Structural biology of antibody:antigen complexes

项目3的总体目标是了解免疫优势的高分辨率结构关联 并为设计免疫原提供结构基础，使免疫反应集中在 特定的、精选的表位。我们的主要假设是，对免疫优势的结构性贡献可以 通过将抗体-抗原复合体的结构与来自 抗体的来源和导致亲和力成熟的突变序列的重建 来自其生殖系前体的抗体。项目1中描述的高吞吐量方法可以为我们提供 所需的有关曲目和从设置中指定的抗体亲和力成熟的信息 接种疫苗VS感染。我们建议在这里提供相应的三维信息。我们 有以下四个目标。[1]与之结合的交叉反应抗体的结构特征是什么 他们的HA表位？我们将确定在项目中研究的抗体的选定代表的结构 1和2，与相关的流感病毒HA形成复合体。我们将从标准结晶学开始 在这方面，我们将继续采用新的方法，但应尽可能迅速地采用更高通量的低温电磁方法(目标2；另见核心C)。 (2)我们会发展方法，把“客体蛋白”(以流感病毒HA为主要目标)安装在 二十面体病毒支架，以创建适合于低温电子显微镜结构确定的对称阵列。我们会 首先以轮状病毒双层颗粒(DLP)为支架，外层糖蛋白VP7为 我们还将测试另一种外层蛋白VP4，作为一种三聚体安装蛋白。[3]什么 让表位成为免疫优势？冷冻EM方法提供的机会来检查一个 系统的和相对公正的复合体集合将使我们能够确定 直接免疫优势。我们将确定选定的HA与抗体的复合体的结构 表示由项目1中的研究揭示的典型曲目的全部光谱，并由 项目2.[4]应用于其他病毒抗原。我们将应用在目标2中制定的安装策略来 其他病毒抗原，结合谱系分析和结构解剖可能导致新的

The overall goals of Project 3 are to understand the high-resolution structural correlates of immunodominance and to provide structural foundations for designing immunogens that can focus an immune response on particular, chosen epitopes. Our principal hypothesis is that structural contributions to immunodominance can be revealed by correlating structures of antibody-antigen complexes with knowledge of the repertoire from which the antibody was derived and reconstruction of the mutational sequence that led to the affinity matured antibody from its germline precursor. The high-throughput approaches described in Project 1 can give us the required information about repertoire and about affinity maturation of antibodies denved from the settings of vaccination vs. infection. We propose here to provide the corresponding three-dimensional information. We have the following four Aims. [1) What are the structural characteristics of cross-reactive antibodies bound with their HA epitopes? We will determine structures of selected representatives of the antibodies studied in projects 1 and 2, in complex with the relevant influenza virus HA. We will begin with standard crystallographic approaches, but proceed as rapidly as possible to higher-throughput cryoEM methods (Aim 2; see also Core C). (2) We will develop approaches for mounting "guest proteins" [with influenza HA as the principal target) onto icosahedral-virus scaffolds, to create a symmetric array suitable for structure determination by cryoEM. We will start with the rotavirus double-layer particle (DLP) as our scaffold, and the outer-layer glycoprotein, VP7, as the mounting device.; we will also test the other outer-layer protein, VP4, as a trimeric mounting protein. [3) What makes an epitope immunodominant? The opportunity afforded by the cryoEM approach to examine a systematic and relatively unbiased set of complexes will allow us to determine the structural correlates of immunodominance directly. We will determine structures of a selected HA in complex with antibodies representing the entire spectrum of a typical repertoire as revealed by the studies in Project 1 and mapped by Project 2. [4) Application to other viral antigens. We will apply to the mounting strategies developed in Aim 2 to other viral antigens for which a combination of repertoire analysis and structural dissection might lead to novel