JP4016054B2 - 組換え乳頭腫ウイルスl1 - Google Patents
組換え乳頭腫ウイルスl1 Download PDFInfo
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- JP4016054B2 JP4016054B2 JP2006187730A JP2006187730A JP4016054B2 JP 4016054 B2 JP4016054 B2 JP 4016054B2 JP 2006187730 A JP2006187730 A JP 2006187730A JP 2006187730 A JP2006187730 A JP 2006187730A JP 4016054 B2 JP4016054 B2 JP 4016054B2
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- protein
- papillomavirus
- recombinant
- hpv6b
- hexahis
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Description
本発明は、L1タンパク質乳頭腫ウイルスに関する。特に、本発明は、組換え乳頭腫ウイルスL1タンパク質、および乳頭腫ウイルス感染を検知し、処置するためのその使用に関する。
乳頭腫ウイルスは、ヒト、ウシ、ヒツジ、イヌおよびネコを含む一連の宿主に感染する。より完全なリストとしては、K. Syrjanen, L. Grissman および L. G. Koss 編集の乳頭腫ウイルスおよびヒトの疾患(Springer Verlag, 1987 年発行)に掲載されている J. P. Sundberg による”Papilloma Virus Infections in Animals"を参照。
本発明は、組換え乳頭腫ウイルスL1タンパク質が多重構造を細胞外に形成し、もとの乳頭腫ウイルスカプシドを認識する免疫応答を起こすとの驚くべき発見から得られたものである。
Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
AGCATGACTGGTGGACAGCAAATGGGTCGGGATCTGTA
CGACGATGACGATAAGGAT
を含む5'ヌクレオチド配列を有し得る。適当な組換えDNA分子を図1に示す。
(1)組換え乳頭腫ウイルスL1タンパク質をコードする組換えDNA分子を
細菌から発現せしめる;
(2)組換え乳頭腫ウイルスL1タンパク質を実質的に精製する;および
(3)細胞外に該多重構造を形成する。
本方法は、(His)6を含むN末端アミノ酸配列を有する組換え乳頭腫ウイルスL1タンパク質を含む多重構造に使用し得る。N末端アミノ酸配列は、配列番号1:
Met Arg Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly
Gly Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp
を含み得る。
(1)乳頭腫ウイルスL1タンパク質を含有している可能性のあるサンプル
でマイクロタイタープレートのウエルをコートする。
(2)組換え乳頭腫ウイルスL1タンパク質に対する抗血清を加えて、乳頭
種ウイルスL1タンパク質-抗体複合体を形成せしめる。
(3)検出試薬で乳頭腫ウイルスL1タンパク質-抗体複合体の存在を検知
する。
(1)乳頭腫ウイルスL1タンパク質でマイクロタイタープレートのウエル
をコートする。
(2)乳頭腫ウイルスL1タンパク質に特異な抗体を含有している可能性の
あるサンプルを加え、組換え乳頭腫ウイルスL1タンパク質−抗体複
合体を形成せしめる。
(3)検出試薬で組換え乳頭腫ウイルスL1タンパク質−抗体複合体の存在
を検知する。
pTRC6bL1の構築
HPV6bのL1開放読み取り枠を、プライマーとして:
GCGGATCCAGATGTGGCGGCCTAGCGACAGCACAGTATATG (配列番号5)
および
CGCCCGGGTTACCTTTTAGTTTTGGCCTCGCTTACGTTTTAGG (配列番号6)
を使用したポリメラーゼ連鎖反応により臨床的単離体からクローン化した。
Met.Arg.Gly.Ser.His.His.His.His.His.His.Gly.Met.Ala.Ser.Met.Thr.Gly.Gly.Gln.Gln.Met.Gly.Arg.Asp.Leu.Tyr.Asp.Asp.Asp.Lys.Asp.(HPV6b L1 aas 1-520)をコードする。
アンピシリン(最終濃度100μg/ml)含有2YTブロス(トリプトン16mg、酵母10mg、NaCl 5mg)10mlに、グリセロール・ストックからの白金耳一杯(loopful)の細菌(エシェリキア・コリDH5)10μlを接種した。培養物を、120rpmで酸素供給しながら37℃で6時間インキュベートした。
細菌をグアニジニウム溶解緩衝液(6Mグアニジニウム塩酸塩および5.8ml/リットルの溶液A[177mM NaH2PO4および5M NaCl]、HClを使用してpH7.8に)50mlに再懸濁し溶解した。懸濁液を2分間30%出力で超音波処理した。細胞残骸を遠心(Beckman JA21ローター、4℃で10000rpm、30分遠心)してペレット化した。HPV6b L1 HEXAHISタンパク質含有上清を保持した。
HPV6b L1 HEXAHISタンパク質を続いて、必須2工程精製工程で精製した。
HPV6b L1 HEXAHISタンパク質に対する抗体を産生させるために、マウス(C57BI/6系)に4週間の間隔で2回、表2の実験プロトコールに従って50μgタンパク質/マウスを皮下注射した。2回目の注射の2週間後、マウスから採血した。血清を標準方法を使用して抽出血液から得た。
