titer, and as shown in FIG. 5, there was a substantially linear correlation between HA titer and ELISA titer. . Further, as is apparent from Table 2, it was found that J12 # 26SF cells produced 2 to 3 times the amount of antigen contained in the culture medium when Japanese encephalitis virus was grown on Vero cells. . The J12 # 26SF cell, which is the transformed cell line of the present invention, expresses a large amount of the antigen continuously, and such a large amount of the sustained antigen is expressed even if the cell passage proceeds (even in the 78th generation). There was no decline.
[0099]
Example 3
Purification of virus-like particles from serum-free medium
J12 # 26SF cells were cultured in a serum-free medium for 72 hours in a culture dish having a diameter of 15 cm. As a serum-free medium for antigen purification, 5 μg / ml blasticidin was added to VP-SFM.
[0100]
The culture solution was centrifuged at 3,000 rpm for 10 minutes, and the culture supernatant was collected. The collected culture supernatant was centrifuged three times at 3,000 × g, 4 ° C. for 30 minutes using Centricon Plus 80 equipped with Biomax-100 ultrafiltration membrane (MWCO100,000) (Millipore, USA). And concentrated to 1/114 volume.
[0101]
The concentrated solution was applied to a Hiprep 16/60 column (1.6 × 60 cm) packed with Sephacryl S-300 (manufactured by Pharmacia Biotech AB, Sweden), and TBS buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl ] Was subjected to molecular sieve chromatography at a flow rate of 1 ml / min and fractionated so that one fraction was 3.75 ml. The antigen titer of each fraction was measured by ELISA, and the peak fraction was collected. The antigen of interest was eluted as a single peak at the exclusion limit.
[0102]
Next, Gradient Master so that the peak fraction is 5% (w / w) at the top and 30% (w / w) at the bottom.^TM(Model 106) (same as BIOCOMP Instruments, Inc., Canada) Overlaid with sucrose density gradient solution, using SW28 rotor and ultracentrifuge L8-70M (both manufactured by Beckman Instruments, Inc., USA) Then, sucrose density gradient centrifugation fractionation was performed for 2 hours under the conditions of 4 ° C. and 26,500 rpm. After centrifugation, a 19-gauge hole was made in the center of the bottom of the tube, and fractionation was performed so that one fraction was 2 ml by the dropping method. The antigen titer of each fraction was measured by ELISA, and the antigen peak fraction was collected. The antigen of interest formed a single peak and was fractionated. The sucrose concentration in each fraction was almost linearly decreasing.
[0103]
Finally, this peak fraction was applied to a Hiprep desalting 26/10 column (2.6 × 10 cm) (manufactured by Pharmacia Biotech AB, Sweden) packed with Sephadex G-25 to remove sucrose and low molecular weight substances. Then, molecular sieve chromatography was performed using PBS (−) at a flow rate of 5 ml / min, and fractionation was performed so that one fraction was 5 ml. By this operation, the antigen was completely separated from sucrose and low-molecular substances, and at the same time, the solvent was exchanged with PBS (−). The antigen fraction from which sucrose was removed and the buffer was exchanged was used as virus-like particles as the final purified product. The purified virus-like particle was named J12 # 26 antigen. For the high performance liquid chromatography system, AKTA explorer 10S (manufactured by Pharmacia Biotech AB, Sweden) was used.
[0104]
At each stage of antigen purification, the amount of protein and the amount of antigen were measured by ELISA. The results are summarized in Table 3.
[0105]
[Table 3]

