乙型肝炎病毒重组腺病毒载体疫苗的构建及发展前景

人类腺病毒基因组Ｅ１区,Ｅ３区及介于Ｅ４区和右侧端插入性末端重复之间的区段存在插入位点,可供插入乙型肝炎病毒保护生免疫原基因,构建重组载体疫苗,本文就Ａｄ基因组分区段阐述了各类ＨＢＶ重组Ａｄ表达戴本构建的一般原则,并指出了辅助细胞依赖性缺陷型Ａｄ载体用作非复制性Ａｄ载体活疫苗的潜力及非缺陷型Ａｄ双表达载体用作复制性Ａｄ载体活疫苗的前景。     

腺病毒重组疫苗载体构建的研究进展

腺病毒（Ad)基因组E1,E3,以及E4区和右侧端插入性末端重复（IRT）之间的区段,均存在外源基因插入位点,本论述了外源保护性免疫原基因插入Ad基因组上述不同位点构建各类重组疫苗载体的研究进展。     

乙型肝炎病毒重组腺病毒载体疫苗的免疫研究进展

以人类腺病毒（Ａｄ）为表达载体发展多价展组口服活疫苗,尤其在乙型肝炎病毒（ＨＢＶ）疫苗研制上成效显著,表达ＨＢｃＡｇ,ＨＢｅＡｇ和ＨＢｓＡｇ的重组人类Ａｄ７型（ｄ７）活载体疫苗在实验动物免疫上取得良好效果。在重组Ａｄ活载体疫苗研制方面是一项重大进展,表达ＨＢｓＡｇ的重组Ａｄ７活载体口服疫苗已有人体初次免疫试验的报道。     

腺病毒载体在基因治疗和疫苗研究中的应用

自从二十世纪五十年代开始认识腺病毒（Adenovirus）以来,对腺病毒的生物学和免疫学特征已经有比较多的认识。腺病毒科包括禽腺病毒属（Aviadenovius）和哺乳动物腺病毒（Mastadenov irus）两个属。除人腺病毒以外,一些哺乳动物的腺病毒和禽腺病毒也成为基因治疗和载体疫苗研究的热点,如猪腺病毒3型（PAV-3）、牛腺病毒3型（BAV-3）、绵羊腺病毒（Ovine adenovirus,OAV）、禽腺病毒（Aviadenovirus）、犬腺病毒（Canine adeno virus,CAV）和黑猩猩腺病毒（Chimpanzee adenovirus）。不同种属的腺病毒虽然基因组序列完全不同,但其结构和功能却非常相似,而且各型腺病毒之间很少有交叉免疫反应,这就为利用这些腺病毒研制基因治疗载体和活载体疫苗提供了丰富的材料。近年来的研究也发现,如果利用一种腺病毒载体来进行重复免疫,宿主针对腺病毒载体蛋白的免疫反应比较强大,造成转基因表达时间缩短,重复免疫的效果较差,换用不同的病毒载体,则可以回避这种免疫反应,提高转基因的表达时间和保护性免疫反应。腺病毒可以感染很多分裂期或静止期细胞,即使在一些高度分化的组织细胞中也可增殖,如可有效的感染肌肉组织、心、肺和脑组织,并能高效的复制、表达其基因产物,因此腺病毒成为基因治疗和活病毒载体疫苗研究的首选工具。     

腺病毒载体在疫苗研究中的应用

以病毒为载体的活疫苗为疾病预防和治疗研究提供了新手段。目前用于疫苗研究的病毒载体主要包括痘苗病毒载体、腺病毒载体、腺相关病毒载体、单纯疱疹病毒载体及逆转录病毒载体等。其中,重组腺病毒载体因其基因组大小适中,易于基因重组操作,繁殖滴度高,易于大量制备和保存,宿主范围广,转导效率高,安全性好,能刺激机体产生强烈的体液和细胞免疫反应等特点,而被广泛应用于重要感染性疾病及恶性肿瘤的疫苗研究。腺病毒载体在人免疫缺陷病毒(HIV)疫苗研究和临床试验中的成败更是备受关注。然而,与其他载体疫苗一样,机体对载体的免疫反应仍是阻碍腺病毒载体疫苗在临床中广泛应用的主要问题。那么,腺病毒载体解决这类问题的优势何在?我们简要综述腺病毒载体的特点及其在疫苗研究中的应用和存在的问题,为进一步优化和利用腺病毒载体在疫苗方面的研究提供参考。     

人类腺病毒载体活疫苗的开发应用研究进展

人类腺病毒载体活疫苗的开发使用近年取得重大进展。文章指明了此类疫苗的研究动向,提出了进一步发展对策,论述了应用前景,从而阐明了腺病毒是重组疫苗的重要载体。     

腺病毒载体在基因治疗和疫苗研究中的应用

自从二十世纪五十年代开始认识腺病毒(Adenovirus)以来,对腺病毒的生物学和免疫学特征已经有比较多的认识.腺病毒科包括禽腺病毒属(Aviadenovius)和哺乳动物腺病毒(Mastadenov irus)两个属.     

