H5N1流感DNA疫苗和腺病毒载体疫苗的免疫原性研究

发布时间：2018-03-02 14:23

本文选题：H5N1　切入点：密码子优化　出处：《中国疾病预防控制中心》2008年硕士论文　论文类型：学位论文

【摘要】： 自1997年H5N1禽流感病毒开始感染人以后,人间病例不断发生,在泰国、印度尼西亚、中国和巴基斯坦还出现过有限的人传人的病例,这表明H5N1流感病毒正在不断进化,一旦H5N1流感病毒获得人传人的能力,引起的世界大流行将给人类带来巨大的灾难。目前防控流感/禽流感最有效的措施仍为接种疫苗。而基于鸡胚生产的季节性流感疫苗的生产能力是有限的,因此,如何缩小当前疫苗生产能力与大流行期间疫苗巨大需求量的差距,提高疫苗产量是全球流感大流行疫苗研究的一个热点问题。提高疫苗产量除了加强、扩大灭活疫苗的生产能力外,提高疫苗主要成分血凝素(HA)蛋白的表达量从而减少疫苗的使用量也是一种有效的方法。本研究拟构建针对H5N1流感病毒的DNA疫苗和腺病毒载体疫苗,通过优化HA基因的密码子,大大提高血凝素的表达水平,从而提高疫苗的免疫效果。另外,目前针对流感大流行疫苗的研究多采用Clade1的H5N1香港株或越南株,然而我国流行的H5N1流感病毒株基本上都是Clade2.3.4的毒株,与Clade1的毒株在抗原性上有很大不同,因此本研究选用中国的分离株具有重要意义。本研究以我国人感染病例中分离到的、WHO推荐的Clade2.3.4疫苗候选株A/Anhui/1/2005(H5N1)为研究对象,首先按照人的偏爱密码子将H5N1(A/Anhui/1/2005)流感病毒的HA基因进行优化改造,以提高其表达量,经全基因合成后,插入到真核表达载体pDC315中,构建了真核表达质粒pDC315-Mod.HA;将此质粒和含野生HA基因的真核表达质粒pDC315-Wt.HA分别转染293T细胞,比较HA蛋白的表达量。在比较密码子优化效果之后,将优化的HA基因Mod.HA插入DNA疫苗载体——pVR-1012,构建了一种H5N1流感病毒的DNA疫苗pVR-Mod.HA;又利用AdMax~(TM)腺病毒包装系统,分别构建了Ad-Wt.HA和Ad-Mod.HA两个腺病毒载体疫苗;最后采用BALB/c小鼠单独或者联合免疫方法,评价不同疫苗和不同免疫策略所诱导的体液免疫及细胞免疫效果,并就疫苗诱导的中和抗体研究了其对8株不同H5N1流感病毒的交叉反应性。结果表明: 1)密码子优化后HA蛋白在293T细胞中的表达水平显著提高,而且疫苗诱导小鼠产生的体液免疫和细胞免疫效果也有明显提高; 2)本研究构建的优化的腺病毒载体疫苗不仅能够有效诱导出中和抗体,而且对中国分离的不同年代不同地域的H5N1流感病毒普遍具有较强的中和作用; 3)单独免疫两次优化的腺病毒载体疫苗能够诱导较强的体液免疫和细胞免疫,而且都显著优于单独免疫两次DNA疫苗; 4) DNA疫苗初免-腺病毒载体疫苗加强的免疫策略是一种相对较好的联合免疫策略,值得进一步研究; 5)从A/Anhui/1/2005(H5N1)HA全序列中筛选到了两个能够刺激BALB/c小鼠CD8+T淋巴细胞分泌IFN-γ的表位,即HA_(212-229):TYISVGTSTLNQRLVPKI和HA_(534-543):IYSTVASSLA,后者比前者强。
[Abstract]:Since 1997, when the H5N1 avian influenza virus began to infect people, human cases have continued to occur, and in Thailand, Indonesia, China and Pakistan, there have been limited cases of human transmission, indicating that the H5N1 influenza virus is evolving. Once the H5N1 influenza virus has acquired the ability to transmit human beings, At present, the most effective measures to prevent and control influenza / avian influenza are vaccination. The production capacity of seasonal influenza vaccine based on chicken embryo is limited, so the production capacity of seasonal influenza vaccine based on chicken embryo is limited. How to narrow the gap between the current vaccine production capacity and the huge demand for vaccines during the pandemic, and how to increase the vaccine production is a hot issue in global influenza pandemic vaccine research. In addition to expanding the production capacity of inactivated vaccines, it is also an effective method to increase the expression of hemagglutinin (HA) protein, which is the main component of the vaccine, so as to reduce the amount of vaccine used. In this study, DNA vaccine and adenovirus vector vaccine against H5N1 influenza virus were constructed. By optimizing the codon of HA gene, the level of hemagglutinin expression was greatly improved, and the immune effect of the vaccine was improved. At present, most of the influenza pandemic vaccine studies use Clade1 H5N1 strain of Hong Kong or Vietnam strain. However, the H5N1 influenza virus strains in China are basically Clade2.3.4 strains, which are very different from Clade1 strains in antigenicity. Therefore, it is of great significance to select Chinese isolates in this study. In this study, the candidate strain of Clade2.3.4 vaccine, A / Anhui / 1 / 2005, recommended by WHO, which was isolated from human infection cases in China, was used as the research object. Firstly, the HA gene of H5N1 N1 / Anhui / 1 / 2005) influenza virus was optimized and modified according to the human preferred codon to increase its expression. After the whole gene was synthesized and inserted into the eukaryotic expression vector pDC315, the eukaryotic expression plasmid pDC315-Mod.HAwas constructed, and the eukaryotic expression plasmid pDC315-Wt.HA containing wild HA gene was transfected into 293T cells, respectively. After comparing the effect of codon optimization, the optimized HA gene Mod.HA was inserted into the DNA vaccine vector pVR-1012 to construct a H5N1 influenza virus DNA vaccine pVR-Mod.HA. Two adenovirus vector vaccines, Ad-Wt.HA and Ad-Mod.HA, were constructed, and the humoral and cellular immunity induced by different vaccines and different immune strategies were evaluated by BALB/c mice alone or in combination. The cross-reactivity of neutralizing antibody to 8 different H5N1 influenza viruses was studied. The results show that:. 1) the expression of HA protein in 293T cells was significantly increased after codon optimization, and the humoral and cellular immunity of mice induced by vaccine was also improved. 2) the optimized adenovirus vector vaccine can not only effectively induce neutralizing antibody, but also have strong neutralizing effect on H5N1 influenza virus isolated in different years and different regions in China. 3) Adenovirus-vector vaccine could induce strong humoral and cellular immunity, and it was significantly better than DNA vaccine twice immunized alone. 4) the immunization strategy of the primary immune-adenovirus vector vaccine reinforced by DNA vaccine is a relatively good strategy of combined immunization, which deserves further study. 5) two epitopes, HA_(212-229):TYISVGTSTLNQRLVPKI and HASP 54-543: IYSTVASSLAs, which can stimulate the secretion of IFN- 纬 by CD8 T lymphocytes in BALB/c mice, were screened from the A / Anhui / 1 / 2005 H _ 5H _ 5N _ 1H _ 1 HA sequence, the latter being stronger than the former.
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2008
【分类号】：R392.1

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