JP5296665B2 - ヒトパピローマウイルスカプソメアワクチン製剤の製造方法 - Google Patents
ヒトパピローマウイルスカプソメアワクチン製剤の製造方法 Download PDFInfo
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- JP5296665B2 JP5296665B2 JP2009292021A JP2009292021A JP5296665B2 JP 5296665 B2 JP5296665 B2 JP 5296665B2 JP 2009292021 A JP2009292021 A JP 2009292021A JP 2009292021 A JP2009292021 A JP 2009292021A JP 5296665 B2 JP5296665 B2 JP 5296665B2
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Description
HPV16 L1の転写解読枠をコードするDNAを、プラスミド16-114/k-L1/L2-pSynxtVI-[Kirnbauerら、Virol. 67巻、6929〜6939頁(1994)]からBglIIを用いて切り出し、得られた断片を、予め固有のBamHI制限部位にて直鎖にしておいたpUC19へとサブクローニングした。先ず、2つの塩基性発現構築体を作製して、融合タンパク質発現を許容すべく引き続いてのDNAの挿入を可能とした。1つの構築体は、9つのアミノ酸の欠失を有する、HPV16L1Δ310をコードしており、欠失された領域は他のパピローマウイルスL1タンパク質すべてと低い相同性を示すことが知られていた。第2の構築体であるHPV16L1ΔCは、カルボキシ末端のL1残基の34アミノ酸の欠失を有するタンパク質をコードしていた。他の構築体には、他のタンパク質の配列をコードするDNAの挿入を容易ならしめるために、欠失位置にEcoRV制限部位を含んでいる。EcoRV制限部位の付加によって、非L1タンパク質のアミノ酸2つ、すなわち、アスパラギン酸およびイソロイシンがコードされることとなっている。
2つのプライマー(配列番号:5および6)を、pUC19ベクターと、配列番号:1のヌクレオチド第916位から942位までを除いた完全なHPV16L1コード配列とを増幅させるように設計した。プライマーはまた、増幅産物の末端で固有のEcoRV制限部位(配列番号:5および6の下線部)も導入するように合成されていた。
(配列番号:5)
ccccgatatctcaaattattttcctacacctagtg
(配列番号:6)
こうして得られたPCR産物をEcoRVで消化して相補性端部を与え、その消化産物を連結反応により環状とした。連結したDNAを標準技術を用いて大腸菌に形質転換し、その結果得られたコロニーからのプラスミドをEcoRV制限部位の存非についてスクリーニングした。HPV16L1Δ310と命名した1つのクローンは、適切な27ヌクレオチドの欠失を有しているものとして同定され、この構築体を下記のとおりEcoRV部位にて他のHPV16タンパク質をコードしているDNA断片を挿入するために使用した。
2つのプライマー(配列番号:7および8)を、プライマーが相互に近接して、前記のとおりpUC19にHPV16L1をコードする配列を含む鋳型DNA上で逆方向の増幅を可能とするように、HPV16L1の転写解読枠に対して相補的に設計した。
(配列番号:7)
aaagatatctaatctacctctacaactgctaaacgcaaaaaacg
(配列番号:8)
各プライマーは、増幅産物の末端にEcoRV制限部位を導入するものであった。下流のプライマー(配列番号:8)では、EcoRV端部を連結して環状とした場合に増幅産物が34カルボキシ末端L1アミノ酸の欠失を含むように、EcoRV部位の後にTAA翻訳終止コドンが配置されていた。PCRを実施して部分的なL1転写解読枠と完全なベクターを増幅させた。増幅産物をEcoRVで切断し、T4 DNAリガーゼで環状として大腸菌DH5α細胞へと形質転換した。生存能力のあるクローン由来のプラスミドを、プラスミドを直鎖状にさせるEcoRV部位の存在について分析した。pUCHPV16L1ΔCと命名した1つの陽性の構築体を同定し、EcoRV部位を利用して他のHPV16タンパク質からDNAを挿入するために使用した。
配列番号:4のアミノ酸1〜50、1〜60、1〜98、25〜75、40〜98、50〜98をコードするHPV6 E7のDNA断片を、HPV16L1Δ310とHPV16L1Δの修飾配列のいずれかへの断片の挿入を容易ならしめるために、端部5' EcoRV制限部位が導入されたプライマーを用いて増幅させた。様々な増幅反応で、プライマーE7.1(配列番号:9)をプライマーE7.2(配列番号:10)と組み合わせて用いてE7アミノ酸1〜50をコードするDNA断片を作製し、プライマーE7.3(配列番号:11)と組み合わせて用いてE7アミノ酸1〜60をコードするDNA断片を作製し、あるいはプライマーE7.4(配列番号:12)と組み合わせて用いてE7アミノ酸1〜98をコードするDNA断片を作製した。他の増幅反応において、E7.5(配列番号:13)とE7.6(配列番号:14)のプライマー対を用いてE7アミノ酸25〜75をコードするDNA断片を増幅させ、E7.7(配列番号:15)とE7.4(配列番号:12)を用いてE7アミノ酸40〜98をコードするDNA断片を増幅させ、そしてE7.8(配列番号:16)とE7.4(配列番号:12)を用いてE7アミノ酸50〜98をコードするDNA断片を増幅させた。
