关于腺病毒载体的免疫原性问题

关于腺病毒载体的免疫原性问题姜志龙陈诗书（上海第二医科大学人类基因治疗研究中心,上海２０００２５）关键词腺病毒载体免疫原性基因治疗近年来,以腺病毒为基因转移载体的基因治疗研究正广泛深入地开展,这是因为腺病毒具有逆转录病毒载体不具备的诸多优点。首先腺病...     

腺病毒载体研究进展

腺病毒载体目前广泛地应用于基因治疗,随着对腺病毒分子结构与功能及感染机制的深入了解,人们在降低其免疫原性,增强基因导入特异性,增强感染和表达效率等方面进行了许多探索,本文就这方面的研究进展进行综述。     

辅助病毒依赖型腺病毒载体研究进展

摘要：辅助病毒依赖型腺病毒载体（helper-dependent adenoviral vector,HDAd）缺乏所有腺病毒的编码序列,与非复制型的第一代腺病毒载体（first-generation adenovirus vector,FGAd）相比,具有载体免疫原性低、安全、转移容量大和持续表达等特点,现广泛用于遗传性疾病、神经退行性疾病和肿瘤等的基因治疗和特异性靶向治疗研究。本文综述了HDAd构建和应用等方面的研究进展及未来的发展方向。     

人类腺病毒受体的研究进展

腺病毒受体的研究近十几年来得到迅速发展,人们对腺病毒受体的研究兴趣主要源于使用腺病毒载体所进行的基因治疗.随着多个腺病毒受体相继被发现和鉴定(表1),对腺病毒感染细胞的过程有了进一步的认识和理解.例如,腺病毒5型(Ad5)已被用作基因治疗的载体,但不能有效地感染受体低表达的细胞[1].人们通过修饰腺病毒载体中与受体结合的相应部位,提高基因传递的效率,使腺病毒载体在基因治疗中的作用不断完善.本文就已经鉴定的腺病毒受体和相应配体在腺病毒进入细胞时所发挥的作用加以综述.     

腺病毒的标记方法

腺病毒载体具有滴度高、容量大及可以同时感染分裂与未分裂细胞等一系列优点,因此在基因治疗领域得到广泛应用。腺病毒颗粒的标记对于腺病毒生物学功能的基础研究及评估腺病毒载体的基因治疗效果具有重要意义。本文通过综述腺病毒标记的方法,回顾腺病毒标记技术的发展过程,同时期望为找到更好的标记腺病毒的方法以及其在基因治疗研究领域的进一步应用提供一些思路。     

腺病毒载体及其基因治疗制品的质量控制

本文介绍了腺病毒与其载体作用相关的基本知识,腺病毒载体的构建方法,从正反两个方面对腺病毒进行评价,综合了腺病毒载体在基因治疗的应用,并对以重组腺病毒为载体的基因治疗制品应考虑的质量问题概括     

人腺病毒工程化应用研究进展

人腺病毒流行广泛,感染常诱发呼吸道疾病、流行性结膜炎等疾病。腺病毒是研究较为深入的一类病原体,并且作为病毒载体已广泛应用,也是基因治疗最常用的病毒载体,尤其在肿瘤基因治疗和病毒溶瘤方面具有较大的研究价值和临床应用潜力。文中对腺病毒的生物学特性以及应用范围等方面进行综述,以期为腺病毒的工程化与临床应用提供参考。     

腺病毒载体衣壳蛋白质的遗传修饰

腺病毒载体是最早用于基因治疗研究的病毒载体之一,也是目前肿瘤基因治疗中最为常见的病毒载体之一,其主要通过靶细胞表面的天然柯萨奇腺病毒受体(coxsackie and adenovirus receptor,CAR)感染宿主细胞。由于大多数肿瘤细胞表面该受体表达水平较低,降低了腺病毒载体对靶细胞的感染效率,从而制约了腺病毒载体在肿瘤基因治疗中的应用。因此,如何提高腺病毒载体对靶细胞的感染效率是腺病毒载体应用于肿瘤基因治疗的关键。目前对腺病毒载体衣壳蛋白质(capsid protein)的遗传修饰是提高其对宿主细胞感染效率的主要途径。本文将对这一领域的主要研究进展作一综述,为该方面的研究提供有用的信息。     

