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

该 P01 计划项目提案的主要目标之一是定义功能和抗原特异性 登革热病毒（DENV）感染前后的记忆体液反应。在这里，我们将确定 类型特异性和交叉反应性人类 B 细胞对 DENV 感染反应的分子和结构基础。 项目4的目标1将研究DENV3类型特异性强中和识别的表位和结构 抗体（Ab）。目标是定义 DENV 中和决定簇的综合抗原图谱 E 蛋白，将为 DENV 疫苗设计和测试工作提供信息。在过去与调查人员的合作中 联盟，我们已经绘制了 DENV1 和 DENV2 上的许多主要抗原决定簇。保护性的 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，我们生成了一组重要的强效 DENV3 特异性中和单克隆抗体 来自尼加拉瓜儿童登革热队列研究 (PDCS) 样本的抗体 (mAb)。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 使用丙氨酸扫描诱变、氢氘进行识别的分子和结构基础 交换质谱、冷冻电镜和晶体学研究。如果抗原图谱看起来不完整， 根据项目 1 在登革热流行人群中的确定，我们将从外周血中产生额外的单克隆抗体 从有重复 DENV3 感染记录的儿童中采集的血液单核细胞 (PBMC)，以及 DENV1 和 DENV2 感染，在 PDCS 中定义完整的抗原图谱。我们假设 大多数 DENV3 特异性抗体针对四级表位，包括靠近 DENV 包膜结构域的表位 I/II (EDI/II) 铰链区，但该复合物的识别具有多种结合姿势和角度 地区。在目标 2 中，我们将为交叉反应抗体定义全面的抗原图谱，以识别和 中和所有 4 种 DENV 血清型的病毒。在初步研究中，我们分离出了广泛且有效的单克隆抗体 与迄今为止报道的有限数量的 E 二聚体表位 (EDE) mAb 具有不同的特征。新的 单克隆抗体表明，还有其他脆弱位点可产生广泛而有效的中和反应，但这些位点并不存在。 却又明白了。这一目标的研究将通过生化、遗传和结构方法来定义新的 与高度中和交叉反应性单克隆抗体相关的表位与 2° DENV 后的 EDE 表位不同 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 PDCS 受试者的 Ab 可变基因库。我们将使用深度测序 先前（2°感染前、疫苗接种前）PBMC 样本中的外周血抗体基因库来自同一样本 个人。这些研究将提供对所有反应的全球看法，这将补充分子生物学 目标 1 和 2 中单克隆抗体研究提供的“快照”。全部研究将阐明复杂性和 人类 B 细胞在更系统的水平上对 DENV 感染反应的特异性。 P01 集成了 几个在 DENV 生物学和适应性免疫不同方面具有专业知识的实验室的工作。的 项目 4 中分离的人单克隆抗体将用于研究中和的分子、结构和遗传基础 响应，还将生成和分发促进项目 1 和 2 中研究的关键 mAb 试剂 在这个P01中。项目 2 产生的嵌合抗原将用于识别具有新型 B 细胞反应的受试者， 并绘制新的单克隆抗体特异性。这里提出的任务不能由任何一个实验室来完成 在 P01 中，由于 Ab 工程和深度序列分析所需的专业知识， 所需的生产以及对独特登革热试剂、队列参与者和分子生物学的需求 接近。了解人类 B 细胞对 DENV 的克隆和整体遗传库的反应 水平有望为类型特异性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。

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.