BPS2026 – Identifying key interactions of monoclonal antibodies that target the HuCoV-HKU1 spike using cryo-EM

Endemic human coronaviruses (EhCoVs) cause seasonal mild respiratory infections accounting for up to 30% of common cold cases during “flu season” each year. While extensive research has focused on pandemic CoVs, like SARS-CoV-2, EhCoVs—EhCoV-229E, EhCoV-NL63, EhCoV-OC43, and EhCoV-HKU1—remain understudied, and no licensed therapies or vaccines are currently available. Thus, to inform therapeutic and vaccine design, we investigated the antigenicity of EhCoV-HKU1 by characterizing key interactions between the EhCoV-HKU1 Spike (S) and three monoclonal antibodies (mAb) identified from HKU1-positive convalescent human sera. The three mAbs, H501-008, H501-018, and H501-022, recognize the S1 C-terminal domain (S1-CTD), where the TMPRSS2 receptor binding domain resides. Our structural and functional analyses revealed three distinct epitopes: (1) the most potently neutralizing (IC50 = 0.009 μg/mL) mAb, H501-022, engages the same interface used by S1-CTD to bind TMPRSS2, thereby blocking receptor engagement. (2) Potently neutralizing (IC50 = 0.05 μg/mL) mAb H501-008 cross-reacts with HuCoV-OC43 but does not neutralize HuCoV-OC43; H501-008 approaches S1-CTD from the top and binds in an epitope outside of the TMPRSS2 binding site. (3) Lesser neutralizing (IC50 = 0.28 μg/mL) EhCoV-HKU1 type-specific mAb H501-018 approaches S1-CTD from the bottom and binds a non-overlapping epitope outside of the TMPRSS2 binding side. Notably, H501-018 recognizes S1-CTD in both “up” and “down” conformational states, whereas H501-022 and H501-008 bind exclusively to the “up” S1-CTD conformation. Together, these mAbs represent the first type-specific EhCoV-HKU1 mAbs isolated. Our findings provide molecular insight into EhCoV-HKU1 antibody recognition and neutralization mechanisms, importantly highlighting antigenic differences comparing EhCoV and pandemic CoVs—a critical step to advancing universal CoV vaccine design.