aaaagatatcatgcatggagatacacctacattgc
プライマーE7.2(配列番号:10)
ttttgatatcggctctgtccggttctgcttgtcc
プライマーE7.3(配列番号:11)
ttttgatatccttgcaacaaaaggttacaatattgtaatgggcc
プライマーE7.4(配列番号:12)
aaaagatatctggtttctgagaacagatggggcac
プライマーE7.5(配列番号:13)
ttttgatatcgattatgagcaattaaatgacagctcag
プライマーE7.6(配列番号:14)
ttttgatatcgtctacgtgtgtgctttgtacgcac
プライマーE7.7(配列番号:15)
tttatcgatatcggtccagctggacaagcagaaccggac
プライマーE7.8(配列番号:16)
ttttgatatcgatgcccattacaatattgtaaccttttg
同様に、インフルエンザマトリックスタンパク質(配列番号:17)をコードするDNA由来のヌクレオチドを、配列番号:19および20に示すプライマー対を使用して増幅させた。両プライマーはいずれも増幅産物中にEcoRV制限部位を導入するものであった。
(配列番号:19)
ttttgatatcgttgtttggatccccattcccattg
(配列番号:20)
各増幅反応から得られたPCR産物は、EcoRVで切断し、同酵素を用いて予め直鎖状にしておいたHPV16L1Δ310とHPV16L1ΔC配列のいずれかのEcoRV部位へと挿入した。E7アミノ酸25〜75および50〜98をコードするプラスミド、そしてインフルエンザマトリックスタンパク質を含むプラスミドでのインサートの方向を調べるために、新しく作られたEcoRV制限部位(gatatcgat)に重複する制限部位の利点を有し、上流プライマーに含められたClaI消化を採用した。HPV16 E7の開始のメチオニンを含む3つの発現構築体について、E7コード領域内のNslI制限部位を利用して挿入方向を調べた。
HPV16L1ΔC配列は、野生型L1ポリペプチドには通常認められないアミノ酸の翻訳を結果的に引き起こすEcoRV部位由来のDNAを含んでいる。しかして、人工的なEcoRV部位が除去された一連の発現構築体を設計した。発現構築体のこのシリーズに対するL1配列は、HPV16L1ΔC*と命名した。
gttatgacatacatacattctatg
プライマーP2(配列番号:22)
ccatgcattcctgcttgtagtaaaaatttgcgtcc
プライマーP3(配列番号:23)
ctacaagcaggaatgcatggagatacacc
プライマーP4(配列番号:24)
catctgaagcttagtaatgggctctgtccggttctg
第1の2つの増幅反応の後、2つの精製産物を、プライマーP1およびP4のみを使用したさらなるPCR反応における鋳型として用いた。こうして得られた増幅産物を、酵素EcoNIおよびHindIIIで消化して前記のHPV16L1ΔC発現構築体へと挿入し、続いて同酵素で消化した。得られた発現構築体は、2つの内部EcoRV制限部位を喪失しているという点でL1およびE7アミノ酸1〜50をコードするDNAを含む元のHPV16L1ΔC構築体と相違していた。第1のEcoRV部位は、本来のこの位置にあるL1のアラニンおよびグリシンアミノ酸をコードするDNAに置換されており、第2の部位は翻訳終止シグナルに置換されていた。加うるに、HPV16L1ΔC*E7 1-52と命名された発現構築体は、第51位のヒスチジンと第52位のチロシンのE7アミノ酸残基もコードするプライマーP4を用いた結果、HPV16 E7の最初の52アミノ酸を含んでいた。次いで、HPV16L1ΔC*E7 1-52を使用し、プライマーP1(配列番号:21)をプライマーP5(配列番号:25)と組み合わせて用いて、E7アミノ酸の1〜55をコードするDNAをさらに包含する、さらなるHPV16L1ΔC発現構築体を作製した。また、プライマー対P1およびP6(配列番号:26)を用いてE7アミノ酸の1〜60を、そしてプライマー対P1およびP7(配列番号:27)を用いてE7アミノ酸の1〜65をコードするDNAをさらに包含するものを作製した。増幅産物中のさらに付加したアミノ酸をコードするDNA配列は、所望のアミノ酸に対する付加的なヌクレオチドを含むようなプライマーの設計から生じるものであった。
catctgaagcttatcaatattgtaatgggctctgtccg
プライマーP6(配列番号:26)
catctgaagcttacttgcaacaaaaggttacaatattgtaatgggctctgtccg
プライマーP7(配列番号:27)
catctgaagcttaaagcgtagagtcacacttgcaacaaaaggttacaatattgtaatgggctctgtccg
同様に、HPV16L1ΔC*E7 1-70を、HPV16L1ΔC*E7 1-66をコードする鋳型DNAとプライマー対P1およびP8(配列番号:28)を用いて作製した。
catctgaagcttattgtacgcacaaccgaagcgtagagtcacacttg
各PCR反応の後、増幅産物をEcoNIおよびHindIIIで消化して、予め同酵素で消化しておいたHPV16L1ΔCに挿入した。各構築体の配列は、蛍光鎖終止ジデオキシヌクレオチドを用い、Applied Biosystems Prism 377配列決定装置を使用して決定した[Proberら、Science、238巻、336〜341頁(1987)]。
