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

SARS-CoV-2在世界各地的持续快速和广泛传播导致了无数的病毒 毒株，但人类免疫是通过接种单一(武汉-胡-1)毒株和/或 以前感染过几种菌株。最近出现的大量替换出现在 S蛋白的受体结合域--一个关键的抗原位点-- 与免疫逃避和假定的物种趋向性扩大有关。免疫逃避可能已经 严重后果，有可能导致感染、再感染和/或进一步 在全球范围内不受控制的传播期间增强病毒适应性。奥米克龙没有直接从 任何以前发现的挥发性有机化合物，都会引发未来高度替代免疫逃避的挥发性有机化合物的幽灵。 潜在变种的空间的先验知识可以使主动和被动的发展成为可能 钝化新型挥发性有机化合物造成损害的免疫策略。然而，科学界 目前缺乏工具来前瞻性地识别能够免疫逃避的新型高度取代的VOCs。在……里面 目的1我们提出了一种流水线，用于发现一组大量替换和免疫逃避的RBD 变种。与之相关的是，医学界缺乏基于绑定的治疗性被动免疫，从而导致 未知的未来变种，可以在发生新的VOC爆发时部署。例如，许多 此前获得FDA紧急使用授权的单抗疗法不再 中和奥米克挥发性有机化合物，减少临床相关药物干预。对于目标2，我们 建议重新设计现有的以小蛋白为基础的治疗实体，以前瞻性地结合几乎所有可能的可能性 鉴定出新的挥发性有机化合物。我们的建议是由PI和CO-I实验室的结果支持的，在这些实验室中，我们 开发了基于结构的高通量计算和实验方法，用于预测和 不同SARS-CoV-2 RBD变异株与ACE2的结合特性及一组中和作用 抗体(NAB)能有效概括S的变异功能和免疫逃避特性。我们将应用我们的 广泛抽样大量取代(10-20个取代)但功能正常的RBD变体并识别的技术 预测将取消与一组具有代表性的中和抗体的结合，这些抗体针对两个 RBD的关键表位区域(1类和2类)。经过实验验证的逃生能力类 RBD变异体将成为设计基于蛋白质的小分子抑制剂的靶标。这一举措的总体影响 该项目将对第1类和第2类表位的可能变异空间进行前瞻性鉴定 这仍然可以通过流行的免疫反应来识别。这将有助于治疗干预和识别 包括在下一代疫苗中的候选RBD变种免疫原。该项目还将提供 多种高度取代的RBD变种的有效抑制剂。随着临床的进一步发展，这些可能会 作为现成的疗法，有效地阻止新的VOC进入其轨道。

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.