高致病性禽流感H5N1康复者体内针对HA的抗体的表位研究

发布时间：2018-03-05 09:02

本文选题：高致病性禽流感病毒H5N1　切入点：酵母展示系统　出处：《清华大学》2015年博士论文　论文类型：学位论文

【摘要】：高致病性禽流感病毒H5N1是一种甲型流感病毒。它致病性强,致死率高,能感染包括人在内的多个物种,是当前公共健康面临的严重威胁。深入地了解宿主体内针对流感病毒表面蛋白血凝素(hemagglutinin,HA)的保护性抗体反应对于研发高致病性禽流感H5N1的疫苗和抗体药物都至关重要,因此我们对来自两位高致病性禽流感H5N1康复者的五株单克隆抗体和两份血清识别的抗原表位进行了全面的研究。我们首先建立了一种新的分析抗原表位的方法。该方法主要包括构建酵母展示的抗原随机切割文库、对文库进行流式筛选和对分选得到的酵母进行序列分析。我们构建了一个H5N1 HA的抗原文库,然后用一株识别构象型表位的单克隆抗体和三份HA短肽免疫的小鼠血清对文库进行了筛选,证明该方法既可以用于分析线性表位,也可以用于分析构象型表位。接着我们还对三份HA重组蛋白免疫的小鼠血清进行了分析,确定了它们主要识别的线性表位,同时也证明该方法可以用于分析复杂的多克隆抗体反应。利用这个方法,我们先对五株单克隆抗体进行了分析,发现它们识别的表位都位于HA头部。接着我们又利用酵母表面展示抗原突变文库进一步确定了五株抗体识别的精细表位,并且还利用点突变假病毒中和实验确定了其中的关键中和位点,证明这五株抗体识别HA头部三个不同的表位。为了更准确地分析这些抗体识别的表位,我们又根据这三个不同的表位挑选了三株单克隆抗体并且分别解析了它们与HA头部的复合物的高分辨率晶体结构。随后我们对这五株中和抗体和其他已经报道的针对H5 HA头部的中和抗体的表位进行了分析和比较,发现可以将它们划分到四个不同的表位区域。最后我们还对两份康复者血清识别的表位进行了分析,发现虽然血清中同时包括针对HA头部和躯干部的抗体,但是其中的中和抗体主要是针对我们定义的四个表位区域。综上所述,我们分析了高致病性禽流感H5N1康复者体内针对HA的单克隆和多克隆抗体识别的表位。这些研究结果不仅能够解释人体内针对高致病性禽流感病毒H5N1的抗体中和病毒的机制,还能够指导这五株单克隆抗体的联用和优化以及新型流感疫苗的设计。
[Abstract]:Highly pathogenic avian influenza virus H5N1 is a type of influenza A virus. It is highly pathogenic, has a high mortality rate and can infect many species, including human beings. This is a serious threat to public health. A thorough understanding of the protective antibody response of the host to the influenza virus surface protein hemagglutinin (HA) is essential to the development of a highly pathogenic avian influenza H5N1 vaccine and antibody drugs. Therefore, we studied five monoclonal antibodies and two antigenic epitopes recognized by serum from two highly pathogenic avian influenza H5N1 patients. We first developed a new method for the analysis of antigenic epitopes. The method mainly consists of constructing a random cut library of antigens displayed by yeast. We constructed an antigenic library of H5N1 HA by flow screening and sequence analysis of the selected yeast. Then the library was screened with a monoclonal antibody which recognized conformational epitopes and three mouse sera immunized with HA short peptides. It was proved that this method could be used to analyze linear epitopes. It can also be used to analyze conformational epitopes. Then we analyzed three sera of mice immunized with HA recombinant proteins and identified their main linear epitopes. It is also proved that this method can be used to analyze complex polyclonal antibody reactions. It was found that the epitopes they recognized were located on the HA head. Then we further identified five fine epitopes recognized by antibodies using the yeast surface display antigen mutation library. The key neutralization sites were determined by neutralization experiments of point mutant pseudoviruses, which proved that the five antibodies recognize three different epitopes of HA head. In order to analyze the epitopes recognized by these antibodies more accurately, We also selected three monoclonal antibodies based on these three different epitopes and analyzed the high-resolution crystal structures of their complexes with HA head, respectively. The epitopes of neutralizing antibody in the head of H5 HA were analyzed and compared. We found that they could be divided into four different epitope regions. Finally, we analyzed the epitopes recognized by two recovered sera, and found that although the serum included antibodies against both head and trunk cadres of HA, But the neutralizing antibodies are mainly directed at the four epitopes that we define. We have analyzed the epitopes recognized by monoclonal and polyclonal antibodies against HA in highly pathogenic avian influenza H5N1 survivors. These results can not only explain the mechanism of neutralization of human antibodies against highly pathogenic avian influenza virus H5N1. It can also guide the combination and optimization of the five monoclonal antibodies and the design of new influenza vaccines.
【学位授予单位】：清华大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R392

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