The Vanderbilt Vaccine Center, Vanderbilt University Medical Center, Nashville, TN 37232.
Department of Pathology, Microbiology, and Immunology, Vanderbilt University, Nashville, TN 37232.
Proc Natl Acad Sci U S A. 2023 Mar 28;120(13):e2213690120. doi: 10.1073/pnas.2213690120. Epub 2023 Mar 24.
Selection and development of monoclonal antibody (mAb) therapeutics against pathogenic viruses depends on certain functional characteristics. Neutralization potency, or the half-maximal inhibitory concentration (IC) values, is an important characteristic of candidate therapeutic antibodies. Structural insights into the bases of neutralization potency differences between antiviral neutralizing mAbs are lacking. In this report, we present cryo-electron microscopy (EM) reconstructions of three anti-Eastern equine encephalitis virus (EEEV) neutralizing human mAbs targeting overlapping epitopes on the E2 protein, with greater than 20-fold differences in their respective IC values. From our structural and biophysical analyses, we identify several constraints that contribute to the observed differences in the neutralization potencies. Cryo-EM reconstructions of EEEV in complex with these Fab fragments reveal structural constraints that dictate intravirion or intervirion cross-linking of glycoprotein spikes by their IgG counterparts as a mechanism of neutralization. Additionally, we describe critical features for the recognition of EEEV by these mAbs including the epitope-paratope interaction surface, occupancy, and kinetic differences in on-rate for binding to the E2 protein. Each constraint contributes to the extent of EEEV inhibition for blockade of virus entry, fusion, and/or egress. These findings provide structural and biophysical insights into the differences in mechanism and neutralization potencies of these antibodies, which help inform rational design principles for candidate vaccines and therapeutic antibodies for all icosahedral viruses.
针对致病病毒的单克隆抗体 (mAb) 治疗药物的选择和开发取决于某些功能特性。中和效力（即半最大抑制浓度 [IC] 值）是候选治疗性抗体的一个重要特性。缺乏针对抗病毒中和 mAb 中和效力差异的基础的结构见解。在本报告中，我们展示了针对 E2 蛋白上重叠表位的三种抗东部马脑炎病毒 (EEEV) 中和人 mAb 的冷冻电子显微镜 (EM) 重建，它们各自的 IC 值差异超过 20 倍。通过我们的结构和生物物理分析，我们确定了几个限制因素，这些限制因素导致了中和效力的差异。与这些 Fab 片段复合物的 EEEV 的冷冻电镜重建揭示了结构限制，这些限制因素规定了免疫球蛋白与其 IgG 对应物在病毒内部或病毒间交联糖蛋白刺突作为中和机制。此外，我们描述了这些 mAb 识别 EEEV 的关键特征，包括表位-抗体结合位相互作用表面、占有率以及与 E2 蛋白结合的结合速率的差异。每个限制因素都有助于 EEEV 抑制程度，从而阻止病毒进入、融合和/或逸出。这些发现为这些抗体的机制和中和效力差异提供了结构和生物物理见解，有助于为所有二十面体病毒的候选疫苗和治疗性抗体提供合理的设计原则。
