WO2002092796A1 - Pseudopapillovirus et son mode de preparation - Google Patents

Pseudopapillovirus et son mode de preparation Download PDF

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Publication number
WO2002092796A1
WO2002092796A1 PCT/CN2002/000187 CN0200187W WO02092796A1 WO 2002092796 A1 WO2002092796 A1 WO 2002092796A1 CN 0200187 W CN0200187 W CN 0200187W WO 02092796 A1 WO02092796 A1 WO 02092796A1
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pseudovirus
papilloma
virus
particles
plasmid
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PCT/CN2002/000187
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French (fr)
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Liang Qiao
Wei Shi
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Liang Qiao
Wei Shi
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Priority to US10/294,087 priority Critical patent/US6878541B2/en
Publication of WO2002092796A1 publication Critical patent/WO2002092796A1/zh
Priority to US11/060,034 priority patent/US7205126B2/en
Priority to US11/784,626 priority patent/US8129144B2/en
Priority to US13/413,386 priority patent/US20120164173A1/en

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    • C—CHEMISTRY; METALLURGY
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    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20023—Virus like particles [VLP]
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    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20061—Methods of inactivation or attenuation

Definitions

  • the invention relates to a papilloma pseudovirus capable of generating an immune response through an oral route and a preparation method thereof.
  • This pseudovirus can be used in vaccines to prevent and treat mucosal pathogens or mucosal tumors.
  • This pseudovirus can also be used in gene therapy.
  • pathogenic microorganisms and human immune function defects are the root cause of many diseases.
  • the human body is often attacked by pathogenic microorganisms and the destruction of hidden tumors inside the human body. Therefore, the human body also needs a kind of body resistance or immunity to resist the invasion and infection of pathogenic microorganisms and diseases inside the body.
  • the current clinically used vaccine is injected subcutaneously or intramuscularly, which results in a specific immune response, leading to resistance to the disease and preventing the disease from occurring in the future.
  • these vaccines are often injected subcutaneously or intramuscularly into the human body. After the vaccine is injected, it can only produce a systemic immune response, not a mucosal immune response. Such vaccines often fail to prevent and treat mucosal infections.
  • many pathogenic bacteria are transmitted by the mucous membranes, such as the Aids virus.
  • An object of the present invention is to develop a virus-like pseudovirus that does not cause disease.
  • a gene or DNA vaccine that is used to treat and prevent disease is injected into the papilloma virus shell, orally or through other methods.
  • the mucosa and systemic lymphoid tissues at the entrance of the mucosa induce the immune response of the human body and play a role in preventing and treating diseases.
  • the papilloma pseudovirus in the present invention is a human papilloma virus-like particle (HPV-VLP) or a bovine (or other animal) papilloma virus-like particle (BPV-VLP) is dissociated and incorporated into plasmids (plasmids). Combination of adult or bovine (or other animal) papilloma pseudoviruses.
  • HPV-VLP human papilloma virus-like particle
  • BPV-VLP bovine (or other animal) papilloma virus-like particle
  • a normal virus is made up of a shell and the DNA inside the shell.
  • the papilloma pseudovirus, which is described in the invention has only a shell of the virus, but no virus DNA in the shell. If a DNA vaccine is placed in the shell of the virus, it can be delivered into the body by mouth.
  • the DNA vaccine described in the present invention is only a piece of DNA fragment, not viral DNA. If this DAN fragment is injected subcutaneously or intramuscularly into a human body, a systemic immune response can be induced. Therefore, the viral shell containing the DNA vaccine is described in the present invention. Fake virus. This pseudovirus is prepared as follows ⁇
