ACE2 Receptor and Antibody Binding to SARS-CoV‑2 Spikes and Virions by Single-Molecule Fluorescence

The envelope (Env) spike (S) trimers of known SARS-CoV-2 strains have evolved to display conformational fluctuations between structural states, which in turn impact host receptor binding efficiency, the mechanism of infection, and susceptibility to antibody binding. Previously, we reported evidence that virion trimers alternate between two end states: one functional and the other nonfunctional. These dynamics, coupled with the close positioning of virion trimers, may constrain the total number of reactive mAb or receptor target sites. Such stoichiometric limits are important considerations for the infection efficiency and susceptibility to various antibody-mediated antiviral mechanisms. Here, we address this question using single-molecule fluorescence detection (SMD) methods and a step-detection algorithm to quantify bound ligands as a function of their stepwise fluorescence photobleaching on a captured virion. Importantly, this approach does not involve genetic or chemical modifications of the virion spike. Single-molecule photobleaching traces provide valuable information regarding the stoichiometry of receptor and/or ligand binding and accessibility of the binding sites present on a single virion, which can prove to be an impetus for improved vaccine designs based on stabilizing common transition state structures in the S trimer that might better elicit more cross-reactive antibodies. We find that the expected three photobleaching steps are detected with soluble trimers of the Wuhan and Omicron variants when reacted with soluble receptors or half-mAbs; i.e., reflecting 3:1 binding stoichiometry. On virions, a range of receptor binding stoichiometries was detected in 37 °C reactions, with a maxima of 12:1 or 9:1 for the Wuhan or Omicron variants, respectively. The Wuhan strain favored higher stoichiometries. The stoichiometries of binding to labeled half-mAbs were also distributed but with less interstrain variance, showing a maxima of 5:1-6:1 mAb/virion. In either case, such values were far lower than the theoretical number of spike binding sites on a virion. Notably, increasing the reaction temperature to 42 °C decreased receptor binding stoichiometry. Overall, these data indicate that a substantial fraction of S trimers on virions are constrained from binding receptors or certain cognate antibodies, either constitutively and/or due to the liganding of neighboring trimers, while a smaller fraction remains capable of achieving or maintaining a functional configuration.