[0106]
In Table 3, the total antigen amount in the culture supernatant using the serum-free medium was 100%, and the recovery rate was calculated based on the yield in each step. The recovery rate of protein was 6.3%, but the recovery rate of virus-like particles was 43.5%. Combined with the result of electrophoresis and the result of Western blot, the antigen could be purified with high purity and high yield.
[0107]
Samples at each purification stage were subjected to SDS-PAGE with the same amount of antigen or protein. After electrophoresis, the proteins fractionated by electrophoresis were stained with the fluorescent dye SYPRO-Orange, which has the same sensitivity as silver staining. The results are shown in FIG. When the antigen amount was electrophoresed equally, as the purification process progressed, a protein band with a molecular weight of 53 kDa showing an equivalent signal intensity was observed equally, while bands of other contaminating proteins decreased. When electrophoresis was carried out with the same amount of protein, the 53 kDa protein band was gradually and strongly observed as purification progressed. Furthermore, it was examined by Western blot whether the major protein band of 53 kDa is the E protein of the neutralizing antigen specific for Japanese encephalitis virus. The results are shown in FIG. A major protein of 53 kDa was recognized by a specific antibody against Japanese encephalitis virus.
[0108]
The protein concentration and HA value of the J12 # 26 antigen solution were measured. As a result, the protein concentration was 44 μg / ml, and the HA value was 25,600 HA. Therefore, the HA activity of J12 # 26 antigen was 582 HA per 1 μg / ml.
[0109]
Example 4
N-terminal amino acid analysis of proteins that make up antigen particles
In order to confirm that the J12 # 26 antigen includes not only the E protein but also the M protein, the N-terminal amino acid sequence of the protein existing downstream of the 53 kDa band corresponding to the E protein was analyzed.
[0110]
To the virus-like particles purified above, 50% trichloroacetic acid was added to a final concentration of 5%, and the mixture was stirred and allowed to stand at room temperature for 30 minutes. The supernatant was discarded after low-speed centrifugation at 3,000 g for 30 minutes, and the remaining precipitate was air-dried and dissolved in 100 μl of 0.1% NaOH to obtain a sample for electrophoresis. The electrophoresis sample was applied to a gel so that the antigen amount was 20 μg / lane and subjected to SDS-PAGE. The gel after electrophoresis was stained with silver to visualize protein bands. Next, the protein band was transferred to the PVDF membrane from the gel after SDS-PAGE. The results are shown in FIG.
[0111]
FIG. 8 shows the results of transfer to SDS-PAGE and PVDF membrane, the left side is a silver-stained polyacrylamide gel, and the right side is a transfer elephant with proteins transferred to the PVDF membrane. Among the bands present downstream of the 53 kDa band corresponding to the E protein, three bands (JEVLP01 to JEVLP03 in FIG. 8) that appear to be derived from JEV, ie, 16.5 kDa, 14.9 kDa, and 6.8 kDa. The band was cut out from the transfer film, and the band was washed in the order of methanol, ultrapure water, and methanol. The amino acid sequence of 5 residues from the N-terminal of the protein constituting each band was analyzed using a sequencer. G1000A (manufactured by Hewlett Packard Company, USA) was used for the protein sequencer, Model 1090 (manufactured by Hewlett Packard Company, USA) was used for the PTH analyzer, and Routine 3.1 PVDF was used for the analysis program. The results are shown in Table 4.
[0112]
[Table 4]

[0113]
As apparent from Table 4 above, JEVLP01 was a mixture of M protein (or fragment thereof) and E protein fragment, JEVLP02 was an E protein fragment, and JEVLP03 was an M protein. From this result, it was clarified that the J12 # 26 antigen contains E protein and M protein.
[0114]
Example 5
Electron microscopy of virus-like particles
The shape of the J12 # 26 antigen was observed with an electron microscope. The purified J12 # 26 antigen was concentrated by centrifugation and negatively stained without prior fixation. Stained virus-like particles were observed under an electron microscope. An electron micrograph is shown in FIG.
[0115]
The virus-like particles were spherical particles having a substantially uniform size, and the average diameter was 25 nm. This result directly proved that the purified antigen formed a particulate structure. In general, fine, irregularly shaped dust-like inclusions caused by the destruction of impurities and virus-like particles are observed under an electron microscope, but since such a thing is hardly seen, the degree of purification is quite high. It is considered a thing.
[0116]
Example 6
Neutralizing antibody induction test with Japanese encephalitis virus antigen
Four-week-old female ddY mice were divided into 10 mice per group, and purified J12 # 26 antigen diluted with PBS was attached to each mouse, and 300 ng of the total protein amount was intraperitoneally administered. Immunization was repeated twice every other week. The control group had 5 PBS (-) inoculated groups and the current vaccinated group. On the seventh day from the second administration, blood was collected aseptically from half of the mice and from the remaining mice on the 14th day, and the blood was allowed to clot by allowing it to stand at 37 ° C. for 1 hour. After removing the clot, the serum was separated by centrifugation at 4 ° C. and 5,000 rpm for 10 minutes. Serum was collected and serum inactivated for 30 minutes at 56 ° C. 50 μl of inactivated serum was pooled from each of the 5 mice and used to measure neutralizing antibody titer. The results are shown in Table 5.
[0117]
[Table 5]

