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Antibodies Recognizing Quaternary Differences in HIV Envelope Glycoproteins (K08)

识别 HIV 包膜糖蛋白四级差异的抗体 (K08)

基本信息

批准号：
8526356
负责人：
Mark Daniel Hicar
金额：
$ 12.78万
依托单位：
STATE UNIVERSITY OF NEW YORK AT BUFFALO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-22 至 2014-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8526356
关键词：
Acids Affinity Amino Acids Antibodies B-Lymphocytes Back Binding Biochemical Biological Assay Cell Line Cell Separation Characteristics Cloning Collection Data Development Enzyme-Linked Immunosorbent Assay Epitope Mapping Epitopes Evolution Exhibits Exposure to Fellowship Genes Germ-Line Mutation Glycoproteins HIV HIV Envelope Protein gp120 HIV Infections HIV Seropositivity HIV vaccine Human Hybrids Immune system Immunoglobulin Somatic Hypermutation Individual Infection Kinetics Knowledge Label Length Light Molecular Mutate Mutation Nucleic Acids Play Population Principal Investigator Process Reporter Research Training Role Sequence Homology Site-Directed Mutagenesis Somatic Mutation Specificity Surface Plasmon Resonance System Vaccine Design Vaccines Viral Virus Virus-like particle Work base complementarity-determining region 3 design driving force env Gene Products expectation expression vector hybrid antibody improved insight monomer neutralizing antibody novel pressure response

项目摘要

DESCRIPTION (provided by applicant): Antibodies (Abs) recognizing unique conformational epitopes only seen on functional HIV trimeric envelope are thought to be important for HIV neutralization and may be the key determinant of selection pressure on envelope (Env) viral sequences. We have created a panel of candidate Env trimer-specific Abs from single-cell sorting B cells from HIV positive subjects. I propose work to define important Ab structural determinants necessary for recognition of HIV trimeric Env and, in doing so, to provide insights for design of an effective HIV vaccine. I hypothesize that continued affinity maturation of Env trimer-specific Abs, rather than Env monomer- specific Abs, is the principal driving force for Env sequence diversity during HIV infection. I will study these novel native human Ab sequences by cloning them into Fab and full-length Ab expression systems. I will then use these constructs to define the biochemical and structural features of neutralizing Abs. In specific aim 1,1 will compare the binding and neutralization characteristics of Env monomer-reactive Abs to Env trimer-specific Abs. 1 will use surface plasmon resonance to compare binding and the infectible reporter TZM-bl cell line to assess neutralization. We expect to show the Env trimer-specific Abs bind and neutralize more effectively than Env monomer-reactive Abs. In specific aim 2,1 will determine the relationship of binding kinetics and neutralization potency to somatic hypermutation. It follows from my hypothesis and preliminary data that Abs possessing large numbers of mutations are likely being created by the process of affinity maturation, and hence, are improved in their binding and neutralization capabilities. Interestingly, most of the observed mutations occur outside of the major determinant of Abs binding, the CDR3 loop. Specific aim 3 is designed to define the role of somatic mutation outside of the CDR3 loop in HIV neutralization. Hybrids of mutagenized Abs with germline Ab sequences will be created by site-directed mutagenesis. I will compare binding and neutralization of the native Abs to these hybrid Abs. Because of the extent of mutation, we expect to show a critical role for regions outside of the CDR3 loop, particularly heavy chain framework 3, in HIV neutralization. RELEVANCE: The overall aim of this work is improvement in design of vaccines to produce higher potency antibodies against HIV. This work will directly study natural antiodies formed during HIV infection to find how they bind virus. Not only may this help improve our knowledge of how the immune system reacts to HIV, but it may shed light on how we can improve components of HIV vaccines and other vaccines to increase their potency.

描述（由申请人提供）：识别仅在功能性HIV三聚体包膜上可见的独特构象表位的抗体（Abs）被认为对HIV中和很重要，并且可能是包膜（Env）病毒序列选择压力的关键决定因素。我们从HIV阳性受试者的单细胞分选B细胞中创建了一组候选Env三聚体特异性抗体。我建议确定识别艾滋病毒三聚体Env所必需的重要Ab结构决定因素，并在此过程中为设计有效的艾滋病毒疫苗提供见解。我推测，在HIV感染过程中，Env三聚体特异性抗体的持续亲和成熟，而不是Env单体特异性抗体，是Env序列多样性的主要驱动力。我将通过将这些新的天然人类Ab序列克隆到Fab和全长Ab表达系统中来研究它们。然后，我将使用这些结构来定义中和抗体的生化和结构特征。在具体目标1中，我将比较Env单体反应性抗体与Env三聚体特异性抗体的结合和中和特性。我将使用表面等离子体共振来比较结合和传染性报告细胞TZM-bl细胞系来评估中和。我们期望证明Env三聚体特异性抗体比Env单体反应性抗体更有效地结合和中和。在特定目标2中，1将确定结合动力学和中和效力与体细胞超突变的关系。根据我的假设和初步数据，具有大量突变的抗体可能是通过亲和成熟的过程产生的，因此，它们的结合和中和能力得到了提高。有趣的是，大多数观察到的突变发生在抗体结合的主要决定因素CDR3环之外。特异性靶3旨在确定CDR3环外体细胞突变在HIV中和中的作用。诱变抗体与种系抗体序列的杂交将通过定点诱变产生。我将比较天然抗体与这些杂交抗体的结合和中和。由于突变的程度，我们希望显示CDR3环外区域，特别是重链框架3在HIV中和中的关键作用。