Antigen recognition and activation of B-cell receptors of HIV-1 broadly neutralizing antibodies

项目总结/摘要 阐明B细胞抗原受体的抗原接合后B细胞表面上的早期事件 (BCR)可以提供抗原（Ag）驱动B细胞活化的体内潜力的评估。相比之下 解离活化模型（DAM）是对“成簇模型”的补充，提出了一种替代的构象状态 以及B细胞膜上的早期事件如何启动B细胞活化。在DAM中，B细胞 活化依赖于Ag解离无活性的静息BCR寡聚体（BCR）的能力 构象开放），并促进BCR与共刺激分子的纳米级重组， 全面激活。HIV-1包膜（Env）蛋白设计的最新进展提供了几个关键的潜力 然而，这些病毒抗原如何被参与抗HIV-1的B细胞上的BCR感知， 产生广泛中和抗体（bnAb）的免疫应答还没有被很好地理解。整体 该建议的目的是桥接定量生物物理和膜动力学测量， 在DAM背景下Ag-BCR与离体和体内B细胞活化的相互作用。为了实现这一目标，我们将 这是第一次在纳米级分辨率上研究和定义银诱导的早期BCR激活事件和 表达指定原型HIV bnAb的BCR的B细胞完全活化所需的共刺激信号传导，或 他们的先驱。我们假设，对于最佳引发HIV Env免疫原，即那些能够最好地 激活B细胞并启动强大的亲和力成熟，BCR暴露于Env抗原导致 首先是纳米级BCR构象开放，其次是BCR重组和共刺激 用于在B细胞膜上信号放大的分子/激酶。在目标1中，我们将执行生物物理 分析和研究B细胞膜动力学，以确定HIV Env Ag-BCR相互作用的性质， 表达bnAb或生殖系前体BCR的B细胞的最佳活化。在目标2中，我们将使用高分辨率 测定诱导纳米级BCR构象开放的Ag结合性质， 共刺激和调节分子以及B细胞膜的时空重组 用HIV Env Ags激活。在目标3中，我们将测试评估的Env Ags激活和驱动的能力， 在“更易于诱导”，（非无反应性或更“轻度”无反应性）bnAb B细胞中的亲和力成熟 敲入（KI）库。这些研究的结果将是第一个允许系统的方法来预测 基于这些确定的Ag诱导的早期B，HIV免疫原引发不同bnAb前体的潜力 细胞激活事件。从长远来看，我们的研究将促进免疫原的设计和预选， 在动物模型中进行测试，并加速HIV-1疫苗的开发。

Project Summary/Abstract Elucidating the early events on the B cell surface following antigen engagement of the B cell antigen receptor (BCR) can provide an assessment of the in vivo potential of an antigen (Ag) to drive B cell activation. In contrast to the “clustering model”, the “Dissociation Activation Model” (DAM) proposes an alternate conformational state of the resting BCR and how early events on the B cell membrane initiates B cell activation. In the DAM, B cell activation is dependent on the ability of an Ag to dissociate the inactive, resting BCR oligomers (BCR conformational opening), and promote nanoscale reorganization of BCR with co-stimulatory molecules, leading to full activation. Recent advances in HIV-1 Envelope (Env) protein designs have provided several key potential immunogens, however, how such viral Ags are sensed by BCRs on B cells that participates in anti-HIV-1 immune responses that generate broadly neutralizing antibodies (bnAbs) is not well understood. The overall objective of this proposal is to bridge the quantitative biophysical and membrane dynamics measurements of Ag-BCR interactions to ex-vivo and in-vivo B cell activation in the context of the DAM. Towards this goal, we will for the first time, study and define at nanoscale resolution, Ag-induced early BCR activation events and the costimulatory signaling required for full activation of B cells expressing BCRs specifying prototype HIV bnAbs or their precursors. We hypothesize that for optimal priming HIV Env immunogens, i.e. those capable of best activating B-cells and initiating robust affinity maturation, the exposure of the BCR to the Env antigens results first in nanoscale BCR conformational opening, followed by reorganization of BCRs and co-stimulatory molecules/kinases for signal amplification on the B cell membrane. In Aim 1, we will perform biophysical analyses and study B cell membrane dynamics to define the properties of HIV Env Ag-BCR interactions for optimal activation of B cells expressing bnAb or germline precursor BCRs. In Aim 2, we will use high resolution assays to determine Ag-binding properties that induce nanoscale BCR conformational opening, the role of costimulatory and regulatory molecules and the spatio-temporal reorganization of the B cell membrane following activation with HIV Env Ags. In Aim 3, we will test evaluated Env Ags for their ability to activate and drive affinity maturation in “more amenable to induce”, (non-anergic or more “mildly” anergic) bnAb B cells in naïve knock-in (KI) repertoires. Results of such studies would be the first to allow a systematic method for predicting the potential of HIV immunogens to prime distinct bnAb precursors based on these defined Ag-induced early B cell activation events. In the long-term our studies will facilitate design and pre-selection of immunogens for testing in animal models and accelerate HIV-1 vaccine development.