遅延型過敏症は細胞媒介免疫応答およびある液性免疫応答を含む。マウス(BALB/c系)を、表3に概説の種々の条件下でHPV6b L1 HEXAHISタンパク質で処理(腹腔内注射)した。11日目、耳を、皮内注射でHPV6b L1 HEXAHISタンパク質または他のHEXAHISタンパク質を投与した。耳の厚さを13および14日目に測定した。14日目に陽性の反応があったマウスを殺し、耳の組織を試験した。
プレートのウェルを、PBS中、pH7.2のエシェリキア・コリ産生HPV6b L1 HEXAHIS、バキュロウイルス産生HPV6 VLP-L1ならびに対照としてバキュロウイルスおよびエシェリキア・コリ調製物(細胞発酵上清)のいずれかで0.2μgタンパク質/ウェルでコートし、一晩室温でインキュベートした。一回の洗浄をpH7.2のPBSで行った。非特異的結合をプレートを1%(w/v)カゼインで、1時間室温でインキュベートすることにより阻止した。
実験の結果は表4に示す。HPV6b L1 HEXAHISタンパク質に対する抗体と結合したHPV6b L1 HEXAHISタンパク質およびHPV6 VLP-L1の両方は、HPV6b L1 が、HPV VLP-L1で示される1個またはそれ以上のエピトープをインビボで正確に示すことを示唆する。HPV6 L1タンパク質に対する血清はまたバキュロウイルスまたはエシェリキア・コリのウェルで陰性であり、反応の特異性を示唆する。これはHPVL1 HEXAHISの、ウイルスと相互作用し、有効に中和できる抗体の誘導に好適なワクチン免疫原としての使用の支持を提供する。更に、本実施例は、ウェルにおける種々のタンパク質のコーティングにより証明された、乳頭腫ウイルスL1タンパク質の検出のための免疫検定および組換えHPV6b L1 HEXAHISタンパク質に対する抗体の使用の支持を提供する。HPV6b由来抗原の何れかを含むウェルは陽性結果であった。この実施例はまた、組換えHPV6b L1 HEXAHISタンパク質でのウェルのコーティングにより証明された乳頭腫ウイルスL1タンパク質に特異的な抗体の検出のための免疫検定およびインフルエンザウイルスA/PR-8に対する血清およびHPV6b L1 HEXAHISタンパク質に対する血清の使用の支持を提供する。この場合、HPV6b L1 HEXAHISタンパク質に対する血清を含むウェルは陽性結果となるが一方インフルエンザウイルスに対する血清は陰性である。
ウエスタン・ブロットおよびELISA実験を前記のようにまたは標準方法に従って行った。ELISA捕獲検定は以下の方法に従って行った:
(1)VLPに特異的なモノクローナル抗体(moAb 8)をマイクロタイタープレートのコーティングに使用した;
(2)HPV VLP L1タンパク質を添加し、好適な条件下でインキュベートし、0.1%トゥイン20含有PBS、pH7.4で洗浄した;
(3)種々の動物(表5のカラム1に示す)における種々の免疫原に対する抗体(表5のカラム2に示す)を添加した;および
(4)好適な検出抗原(ウサギ抗血清の場合、ヤギ−抗ウサギペルオキシダーゼコンジュゲートを使用した)を添加し、多重構造/VLP−抗体複合体を検出した。
ウェル当たりの捕獲組換え乳頭腫ウイルスHEXAHISの量を表5に示す。これらの実験は、組換え乳頭腫ウイルスL1 HEXAHISタンパク質に対する抗血清は、L1タンパク質を含む乳頭腫ウイルスVLPを認識する免疫応答を誘発することを証明する。
L1タンパク質を含む乳頭腫ウイルスVLPを認識する免疫応答を起こす乳頭腫ウイルスL1タンパク質の活性には、適切なエピトープの正しい提示を必要とする。VLPを形成しない組換え乳頭腫ウイルスL1タンパク質は、L1タンパク質を含む乳頭腫ウイルスVLPを認識する免疫応答を惹起しない。組換えGST乳頭腫ウイルスL1タンパク質、組換えMS2乳頭腫ウイルスL1タンパク質および変質乳頭腫ウイルスL1タンパク質は、L1タンパク質を含む乳頭腫ウイルスVLPを認識する免疫応答を起こさない。今まですべてのVLPは、乳頭腫ウイルスL1またはL1およびL2遺伝子の発現で細胞内に生産される。本発明の組換え乳頭腫ウイルスL1タンパク質は、L1タンパク質を含む乳頭腫ウイルスVLPを認識する免疫応答を起こす1またはそれ以上のエピトープを正しく提示する。本発明の組換え乳頭腫ウイルスL1タンパク質は、多重構造またはVLPを細胞外に形成し得る。組換え乳頭腫ウイルスL1タンパク質から形成されたVLPの多重構造は、L1タンパク質を含む乳頭腫ウイルスVLPを認識する免疫応答を起こす1またはそれ以上のエピトープを正しく提示すると、思われている。従って、本発明は、L1タンパク質を含む乳頭腫ウイルスVLPを認識する免疫応答を起こし得る多重構造またはVLPを細胞外に形成し得る組換え乳頭腫ウイルスL1タンパク質を提供する。なお、この多重構造は、複数の組換え乳頭腫ウイルスL1タンパク質からなっている。
a 各群のマウスは4匹のマウスを含む
b 6b L1 HEXAHISタンパク質をマウス当たり50μgタンパク質で投与した
b L1は6b L1 HEXAHISタンパク質を意味し、IRRは不適切なHEXAHISタンパク質を意味する。
c PBSはリン酸緩衝化食塩水およびCFAは完全フロインドアジュバントである。
d 6b L1 HEXAHISタンパク質を最大容量2μl中10μgで投与した。
e 30μgの百日咳原を添加した。
f 耳測定(μm x10)
Claims (8)
- 5分子の乳頭腫ウイルスL1タンパク質分子からなる5量体であって、前記各L1タンパク質分子が細菌によって発現されたものであり、前記L1タンパク質の変性条件を経て取得され得る、前記5量体。
