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

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.