Ad35腺病毒载体构建及病毒蛋白免疫原性研究
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摘要
目前以C组5型腺病毒(Ad5)载体为基础构建的腺病毒载体在基因治疗和疫苗研究中得到广泛应用,但是由于其转导作用依赖于细胞表面的柯萨奇-腺病毒受体(CAR),许多重要的细胞如造血干细胞和树突状细胞表面Ad5吸附受体CAR表达很少,一些恶性肿瘤细胞CAR的表达比相应的正常组织细胞要少,Ad5载体难以转导外源基因；同时正常人群Ad35的感染率在90%左右,体内预存的中和抗体可迅速清除腺病毒载体,导致治疗基因消失。这些问题限制了5型腺病毒(Ad5)载体的广泛应用。为此人们在试图利用其他型别的腺病毒开发新型载体,以克服这些不足。
35型腺病毒(Ad35)以几乎在所有的人类体细胞表面都有表达的CD46作为吸附受体,可以转导不易被Ad5感染造血细胞、树突状细胞和恶性肿瘤细胞等靶细胞。而且世界范围内正常人群中Ad35感染率不到5%,病毒载体被抗体清除的概率小,所以Ad35腺病毒作为颇具吸引力的基因治疗载体近年来受到人们的青睐。但目前Ad35载体尚未商品化,且迄今为止仅有的几例有关成功构建Ad35载体一般采用体外连接法,载体构建过程繁琐,病毒包装成功率低。有鉴于此,本文拟利用体外同源重组方法来构建Ad35载体包装系统,以期建立一种操作简便的Ad35载体系统。
为了评价Ad35载体在实际应用中的可行性,为构建Ad35载体提供可靠的理论依据,本文首先对人6个组10种不同型腺病毒基因组序列同源性进行了比较,发现人群普遍感染的C组Ad5腺病毒与人群感染率很低的B组Ad35腺病毒在进化上亲缘关系比较远,六邻体蛋白氨基酸同源性比较显示Ad5和Ad35六邻体的同源性只有75.3%,这提示Ad5和Ad35六邻体之间存在交叉反应的可能性比较小；之后对中国1157份0--80岁健康人群进行了Ad4、Ad5和Ad35抗体水平的本底筛查,结果显示在中国正常人群感染Ad4和Ad5的比例分别为70%和90%左右,而Ad35的感染要远远低于Ad4和Ad5,只有不到5%。
根据细菌内同源重组的原理,克隆了Ad35的全基因组DNA,构建了病毒包装所需的穿梭质粒、骨架质粒和表达Ad35腺病毒E1B的细胞系293-E1B,并通过同源重组的方法成功在293-E1B细胞系中包装出复制缺陷型的Ad35腺病毒,病毒的包装滴度达到106TCID50/ml,接近于野生型腺病毒。同时还对影响病毒包装效率的pXI蛋白进行了初步研究,发现pXI蛋白是影响重组病毒包装效率的主要因素。
目前对Ad35的基因组编码产物研究的还很少,为了阐明Ad35基因组编码产物中与诱导体液免疫有关的蛋白,为今后改进Ad35载体系统打下基础,利用蛋白质组学的方法对Ad35腺病不同毒蛋白免疫原性进行了初步鉴别,首次发现Ad35腺病毒非结构蛋白E2A (DNA binding protein)能够诱导机体产生抗体。
综上所述,本文首次调查了我国人群中Ad35的感染情况、利用细菌内同源重组机制构建了Ad35载体系统并鉴别出一个以前未报道的体液免疫原,这些结果的取得为Ad35载体的开发、应用打下了基础。
Adenovirus (Ads) is a nonenveloped double-stranded, linear DNA virus with icosahedral symmetry. The human adenovirus constitutes a large family with at least 51 identified serotypes, which are classified into six subgroup on the base of hemagglutination.