- 1以上の乳頭腫ウイルスL1タンパク質分子が、ニッケルに結合し得るN末端アミノ酸配列を含む組換え融合タンパク質である、請求項1記載の5量体。
- 各々の乳頭腫ウイルスL1タンパク質分子が、ニッケルに結合し得るN末端アミノ酸配列を含む組換え融合タンパク質である、請求項1記載の5量体。
- 1以上のまたは各々の乳頭腫ウイルスL1タンパク質分子が、(His)6を含むN末端アミノ酸配列を有する、請求項2または3記載の5量体。
- 1以上のまたは各々の乳頭腫ウイルスL1タンパク質分子が、配列番号1または配列番号4のアミノ酸配列を含む、請求項1〜4のいずか1項記載の5量体。
- 乳頭腫ウイルスVLPを認識する免疫応答を誘発し得る、請求項1〜5のいずれか1項記載の5量体。
- 請求項1〜6のいずれか1項記載の5量体を含む治療的または予防的ワクチン。
- さらにアジュバントを含む、請求項7記載の治療的または予防的ワクチン。
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JP7529247A Withdrawn JPH10500295A (ja) | 1994-05-17 | 1995-05-17 | 組換え乳頭腫ウイルスl1 |
JP2005196551A Expired - Lifetime JP3878197B2 (ja) | 1994-05-17 | 2005-07-05 | 組換え乳頭腫ウイルスl1 |
JP2006187730A Expired - Lifetime JP4016054B2 (ja) | 1994-05-17 | 2006-07-07 | 組換え乳頭腫ウイルスl1 |
Family Applications Before (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
JP7529247A Withdrawn JPH10500295A (ja) | 1994-05-17 | 1995-05-17 | 組換え乳頭腫ウイルスl1 |
JP2005196551A Expired - Lifetime JP3878197B2 (ja) | 1994-05-17 | 2005-07-05 | 組換え乳頭腫ウイルスl1 |
Country Status (13)
Country | Link |
---|---|
US (1) | US6013262A (ja) |
EP (2) | EP1325957B1 (ja) |
JP (3) | JPH10500295A (ja) |
KR (1) | KR100384922B1 (ja) |
AT (2) | ATE340859T1 (ja) |
AU (1) | AUPM566794A0 (ja) |
CA (1) | CA2190473C (ja) |
DE (2) | DE69531297T2 (ja) |
DK (1) | DK0759935T3 (ja) |
ES (2) | ES2275965T3 (ja) |
NZ (1) | NZ285339A (ja) |
PT (2) | PT1325957E (ja) |
WO (1) | WO1995031476A1 (ja) |
Families Citing this family (38)
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US8062642B1 (en) | 1993-03-09 | 2011-11-22 | University Of Rochester | Production of papillomavirus capsid protein and virus-like particles |
SI0757717T1 (sl) * | 1994-05-16 | 2006-08-31 | Merck & Co Inc | Cepivo proti papilomavirusu |
GB9621091D0 (en) | 1996-10-09 | 1996-11-27 | Fondation Pour Le Perfectionem | Attenuated microorganisms strains and their uses |
CA2286294A1 (en) * | 1997-04-08 | 1998-10-15 | Merck & Co., Inc. | Stabilized human papillomavirus formulations |
US6228368B1 (en) * | 1997-10-06 | 2001-05-08 | Loyola University Of Chicago | Papilloma virus capsomere formulations and method of use |
US6689366B1 (en) | 1998-12-17 | 2004-02-10 | Merck & Co., Inc. | Synthetic virus like particles with heterologous epitopes |
DE69930960T2 (de) * | 1998-12-17 | 2007-04-19 | Merck & Co., Inc. | Synthetische virus-ähnliche partikel mit heterologen epitopen. |
AU763853B2 (en) * | 1998-12-23 | 2003-07-31 | Merck & Co., Inc. | Neutralizing assay using human papillomavirus virus-like particles |