重组腺病毒载体疫苗黏膜免疫机制与途径研究

腺病毒作为载体具有许多优点,因此被广泛地应用于体外基因转导、体内接种疫苗和基因治疗等领域。近年来,国内外学者已经构建了表达不同抗原的腺病毒载体,如人(猿)免疫缺陷病毒Gag,pol,Nef,Env,狂犬病毒糖蛋白,登革热病毒包膜蛋白,乙型肝炎病毒表面抗原,丙型肝炎病毒E1、E2、core、NS3,麻疹病毒核衣壳、凝血素,呼吸道合胞病毒糖蛋白,2型单纯疱疹病毒糖蛋白B、炭疽杆菌保护性抗原等。其中有不少载体疫苗通过黏膜免疫接种,诱导机体产生具有保护作用的免疫反应。本文就重组腺病毒载体疫苗黏膜免疫机制和黏膜免疫途径的研究作一回顾。     

含乙型肝炎病毒S-PreS1 基因的DNA疫苗与蛋白颗粒疫苗联合免疫可增强免疫应答

为研制新型有效的HBV治疗性疫苗,构建了含PreS1与S融合基因的HBV DNA疫苗,即pVRC-HBSS1 (PreS1 21–47 aa融合在S抗原1–223的羧基端),并制备了CHO表达相同结构的蛋白颗粒亚单位疫苗HBSS1。在Balb/C小鼠中采用不同的DNA免疫方式 (即肌肉注射、皮内注射加电转) 初免3次,蛋白颗粒亚单位疫苗 (不同佐剂) 肌肉注射加强免疫1次,然后我们分析比较了各组疫苗所引起的免疫应答特点。抗体检测结果表明：皮内注射结合电转初免组产生的PreS1与 S特异性抗体水平皆高于肌肉直接注射组。进一步还发现DNA疫苗与蛋白颗粒亚单位疫苗两种疫苗联合应用后S抗原特异的细胞免疫应答 (IFN-γ ELISpot分析) 明显高于DNA疫苗或蛋白颗粒亚单位单独应用,其中皮内注射+电转结合蛋白颗粒亚单位疫苗联合免疫组可产生最强的细胞免疫应答。这些研究为新型HBV 治疗性疫苗的优化设计与合理应用提供了依据。     

Molecular analysis of the modulatory factors of the response to HBsAg in mice as an approach to HBV vaccine enhancement

Abstract Mice were injected with immune complexes containing the recombinant hepatitis B surface antigen (HBsAg) vaccine (S + preS2) bound to different monoclonal antibodies (mAbs), in order to determine whether an enhancement of the response to a human vaccine could be obtained and observed. Enhancement and indifference were observed, as well as a decrease in immunogenicity. N relationship could be established between any effect and affinity or isotypy of the bound mAbs. The preS2 region was rendered more immunogenic when an IgG2a mAb was bound to the S region of the HBsAg. The response to the S region was not modulated, whereas immunogenicity of the preS2 collinear region was decreased by antibody shielding. The mAb which was the most efficient as an enhancer of the antibody response also increased binding of the complexed immunogen to antigen presenting cells. The binding of a human mAb to the sole S region, but not to the preS2 region, should be tested as a potentiating agent of the anti-preS2 human immune response.     

Adenoviral-vector mediated transfer of HBV-targeted ribonuclease can inhibit HBV replication in vivo

Hepatitis B virus (HBV)-targeted ribonuclease (HBV-TR) is a fused protein of HBV core protein and a ribonuclease, human eosinophil-derived neurotoxin (hEDN). Our previous results showed that HBV-TR could effectively inhibit HBV replication in vitro. To test whether HBV-TR can inhibit HBV replication in vivo, we constructed a recombinant adenoviral vector expressing HBV-TR (Ad-TR) and used it to treat HBV-transgenic mice. Immunohistochemical staining showed that TR was expressed at varied levels in different tissues of Ad-TR-treated mice. Serum HBsAg concentration was decreased by 64.8% for the Ad-TR-treated mice compared with empty adenoviral vector-treated control mice. The amount of HBV-DNA in the livers of the Ad-TR-treated mice was 0.74 × 10⁷ copies/μg of genomic DNA while the amount of HBV-DNA in the livers of the empty adenoviral vector-treated control mice was 2.86 × 10⁷ copies/μg of genomic DNA. Serum HBV-DNA of Ad-TR-treated mice was also decreased by 71.4% compared with empty adenoviral vector-treated control mice. In addition, for some Ad-TR-treated mice, the expression of HBsAg in the liver cells turned negative. No discernible adverse effects were observed for Ad-TR-treated mice. Taken together, our results indicated that adenovirus mediated transfer of HBV-TR can inhibit HBV replication in vivo.     