E1/E3缺失型腺病毒载体引起细胞周期G_2/M阻滞

腺病毒载体广泛应用于基因治疗和转基因研究 ,目前常用的E1 E3缺失型复制缺陷腺病毒载体虽然失去了病毒复制必需的E1基因 ,但载体上的其它病毒基因仍能在宿主细胞内表达 .为研究这些基因对细胞的毒性作用 ,选择了 3种携带没有明显细胞毒性外源基因的腺病毒载体 ,观察感染 2种肿瘤细胞后细胞核形态改变 ,并用流式细胞仪检测细胞周期及凋亡情况 .发现大剂量重组缺陷型腺病毒感染细胞后引起细胞变圆 ,核增大 ,细胞周期阻滞于G2 M期 ,继而染色质凝聚 ,细胞发生坏死或凋亡 ;各种腺病毒载体造成G2 M阻滞所需感染量不同 ,但都随时间延长和感染量增加而加重 .这些结果提示腺病毒基因对细胞的影响是多方面的 ,在以此类病毒载体进行基因转移和基因治疗的研究中 ,精确滴定病毒滴度和转导效率非常重要 ,腺病毒基因表达造成的毒副作用给此类研究增加了变数     

腺病毒载体及其在基因治疗研究中的应用

腺病毒载体在基因治疗研究中初露锋芒,引起关注,文章就腺病毒基因组的基本结构、腺病毒载体的类型及构建、重组腺病毒在基因治疗研究中的应用及其主要优、缺点等方面进行了较为全面的综述．     

HIV-1 gagpol重组腺病毒疫苗的构建及其免疫原性的研究

目的：构建HIV-1重组腺病毒疫苗并初步鉴定其免疫原性。方法：用Adeno-XExpressionSystem试剂盒将HIV—lgagpol基因片段插入到腺病毒载体上,通过脂质体介导将获得的重组腺病毒质粒Adeno—X-gagpol转染至293细胞,使之自主包装成有感染活性的重组腺病毒tad—gagpol,用western-blotting法鉴定其表达情况;用CsCl密度梯度离心法纯化该重组腺病毒,以5×10^8 pfu／mL的滴度lmL单次免疫小鼠,15天后测小鼠体液免疫效果。结果：克隆序列与设计相符,重组腺病毒疫苗能稳定表达gag—pol蛋白,体液免疫中检测出抗p55和p24的特异性抗体。结论：HIV—lgagpol重组腺病毒构建成功,具有一定的体液免疫原性。     

Long-lasting adenovirus transgene expression in mice through neonatal intrathymic tolerance induction without the use of immunosuppression.

The major barrier to the clinical application of adenovirus gene therapy for diseases that require stable transgene expression is the immunogenicity of recombinant adenovirus, which ordinarily limits the duration of its effects to a period of about 2 weeks. We postulated that tolerance to adenovirus could be induced and transgene expression could be prolonged if T lymphocytes underwent thymic selection in the presence of adenovirus antigens. Mice were inoculated in the thymus with a recombinant adenovirus containing the lacZ marker gene during the neonatal period at a time before T-cell maturation had occurred. When the virus was administered intravenously to these mice in adulthood, they were found to have an impaired adenovirus-specific cytotoxic T-lymphocyte response which allowed prolonged hepatic lacZ expression, for up to 260 days. The ability to achieve unresponsiveness to a recombinant adenovirus after inoculation of the thymus in neonates extends the paradigm of intrathymic tolerance induction. Furthermore, this model will enable the study of stable adenovirus transgene expression in vivo without the use of immunosuppression and ultimately may have clinical utility.     

Expanded-capacity adenoviral vectors--the helper-dependent vectors

Significant advances have recently been made in the development of vectors and gene-delivery systems for gene therapy. Experiments performed over the past decade have revealed how vectors will have to be modified to make them a clinically viable treatment option. In the case of adenovirus (Ad) vectors, which have been particularly useful as gene delivery vehicles, the main drawback associated with their use is vector-mediated immunogenicity. Recent modifications of the Ad backbone have led to the development of helper-dependent (HD) Ad vectors, which are completely devoid of all viral protein-coding sequences. These modifications have significantly reduced the immunogenicity of Ad vectors and have enhanced their safety. It is expected that HD vectors will become important tools for future clinical gene therapy.     