SI1150712T1 (sl) | 1999-02-05 | 2009-02-28 | Merck & Co Inc | Pripravek cepiva proti humanem papiloma virusu |
US8128922B2 (en) | 1999-10-20 | 2012-03-06 | Johns Hopkins University | Superior molecular vaccine linking the translocation domain of a bacterial toxin to an antigen |
US7026443B1 (en) | 1999-12-10 | 2006-04-11 | Epimmune Inc. | Inducing cellular immune responses to human Papillomavirus using peptide and nucleic acid compositions |
KR100366608B1 (ko) * | 2000-02-15 | 2003-01-09 | 마스터진(주) | 형질전환 식물체로부터 생산된 재조합 인간 파필로마바이러스 백신 |
EP1305039B1 (en) * | 2000-07-06 | 2008-12-31 | Georgetown University | Stable (fixed) forms of viral l1 capsid proteins, fusion proteins and uses thereof |
US7318928B2 (en) * | 2000-08-01 | 2008-01-15 | The Johns Hopkins University | Molecular vaccine linking intercellular spreading protein to an antigen |
JP5087201B2 (ja) * | 2000-08-03 | 2012-12-05 | ジョンズ・ホプキンス・ユニバーシティ | 抗原に小胞体シャペロンポリペプチドを連結した分子ワクチン |
AUPR446801A0 (en) | 2001-04-18 | 2001-05-17 | University Of Queensland, The | Novel compositions and uses therefor |
AU2003213065A1 (en) * | 2002-02-14 | 2003-09-04 | Novavax, Inc. | Novel insect cell line |
CA2497798A1 (en) * | 2002-09-03 | 2004-04-22 | Large Scale Biology Corporation | Production of peptides in plants as viral coat protein fusions |
US9701725B2 (en) * | 2003-05-05 | 2017-07-11 | The Johns Hopkins University | Anti-cancer DNA vaccine employing plasmids encoding signal sequence, mutant oncoprotein antigen, and heat shock protein |
ATE476528T1 (de) | 2004-12-08 | 2010-08-15 | Gen Probe Inc | Nukleinsäuredetektion aus verschiedenen typen des humanen papillomavirus |
CA2594040A1 (en) * | 2005-01-06 | 2006-07-13 | The Johns Hopkins University | Rna interference that blocks expression of pro-apoptotic proteins potentiates immunity induced by dna and transfected dendritic cell vaccines |
CA2595726A1 (en) * | 2005-01-26 | 2006-08-03 | The Johns Hopkins University | Anti-cancer dna vaccine employing plasmids encoding mutant oncoprotein antigen and calreticulin |
US7691579B2 (en) * | 2005-04-15 | 2010-04-06 | The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services | Methods and compositions for producing an enhanced immune response to a human papillomavirus immunogen |
US7972776B2 (en) * | 2005-11-15 | 2011-07-05 | Oncohealth Corporation | Protein chips for HPV detection |
US7732166B2 (en) * | 2005-11-15 | 2010-06-08 | Oncohealth Corporation | Detection method for human pappilomavirus (HPV) and its application in cervical cancer |
