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

本P01项目提案的主要目标之一是定义功能和抗原特异性 登革病毒（DENV）感染前后记忆性体液反应的变化。在这里，我们将确定 型特异性和交叉反应性人B细胞对DENV感染应答的分子和结构基础。 项目4的目的1将研究DENV 3型特异性强效中和抗体识别的表位和结构， 抗体（Abs）。目的是确定DENV中和决定簇的全面抗原图谱， E蛋白，这将为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， 在DENV 1和DENV 2上，我们已经定位了许多主要的抗原决定簇。保护 DENV 3的决定因素研究较少。幸运的是，在初步的实验中， 项目1和核心C，我们已经产生了一组有效的DENV 3特异性中和单克隆抗体， Abs（mAb）来自中国儿童登革热队列研究（PDCS）样本。很明显，其中许多 抗体识别先前未知的新表位。我们将使用这些试剂来鉴定 使用丙氨酸扫描诱变识别的分子和结构基础，氢氘 交换质谱、冷冻电镜和晶体学研究。如果抗原图谱不完整， 正如项目1在登革热流行人群中所确定的那样，我们将从外周血中产生额外的mAb。 血液单核细胞（PBMC）收集自记录重复DENV 3感染的儿童，以及 DENV 1和DENV 2感染，以定义完整的抗原景观。我们假设 大多数DENV 3特异性Ab针对四元表位，包括靠近DENV包膜结构域的那些 I/II（EDI/II）铰链区，但该复合物的识别存在多种结合位姿和角度 地区在目标2中，我们将定义交叉反应性Ab的全面抗原图谱，这些Ab识别并 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而有效的mAb， 具有与迄今报道的有限数量的E二聚体表位（EDE）mAb不同的特征。新 单克隆抗体表明，还有其他部位的脆弱性，广泛和有效的中和反应， 但却被理解在这一目标的研究将定义与生物化学，遗传和结构的方法， 与2° DENV后不同于EDE表位的高度中和交叉反应性mAb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PDCS受试者中的Ab可变基因库。我们将使用深度测序 来自相同样本的既往（2°感染前、接种前）PBMC样本中的外周血Ab基因库 个体这些研究将提供一个全面的剧目反应，将补充分子 目标1和2中mAb研究提供的“快照”。剧目研究将阐明复杂性， 在更系统的水平上，人B细胞应答DENV感染的特异性。P01集成了 在DENV生物学和适应性免疫的不同方面具有专业知识的几个实验室的工作。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。 此外，还将生产和分发促进项目1和2研究的关键mAb试剂 在P01。项目2产生的嵌合抗原将用于鉴定具有新型B细胞应答的受试者， 并绘制新的mAb特异性。这里提出的任务不可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， 生产所需，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细胞在克隆和整体遗传库中对DENV的反应 水平有望为类型特异性和交叉反应性的分子和遗传基础提供重要的新见解。 人B细胞应答。

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.