Among the 51 serotypes, the conventional Ad vectors that are most widely used, including for human clinical trials, are constructed based on the subgroup C Ad serotype 5 (Ad5). Ad5 vector has several advantages as a gene delivery. For example, Ad5 can infect many different types of cells, the virus particle is more stable and easy to get high titer of adenovirus. But clinical and preclinical studies have revealed two major disadvantages of Ad5 vector. First, target cells that are important for gene therapy, including malignant tumor cells and dendritic cells, express nil or insufficient level of a cellular receptor for Ad5, the coxsackievirus and adenovirus receptor. Second, about 90% of adults are seropositive for Ad5 because natural infection with Ad5 is common. Pre-exsiting anti-Ad5 antibodies not only largely inhibit Ad5 vector-mediated transduction, but may also enhance the toxicities induced by Ad5 vector.
In contrast, human species B Ad serotype 35(Ad35) utilizes human CD46 as a cellular receptor. Human CD46 is ubiquitously express on almost all human cells, leading to a wide tropism of Ad35 vector. In addition, pre-exsiting anti-Ad5 immunity does not hamper Ad35 vector mediated transduction, and seroprevalence for Ad35 is much lower than that for Ad5. All these demonstrate that Ad35 vector is a promising vector candidate. Till now, there have been very few successful cases regarding the development of the Ad35 vector. Thus, it is essential to develop the Ad35 vector In the paper, we compare the evolution relationship of the human six subgroup adenovirus and concluded that the species B and species C are far from each other on the evolution relationship and at the same time we detected anti-Ad (including Ad4、Ad5、Ad 35)antibody in 1157 normal people serum samples. The results show that people infected by Ad4 and Ad5 are about 70% and 90% respectively, while less than 5% by Ad35. After the detection, we successfully made the Ad5 vector packaging system, including backbone plasmid, shuttle plasmid and cell line expressing Ad35 E1B constructively. With vitro recombination, we get replication-defective Ad35, the titer of which is 106TCID50/ml. In addition, we found that protein pXI is important for the package of replication-defective Ad35.
In order to optimize the packaging system we analysis the immunogen ability of the adenovirus protein with proteomic assay, and the results show that the non-structure protein E2A (DNA binding protein) has strong immunogen besides some structure protein such and hexon、penton and fiber.
35型腺病毒(Ad35)以几乎在所有的人类体细胞表面都有表达的CD46作为吸附受体,可以转导不易被Ad5感染造血细胞、树突状细胞和恶性肿瘤细胞等靶细胞。而且世界范围内正常人群中Ad35感染率不到5%,病毒载体被抗体清除的概率小,所以Ad35腺病毒作为颇具吸引力的基因治疗载体近年来受到人们的青睐。但目前Ad35载体尚未商品化,且迄今为止仅有的几例有关成功构建Ad35载体一般采用体外连接法,载体构建过程繁琐,病毒包装成功率低。有鉴于此,本文拟利用体外同源重组方法来构建Ad35载体包装系统,以期建立一种操作简便的Ad35载体系统。
为了评价Ad35载体在实际应用中的可行性,为构建Ad35载体提供可靠的理论依据,本文首先对人6个组10种不同型腺病毒基因组序列同源性进行了比较,发现人群普遍感染的C组Ad5腺病毒与人群感染率很低的B组Ad35腺病毒在进化上亲缘关系比较远,六邻体蛋白氨基酸同源性比较显示Ad5和Ad35六邻体的同源性只有75.3%,这提示Ad5和Ad35六邻体之间存在交叉反应的可能性比较小；之后对中国1157份0--80岁健康人群进行了Ad4、Ad5和Ad35抗体水平的本底筛查,结果显示在中国正常人群感染Ad4和Ad5的比例分别为70%和90%左右,而Ad35的感染要远远低于Ad4和Ad5,只有不到5%。
根据细菌内同源重组的原理,克隆了Ad35的全基因组DNA,构建了病毒包装所需的穿梭质粒、骨架质粒和表达Ad35腺病毒E1B的细胞系293-E1B,并通过同源重组的方法成功在293-E1B细胞系中包装出复制缺陷型的Ad35腺病毒,病毒的包装滴度达到106TCID50/ml,接近于野生型腺病毒。同时还对影响病毒包装效率的pXI蛋白进行了初步研究,发现pXI蛋白是影响重组病毒包装效率的主要因素。
