Mass Photometry Reveals Distinct ACE2 Binding Stoichiometries across SARS-CoV-2 Omicron Subvariants

Since late 2021, the SARS-CoV-2 Omicron variant has rapidly accumulated mutations in its spike (S) protein, leading to increased transmissibility and immune evasion. The COVID-19 pandemic caused by SARS-CoV-2 infections poses a significant global health challenge. While the binding affinity of Omicron S proteins to host receptor ACE2 has been extensively characterized, the binding stoichiometry across subvariants remains unclear. We used mass photometry (MP) to determine the ACE2 binding stoichiometry to different Omicron S subvariants. MP revealed diverse stoichiometries across subvariants, indicating that S protein mutations modulate ACE2 engagement beyond mere affinity. These findings reveal a nonlinear evolutionary trajectory of ACE2 engagement among Omicron subvariants, underscoring stoichiometry as a key variable in viral adaptation.