串联表达PRRSV M与GP5蛋白重组腺病毒的构建及其免疫特性研究

利用PCR扩增出猪繁殖与呼吸综合征病毒的M基因,按正确的读码框与GP5基因串联,成功构建穿梭载体pShuttle-CMV-M-GP5,经PCR、测序鉴定正确。PmeⅠ线性化后在BJ5183大肠杆菌内与腺病毒骨架载体pAdEasy-1同源重组,产生重组腺病毒DNA。重组腺病毒DNA经PacⅠ线性化后用脂质体转染HEK-293A细胞,在细胞内包装成完整的腺病毒,通过IFA可以检测到M与GP5串联的重组腺病毒构建成功,可以正确地表达目的蛋白。将构建好的重组腺病毒免疫小鼠,结果表明可以诱导产生较强的体液免疫应答(ELISA抗体和中和抗体)和细胞免疫应答(淋巴细胞增殖和CTL反应)。证明该重组腺病毒具有较好的免疫原性,为下一步猪体免疫试验奠定了基础。     

"Stealth" adenoviruses blunt cell-mediated and humoral immune responses against the virus and allow for significant gene expression upon readministration in the lung

Most of the early gene therapy trials for cystic fibrosis have been with adenovirus vectors. First-generation viruses with E1a and E1b deleted are limited by transient expression of the transgene and substantial inflammatory responses. Gene transfer is also significantly curtailed following a second dose of virus. In an effort to reduce adenovirus-associated inflammation, capsids of first-generation vectors were modified with various activated monomethoxypolyethylene glycols. Cytotoxic T-lymphocyte production was significantly reduced in C57BL/6 mice after a single intratracheal administration of modified vectors, and length of gene expression was extended from 4 to 42 days. T-cell subsets from mice exposed to the conjugated vectors demonstrated a marked decrease in Th1 responses and slight enhancement of Th2 responses compared to animals dosed with native virus. Neutralizing antibodies (NAB) against adenovirus capsid proteins were reduced in serum and bronchoalveolar lavage fluid of animals after a single dose of modified virus, allowing significant levels of gene expression upon rechallenge with native adenovirus. Modification with polyethylene glycol (PEG) also allowed substantial gene expression from the new vectors in animals previously immunized with unmodified virus. However, gene expression was significantly reduced after two doses of the same PEG-conjugated vector. Alternating the activation group of PEG between doses did produce significant gene expression upon readministration. This technology in combination with second-generation or helper-dependent adenovirus could produce dosing strategies which promote successful readministration of vector in clinical trials and marked expression in patients with significant anti-adenovirus NAB levels and reduce the possibility of immune reactions against viral vectors for gene therapy.     

Vaccine-induced immunity in baboons by using DNA and replication-incompetent adenovirus type 5 vectors expressing a human immunodeficiency virus type 1 gag gene

The cellular immunogenicity of formulated plasmid DNA and replication-defective human adenovirus serotype 5 (Ad5) vaccine vectors expressing a codon-optimized human immunodeficiency virus type 1 gag gene was examined in baboons. The Ad5 vaccine was capable of inducing consistently strong, long-lived CD8(+)-biased T-cell responses and in vitro cytotoxic activities. The DNA vaccine-elicited immune responses were weaker than those elicited by the Ad5 vaccine and highly variable; formulation with chemical adjuvants led to moderate increases in the levels of Gag-specific T cells. Increasing the DNA-primed responses with booster doses of either Ad5 or modified vaccinia virus Ankara vaccines suggests a difference in the relative levels of cytotoxic and helper responses. The implications of these results are discussed.     

Gene Delivery by Subconjunctival Injection of Adenovirus in Rats: A Study of Local Distribution,Transgene Duration and Safety