[0118]
As is clear from Table 5, neutralizing antibody titers about 3 times higher than the current vaccine were induced in the group inoculated with the J12 # 26 antigen in the same manner as the current vaccine. From the above results, it became clear that the Japanese encephalitis virus antigen of the present invention exhibits immunogenicity equivalent to that of the current vaccine.
[0119]
Example 7
Infection protection by Japanese encephalitis virus antigen
As in Example 6, 4-week-old female ddY mice were divided into 10 groups, and immunized intraperitoneally with Japanese encephalitis virus antigen diluted with PBS. As a Japanese encephalitis virus antigen, J12 # 26 antigen diluted with PBS was used, and 300 ng was administered in an antigen amount (amount determined by ELISA) per animal. The control group had PBS (-) inoculation group and current vaccination group. When the immunized mice are 6 weeks old, 2 × 10⁶PFU's Japanese encephalitis virus [special virus strain for production of Japanese encephalitis vaccine (Beijing-1 strain, JWS-P-4), which was distributed from the Osaka University Microbial Disease Research Society) was inoculated intraperitoneally. Simultaneously with intraperitoneal inoculation, mice were injected with 20 μl of PBS intracerebrally to break the blood brain barrier. During the 21 days after inoculation, the mice were examined for survival or death, and the survival rate (%) was calculated. The results are shown in Table 6.
[0120]
[Table 6]

[0121]
As is apparent from Table 6, mice immunized with the antigen particles of the present invention showed 100% survival as did mice immunized with the current vaccine. Therefore, the antigen-like particles of the present invention had sufficient infection protection ability as an antigen for Japanese encephalitis vaccine.
[0122]
Example 8
Diagnosis of Japanese encephalitis using Japanese encephalitis virus antigen
The J12 # 26 antigen and tissue culture Japanese encephalitis virus purified antigen (BM3) were diluted with a carbonate buffer (pH 9.6) so that the protein amount was 1.0 μg / ml, respectively, and used as a solid-phased antigen. The antigen was dispensed at 100 μl / well on an ELISA plate and allowed to stand overnight at 4 ° C. to immobilize the antigen. The wells were washed with PBS-T, PBS containing 1% FBS was dispensed at 200 μl / well, and blocked at 37 ° C. for 2 hours. The wells were washed with PBS-T, and 100 μl of human serum diluted 200-fold was added to each well and incubated at 37 ° C. for 60 minutes. As human sera, two sera (S1 and S2) negative for neutralizing antibody titer against the Japanese encephalitis virus Beijing strain and two positive sera (S3 and S4) were used.
[0123]
The wells were washed with PBS-T, and labeled antibody HRPO-anti human IgG goat IgG [US, Rockland Immunochemicals (USA)] diluted 1000-fold with PBS-T was dispensed into each well in an amount of 100 μl. Incubated for 60 minutes at ° C. After washing, 100 μl of coloring solution was added to each well and allowed to react for 20 minutes. The coloring solution contains 25 μl of 30% hydrogen peroxide as a substrate and 20 mg of ABTS [2,2′-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) Diammonium Salt] (manufactured by Sigma, USA) as a coloring agent. A solution dissolved in 100 ml of citrate buffer (pH 4.0) was used. Next, 100 μl of 1.5% oxalic acid was added to each well to stop the reaction, and the absorbance at 415 nm was measured. A sample having an absorbance of 0.2 or more was determined as positive. The results are shown in Table 7.
[0124]
[Table 7]

[0125]
When the presence or absence of reactivity with an antibody against Japanese encephalitis virus in human serum was tested using the antigen of the present invention, the results correlated with both the neutralizing antibody titer and the test result using tissue culture Japanese encephalitis virus purified antigen was gotten. Therefore, the antigen of the present invention can also be used as an antigen for a diagnostic agent.
[0126]
【The invention's effect】
According to the present invention, there is a Japanese encephalitis virus antigen comprising Japanese encephalitis virus-like particles, wherein the virus-like particles include M protein and E protein of Japanese encephalitis virus, and RNA is contained in the particles. There is provided a Japanese encephalitis virus antigen characterized in that the virus-like particle exhibits a hemagglutination activity. By using the antigen of the present invention, a substitute for the inactivated Japanese encephalitis virus used in the current Japanese encephalitis vaccine, no mouse brain, no virus, low cost Japanese encephalitis vaccine and diagnostic agent Manufacture is possible.
[0127]
[Sequence Listing]