目前对Ad35的基因组编码产物研究的还很少,为了阐明Ad35基因组编码产物中与诱导体液免疫有关的蛋白,为今后改进Ad35载体系统打下基础,利用蛋白质组学的方法对Ad35腺病不同毒蛋白免疫原性进行了初步鉴别,首次发现Ad35腺病毒非结构蛋白E2A (DNA binding protein)能够诱导机体产生抗体。
综上所述,本文首次调查了我国人群中Ad35的感染情况、利用细菌内同源重组机制构建了Ad35载体系统并鉴别出一个以前未报道的体液免疫原,这些结果的取得为Ad35载体的开发、应用打下了基础。
Adenovirus (Ads) is a nonenveloped double-stranded, linear DNA virus with icosahedral symmetry. The human adenovirus constitutes a large family with at least 51 identified serotypes, which are classified into six subgroup on the base of hemagglutination.
Among the 51 serotypes, the conventional Ad vectors that are most widely used, including for human clinical trials, are constructed based on the subgroup C Ad serotype 5 (Ad5). Ad5 vector has several advantages as a gene delivery. For example, Ad5 can infect many different types of cells, the virus particle is more stable and easy to get high titer of adenovirus. But clinical and preclinical studies have revealed two major disadvantages of Ad5 vector. First, target cells that are important for gene therapy, including malignant tumor cells and dendritic cells, express nil or insufficient level of a cellular receptor for Ad5, the coxsackievirus and adenovirus receptor. Second, about 90% of adults are seropositive for Ad5 because natural infection with Ad5 is common. Pre-exsiting anti-Ad5 antibodies not only largely inhibit Ad5 vector-mediated transduction, but may also enhance the toxicities induced by Ad5 vector.
In contrast, human species B Ad serotype 35(Ad35) utilizes human CD46 as a cellular receptor. Human CD46 is ubiquitously express on almost all human cells, leading to a wide tropism of Ad35 vector. In addition, pre-exsiting anti-Ad5 immunity does not hamper Ad35 vector mediated transduction, and seroprevalence for Ad35 is much lower than that for Ad5. All these demonstrate that Ad35 vector is a promising vector candidate. Till now, there have been very few successful cases regarding the development of the Ad35 vector. Thus, it is essential to develop the Ad35 vector In the paper, we compare the evolution relationship of the human six subgroup adenovirus and concluded that the species B and species C are far from each other on the evolution relationship and at the same time we detected anti-Ad (including Ad4、Ad5、Ad 35)antibody in 1157 normal people serum samples. The results show that people infected by Ad4 and Ad5 are about 70% and 90% respectively, while less than 5% by Ad35. After the detection, we successfully made the Ad5 vector packaging system, including backbone plasmid, shuttle plasmid and cell line expressing Ad35 E1B constructively. With vitro recombination, we get replication-defective Ad35, the titer of which is 106TCID50/ml. In addition, we found that protein pXI is important for the package of replication-defective Ad35.
In order to optimize the packaging system we analysis the immunogen ability of the adenovirus protein with proteomic assay, and the results show that the non-structure protein E2A (DNA binding protein) has strong immunogen besides some structure protein such and hexon、penton and fiber.
引文
[1].分子病毒学 黄文林 主编 人民卫生出版社 2002年8月
[2].Rekosh DM, Russell WC, Bellet AJ, Robinson AJ Identification of a protein linked to the ends of adenovirus DNA.Cell.1977 Jun;11(2):283-95.
