表达人呼吸道合胞病毒融合蛋白的重组腺病毒的免疫保护作用

发布时间：2018-06-28 02:21

本文选题：表达 + 呼吸道　；参考：《中国疾病预防控制中心》2009年博士论文

【摘要】： 人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染的最重要的病毒病原之一,老年人和免疫缺陷病人也易遭受RSV引起的严重感染。RSV属于副粘病毒科肺病毒属,基因组为15.2 Kb的单股负链RNA,编码11种蛋白,其中G、F系包膜糖蛋白,为RSV中和抗原。与G蛋白相比,F蛋白不但具有B细胞抗原表位也有CD8+细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)抗原表位,且其抗原性极为保守,可产生交叉保护性免疫。从福尔马林灭活疫苗开始,人们已经研制了多种形式的RSV疫苗,但由于安全性和免疫原性等问题,至今尚未有能够用于预防人RSV感染的疫苗问世,因此世界卫生组织将发展RSV疫苗列为二十一世纪最优先发展的疫苗项目之一。非复制型第一代腺病毒载体(first generation replication deficient recombinantadenoviral vector,FGAd)具有繁殖滴度高、易纯化、能够感染分裂和非分裂细胞等优点而被广泛用于疫苗研究。FGAd曾被用于表达经基因工程改造的RSV G蛋白,在动物实验中获得了较好的免疫保护作用,然而以FGAd为基础开展RSVF蛋白疫苗研究尚未见报道。与FGAd相比,辅助病毒依赖型腺病毒载体(helper-dependent adenoviral vector,HDAd)可大大降低腺病毒(Adenovirus,Ad)特异性的细胞免疫反应,增强转染DC细胞能力,且转基因表达时间更加持久。以HDAd作为黏膜疫苗载体,开展RSV F蛋白疫苗研究有待深入研究。本课题首先用表达RSV F蛋白的FGAd重组腺病毒(FGAd-F)滴鼻免疫BALB/c小鼠,观察免疫保护效果和保护作用特点。结果表明:FGAd-F滴鼻免疫BALB/c小鼠能够诱导产生RSV特异性的血清IgG、黏膜IgA和CD8+CTL,抗体亚类分析显示诱导产生了Th1和Th2平衡的CD4+T细胞免疫应答,没有产生疾病增强,对RSV攻击具有一定的免疫保护作用。随后,我们用表达EGFP蛋白的HDAd重组腺病毒(HDAd-EGFP)滴鼻免疫BALB/c小鼠,探讨HDAd作为黏膜疫苗载体的可行性和合理性。结果表明:尽管与等量的FGAd-EGFP诱导产生相似的anti-Ad抗体,但HDAd-EGFP经鼻免疫小鼠可诱导产生更高的EGFP特异性的血清IgG、黏膜IgA和细胞免疫,且抗体亚类分析显示诱导产生了Th1和Th2平衡的CD4+T细胞免疫应答。另外HDAd-EGFP同源加强免疫也能提高EGFP特异性的体液免疫和细胞免疫。在上述研究的基础上,我们用表达RSV F蛋白的HDAd重组腺病毒(HDAd-F)经滴鼻途径免疫BALB/c小鼠,初步探讨了HDAd-F的免疫效果。结果表明:与等量的FGAd-F相比,HDAd-F经鼻两次免疫小鼠可诱导更好的RSV F特异性的黏膜免疫,HDAd-F经鼻一次免疫小鼠可诱导更高的RSV F特异性的血清抗体。综上所述,FGAd-F能够诱导有效的免疫保护作用,以FGAd为载体的疫苗是预防RSV等通过黏膜途径感染的病毒疫苗的一个重要方法。HDAd是一种理想的黏膜疫苗载体,适于经呼吸道黏膜感染的RSV疫苗的研制,通过提高转基因表达量、降低载体用量等措施,有望获得较好的免疫效果和免疫保护作用。
[Abstract]:Human respiratory syncytial virus (Human Respiratory Syncytial Virus, RSV) is widely distributed all over the world. It is one of the most important viral pathogens causing severe lower respiratory infection in infants and young children. The elderly and immunodeficiency patients are also vulnerable to RSV caused by severe infection of.RSV belonging to the paramyxovirus family, a single strand of 15.2 Kb. Negative chain RNA, encoding 11 proteins, of which G, F envelope glycoprotein is RSV neutralizing antigen. Compared with G protein, F protein not only has the epitopes of B cell antigen but also CD8+ cell toxic T lymphocyte (cytotoxic T lymphocyte) antigen epitope, and its antigenicity is extremely conservative, and can produce cross protective immunity.
Since the formalin inactivated vaccine has begun, many forms of RSV vaccines have been developed, but because of the safety and immunogenicity of the vaccine, there has not yet been a vaccine to prevent human RSV infection. Therefore, the WHO is developing the RSV vaccine as one of the top priority vaccine projects in twenty-first Century.
