3.1. The binding of S glycoprotein homotrimers to hACE2-Fc and
mvACE2-Fc
To identify potential differences in the binding affinity of S
glycoproteins to hACE2-Fc or mvACE2-Fc glycoproteins, we performed
bio-layer interferometry (BLI) assays. We determined the equilibrium
dissociation constants (Kd) for trimeric S stabilized by 2 proline
substitutions (K986P and V987P)35 and harboring either
the human (SD614G) or American mink-associated
mutations (SD614G+Y453F or
SD614G+Δ69-70+Y453F) from immobilized dimeric hACE2-Fc
or mvACE2-Fc receptors. The SD614G was chosen as it
represents the predominant SARS-CoV-2 viruses circulating in humans when
the variants infecting American mink emerged. The trimeric S
glycoproteins SD614G, SD614G+Y453F,
and SD614G+Δ69-70+Y453F all bound to dimeric hACE2-Fc
receptor with similar affinity, and no statistically significant
differences were observed (Fig. 1). However, the
SD614G displayed a 5-fold reduction in affinity when
the hACE2-Fc was substituted with mvACE2-Fc in the assay. The affinity
of S for mvACE2-Fc was restored to SD614G-hACE2-Fc
levels by the Y453F mutation in the spike, as both
SD614G+Y453F and SD614G+Δ69-70+Y453Fshowed a >4-fold increase of affinity to mvACE2-Fc,
compared to SD614G (p<0.0001). The Δ69-70
deletion in NTD didn’t further increase the binding affinity to
mvACE2-Fc beyond the effect of the Y453F RBD mutation (Fig. 1).