[3].Matthews DA, Russell WC.Adenovirus protein-protein interactions:molecular parameters governing the binding of protein VI to hexon and the activation of the adenovirus 23 K protease. J Gen Virol.1995 Aug;76 (Pt 8):1959-69.
[4].Rekosh DM, Russell WC.Processing of the precursor to the major core polypeptide of adenovirus type 5 removes a region near the amino terminus.Virology.1977 Oct 15;82(2):513-7
[5].Debbas M and White E Wide-type p53 mediate apoptosis by E1A, which is inhibited by E1B. Genes Dev,1993,7:546-554
[6]. VALENTINE RC, HOPPER PKPolyhedral shape of adenovirus particles as shown by electron microscopy. Nature.1957 Nov 2;180(4592):928
[7].Dyson N and Harlow E Adenovirus E1A targets key regulators of cell proliferation. Cancer Surg,1992,12:161-195
[8].Gooding LR, Ranheim TS The 10400-and 14500-dalton protein encoded by region E3of adenovirus function together to protect many but not all mouse cell lines against lysis by tutor necrosis factor. J Virol,1991,65:4114-4123
[9].Hehir KM, Amentano D Molecular characterization of replication -competent variants of adenovirus vectors and genome modification to prevent their occurrence J Virol,1996,70: 8459-8467
[10]. Rux JJ, Burnett RMType-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexonMol Ther. 2000 Jan; 1(1):18-30
[11].Lusky M, Christ M In vitro and in vivo biology of recombinant adenovirus vectors with E1,E1/E2A, or E1/E4deleted J Virol,1998,72:2022-2032
[12]. Burgert HG, Blusch JH Immunomodulatory functions encoded by the E3 transcription unit of adenoviruses Virus Genes.2000;21 (1-2):13-25
[13].Hu H, Serra D Persistence of an [E1-,polymerase-] adenovirus vector despite transduction of a neoantigen into immune-competent mice Hum Gene Ther 1999.10:355-364
[14].Morsy MA,Cu MC An adenovirus vector deleted for all viral coding sequence results an enhanced safety and extended expression of a leptin transgene PNAS 1998,95:7866-7871
[15]. Matthews DA, Russell WC.Adenovirus protein-protein interactions:hexon and protein VI.J Gen Virol.1994 Dec;75 (Pt 12):3365-74
[16]. MILOCHEVITCH R Adenovirus infections of the respiratory tract Gaz Med Fr.1957 Oct 25;64(20):1679
[17].ANDREWS BE, MCDONALD JC, PEREIRA HG, KELLY B Discussion on adenovirus infections Proc R Soc Med.1957 Oct;50(10):753-60
[18].Imler JL. Adenovirus vector as recombinant viral vaccine Vaccine 1995,13:1143-1151
[19].Reddy PS, Idamakanti N Development of porcine adenovirus 3 as an expression vector J Gene Virol 1990,80:563-570
[20].Benibond K, Yeh P Adenovirus vectors for gene delivery Current Opinion Biotechnol 1999,10:440-447
[21].Lieber A,He CY Recombinant adenovirus with large deletion generated by cre-mediated excision exhibit different biological property compared with first-generated vectors in vitro and in vivo J Virol 1996,70:8944-8960
[22].韦芳黄倩腺病毒载体研究进展与展望Tumor Sep.2005 Vol 25 No 5
[23].Replication-deficient human adenovirus type 35 vector for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity J Virol 2003 Vol 77, No 15
[24].Wickham TJ, Mathias P Integrinsavβ3 and αvβ5 promote adenovirus internalization but not virus attachment cell 199373:309-319
[25].Sebastian Tuve, Hongjie Wang A new group B adenovirus receptor is expressed at high levels on human stem and tumor cell J Virol Dec.2006 Vol 80 No 2412109-12120
[26]. Shayakhmetov DM Efficient gene transfer into human CD34(+) cell by a retargeted adenovirus vector J Virol 200074:2567-2583
[27].Nilsson,M, Stefan Karlsson S Function distinct subpopulation of cord blood CD34+cell are transduced by adenoviral vector with serotype 5 or 35 tropism Mol Ther 20049(3):377-388
[28].Rea D, Havenga MJ Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber modified adenovirus vector enhances transgene-encode antigen presentation to cytotoxic T cells J Immunol 2001 166:5236-5244
[29].Havenga MJ Lemckert AA Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease J Virol 2002 76:4612-4620
[30].Jung D, Neron S Efficient gene transfer into normal human B lymphocytes with the chimeric adenovirus vector Ad5F35 J Immunol Meth 2005 304:78-87
[31].Bollard CM, Straathof KCM The generation and characterization of LMP2-Specific CTLs for use as adaptive transfer from patients with relapsed EBV-positive Hodgkin disease J Immunol Ther 200427:317-327
[32]. England JJ, McChesney AE, Chow TL Characterization of an equine adenovirus.Am J Vet Res.1973 Dec;34(12):1587-90.
