CN1333338A - 乳头瘤假病毒及其制备方法 - Google Patents
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Abstract
本发明涉及一种能通过口服途径产生免疫反应的乳头瘤假病毒及其制备方法。其特点,是将人乳头瘤病毒样颗粒(HPV－VLP)或牛乳头瘤病毒样颗粒(BPV－VLP),解离,掺入质粒(plasmids或DNA疫苗),从新组合成人或牛乳头瘤假病毒,通过口服送入人体的粘膜和系统性淋巴组织,激活人体的免疫反应,起到防病治病的作用,此假病毒可以诱导出比基因疫苗更强的免疫反应。之外,此假病毒可以将基因送到体内淋巴组织,可以用于基因治疗。
Description
本发明涉及一种能通过口服途径产生免疫反应的乳头瘤假病毒及其制备方法。此假病毒可以用于疫苗来预防和治疗粘膜致病菌感染或粘膜肿瘤，此假病毒也可用于基因治疗。
我们都知道病原微生物和人体免疫机能缺陷是导致许多疾病的根源，人体经常遭受病原微生物的侵袭及人体内部暗藏的肿瘤的破坏。因此，人体也需要一种机体抵抗力或免役力来抵抗病原微生物的入侵感染和机体内部的病变。目前临床使用的疫苗是通过皮下或肌肉注射，从而产生特异的免疫反应，导致对这种疾病的抵抗能力，使以后不再得这种病。但是，这些疫苗往往是皮下或肌肉注入人体后，只能产生系统性免疫反应，而不能产生粘膜免疫反应。这样的疫苗往往不能预防和治疗粘膜传染的致病菌。但是，许多致病菌是由粘膜传染的，比如：艾兹病。
本发明的目的就是研究出一种不会致病的类似病毒的假病毒，将用于治疗和预防疾病的基因或DNA疫苗注入此病毒外壳内，通过口服的途径送入人体的粘膜和系统性淋巴组织，激活人体的免疫反应，起到防病治病的作用。
本发明所说的乳头瘤假病毒是将人乳头瘤病毒样颗粒(HPV-VLP)或牛乳头瘤病毒样颗粒(BPV-VLP)，解离，掺入质粒(plasmids)，从新组合成人或牛乳头瘤假病毒。正常病毒是由一个外壳和壳内的DNA构成，而发明所说的这个乳头瘤假病毒只有一个病毒的外壳，而无壳内的病毒的DNA。如果将DNA疫苗放入这个病毒外壳内，就可以通过口服送入人体内。本发明所述的DNA疫苗只是一段DNA片段，不是病毒的DNA，若把这个DAN片段皮下或肌肉注射人体内，就可以诱导系统免疫反应，因此含有DNA疫苗的病毒外壳就是本发明所述的假病毒。此假病毒的制备方法如下：
(1)将乳头瘤病毒样颗粒与解离缓冲液混合，体积混合比为1∶1，培养60min，室温；解离缓冲液：EGTA 20mM、DTT 40mM、NaCl 300mM、Tris-HCL(PH8.0)100mM；
(2)加入1/10体积的质粒，0.5-1μg/μl；
(3)逐步加入终止缓冲液，终止缓冲液：Cacl2 25mM，DMSO20％(体积百分比)
(4)将混合液放在4℃过夜。
此假病毒不致病，可用于基因治疗，即将所要表达的基因插入质粒，装入病毒样颗粒，此假病毒即可将此基因经口服送入肠粘膜及系统淋巴组织，还有粘膜上皮组织。更重要的是此假病毒可以用作疫苗，将抗原的基因装入质粒，然后合成假病毒，经口服，可产生保护性的免疫反映。这一点与其他疫苗不同，是因为许多疫苗只能皮下或肌肉注射而不能口服。只能产生系统性免疫反应但不能产生粘膜免疫反应。因为许多致病菌是通过粘膜传染，所以只有此假病毒才可能产生有效的免疫反应来防止或治疗粘膜的致病菌的感染。这些致病菌包括细菌，比如沙门氏菌等。还有病毒，比如HIV(人免疫缺陷病毒)等。同时此病毒还可以用于诱导抗肿瘤免疫反应来治疗肿瘤，特别是粘膜肿瘤，比如结肠肿瘤。假病毒能诱导比基因疫苗更强的免疫反应。
实例1
首先将人乳头瘤病毒的外壳与解离缓冲液按1∶1体积混合、培养60min，室温，解离缓冲液为：EGTA 20mM，DTT 40mM，NaCl300mM，Tris-HCL(PH8.0)100mM，然后加入1/10体积的0.5μg/μl的pCI-GLP-LCMV质粒，再将终止缓冲液逐步加入，终止缓冲液为：CaCl2 25mM，DMSO 20％体积百分比，将混合好的混合液放在4℃过夜。用假病毒对C57BL6小鼠做免疫试验，用皮下注射方式注入小鼠体内，同时也将此种质粒直接注入其他小鼠的体内与其作对比试验，用Cr51释放试验，或r干扰素分泌试验(Elispot)。测出假病毒可以诱导出比质粒诱导出更多的特异性细胞毒细胞。因此在诱导细胞免疫反应上，假病毒疫苗比基因疫苗效用高。实例2
首先将牛乳头瘤病毒的外壳与解离缓冲液按1∶1体积混合、培养60min，室温，解离缓冲液为：EGTA 20mM，DTT 40mM，NaCl300mM，Tris-HCL(PH8.0)100mM，然后加入1/10体积的1.0μg/μl的携带绿色荧光蛋白GLP的基因的质粒，再将终止缓冲液逐步加入，终止缓冲液为：CaCl2 25mM，DMSO 20％体积百分比，将混合好的混合液放在4℃过夜。用此假病毒让小鼠口服(使用胃管)，然后检测GLP的表达。我们可以在肠粘膜、肠粘膜淋巴结，脾脏检测出GLP此假病毒可以携带基因到粘膜及系统淋巴系统。因此，可以用于基因治疗。若用质粒本身口服则测不到GLP。实例3用与实例1同样的方法制备含有HPV16E7质粒的假病毒经口服来免疫动物，可诱导出粘膜及系统E7特异性的细胞毒细胞，然而直接用此质粒本身，则诱导不出免疫反应。因此此假病毒可以用于诱导粘膜及系统免疫反应。实例4用于实例1、2同样的方法制备含有HPV和BPV质粒的假病毒，然后用含有HPV的假病毒经口服免疫小鼠，然后用含有BPV的假病毒来感染小鼠，发现HPV假病毒可以防止BPV假病毒的感染。此假病毒可以用于提供免疫保护。实例5用实例1或实例2的方法制备含有白介素-II的假病毒，用于提高粘膜反应的效率。经过口服进入肠粘膜淋巴组织和其他淋巴组织，提高对粘膜免疫诱导的效率。
Claims (4)
1.一种乳头瘤假病毒，其特征在于：它是由人乳头瘤病毒样颗粒或牛乳头瘤病毒样颗粒解离然后参入质粒，重新组合而成的人或牛乳头瘤假病毒。
2.一种乳头瘤假病毒的制备方法，其特征在于：
(1)首先将人乳头瘤病毒样颗粒或牛乳头瘤病毒样颗粒与解离缓冲液的1∶1体积混合，解离缓冲液为：EGTA 20mM，DTT 40mM，NaCl2 300mM，Tris-HCL(PH8.0)100mM；
(2)加入1/10体积的质粒：0.5-1μg/μl的质粒；
(3)逐渐加入终止缓冲液，终止缓冲液为：CaCl2 25mM，DMSO20％体积百分比；
(4)混合好的混合液放在4℃过夜；