WO2008024844A2 (en) * | 2006-08-22 | 2008-02-28 | The Johns Hopkins University | Anticancer combination therapies |
US8916342B2 (en) | 2006-11-13 | 2014-12-23 | Oncohealth Corp. | Identification of high grade or ≧ CIN2 for early stages and late stages detection, screening, and diagnosis of human papillomavirus (HPV) and HPV-associated cancers |
US8278056B2 (en) | 2008-06-13 | 2012-10-02 | Oncohealth Corp. | Detection of early stages and late stages HPV infection |
US8968995B2 (en) * | 2008-11-12 | 2015-03-03 | Oncohealth Corp. | Detection, screening, and diagnosis of HPV-associated cancers |
US9085638B2 (en) | 2007-03-07 | 2015-07-21 | The Johns Hopkins University | DNA vaccine enhancement with MHC class II activators |
US20080260765A1 (en) * | 2007-03-15 | 2008-10-23 | Johns Hopkins University | HPV DNA Vaccines and Methods of Use Thereof |
US9364529B2 (en) | 2007-04-29 | 2016-06-14 | Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. | Truncated L1 protein of human papillomavirus type 18 |
US9428555B2 (en) * | 2007-04-29 | 2016-08-30 | Beijing Wantai Biological Pharmacy Enterprise Co., Ltd. | Truncated L1 protein of Human Papillomavirus type 16 |
EP2154149B1 (en) * | 2007-05-29 | 2019-07-10 | Xiamen University | A truncated l1 protein of human papillomavirus 6 |
US20090285861A1 (en) * | 2008-04-17 | 2009-11-19 | Tzyy-Choou Wu | Tumor cell-based cancer immunotherapeutic compositions and methods |
EP2521914A4 (en) | 2010-01-08 | 2013-07-10 | Oncohealth Corp | CELL-BASED HPV IMMUNOTESTS HAVE A HIGH PERFORMANCE FOR DIAGNOSIS AND SCANNING OF HPV ASSOCIATED CANCER |
AU2011258501B2 (en) | 2010-05-25 | 2016-07-07 | Qiagen Gaithersburg, Inc. | Fast results hybrid capture assay and associated strategically-truncated probes |
EP4141110A1 (en) | 2020-04-24 | 2023-03-01 | Obshchestvo S Ogranichennoy Otvetstvennost'yu "Ingenik" | Method for producing particles of bacteriophages of the genus levivirus |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0133123A1 (en) * | 1983-07-25 | 1985-02-13 | Mgi Pharma, Inc. | Immunogens of papilloma viruses |
SG48769A1 (en) * | 1991-07-19 | 1998-05-18 | Univ Queensland | Papilloma virus vaccine |
DE122007000099I1 (de) * | 1992-06-25 | 2008-03-27 | Papillomavirus vakzine | |
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 |
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 |
ES2263406T3 (es) * | 1993-03-09 | 2011-06-06 | The University Of Rochester | Producción de la proteína de capside l1 del papilomavirus humano hpv-11 y partículas de tipo virus. |
-