The non replicating first generation adenovirus vector (first generation replication deficient recombinantadenoviral vector, FGAd) has the advantages of high breeding titer, easy purification, and the ability to infect split and non cleavage cells and is widely used in vaccine research..FGAd has been used to express the RSV G protein that has been genetically engineered. It has been obtained in animal experiments. Better immune protection has been achieved. However, the study of RSVF vaccine based on FGAd has not yet been reported. Compared with FGAd, the auxiliary virus dependent adenovirus vector (helper-dependent adenoviral vector, HDAd) can greatly reduce the specific cellular immune response of adenovirus (Adenovirus, Ad), enhance the transfection of DC cells, and turn the base of the transfected DC cells. Because of the longer expression time, the study of RSV F protein vaccine needs to be further studied with HDAd as a mucosal vaccine carrier.
In this study, we first immunized BALB/c mice with FGAd recombinant adenovirus (FGAd-F) expressing RSV F protein (FGAd-F), and observed the protective effects and protective effects of BALB/c. The results showed that BALB/c mice immunized with FGAd-F nose drops could induce RSV specific serum IgG, mucous membrane IgA and CD8+CTL. Antibody subclass analysis showed that the inducible production of Th1 and equilibrium was induced by the antibody subclass analysis. CD4+T cell immune response did not produce disease enhancement, and had a certain immune protection against RSV attack.
Subsequently, we immunized the BALB/c mice with the recombinant adenovirus expressing EGFP protein (HDAd-EGFP) in the nose drops to explore the feasibility and rationality of HDAd as a mucosal vaccine carrier. The results showed that, despite the similar anti-Ad antibody induced by the equivalent FGAd-EGFP induction, the HDAd-EGFP transnasal immune mice could induce higher EGFP specific blood. IgG, mucosal IgA and cellular immunity, and the antibody subclass analysis showed that the induced CD4+T cell immune response was induced by the balance of Th1 and Th2. In addition, HDAd-EGFP homologous immunization could also improve the humoral and cellular immunity of EGFP specific body.
On the basis of the above study, we immunized BALB/c mice with the HDAd recombinant adenovirus expressing RSV F protein (HDAd-F) through the nasal drip route. The immune effect of HDAd-F was preliminarily discussed. The results showed that the two mice immunized with HDAd-F through the nose could induce better RSV F specific mucosal immunity compared with the equal amount of FGAd-F, and the HDAd-F via the nose was immune to one time. Mice could induce higher RSV F specific serum antibodies.
To sum up, FGAd-F can induce effective immune protection. FGAd based vaccine is an important way to prevent RSV and other viral vaccines through mucosal pathway..HDAd is an ideal carrier for mucosal vaccine. It is suitable for the research of RSV vaccine that is infected by respiratory tract mucosa. By improving the expression of transgene and reducing the carrier use And other measures are expected to achieve better immune effects and immune protection.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392.1

【共引文献】