3.根据权利要求2所述的一种乳头瘤假病毒的制备方法，其特征在于：可将抗原基因插入质粒合成假病毒来制作疫苗；
4.根据权利要求2所述的一种乳头瘤假病毒的制备方法，其特征在于：可将治疗基因插入质粒合成假病毒，用于基因治疗。
Priority Applications (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN01118003A CN1114690C (zh) | 2001-05-15 | 2001-05-15 | 乳头瘤假病毒及其制备方法 |
| JP2001321828A JP4511090B2 (ja) | 2001-05-15 | 2001-10-19 | Ｄｎａプラスミドワクチン用アジュバンド及びその調製方法 |
| PCT/CN2002/000187 WO2002092796A1 (fr) | 2001-05-15 | 2002-03-22 | Pseudopapillovirus et son mode de preparation |
| US10/294,087 US6878541B2 (en) | 2001-05-15 | 2002-11-14 | Papilloma pseudo-virus and preparation |
| US11/060,034 US7205126B2 (en) | 2001-05-15 | 2005-02-17 | Papilloma pseudovirus and preparation |
| US11/784,626 US8129144B2 (en) | 2001-05-15 | 2007-04-09 | Papilloma pseudovirus and preparation |
| JP2007124042A JP4472724B2 (ja) | 2001-05-15 | 2007-05-09 | パピロマウイルス偽ウイルス及びその調製方法 |
| US13/413,386 US20120164173A1 (en) | 2001-05-15 | 2012-03-06 | Papilloma Pseudovirus and Preparation |
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| US8129144B2 (en) | 2001-05-15 | 2012-03-06 | Loyola University Chicago | Papilloma pseudovirus and preparation |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120164173A1 (en) * | 2001-05-15 | 2012-06-28 | Liang Qiao | Papilloma Pseudovirus and Preparation |
| 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 |
| AU2008251615B2 (en) | 2007-05-08 | 2014-09-18 | The United States of America, as represented by The Secretary, Department of Health and Human Services Office of Technology Transfer National Institute of Health | Papillomavirus pseudoviruses for detection and therapy of tumors |
| CN102481378A (zh) | 2009-04-13 | 2012-05-30 | 法国健康和医学研究院 | Hpv颗粒及其用途 |
| WO2011039646A2 (en) | 2009-09-30 | 2011-04-07 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Papilloma virus -like particles for targeted gene delivery |
| CA2778678A1 (en) | 2009-10-30 | 2011-05-05 | Cns Therapeutics, Inc. | Improved neurturin molecules |
| US9700639B2 (en) | 2012-02-07 | 2017-07-11 | Aura Biosciences, Inc. | Virion-derived nanospheres for selective delivery of therapeutic and diagnostic agents to cancer cells |
| CN104334196B (zh) | 2012-02-16 | 2018-04-10 | Atyr 医药公司 | 用于治疗自身免疫疾病和炎性疾病的组氨酰‑tRNA合成酶 |
| DK3046583T3 (da) | 2013-09-18 | 2019-05-20 | Aura Biosciences Inc | Viruslignende partikelkonjugater til behandling af tumorer |
| JP2018532801A (ja) | 2015-10-30 | 2018-11-08 | ザ ユナイテッド ステイツ オブ アメリカ， アズ リプレゼンテッド バイ ザ セクレタリー， デパートメント オブ ヘルス アンド ヒューマン サービシーズ | 標的化がん療法 |
| KR20180081532A (ko) | 2015-10-30 | 2018-07-16 | 알레타 바이오쎄라퓨틱스, 인크. | 암 치료용 조성물 및 방법 |
| CN108472365A (zh) | 2015-10-30 | 2018-08-31 | 艾丽塔生物治疗剂公司 | 用于肿瘤转导的组合物和方法 |
| US10751398B2 (en) * | 2016-10-07 | 2020-08-25 | Loyola University Chicago | Inflammasome activators and methods of use to treat tumors |