1994
- 1994-05-17 AU AUPM5667A patent/AUPM566794A0/en not_active Abandoned
-
1995
- 1995-05-17 AT AT03006355T patent/ATE340859T1/de active
- 1995-05-17 EP EP03006355A patent/EP1325957B1/en not_active Expired - Lifetime
- 1995-05-17 DE DE69531297T patent/DE69531297T2/de not_active Expired - Lifetime
- 1995-05-17 US US08/737,336 patent/US6013262A/en not_active Expired - Lifetime
- 1995-05-17 WO PCT/AU1995/000292 patent/WO1995031476A1/en active IP Right Grant
- 1995-05-17 DE DE69535249T patent/DE69535249T2/de not_active Expired - Lifetime
- 1995-05-17 ES ES03006355T patent/ES2275965T3/es not_active Expired - Lifetime
- 1995-05-17 PT PT03006355T patent/PT1325957E/pt unknown
- 1995-05-17 EP EP95918467A patent/EP0759935B1/en not_active Expired - Lifetime
- 1995-05-17 CA CA2190473A patent/CA2190473C/en not_active Expired - Lifetime
- 1995-05-17 KR KR1019960706525A patent/KR100384922B1/ko not_active IP Right Cessation
- 1995-05-17 DK DK95918467T patent/DK0759935T3/da active
- 1995-05-17 AT AT95918467T patent/ATE245164T1/de active
- 1995-05-17 ES ES95918467T patent/ES2203640T3/es not_active Expired - Lifetime
- 1995-05-17 PT PT95918467T patent/PT759935E/pt unknown
- 1995-05-17 JP JP7529247A patent/JPH10500295A/ja not_active Withdrawn
- 1995-05-17 NZ NZ285339A patent/NZ285339A/en not_active IP Right Cessation
-
2005
- 2005-07-05 JP JP2005196551A patent/JP3878197B2/ja not_active Expired - Lifetime
-
2006
- 2006-07-07 JP JP2006187730A patent/JP4016054B2/ja not_active Expired - Lifetime
Also Published As
Publication number | Publication date |
---|---|
NZ285339A (en) | 1998-04-27 |
JP2006321806A (ja) | 2006-11-30 |
WO1995031476A1 (en) | 1995-11-23 |
ES2275965T3 (es) | 2007-06-16 |
PT1325957E (pt) | 2007-01-31 |
ATE340859T1 (de) | 2006-10-15 |
KR100384922B1 (ko) | 2003-08-21 |
ES2203640T3 (es) | 2004-04-16 |
EP0759935B1 (en) | 2003-07-16 |
ATE245164T1 (de) | 2003-08-15 |
US6013262A (en) | 2000-01-11 |
JP2006001936A (ja) | 2006-01-05 |
CA2190473C (en) | 2011-08-30 |
EP0759935A1 (en) | 1997-03-05 |
DE69535249T2 (de) | 2007-05-10 |
PT759935E (pt) | 2003-11-28 |
DE69535249D1 (de) | 2006-11-09 |
JPH10500295A (ja) | 1998-01-13 |
DK0759935T3 (da) | 2003-11-10 |
JP3878197B2 (ja) | 2007-02-07 |
DE69531297T2 (de) | 2004-05-27 |
EP1325957B1 (en) | 2006-09-27 |
EP0759935A4 (en) | 1999-07-07 |
EP1325957A2 (en) | 2003-07-09 |
DE69531297D1 (de) | 2003-08-21 |
CA2190473A1 (en) | 1995-11-23 |
AUPM566794A0 (en) | 1994-06-09 |
EP1325957A3 (en) | 2003-11-26 |
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