Spodoptera frugiperda(Sf9)細胞を、10%胎児仔ウシ血清および2mMグルタミンを追加したTNMFH培地(Sigma社)中で27℃で懸濁または単層培養にて生育した。HPV16L1に基づく組換えバキュロウイルス構築体に対し、2μgのBaculo-Gold DNA(PharMingen、サンディエゴ、カリホルニア州)と共に10μgの伝達プラスミドを用いて、Sf9細胞をトランスフェクトした。製造業者が提示したプロトコルに従うことにより組換えウイルスを精製した。
トリコプルシアニ(Trichopulsiani、TN)High Five細胞を、Ex-Cell405無血清培地(JRH Biosciences)中でおよそ2×106細胞/mlの密度で生育した。約2×108の細胞を、1000×gで15分間遠心分離することによりペレット化し、20mlの培地に再懸濁し、そして2乃至5のm.o.iで1時間室温にて組換えバキュロウイルスで感染させた。200mlの培地を添加した後、細胞を播種し、27℃にて3乃至4日間インキュベートした。インキュベーションの後、細胞を回収してペレット化し、そして10mlの抽出用緩衝液に再懸濁させた。
60μgのHPVキメラカプソメアを、完全または不完全フロインドアジュバントと1:1で混合して総容量100μlとしたものを用いて、4週間おきに3回、Balb/cマウスに対し皮下に免疫付与する。第3回目の免疫付与から6週間後にマウスを屠殺し、心臓穿刺により血液を集める。
PBS中10μg/mlの濃度のペプチドE701[Mullarら、1982]50μで、微量定量プレート(Dynatech)を終夜被覆する。PBS中に5%BSAおよび0.05%Tween 20を含有する緩衝液100μlで、37℃にて2時間ブロッキングを行い、そして0.05%Tween 20を含有するPBSで3回洗浄する。第3回目の洗浄の後、BSA/Tween 20/PBSで1:5000に希釈した50μlの血清を各ウェルに添加し、そしてインキュベーションを1時間実施する。プレートを前回と同様に再度洗浄し、そして1:5000希釈にて、ヤギ−抗マウスペルオキシダーゼ接合物50μlを添加する。1時間後にプレートを洗浄し、ABTS基質(0.1Mナトリウム-酢酸-リン酸緩衝液(pH4.2)中に、0.2mg/ml、2,2'-アジノ-ビス(3-エチルベンズヒアゾリン-β-スルホン酸を、10mlに対し4μlの30%H2O2と共に含むもの)を用いて染色する。Dynatech自動式プレートリーダーにて、490nmで1時間後に吸光度を測定する。
CsCl密度勾配でのウイルス様粒子およびカプソメアの相対的な定量を許容するために、抗原捕獲ELISAを利用した。微量定量プレートを、HPV16L1に対して免疫特異性を有する、プロテインAで精製されたマウスモノクローナル抗体(抗体25/C、MM07およびRitti1は、HPV16 VLPを用いて免疫付与したマウスから得たもの)の1:500希釈液(PBS中、最終濃度は1ml当たり2μg)50μl/ウェルで終夜被覆した。プレートを5%ミルク/PBSで1時間ブロッキングし、そしてCsCl密度勾配の画分50μlを1:300の希釈液(5%ミルク/PBS溶液)にて37℃で1時間添加した。PBS/0.05%Tween 20で3回洗浄した後、HPV16 VLPに対して作製したポリクローナルウサギ抗血清(ミルク/PBSを用いた1:3000希釈液)50μlを添加し、このプレートを37℃にて1時間インキュベートした。プレートを再度洗浄し、さらに5%のミルクを含有するPBSで1:5000に希釈したヤギ−抗ウサギペルオキシダーゼ接合物(Sigma社)50μlとさらに1時間インキュベートした。最後に洗浄してから、プレートをABTS基質で30分間染色し、Dynatech自動化プレートリーダーで490nmにて吸光度を測定した。負の対照として、アッセイにはPBSのみを被覆したウェルも含めておいた。
キメラカプソメアワクチンが中和抗体の生産を惹起する程度を調べるために、赤血球凝集阻害アッセイを、下記に概略説明するとおりに行う。このアッセイは、ウイルス様粒子が赤血球を凝集させることができるとの過去の観察に基づいたものである。
Claims (9)
- ヒトパピローマウイルス(HPV)カプソメアを含むワクチン製剤の製造方法であって、当該カプソメアが、第2タンパク質由来のアミノ酸残基に近接するヒトパピローマウイルスL1タンパク質を含む融合タンパク質を含み、当該L1タンパク質と第2タンパク質由来の当該アミノ酸残基とが、ウィルス様粒子の形成を阻害する位置にあり、当該製剤が、ヒトパピローマウイルスビリオンを中和する免疫応答性を惹起し、および、当該方法が、第2タンパク質由来の当該アミノ酸残基に近接しているヒトパピローマウイルスL1タンパク質を発現および精製する工程を含む、ことを特徴とするワクチン製剤の製造方法。
- 前記L1タンパク質が、HPV6、HPV11、HPV16、HPV18、HPV33、HPV35、および、HPV45よりなる群から選択されるヒトパピローマウイルスのゲノムによってコードされる請求項1に記載の製造方法。
- 前記パピローマウイルスが、HPV16である請求項2に記載の製造方法。
- 前記L1タンパク質が、そのカルボキシ末端アミノ酸残基を欠失している請求項3に記載の製造方法。
