PROJECT 4: Genetic and Structural Basis for Human Antibody Inhibition of Dengue Viruses

项目 4：人类抗体抑制登革热病毒的遗传和结构基础

• 批准号: 10458132
• 负责人: Eva Harris
• 金额: $ 59.66万
• 依托单位: UNIVERSITY OF CALIFORNIA BERKELEY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-29 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10458132
• 关键词: Antibodies; Dengue Virus; Genetic; Human; Immunity; Infection; Vaccination

One of the primary goals of this P01 program project proposal is to define the functional and antigenic specificity of the memory humoral response before and after dengue virus (DENV) infection. Here, we will determine the molecular and structural basis for type-specific and cross-reactive human B cell responses to DENV infection. Aim 1 of Project 4 will study the epitopes and structures recognized by DENV3 type-specific potently neutralizing antibodies (Abs). The goal is to define a comprehensive antigenic map of neutralizing determinants on the DENV E protein, which will inform DENV vaccine design and testing efforts. In past work with investigators in this consortium, we have mapped many of the major antigenic determinants on DENV1 and DENV2. The protective determinants on DENV3 are less well studied. Fortunately, in preliminary experiments, in collaboration with Project 1 and Core C, we have generated a significant panel of potent DENV3-specific neutralizing monoclonal Abs (mAbs) from the Nicaraguan Pediatric Dengue Cohort Study (PDCS) samples. It is clear that many of these antibodies recognize novel epitopes that are not previously known. We will use these reagents to identify the molecular and structural basis for recognition using alanine scanning mutagenesis, hydrogen deuterium exchange mass spectrometry, cryo-EM and crystallography studies. If the antigenic maps appear incomplete, as determined by Project 1 in dengue-endemic populations, we will generate additional mAbs from the peripheral blood mononuclear cells (PBMCs) collected from children with documented repeat DENV3 infections, as well as DENV1 and DENV2 infections, in the PDCS to define the complete antigenic landscape. We hypothesize that most DENV3-specific Abs are directed to quaternary epitopes, including those near the DENV envelope domain I/II (EDI/II) hinge region, but that there is a diversity of binding poses and angles for recognition of this complex region. In Aim 2, we will define comprehensive antigenic maps for cross-reactive Abs that recognize and neutralize viruses of all 4 DENV serotypes. In preliminary studies, we have isolated broad and potent mAbs that have distinct profiles from those of the limited number of E dimer epitope (EDE) mAbs reported to date. The new mAbs suggest there are additional sites of vulnerability for broad and potent neutralizing responses that are not yet understood. Studies in this aim will define with biochemical, genetic and structural approaches the novel epitopes associated with highly neutralizing cross-reactive mAbs that differ from EDE epitopes after 2° DENV infection. Finally, in Aim 3, we will use emerging techniques in adaptive immune receptor next generation sequencing to interrogate the Ab variable gene repertoires in PDCS subjects. We will use deep sequencing of peripheral blood Ab gene repertoires in prior (pre-2° infection, pre-vaccination) PBMC samples from the same individuals. These studies will provide a global view of repertoire responses that will complement the molecular ‘snapshots” afforded by mAb studies in Aims 1 and 2. The repertoire studies will elucidate the complexity and specificity of the human B cell response to DENV infection on a more systematic level. The P01 integrates the work of several laboratories with expertise in different aspects of DENV biology and adaptive immunity. The human mAbs isolated in Project 4 will be used to study the molecular, structural and genetic basis for neutralizing responses but also will generate and distribute key mAb reagents that facilitate the studies in Projects 1 and 2 in this P01. Chimeric antigens produced on Project 2 will be used to identify subjects with novel B cell responses, and also to map new mAb specificities. The tasks proposed here could not be performed by any single laboratory in the P01 because of the expertise needed in Ab engineering and deep sequence analysis, the scale of production required, and the need for unique dengue reagents, cohort participants, and molecular biology approaches. Understanding human B cell responses to DENV at both the clonal and overall genetic repertoire level promises to give significant new insights into the molecular and genetic basis for type-specific and crossreactive human B cell responses.

本P01计划项目提案的主要目标之一是确定功能和抗原特异性 研究登革病毒(DENV)感染前后的记忆体液反应。在这里，我们将确定 人类B细胞对DENV感染的类型特异性和交叉反应的分子和结构基础。 项目4的目标1将研究DENV3型特异性有效中和识别的表位和结构 抗体(Abs)。目标是定义DENV上中和决定簇的全面抗原图。 E蛋白，这将为DENV疫苗的设计和测试工作提供信息。在过去与调查人员的合作中 我们已经绘制了DENV1和DENV2上的许多主要抗原决定簇。保护者 对DENV3的决定因素研究较少。幸运的是，在初步实验中，与 项目1和核心C，我们已经产生了一组有效的DENV3特异性中和单抗 来自尼加拉瓜儿童登革热队列研究(PDCS)样本的ABS(MAbs)。很明显，其中许多 抗体识别以前未知的新表位。我们将使用这些试剂来鉴定 丙氨酸扫描诱变识别的分子和结构基础 交换质谱学、低温EM和结晶学研究。如果抗原图看起来不完整， 正如项目1在登革热流行人群中所确定的那样，我们将从外周血细胞中产生额外的单抗 从有记录的重复感染DENV3的儿童身上收集血液单个核细胞(PBMC)，以及 DENV1和DENV2感染，在pDC中定义完整的抗原图。我们假设 大多数DENV3特异性抗体针对四个表位，包括DENV包膜结构域附近的表位 I/II(EDI/II)铰链区，但有多种结合姿势和角度来识别这个复合体 区域。在目标2中，我们将为识别和识别的交叉反应抗体定义全面的抗原图 中和所有4种DENV血清型的病毒。在初步研究中，我们已经分离出广泛和有效的单抗 与迄今报道的数量有限的E二聚体表位(EDE)单抗的特征截然不同。新的 单抗表明，广泛而有效的中和反应还有更多的脆弱性部位，而不是 但还不能理解。这一目标的研究将用生化、遗传和结构方法定义这部小说 与2°DENV后不同于EDE表位的高度中和交叉反应单抗相关的表位 感染。最后，在目标3中，我们将在下一代适应性免疫受体中使用新兴技术 测序以询问pDC受试者的抗体可变区基因谱。我们将使用深度测序技术 2°感染前、接种前PBMC样本中的外周血抗基因谱 个人。这些研究将提供补充分子反应的谱系反应的全球视角 AIMS 1和2中的单抗研究提供的“快照”。曲目研究将阐明 更系统的人类B细胞对DENV感染反应的特异性。P01集成了 在DENV生物学和适应性免疫的不同方面拥有专业知识的几个实验室的工作。这个 在项目4中分离的人单抗将用于研究中和的分子、结构和遗传基础 还将产生和分发关键的单抗试剂，以促进项目1和2中的研究 在本P01中。项目2产生的嵌合抗原将用于识别具有新的B细胞反应的受试者， 也是为了绘制新的mAb特异性图谱。这里提出的任务不是任何一个实验室可以完成的 在P01中，由于抗体工程和深层序列分析所需的专业知识， 所需的生产，以及需要独特的登革热试剂、队列参与者和分子生物学 接近了。从克隆和整体遗传学角度了解人类B细胞对DENV的反应 Level承诺为特定类型和交叉反应的分子和遗传基础提供重要的新见解 人类B细胞反应。