HIV-1广谱中和抗体PGT135的结构和识别的抗原表位研究

发布时间：2019-02-17 19:37

【摘要】：人类免疫缺陷病毒(Human Immunodeficiency Virus, HIV)分为HIV-1与HIV-2。绝大多数HIV感染是由HIV-1造成的,并且90%以上的HIV-1感染者会在10-12年内发展成为艾滋病。HIV-1极易在感染过程中发生突变,逃逸人体免疫系统的监控,通过疫苗诱导HIV-1广谱中和抗体的产生是很困难的,因此设计保护性HIV-1疫苗一直是人类的梦想。尽管抗病毒药物在治疗HIV-1方面取得了显著的成果,但目前还没有疫苗能有效地预防和控制HIV-1感染。HIV-1自然感染过程中,会产生广谱中和抗体,近年来分离的VRC01类抗体、PT系列和PGT系列抗体能中和目前世界范围内流行的大多数毒株,尤其是VRC01类抗体能中和90%以上的世界流行株。但HIV-1广谱中和抗体,在HIV-1感染后数月甚至1-2年后才会产生。HIV-1自然感染过中,广谱中和抗体产生相对比较滞后,因此不能有效地清除体内感染的HIV-1病毒。最近,人们通过结构生物学的方法,研究HIV-1广谱中和抗体与HIV-1膜蛋白gp120或者gp41复合物的结构,对HIV-1广谱中和抗体以及这些抗体所识别的HIV-1膜蛋白的保守性表位有了更加深入的了解。了解HIV-1广谱中和抗体以及这些抗体所识别的抗原表位,有助于HIV-1疫苗的研究。我们可以根据HIV-1广谱中和抗体的表位进行HIV-1疫苗的免疫原设计,同时也可以将HIV-1广谱中和抗体用于被动免疫。本文选取HIV-1广谱中和抗体PGT135进行研究,对已有的PGT135抗体基因进行密码子优化,在293T细胞中进行抗体的大量表达和纯化,对PGT135进行晶体结构解析；检测PGT135抗体对HIV-1假病毒中国流行株的中和敏感性；用酵母展示随机抗原片段库,筛选PGT135抗体所识别的抗原表位；用酵母展示抗原随机突变库,筛选PGT135抗体所识别的抗原表位的重要氨基酸位点,用假病毒中和抑制实验验证PGT135抗体所识别抗原表位中的关键性位点；表达PGT135抗体所识别的抗原表位片段,通过筛选人类非免疫的单链抗体(single chain fragments variablescFvs)酵母库,寻找与PGT135抗体类似的HIV-1广谱中和抗体。通过解析PGT135抗体的晶体结构,发现PGT135抗体比VRC01类抗体的重链有较长的互补决定区(complementarity determining region, CDRH3),可能对这类抗体识别隐蔽的糖基化位点有一定的帮助；PGT135抗体对HIV-1中国流行株病毒大多数有中和抑制作用；用酵母展示抗原随机突变库,筛选出PGT135抗体的抗原表位在HIV-1膜蛋白gp120的V3loop区和V4loop区之间；利用酵母表面展示抗原随机突变库,筛选出PGT135抗体所识别的重要氨基酸位点。用假病毒中和抑制实验,验证了HIV-1膜蛋白上仅仅一个糖基化位点的改变,就可以改变抗体对HIV-1的中和敏感性。用昆虫细胞表达的PGT135识别的抗原表位,从人类未免疫的scFvs,筛选出对PGT135抗体表位的抗原片段有结合能力的抗体。对HIV-1广谱中和抗体PGT135晶体结构的解析以及它所识别的抗原表位研究,可以为HIV-1疫苗的设计提供帮助。可以将PGT135抗体所识别的表位用于设计HIV-1疫苗的免疫原,也可以将PGT135用于被动免疫中疫苗的设计。
[Abstract]:Human Immunodeficiency Virus (HIV) is divided into HIV-1 and HIV-2. Most of the HIV infections are caused by HIV-1, and more than 90% of HIV-1 infections will develop into AIDS within 10-12 years. HIV-1 is very easy to mutate in the course of infection, to escape the monitoring of the immune system of the human body, and to induce the production of the HIV-1 broad-spectrum neutralizing antibody by the vaccine is very difficult, so the design of the protective HIV-1 vaccine has been a human dream. Although antiviral drugs have achieved significant results in the treatment of HIV-1, no vaccine is currently available to effectively prevent and control HIV-1 infection. In the course of HIV-1 natural infection, a broad-spectrum neutralizing antibody is generated, and in recent years, the isolated VRC01 antibody, the PT-series and the PGT-series antibody can be used for neutralizing more than 90% of the world-wide strains in the current world. However, the HIV-1 broad-spectrum neutralizing antibody can only be produced in the months after HIV-1 infection and even after 1-2 years. The HIV-1 naturally-infected, broad-spectrum neutralizing antibody is relatively late, so it is not possible to effectively remove the in-vivo infected HIV-1 virus. Recently, by means of structural biology, the structure of HIV-1 broad-spectrum neutralizing antibody and HIV-1 membrane protein gp120 or gp41 complex is studied, and the conservative table position of HIV-1 membrane protein identified by the anti-HIV-1 broad-spectrum neutralizing antibody and these antibodies has been better understood. To understand