| BR112019027488A2 (pt) | 2017-06-23 | 2020-07-07 | Pathovax Llc | partículas semelhantes a vírus quiméricas e usos destas como redirecionadores de respostas imunológicas antígeno-específicos |
| CN111315404A (zh) | 2017-11-06 | 2020-06-19 | 美利坚合众国, 由健康及人类服务部部长代表 | 利用预先存在的微生物免疫的癌症治疗 |
| WO2019096796A1 (en) | 2017-11-14 | 2019-05-23 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | Non-human papillomaviruses for gene delivery in vitro and in vivo |
| US11560408B2 (en) | 2018-12-27 | 2023-01-24 | Verimmune Inc. | Conjugated virus-like particles and uses thereof as anti-tumor immune redirectors |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19526752C2 (de) * | 1995-07-21 | 1997-08-07 | Lutz Prof Dr Gissmann | Hocheffiziente Bildung von Papillomavirusähnlichen Partikeln |
| FR2749323B1 (fr) * | 1996-06-04 | 1998-07-10 | Pasteur Merieux Serums Vacc | Pseudo-particules virales utiles en tant que vecteur de delivrance d'acide nucleique |
| JP2000515741A (ja) * | 1996-07-17 | 2000-11-28 | アメリカ合衆国 | 感染性パピローマウイルス偽ウイルス粒子 |
| ATE333501T1 (de) * | 1997-09-05 | 2006-08-15 | Medimmune Inc | Methode zur in vitro auseinander- und wiederzusammensetzung von papillomavirus virus- ähnlichen tielchen (vlps) |
| GB9806666D0 (en) * | 1998-03-27 | 1998-05-27 | Stanley Margaret | Antigen preparation and use |
| CN1241636A (zh) * | 1999-07-05 | 2000-01-19 | 第一军医大学珠江医院 | 抗hpv16e6核酶质粒及其在人乳头瘤病毒与肿瘤治疗中的应用 |
| GB0007231D0 (en) * | 2000-03-24 | 2000-05-17 | Chiron Spa | Modified rna for gene delivery |
| CN1114690C (zh) | 2001-05-15 | 2003-07-16 | 乔良 | 乳头瘤假病毒及其制备方法 |
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2001
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- 2001-10-19 JP JP2001321828A patent/JP4511090B2/ja not_active Expired - Fee Related
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2002
- 2002-03-22 WO PCT/CN2002/000187 patent/WO2002092796A1/zh not_active Application Discontinuation
- 2002-11-14 US US10/294,087 patent/US6878541B2/en not_active Expired - Lifetime
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2005
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8129144B2 (en) | 2001-05-15 | 2012-03-06 | Loyola University Chicago | Papilloma pseudovirus and preparation |
Also Published As
| Publication number | Publication date |
|---|---|
| JP4472724B2 (ja) | 2010-06-02 |
| WO2002092796A1 (fr) | 2002-11-21 |
| JP4511090B2 (ja) | 2010-07-28 |
| US20030129728A1 (en) | 2003-07-10 |
| US7205126B2 (en) | 2007-04-17 |
| US8129144B2 (en) | 2012-03-06 |
| JP2007211026A (ja) | 2007-08-23 |
| CN1114690C (zh) | 2003-07-16 |
| US6878541B2 (en) | 2005-04-12 |
| JP2003024058A (ja) | 2003-01-28 |
| US20050142115A1 (en) | 2005-06-30 |
| US20100255027A1 (en) | 2010-10-07 |
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