Subconjunctival injection is a minimally invasive route for gene delivery to ocular tissues, but has traditionally been limited to use in the cornea. The accurate ocular distribution of virus has not, however, been previously investigated. Adenovirus is an attractive gene vector as it can deliver large genes and allow for short-term gene expression, but how safe it is when delivered via subconjunctival injection remains to be established. We have characterized the bio-distribution and safety of subconjunctivally administered adenovirus in Brown Norway rats. The bio-distribution and transgene duration of adenovirus carrying luciferase gene (Ad-Luci) at various time intervals were evaluated via bioluminescence imaging after subconjunctival injection. Adenovirus carrying a reporter gene, β-galactosidase (Ad-LacZ) or hrGFP (Ad-hrGFP) was administered subconjunctivally and the viral distribution in various ocular tissues was assessed by histological analysis and quantitative PCR (qPCR). Hepatic damage was assessed by biochemical and immunohistological analysis with TUNEL stain. Systemic immunogenicity was assessed by measuring serum level of TNF-α via ELISA, 2 hours and 14 days after administration of adenovirus. Retinal function was examined by electroretinography. Subconjunctival injection of Ad-Luci induced luciferase expression in the injected eyes within 24 hours, for at least 64 days. Histological analysis showed adenovirus distributed across anterior and posterior ocular tissues. qPCR demonstrated different amounts of adenovirus in different ocular tissues, with the highest amounts closest to the injection site Unlike the intravenous route, subconjunctivally delivered adenovirus did not elicit any detectable hepatic injury or systemic immunogenicity. Retinal function was unaffected by adenovirus irrespective of administration route. In conclusion, an adenoviral vector administered subconjunctivally can infiltrate into different ocular tissues and lead to short-term ocular transgene expression, without causing hepatic injury and immune activation. Therefore, subconjunctivally administered adenovirus may be a promising gene delivery approach for managing anterior and posterior segment eye diseases requiring short-term therapy.     

表达猪瘟病毒E2蛋白的重组腺病毒的构建及其在兔体内的免疫原性分析

为了构建猪瘟重组腺病毒载体疫苗,通过细菌内同源重组法构建了含有猪瘟病毒E2基因的重组腺病毒rAdV-E2.测定其一步生长曲线,同时用间接免疫荧光试验和Western blotting检测外源基因表达,然后用rAdV-E2免疫家兔,免疫后6周用猪瘟兔化弱毒疫苗株(c株)进行攻击,攻毒后3 d取其脾脏,用实时荧光定量RT-PCR检测C株病毒RNA.结果表明,该重组腺病毒传至第10代时,毒价可达1.0×1010TCID<,50/mL;外源基因可在其中得到稳定表达;rAdV-E2接种兔免疫后2周产生猪瘟特异性抗体,免疫后5 W抗体达到峰值,攻毒后rAdV-E2接种兔和C株接种兔均未出现定型热反应,从其脾脏也未检测到C株病毒RNA,而野生型腺病毒接种兔均出现了定型热反应,并且从其脾脏检测大量C株病毒RNA,其含量达到了103拷贝/μL以上.由此表明,rAdV-E2可望开发为猪瘟候选疫苗.     

The Effect of Fiber Truncations on the Stability of Adenovirus Type 5

While fiberless adenovirus has the potential for use as a vaccine or gene delivery vector, some groups have observed instability issues associated with the modified virus. To investigate the effect of fiber modification on adenovirus stability, we produced mutant adenovirus particles that contained the tail and a portion of the shaft domain without the knob. The shaft domain was either completely removed (i.e., fiberless) or truncated to 7-, 14-, or 21-repeats. The mutants were evaluated by biophysical characterization techniques to determine their relative stabilities based on temperature-induced changes to the secondary, tertiary, and quaternary structures of the virus and its constituent proteins. Data acquired using circular dichroism, intrinsic/extrinsic fluorescence, and static/dynamic light scattering were compiled into a comprehensive empirical phase diagram, which showed that native adenovirus was the most stable followed by fiberless adenovirus and then the mutants with truncated fiber protein. In summary, the individual biophysical measurements and the empirical phase diagram showed that providing several repeats of shaft protein negatively impacted the structural stability of the virus more so than completely removing the fiber protein.     

Genetically targeted adenovirus vector directed to CD40-expressing cells

The success of gene therapy depends on the specificity of transgene delivery by therapeutic vectors. The present study describes the use of an adenovirus (Ad) fiber replacement strategy for genetic targeting of the virus to human CD40, which is expressed by a variety of diseased tissues. The tropism of the virus was modified by the incorporation into its capsid of a protein chimera comprising structural domains of three different proteins: the Ad serotype 5 fiber, phage T4 fibritin, and the human CD40 ligand (CD40L). The tumor necrosis factor-like domain of CD40L retains its functional tertiary structure upon incorporation into this chimera and allows the virus to use CD40 as a surrogate receptor for cell entry. The ability of the modified Ad vector to infect CD40-positive dendritic cells and tumor cells with a high efficiency makes this virus a prototype of choice for the derivation of therapeutic vectors for the genetic immunization and targeted destruction of tumors.