  • Dissociation buffer ethylene glycol bis (e-aminoethyl ether) tetraacetic acid (EGTA) 20mM, dithiothreitol (DTT) 40mM, sodium chloride (NaCl) 300mM, tris (hydroxymethyl) ) Carbamate hydrochloride (Tris—HC1) (PH 8.0) 100 mM;
  • stop buffer calcium chloride (CaCb) 25mM, dimethyl sulfoxide (DMSO) 20% (total volume of stop buffer);
  • the dissociation buffer was: ethylene glycol di (em-based ether) tetraacetic acid (EGTA) 20 mM, dithiothreo Sugar alcohol (DTT) 40 mM, sodium chloride (NaCl) 300 mM, tris (hydroxymethyl) 'carbamidine hydrochloride (Tris-HC1) (PH 8.0) 100 mM, then add 1/10 volume of 0.5 g / 4l PCI-GLP-LCMV plasmid, and then gradually add an equal volume of stop buffer, the stop buffer is: calcium chloride (CaCl 2 ) 25mM, dimethyl sulfoxide (DMSO) 20% by volume, mix well The solution was left at 4 ° C overnight.
  • EGTA ethylene glycol di (em-based ether) tetraacetic acid
  • DTT dithiothreo Sugar alcohol
  • Tris-HC1 tris (hydroxymethyl) 'carbamidine hydrochloride (Tris-HC1) (PH
  • pseudovirus vaccine is more effective than the gene vaccine in inducing cellular immune response.
  • the shell of bovine papilloma virus and dissociation buffer were mixed in a volume of 1: 1 and cultured for 60 minutes at room temperature.
  • the dissociation buffer was: EGTA 20mM, DTT 40mM, NaCl 300mM, tris (hydroxymethyl) aminomethane salt.
  • Tris—HC1) (PH 8.0) .00mM then add 1/10 volume of 1.0 ⁇ ⁇ / ⁇ 1 plasmid carrying the green fluorescent protein GLP gene, and then gradually add equal volume of stop buffer to stop buffer
  • Stop buffer For: CaCh25mM, DMSO 20% by volume, put the mixed mixture at 4'C overnight. Use this fake disease.
  • the mice were given orally (using a gastric tube) and then tested for GLP expression.
  • GLP cannot be detected if the plasmid is taken orally.
  • a pseudovirus containing the HPV 16E7 plasmid prepared by the same method as in Example 1 was orally used to immunize animals, and mucosal and systemic E7-specific cytotoxic cells were induced.
  • the plasmid itself was used directly, and no immunity was induced after oral administration. reaction. This pseudovirus can therefore be used to induce mucosal and systemic immune responses.
  • HPV and BPV pseudoviruses containing the HPV16E7 plasmid were prepared in the same way as in Examples 1 and 2.
  • the mice were first orally immunized with the HPV pseudovirus and then infected with the BPV pseudovirus.
  • the HPV pseudovirus was found to prevent BPV. Pseudovirus infection. This pseudovirus can be used to provide immune protection.
  • Example 1 The method of Example 1 or Example 2 was used to prepare the interleukin-II-containing pseudovirus, and the intestinal mucosal lymphatic tissue and systemic lymphoid tissue were orally administered to improve the efficiency of mucosal or systemic immune induction.
  • This pseudovirus is not pathogenic and can be used for gene therapy. That is, the gene plasmid to be expressed is incorporated into virus-like particles. This pseudovirus can orally deliver this gene into the intestinal mucosa and systemic lymphoid tissues, as well as mucosal epithelial tissues. More importantly, the pseudovirus can be used as a vaccine, and a plasmid expressing the antigen gene is incorporated to synthesize the pseudovirus, which can produce a protective immune response when taken orally. This differs from other vaccines in that many vaccines can only be injected subcutaneously or intramuscularly and cannot be taken orally. It can only produce a systemic immune response but not a mucosal immune response.
  • pathogenic bacteria are transmitted through the mucous membranes, only this pseudovirus can produce an effective immune response to prevent or treat mucosal pathogenic infections.
  • pathogenic bacteria include bacteria such as Salmonella.
  • viruses such as HIV (human immunodeficiency virus).
  • HIV human immunodeficiency virus
  • the virus can also be used to induce anti-tumor immune responses to treat tumors, especially mucosal tumors, such as colon tumors.
  • Pseudoviruses can induce a stronger immune response than genetic vaccines.

