Seeing the Unseen: High-Throughput Prospective Profiling and inhibition of SARS-CoV-2 receptor-binding domain variants

SARS-CoV-2在全世界的持续快速和广泛传播导致了无数的病毒感染， 菌株，但通过接种抗单一（武汉-Hu-1）菌株获得了人体免疫力，和/或 之前感染过几种菌株最近出现的大量替代品 Omicron关注变体（VOC）刺突（S）蛋白的受体结合域（RBD）-一个关键抗原位点- 与免疫逃避和物种趋向性的推定扩大有关。免疫逃避可能 严重后果，可能导致感染、再感染和/或进一步 在地球仪不受控制的传播过程中增强病毒适应性。欧米克隆并非直接起源于 任何以前确定的挥发性有机化合物提出了未来的幽灵，高度取代免疫逃避挥发性有机化合物。 潜在变体空间的先验知识可以使主动和被动的开发成为可能。 免疫策略，以减轻新型VOCs造成的损害。然而，科学界 目前缺乏前瞻性地鉴定能够免疫逃避的新型高度取代的VOC的工具。在 目的1我们提出了一个管道，用于发现一组不同的严重取代和免疫逃避的RBD 变体。与此相关的是，医学界缺乏基于结合的治疗性被动免疫， 未知的未来变体，可以在新的VOC爆发的情况下部署。比如很多 以前获得FDA紧急使用许可的单克隆抗体疗法不再 中和Omicron VOC，从而减少临床相关的药物干预。对于目标2， 建议重新设计现有的基于小蛋白的治疗实体，以结合几乎所有可能的前瞻性 发现了新的VOC。我们的建议是由PI和co-I实验室的结果，其中我们 开发了高通量的基于结构的计算和实验方法，用于预测和 表征不同SARS-CoV-2 RBD变体与ACE 2的结合，以及一组中和抗体， 抗体（nAb）来有效地再现变体S功能和免疫逃避特性。我们将应用我们的 技术来广泛地采样高度取代（10-20个取代）但功能性RBD变体，并鉴定 这些抗体被预测为消除与针对两种抗体产生的代表性中和抗体的结合， RBD的关键（1类和2类）表位区。经过实验验证的逃生能力等级 RBD变体将作为设计基于小蛋白的抑制剂的靶标。这件事的总体影响 项目将是对1类和2类表位可能变异空间的前瞻性鉴定 仍然可以被普遍的免疫反应所识别。这将有助于治疗干预和识别 候选RBD变体免疫原用于包含在下一代疫苗中。该项目还将提供 多种高度取代的RBD变体的有效抑制剂。随着进一步的临床发展，这些可能 作为现成的治疗方法，有效地阻止新的VOC的发展。

The ongoing rapid and widespread transmission of SARS-CoV-2 across the world has resulted in myriad viral strains, but human immunity has been acquired from vaccination against a single (Wuhan-Hu-1) strain and/or previous infection with a handful of strains. The large number of substitutions present in the recently emerged Omicron Variant of Concern (VOC) spike (S) protein's receptor-binding domain (RBD) – a key antigenic site – are associated with immune evasion and a putative broadening of species tropism. Immune evasion may have serious consequences, potentially leading to increased incidence of infection, reinfection and/or further enhancing viral fitness during uncontrolled spread around the globe. Omicron has not directly descended from any of the previously identified VOCs raising the specter of future, highly-substituted immune-evasive VOCs. Prior knowledge of the space of potential variants may enable the development of active and passive immunization strategies for blunting the damage caused by novel VOCs. However, the scientific community currently lacks the tools to prospectively identify novel highly-substituted VOCs capable of immune evasion. In Aim 1 we propose a pipeline for discovering a diverse set of heavily substituted and immune-evasive RBD variants. Relatedly, the medical community lacks binding-based therapeutic passive immunization leads against unknown future variants that can be deployed in the event of a novel VOC outbreak. For example, many monoclonal antibody therapies that previously received emergency use authorization from FDA no longer neutralize the Omicron VOC, leading to fewer clinically relevant pharmaceutical interventions. For Aim 2, we propose to redesign existing small-protein-based therapeutic entities to bind nearly all possible prospectively identified novel VOCs. Our proposal is enabled by results from the PI and co-I laboratories, in which we developed high-throughput structure-based computational and experimental approaches for predicting and characterizing the binding of diverse SARS-CoV-2 RBD variants with the ACE2 and a set of neutralizing antibodies (nAbs) to effectively recapitulate variant S function and immune evasion properties. We will apply our technologies to broadly sample heavily substituted (10-20 substitutions) but functional RBD variants and identify those that are predicted to abrogate binding to a representative set of neutralizing antibodies raised against two key (Class 1 and Class 2) epitope regions of the RBD. Experimentally validated classes of escape-competent RBD variants will serve as targets for the design of small protein-based inhibitors. The overall impact of this project will be the prospective identification of the space of possible variation in Class 1 and Class 2 epitopes that can still be recognized by the prevalent immune response. This will aid therapeutic intervention and identify candidate RBD variant immunogens for inclusion in next-generation vaccines. This project will also provide potent inhibitors of the diverse highly substituted RBD variants. With further clinical development, these may serve as off-the-shelf therapeutics for effectively stopping a new VOC in its tracks.