[33].Gugalal Z Ol msted-David EA Osteoinduction by ex vivo adenovirus-mediated BMP2 delivery is independent of cell type Gene Ther 2003 10(16):1289-1296
[34].Anna Segerman Kristina Lindman Adenovirus type 11p and 35 attach to and infect primary lymphocytes and monocytes,but hexon expression in T-cell requires prior activation Virology 349(2006):96-111
[35]Sakurai,F etal(2006). The short consensus repeats 1 and 2, not the cytoplasmic domain, of human CD46 are crucial for infection of subgroup B adenovirus serotype 35. J control Release 113:271-278
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[2].Rekosh DM, Russell WC, Bellet AJ, Robinson AJ Identification of a protein linked to the ends of adenovirus DNA.Cell.1977 Jun;11(2):283-95.
[3].Matthews DA, Russell WC.Adenovirus protein-protein interactions:molecular parameters governing the binding of protein VI to hexon and the activation of the adenovirus 23 K protease. J Gen Virol.1995 Aug;76 (Pt 8):1959-69.
[4].Rekosh DM, Russell WC.Processing of the precursor to the major core polypeptide of adenovirus type 5 removes a region near the amino terminus.Virology.1977 Oct 15;82(2):513-7
[5].Debbas M and White E Wide-type p53 mediate apoptosis by E1A, which is inhibited by E1B. Genes Dev,1993,7:546-554
[6]. VALENTINE RC, HOPPER PKPolyhedral shape of adenovirus particles as shown by electron microscopy. Nature.1957 Nov 2;180(4592):928
[7].Dyson N and Harlow E Adenovirus E1A targets key regulators of cell proliferation. Cancer Surg,1992,12:161-195
[8].Gooding LR, Ranheim TS The 10400-and 14500-dalton protein encoded by region E3of adenovirus function together to protect many but not all mouse cell lines against lysis by tutor necrosis factor. J Virol,1991,65:4114-4123
[9].Hehir KM, Amentano D Molecular characterization of replication -competent variants of adenovirus vectors and genome modification to prevent their occurrence J Virol,1996,70: 8459-8467
[10]. Rux JJ, Burnett RMType-specific epitope locations revealed by X-ray crystallographic study of adenovirus type 5 hexonMol Ther. 2000 Jan; 1(1):18-30
[11].Lusky M, Christ M In vitro and in vivo biology of recombinant adenovirus vectors with E1,E1/E2A, or E1/E4deleted J Virol,1998,72:2022-2032
[12]. Burgert HG, Blusch JH Immunomodulatory functions encoded by the E3 transcription unit of adenoviruses Virus Genes.2000;21 (1-2):13-25
[13].Hu H, Serra D Persistence of an [E1-,polymerase-] adenovirus vector despite transduction of a neoantigen into immune-competent mice Hum Gene Ther 1999.10:355-364
[14].Morsy MA,Cu MC An adenovirus vector deleted for all viral coding sequence results an enhanced safety and extended expression of a leptin transgene PNAS 1998,95:7866-7871
[15]. Matthews DA, Russell WC.Adenovirus protein-protein interactions:hexon and protein VI.J Gen Virol.1994 Dec;75 (Pt 12):3365-74
[16]. MILOCHEVITCH R Adenovirus infections of the respiratory tract Gaz Med Fr.1957 Oct 25;64(20):1679