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Description

技术领域：
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。
技术背景：
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疫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： 艾兹病毒。
发明内容: 一 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， 即将用于治疗和预防疾病的基因或 DNA 疫苗注入乳头瘤病毒外壳 内， 通过口服的途径或经过其他粘膜入口处送入人体的粘膜和系统 性淋巴组织， 诱导人体的免疫反应， 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 （或其他动物） 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 （或其他动物） 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 片段经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： 乙二醇二 （e-氨基乙醚） 四 乙酸（EGTA)20mM ，二硫苏糖醇（ DTT) 40mM，氯化钠（NaCl) 300mM， 三 （羟甲基） 氨基甲垸盐酸盐 （Tris— HC1) (PH 8.0) lOOmM;
(说明： mM 中文叫 每升中以分子量作为它的克数）
(2) 加入 1/10体积的质粒， 0.5— ΐμβ/μΐ;
(3) 逐步加入等容积的终止缓冲液， 终止缓冲液： 氯化钙 （CaCb) 25mM，二甲基亚砜 （DMSO) 20% (终止缓冲液总体积百分比）；
(4) 将混合液在 4'C放置 4—12小时。
具体实施方式：
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： 乙二醇二 （e-m基乙醚） 四乙 酸（EGTA)20mM， 二硫苏糖醇（ DTT)40mM,氯化钠（ NaCl) 300mM, 三 （羟甲基）'氨基甲垸盐酸盐 (Tris— HC1) (PH 8.0) lOOmM，然后加 入 1/10体积的 0.5 g/4l 的 PCI— GLP— LCMV质粒， 再将等容积终 止缓冲液逐步加入， 终止缓冲液为： 氯化钙 （CaCl2) 25mM, 二甲基 亚砜 (DMSO) 20%体积百分比， 将混合好的混合液放在 4°C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 60 min， 室温， 解离缓冲液为： EGTA 20mM, DTT 40mM, NaCl 300mM, 三（羟甲基）氨基甲烷盐酸盐 （Tris— HC1) (PH 8.0).00mM, 然后加入 1/10体积的 1.0μδ/μ1 的携带绿色荧光蛋白 GLP 的基因的 质粒， 再将等容积终止缓冲液逐步加入， 终止缓冲液为： CaCh25mM， DMSO 20%体积百分比， 将混合好的混合液放在 4'C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。 这就证明此假病毒可以携带基 因到粘膜及系统淋巴组织。 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 HPV 16E7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， 经口服之后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 HPV16E7质粒的 HPV和 BPV的 假病毒， 先用 HPV假病毒经口服免疫小鼠， 然后用 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， 经过口服进 入肠粘膜淋巴组织和系统淋巴组织，提高对粘膜或系统免疫诱导的效 率。
工业应用性：
此假病毒不致病， 可用于基因治疗， 即将所要表达的基因质粒， 掺入病毒样颗粒，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 表达抗原基因的质粒掺入， 合成假病毒， 经口服， 可产生保护性的免 疫反应。 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.。

Claims

权利要求
1. 一种乳头瘤假病毒， 其特征在于： 它是由入乳头瘤病毒样颗粒或 牛 或其他动物 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 ：或其他动物 乳头瘤假病毒。
2. —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 1 ： 1 混合，解离缓冲液为： 乙二醇二 （ e -氨基乙醚） 四乙酸
(EGTA)20mM, 二硫苏糖醇（DTT) 40mM， 氯化钠（NaCl) 300mM， 三 (羟甲基） 氨基甲垸盐酸盐（Tris— HC1) (PH 8.0) lOOm ;
(2) 加入 1/10体积的质粒： 0.5— ΐμδ/μ1 的质粒；
(3) 逐渐加入终止缓冲液， 终止缓冲液为.： 氯化钙 （CaCl2).25mM, 二甲基亚砜（DMSO) 20%体积百分比；
(.4) 混合好的混合液放在 4.Ό放置 4一 12小时。
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： '可将表达抗原基因的质粒掺入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： 可将表达治疗基因的质粒掺入合成假病毒， 用于基因治疗。
5.根据权力要求 2 所述的一种乳头瘤假病毒的制备方法， 其特征在 于：所述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 1 ： 1体积混合；
6. 根据权力要求 2所述的一种乳头瘤假病毒的制备方法， 其特征在 于： 所述终止缓冲液的加入量与所述的 1、 2、 3步骤得到的中间物 等体积。
PCT/CN2002/000187 2001-05-15 2002-03-22 Pseudopapillovirus et son mode de preparation WO2002092796A1 (fr)

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US10/294,087 US6878541B2 (en) 2001-05-15 2002-11-14 Papilloma pseudo-virus and preparation
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US11/784,626 US8129144B2 (en) 2001-05-15 2007-04-09 Papilloma pseudovirus and preparation
US13/413,386 US20120164173A1 (en) 2001-05-15 2012-03-06 Papilloma Pseudovirus and Preparation

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JP4511090B2 (ja) 2010-07-28
US8129144B2 (en) 2012-03-06
US20050142115A1 (en) 2005-06-30
JP2003024058A (ja) 2003-01-28
JP4472724B2 (ja) 2010-06-02
US7205126B2 (en) 2007-04-17
US6878541B2 (en) 2005-04-12
CN1114690C (zh) 2003-07-16
JP2007211026A (ja) 2007-08-23
CN1333338A (zh) 2002-01-30
US20100255027A1 (en) 2010-10-07

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