[Brief description of the drawings]
FIG. 1 is a schematic diagram of recombinant DNApCAGJ12bsr.
FIG. 2 shows the relationship between the passage number of serum-free medium-unconditioned cells (J12 # 26 cells) passaged with 10% FBS-MEM and the amount of virus-like particles expressed in the culture supernatant. It is a graph, a vertical axis | shaft is the amount of virus-like particles (microgram / ml) measured by ELISA method, and a horizontal axis | shaft is a passage number.
FIG. 3 shows the relationship between the number of passages of serum-free medium-conditioned cells (J12 # 26SF cells) passaged with VP-SFM (serum-free medium) and the amount of virus-like particles expressed in the culture supernatant. The vertical axis is the amount of virus-like particles (μg / ml) measured by ELISA, and the horizontal axis is the passage number.
FIG. 4 is a graph showing time-dependent changes in the HA titer and ELISA titer of the culture supernatant of Japanese encephalitis virus-infected Vero cells, and the left vertical axis represents the purified inactivated virus antigen TJP ** 012 The ELISA value of the culture supernatant with an ELISA value of 100. The vertical axis on the right is the HA value (maximum dilution rate at which HA activity was observed), the horizontal axis is the number of culture days, and ◯ represents the ELISA value. , ◆ represents the HA value.
FIG. 5 is a graph showing time-dependent changes in the HA titer and ELISA titer of the culture supernatant of J38 # 26SF cells cultured for 38 generations, and the left vertical axis represents purified inactivated virus antigen TJP **. The ELISA titer of the culture supernatant with the ELISA titer of 012 as 100, the right vertical axis is the HA titer (maximum dilution rate at which HA activity was observed), the horizontal axis is the number of days of culture, and ◯ is the ELISA titer And ◆ represents the HA value.
FIG. 6 is an SDS-PAGE of virus-like particles. In the figure, lanes 2 to 6 are the results of applying the same amount of antigen, lanes 8 to 12 are the results of applying the same amount of protein, lanes 1 and 7 are molecular weight markers, and lanes 2 and 8 are ultrafiltration. Concentrated culture supernatants, lanes 3 and 9 are peak fractions of Sephacryl S-300 chromatography, lanes 4 and 10 are peak fractions obtained by sucrose gradient centrifugation, and lanes 5 and 11 are of Sephadex G-25 chromatography. Peak fractions and lanes 6 and 12 are commercially available Japanese encephalitis vaccines.
FIG. 7 shows the results of Western blotting using rabbit anti-JEV antiserum against the SDS-PAGE of FIG. In the figure, lanes 2 to 6 are the results of applying the same amount of antigen, lanes 8 to 12 are the results of applying the same amount of protein, lanes 1 and 7 are molecular weight markers, and lanes 2 and 8 are ultrafiltration. Concentrated culture supernatants, lanes 3 and 9 are peak fractions of Sephacryl S-300 chromatography, lanes 4 and 10 are peak fractions obtained by sucrose gradient centrifugation, and lanes 5 and 11 are of Sephadex G-25 chromatography. Peak fractions and lanes 6 and 12 are commercially available Japanese encephalitis vaccines.
FIG. 8 is a result of transferring a protein band fractionated by SDS-PAGE and SDS-PAGE of virus-like particles treated with TCA to a PVDF membrane, and a right side is a polyacrylamide gel stained with silver. The left hand side is a transcription image obtained by transferring the protein to the PVDF membrane, the number on the left end is a molecular weight marker, and there are three bands (JEVLP01 to JEVLP03) downstream of the 20.9 kDa band.
FIG. 9 is an electron micrograph of negative-stained virus-like particles, and the horizontal line indicating the scale corresponds to 100 nm.

Claims (4)

(A) providing a cDNA prepared from the genomic RNA of Japanese encephalitis virus, the cDNA comprising a cDNA fragment encoding prM protein and a cDNA fragment encoding E protein in this order;
(B) constructing a replicable recombinant DNA obtained by incorporating the cDNA into a replicable expression vector containing a β-actin promoter so that the cDNA can be expressed;
(C) transforming animal cells with the replicable recombinant DNA to form transformed cells;
(D) A transformed cell obtained by a method comprising selecting the transformed cell from a parent cell, and (e) acclimating the transformed cell to a serum-free medium. The transformed cell according to claim 1, wherein the cDNA is the base sequence of SEQ ID NO: 4 in the sequence listing. The transformed cell according to claim 1 or 2, wherein the animal cell is an RK13 cell. (A) culturing the transformed cell according to any one of claims 1 to 3 in a serum-free medium, and releasing Japanese encephalitis virus-like particles into the culture solution;
(B) obtaining a culture supernatant from the culture solution;
(C) isolating and purifying the virus-like particles from the culture supernatant,
And a method for producing a Japanese encephalitis virus antigen.

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