Antibodies Recognizing Quaternary Differences in HIV Envelope Glycoproteins (K08)

描述（由申请人提供）：识别仅在功能性 HIV 三聚体包膜上看到的独特构象表位的抗体 (Abs) 被认为对于 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。我建议努力定义识别 HIV 三聚体 Env 所需的重要抗体结构决定因素，并在此过程中为设计有效的 HIV 疫苗提供见解。我假设，Env 三聚体特异性抗体的持续亲和力成熟，而不是 Env 单体特异性抗体，是 HIV 感染期间 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抗体序列克隆到 Fab 和全长抗体表达系统中来研究它们。然后我将使用这些构建体来定义中和抗体的生化和结构特征。具体目标 1,1 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。图 1 将使用表面等离振子共振来比较结合，并使用可感染的报告 TZM-bl 细胞系来评估中和作用。我们期望展示 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。具体目标 2,1 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很可能是通过亲和力成熟过程产生的，因此其结合和中和能力得到了改善。有趣的是，大多数观察到的突变发生在 Abs 结合的主要决定因素 CDR3 环之外。具体目标 3 旨在定义 CDR3 环外的体细胞突变在 HIV 中和中的作用。诱变抗体与种系抗体序列的杂交体将通过定点诱变产生。我将比较天然抗体与这些混合抗体的结合和中和作用。由于突变的程度，我们预计 CDR3 环之外的区域（特别是重链框架 3）在 HIV 中和中发挥关键作用。 相关性：这项工作的总体目标是改进疫苗设计，以产生更高效的抗 HIV 抗体。这项工作将直接研究艾滋病毒感染过程中形成的天然抗体，以了解它们如何结合病毒。这不仅有助于提高我们对免疫系统如何对艾滋病毒作出反应的了解，而且可能有助于我们了解如何改进艾滋病毒疫苗和其他疫苗的成分以提高其效力。

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.