the HIV-1 broad spectrum neutralizing antibodies, and the epitope identified by these antibodies, contribute to the study of the HIV-1 vaccine. The HIV-1 vaccine's immunogen design can be carried out according to the HIV-1 broad spectrum and the epitope of the antibody, and the HIV-1 broad spectrum neutralizing antibody can also be used for passive immunization. In this paper, an HIV-1 broad-spectrum neutralizing antibody, PGT135, is selected to be used for codon optimization of the existing PGT135 antibody gene. The expression and purification of the antibody in the 293T cell are carried out, and the crystal structure of the PGT135 is analyzed; and the neutralization sensitivity of the PGT135 antibody to the HIV-1 pseudovirus Chinese epidemic strain is detected. sex; using a yeast to display a random antigen fragment library to screen the epitope of the antigen identified by the PGT135 antibody; displaying an antigen random mutation library with a yeast, screening an important amino acid position of the epitope of the antigen identified by the PGT135 antibody, The key site in the epitope of the antigen identified by the PGT135 antibody was verified by the false virus neutralization inhibition assay; the epitope fragment of the antigen identified by the PGT135 antibody was expressed, and the HIV-1 broad-spectrum neutralizing anti-HIV-1 similar to the PGT135 antibody was found by screening the yeast library of the human non-immune single chain antibody. By analyzing the crystal structure of the PGT135 antibody, it is found that the PGT135 antibody has a longer complementary determining region (CDRH3) than the heavy chain of the VRC01 antibody, and it is possible to identify the hidden glycosylation site for the antibody. The PGT135 antibody has a neutralizing effect on most of the HIV-1 Chinese epidemic strains, and the yeast shows the antigen random mutation library, and the antigen table of the PGT135 antibody is screened between the V3loop region and the V4loop region of the HIV-1 membrane protein gp120, and the antigen is displayed on the surface of the yeast. Mutant library for screening important amino groups identified by the PGT135 antibody Acid sites. The alteration of only one glycosylation site on the HIV-1 membrane protein can be altered by neutralizing the inhibition assay with a pseudovirus, and the neutralization of the antibody against HIV-1 can be altered Sensitivity. The epitope of the antigen identified by the PGT135 expressed by the insect cells, the ability to bind to the antigen fragment of the epitope of the PGT135 antibody from the human unimmunized scFvs. The analysis of the crystal structure of the HIV-1 broad spectrum and the antibody PGT135, as well as the epitope of the antigen identified by it, can be the design of the HIV-1 vaccine The meter provides help. The table bits identified by the PGT135 antibody can be used to design the immunogen for the HIV-1 vaccine or the PGT135 can be used in passive immunization
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R512.91

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