[17].ANDREWS BE, MCDONALD JC, PEREIRA HG, KELLY B Discussion on adenovirus infections Proc R Soc Med.1957 Oct;50(10):753-60
[18].Imler JL. Adenovirus vector as recombinant viral vaccine Vaccine 1995,13:1143-1151
[19].Reddy PS, Idamakanti N Development of porcine adenovirus 3 as an expression vector J Gene Virol 1990,80:563-570
[20].Benibond K, Yeh P Adenovirus vectors for gene delivery Current Opinion Biotechnol 1999,10:440-447
[21].Lieber A,He CY Recombinant adenovirus with large deletion generated by cre-mediated excision exhibit different biological property compared with first-generated vectors in vitro and in vivo J Virol 1996,70:8944-8960
[22].韦芳黄倩腺病毒载体研究进展与展望Tumor Sep.2005 Vol 25 No 5
[23].Replication-deficient human adenovirus type 35 vector for gene transfer and vaccination: efficient human cell infection and bypass of preexisting adenovirus immunity J Virol 2003 Vol 77, No 15
[24].Wickham TJ, Mathias P Integrinsavβ3 and αvβ5 promote adenovirus internalization but not virus attachment cell 199373:309-319
[25].Sebastian Tuve, Hongjie Wang A new group B adenovirus receptor is expressed at high levels on human stem and tumor cell J Virol Dec.2006 Vol 80 No 2412109-12120
[26]. Shayakhmetov DM Efficient gene transfer into human CD34(+) cell by a retargeted adenovirus vector J Virol 200074:2567-2583
[27].Nilsson,M, Stefan Karlsson S Function distinct subpopulation of cord blood CD34+cell are transduced by adenoviral vector with serotype 5 or 35 tropism Mol Ther 20049(3):377-388
[28].Rea D, Havenga MJ Highly efficient transduction of human monocyte-derived dendritic cells with subgroup B fiber modified adenovirus vector enhances transgene-encode antigen presentation to cytotoxic T cells J Immunol 2001 166:5236-5244
[29].Havenga MJ Lemckert AA Exploiting the natural diversity in adenovirus tropism for therapy and prevention of disease J Virol 2002 76:4612-4620
[30].Jung D, Neron S Efficient gene transfer into normal human B lymphocytes with the chimeric adenovirus vector Ad5F35 J Immunol Meth 2005 304:78-87
[31].Bollard CM, Straathof KCM The generation and characterization of LMP2-Specific CTLs for use as adaptive transfer from patients with relapsed EBV-positive Hodgkin disease J Immunol Ther 200427:317-327
[32]. England JJ, McChesney AE, Chow TL Characterization of an equine adenovirus.Am J Vet Res.1973 Dec;34(12):1587-90.
[33].Gugalal Z Ol msted-David EA Osteoinduction by ex vivo adenovirus-mediated BMP2 delivery is independent of cell type Gene Ther 2003 10(16):1289-1296
[34].Anna Segerman Kristina Lindman Adenovirus type 11p and 35 attach to and infect primary lymphocytes and monocytes,but hexon expression in T-cell requires prior activation Virology 349(2006):96-111
[35]Sakurai,F etal(2006). The short consensus repeats 1 and 2, not the cytoplasmic domain, of human CD46 are crucial for infection of subgroup B adenovirus serotype 35. J control Release 113:271-278
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