- 前記L1タンパク質が、そのカルボキシ末端の第1位〜第34位のアミノ酸残基を欠失している請求項4に記載の製造方法。
- 前記L1タンパク質が、そのカルボキシ末端アミノ酸残基から34個のアミノ酸を欠失している請求項5に記載の製造方法。
- 前記L1タンパク質が、そのアミノ末端アミノ酸残基を欠失している請求項3に記載の製造方法。
- 前記L1タンパク質が、その内部アミノ酸残基を欠失している請求項3に記載の製造方法。
- 前記内部アミノ酸残基が、核局在化シグナルを含む請求項8に記載の製造方法。
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US08/944,368 US6228368B1 (en) | 1997-10-06 | 1997-10-06 | Papilloma virus capsomere formulations and method of use |
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JP2009292021A Expired - Fee Related JP5296665B2 (ja) | 1997-10-06 | 2009-12-24 | ヒトパピローマウイルスカプソメアワクチン製剤の製造方法 |
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EP (2) | EP1690941B1 (ja) |
JP (3) | JP4520034B2 (ja) |
KR (1) | KR20010030969A (ja) |
AT (2) | ATE349536T1 (ja) |
AU (1) | AU9684698A (ja) |
BR (1) | BR9814606A (ja) |
CA (1) | CA2305382A1 (ja) |
CY (1) | CY1105963T1 (ja) |
CZ (1) | CZ302351B6 (ja) |
DE (2) | DE69841342D1 (ja) |
DK (1) | DK1021547T3 (ja) |
ES (2) | ES2337492T3 (ja) |
HU (1) | HU225893B1 (ja) |
IL (3) | IL135528A0 (ja) |
NO (2) | NO328128B1 (ja) |
NZ (1) | NZ503830A (ja) |
PL (1) | PL195332B1 (ja) |
PT (1) | PT1021547E (ja) |
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Families Citing this family (44)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ES2264563T3 (es) * | 1994-10-07 | 2007-01-01 | Loyola University Of Chicago | Particulas semejantes al virus del papiloma, proteinas de fusion y procedimiento para su preparacion. |
WO1998054325A1 (en) * | 1997-05-29 | 1998-12-03 | The Government Of The United States Of America,Re Presented By The Secretary, Department Of Health And Human Services | Human frp and fragments thereof including methods for using them |
US6228368B1 (en) * | 1997-10-06 | 2001-05-08 | Loyola University Of Chicago | Papilloma virus capsomere formulations and method of use |
US7494658B2 (en) * | 1998-02-20 | 2009-02-24 | Medigene Ag | Papilloma virus truncated L1 protein and fusion protein constructs |
US7182947B2 (en) * | 1998-02-20 | 2007-02-27 | Medigene Ag | Papillomavirus truncated L1 protein and fusion protein constructs |
CA2229955C (en) * | 1998-02-20 | 2003-12-09 | Medigene Gmbh | Papilloma virus capsomere vaccine formulations and methods of use |
US20020039584A1 (en) * | 1998-02-20 | 2002-04-04 | Medigene Ag | Papilloma virus capsomere vaccine formulations and methods of use |
GB9806666D0 (en) * | 1998-03-27 | 1998-05-27 | Stanley Margaret | Antigen preparation and use |