乙型肝炎病毒全S基因疫苗诱导小鼠的特异性免疫应答

乙型肝炎病毒 (ＨＢＶ)感染是我国常见病及多发病。ＨＢＶ难以清除的原因之一就是机体的免疫功能障碍。目前虽然基因重组ＨＢＶ表面抗原 (ＨＢｓＡｇ)疫苗预防ＨＢＶ感染取得了较好的效果 ,但基因重组ＨＢｓＡｇ疫苗主要能诱导特异性体液免疫 ,不能刺激机体的细胞免疫应答。近年来发现基因疫苗可诱导机体产生细胞及体液免疫反应 ,特别是诱导细胞免疫反应的能力优于蛋白、多肽类疫苗 ,更适应于慢性病毒感染的预防与治疗[1,2 ] 。为了探讨应用ＨＢＶ基因疫苗预防ＨＢＶ感染的可能性 ,本文构建了ＨＢＶ全Ｓ基因和ＨＢｓＡｇ基因疫苗 ,观察和比…     

少年人群接种乙肝疫苗后七年随访观察

对７９名ＨＢＶ标志阴性少年人群,以乙肝疫苗１０μｇ×３的免疫剂量和０、１、２月的免疫程序进行接种,对其抗－ＨＢｓ免疫应答和临床保护效果作了为期７年的定人随访。结果表明,抗－ＨＢｓ阳转率在免后三个月时为１００％,均值为３０８４ＭＩＵ／ｍｌ。至免后８４个月时疫苗接受者中仍有５５．７％的抗－ＨＢｓ水平≥１０ＭＩＵ／ｍｌ。６例检出抗－ＨＢｓ,其中５例的抗－ＨＢｓ持续处于高水平。全部观察对象无一例检出ＨＢｓＡｇ或发生临床肝炎。少年接种乙肝血源疫苗至少７年内可具有保护性抗体。故在此期内不需加强免疫。     

Production of adenovirus vector for gene therapy

The field of gene therapy is rapidly expanding with a major focus on the treatment of cancer. Replication-defective adenoviruses are vectors of choice for delivering corrective genes into human cells. Major efforts are directed to design new generations of adenoviral vectors that feature reduced immunogenicity and improved targeting ability. However, the production of adenoviral vectors for gene therapy applications faces a number of challenges that limit the availability of high quality material at the early stages of research and development in the gene therapy field. Moreover, very few papers have been published on the subject and information on large-scale production methods are only available through specialized conference proceedings. This review outlines the problems associated with mass production of adenovirus vectors and describes research efforts by a number of groups who have contributed to optimize production methods. Better understanding of the adenovirus infection and replication kinetics as well as better understanding of complementing cell line physiology and metabolism greatly contributed to improving vector titers and volumetric productivity at higher cell densities. Also, the critical aspect of viral vector quantitation is discussed.     

The improvement of adenovirus vector production by increased expression of coxsackie adenovirus receptor

Adenoviruses are promising vectors for gene therapy. The production of adenoviral vectors (AdV), however, is limited by the cell density effect, namely when cell infection is performed at above 10⁶ cells/ml, a drop in cell-specific adenovirus productivity occurs. Our results also show that the coxsackie adenovirus receptor (CAR) plays an important role in AdV production. CAR expression of infected cells varied with culture time and the cell-specific AdV productivity dropped rapidly along with decreased CAR expression. Furthermore, CAR expression of cells was maintained at a high level by replacing the medium or supplementing it with trichostatin A, which could improve the cell-specific productivity. Thus, a higher CAR expression level at infection time could improve cell-specific AdV productivity at high cell densities.     

热休克蛋白HSP70和gp96增强乙肝DNA疫苗的细胞和体液免疫应答

旨在以乙肝病毒 (HBV) 的主要结构蛋白-表面蛋白 (HBsAg) 和核心蛋白 (HBcAg) 作为抗原设计DNA疫苗,研究热休克蛋白HSP70和gp96作为新型免疫佐剂增强疫苗的细胞免疫和体液免疫水平。利用酶联免疫斑点实验、流式细胞内因子染色、3H-TdR实验、酶联免疫吸附实验技术分析,结果显示HSP70和gp96可使疫苗的细胞免疫水平提高1~6倍,提高体液免疫水平20％~60％。研究结果为设计以HSP70和gp96作为免疫佐剂的新型乙肝治疗性疫苗提供了依据。     

双表达小干扰RNA抑制小鼠体内HBV感染

目的运用水压法复制HBV小鼠急性模型,用于研究双表达siRNA在小鼠体内对HBV复制的抑制作用。方法使用HBV质粒DNA对BALB/c小鼠进行尾静脉大剂量快速推注,获得急性HBV体内表达模型。同时使用相同方法对推注后的小鼠进行RNAi干预。对不同处理组动物的HBsAg、HBeAg、HBcAg和HBV DNA进行ELISA、免疫组织化学和实时定量RT-PCR检测。结果水压法复制HBV小鼠急性模型与HBV DNA感染剂量无明显相关。与单表达siRNA相比较,双表达siRNA对HBV抗原表达抑制较为明显。结论双表达siRNA不失为一种体内抑制HBV感染的一种策略。