AUPP765398A0 (en) | 1998-12-11 | 1999-01-14 | University Of Queensland, The | Treatment of papillomavirus infections |
DE19905883C2 (de) * | 1999-02-11 | 2001-05-23 | Deutsches Krebsforsch | Chimäre Virus-ähnliche Partikel bzw. chimäre Capsomere von BPV |
FI107932B (fi) * | 1999-02-16 | 2001-10-31 | Mikael Paronen | Polymeerikalvo ja menetelmä sen valmistamiseksi |
US6551597B1 (en) * | 1999-03-18 | 2003-04-22 | President & Fellows Of Harvard College | Vaccine compositions for human papillomavirus |
DE19925234A1 (de) * | 1999-06-01 | 2000-12-14 | Medigene Ag | Capsomere, stabile Capsomere, Capside, VLPs, oder CVLPs bzw. damit beladenen Zellen und ihre Verwendung in Diagnostik und Therapie |
DE19925199A1 (de) | 1999-06-01 | 2000-12-07 | Medigene Ag | Zytotoxische T-Zellepitope des Papillomavirus L1-Proteins und ihre Verwendung in Diagnostik und Therapie |
DE19925235A1 (de) | 1999-06-01 | 2000-12-07 | Medigene Ag | Zytotoxische T-Zellepitope des Papillomavirus L1-Proteins und ihre Verwendung in Diagnostik und Therapie |
ATE284898T1 (de) * | 1999-08-25 | 2005-01-15 | Merck & Co Inc | Synthetische papillomavirus gene, welche für expression in menschlichen zellen optimiert sind |
DE60041889D1 (de) * | 1999-12-09 | 2009-05-07 | Medimmune Inc | In-vitro-methode zur zerlegung und zum wiederzusammenfügen von papillomavirusartigen partikeln |
US6600018B1 (en) * | 2000-04-10 | 2003-07-29 | The United States Of America As Represented By The Department Of Health And Human Services | Secreted frizzled related protein, sFRP, fragments and methods of use thereof |
US6908613B2 (en) * | 2000-06-21 | 2005-06-21 | Medimmune, Inc. | Chimeric human papillomavirus (HPV) L1 molecules and uses therefor |
DE10055545A1 (de) * | 2000-11-09 | 2002-07-25 | Deutsches Krebsforsch | Für Expression in Eukaryonten optimierte HPV 16-L1 und HPV 16-L2 kodierende DNA Sequenzen |
DE10059630A1 (de) * | 2000-12-01 | 2002-06-06 | Medigene Ag | Arzneimittel zur Vermeidung oder Behandlung von durch humanen Papillomavirus-Typ 18-hervorgerufenem Tumor |
AU2002367977B2 (en) | 2001-08-13 | 2008-06-19 | University Of Rochester | Transcutaneous immunization against papillomavirus with papillomavirus virus-like particles |
CN100424094C (zh) * | 2001-08-31 | 2008-10-08 | 开普敦大学 | 药物组合物,制备和分离其的方法,及其在制备预防性治疗损害和癌的药物中的应用 |
US20040063188A1 (en) * | 2002-02-14 | 2004-04-01 | Novavax, Inc. | Kit for treating gastrointestinal tract |
PT1506222E (pt) † | 2002-05-17 | 2009-06-12 | Univ Cape Town | Proteínas l1 quiméricas do papilomavírus humano 16 incluindo um péptido l2, partículas semelhantes a vírus preparadas a partir daquelas e um método de preparação das partículas |
US8101189B2 (en) | 2002-07-05 | 2012-01-24 | Folia Biotech Inc. | Vaccines and immunopotentiating compositions and methods for making and using them |
ES2431963T3 (es) | 2002-07-05 | 2013-11-28 | Folia Biotech Inc. | Partícula viral adyuvante |
SE0202897D0 (sv) * | 2002-10-01 | 2002-10-01 | Quantovir Ab | Method and kit for quantitative and qualitative determination of human papillomavirus |
SE0202896D0 (sv) * | 2002-10-01 | 2002-10-01 | Quantovir Ab | Method for estimating the risk of carcinoma development |
WO2004062584A2 (en) * | 2003-01-10 | 2004-07-29 | Regents Of The University Of Colorado | Therapeutic and prophylactic vaccine for the treatment and prevention of papillomavirus infection |
US7172773B2 (en) * | 2004-04-26 | 2007-02-06 | Renew Life Inc. | Food supplement formulation |
KR101187625B1 (ko) * | 2004-09-10 | 2012-10-05 | 도쿠리츠다이가쿠호징 가나자와다이가쿠 | 경구 투여 백신 |
US7354719B2 (en) | 2004-12-08 | 2008-04-08 | Gen-Probe Incorporated | Detection of nucleic acids from multiple types of human papillomaviruses |
US7314630B2 (en) * | 2005-01-07 | 2008-01-01 | Yao-Xiong Hu | Compounds and methods of early diagnosis of cervical cancer and genital condyloma with HPV, CHSP60 tumor suppressor H-Ras, K-Ras and PTEN derived peptides modified |
CN101570571B (zh) | 2007-04-29 | 2012-07-11 | 北京万泰生物药业股份有限公司 | 截短的人乳头瘤病毒18型l1蛋白 |
BRPI0811016B1 (pt) * | 2007-04-29 | 2021-09-21 | Xiamen Innovax Biotech Co., Ltd. | Proteína li truncada do papiloma vírus humano tipo 16 |
DK2154149T3 (da) * | 2007-05-29 | 2019-10-14 | Univ Xiamen | En trunkeret l1-protein af human papillomavirus 6 |
US8507811B2 (en) * | 2009-02-02 | 2013-08-13 | Apple Inc. | Touch sensor panels with reduced static capacitance |
EP2377879A1 (en) * | 2010-04-14 | 2011-10-19 | Deutsches Krebsforschungszentrum | N-terminal HPV E7 fusion proteins |
US10413603B2 (en) * | 2014-06-19 | 2019-09-17 | The Regents Of The University Of Colorado, A Body Corporate | Compositions, methods and uses for improved human papilloma virus constructs |
WO2016026919A1 (en) * | 2014-08-20 | 2016-02-25 | Ruprecht-Karls-Universität Heidelberg | Vaccines and manufacturing methods for treatment and prevention of disease |
CN114127099B (zh) * | 2019-07-19 | 2024-04-19 | 神州细胞工程有限公司 | 嵌合的人乳头瘤病毒6型l1蛋白 |
US11382700B2 (en) | 2020-05-08 | 2022-07-12 | Globus Medical Inc. | Extended reality headset tool tracking and control |
US11510750B2 (en) | 2020-05-08 | 2022-11-29 | Globus Medical, Inc. | Leveraging two-dimensional digital imaging and communication in medicine imagery in three-dimensional extended reality applications |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ATE332973T1 (de) | 1991-07-19 | 2006-08-15 | Univ Queensland | Polynukleotidabschnitt des hpv16-genoms |
GB9207701D0 (en) | 1992-04-08 | 1992-05-27 | Cancer Res Campaign Tech | Papillomavirus e7 protein |
PT647140E (pt) | 1992-06-25 | 2007-12-27 | Univ Georgetown | Vacinas de papilomavírus |
US5437951A (en) * | 1992-09-03 | 1995-08-01 | The United States Of America As Represented By The Department Of Health And Human Services | Self-assembling recombinant papillomavirus capsid proteins |
US5618536A (en) * | 1992-09-03 | 1997-04-08 | The United States Of America As Represented By The Department Of Health And Human Services | Chimeric papillomavirus-like particles |
CA2157932C (en) * | 1993-03-09 | 2011-10-11 | Robert C. Rose | Production of human papillomavirus capsid protein and virus-like particles |
AUPM566794A0 (en) * | 1994-05-17 | 1994-06-09 | University Of Queensland, The | Process and product |
DE4447664C2 (de) | 1994-10-07 | 1999-04-15 | Lutz Prof Dr Gissmann | Fusionsproteine, Verfahren zu deren Herstellung sowie deren Anwendung |
ES2264563T3 (es) * | 1994-10-07 | 2007-01-01 | Loyola University Of Chicago | Particulas semejantes al virus del papiloma, proteinas de fusion y procedimiento para su preparacion. |
JP3413020B2 (ja) * | 1996-07-17 | 2003-06-03 | 株式会社東芝 | 半導体装置の製造方法 |
DE19712541C1 (de) | 1997-03-25 | 1998-11-05 | Deutsches Krebsforsch | Papillomviren, Mittel zu deren Nachweis sowie zur Therapie von durch sie verursachten Erkrankungen |
CA2295316C (en) * | 1997-07-03 | 2007-06-26 | University Of Colorado, University Technology Corporation | Homogeneous human papilloma virus capsomere containing compositions, methods for manufacture, and the use thereof as diagnostic, prophylactic or therapy |
JP3559048B2 (ja) | 1997-08-26 | 2004-08-25 | 日本精工株式会社 | 転がり軸受の製造方法 |
US6228368B1 (en) * | 1997-10-06 | 2001-05-08 | Loyola University Of Chicago | Papilloma virus capsomere formulations and method of use |
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