Flow cytometry assay shows that Nanosota-5-Fc increases ACE2 binding by prototypic spike (A) but decreases ACE2 binding by XBB.1.5 spike (B). A mixture of recombinant ACE2 ectodomain and recombinant Nanosota-5-Fc was incubated with spike-expressing cells, and the binding between the recombinant ACE2 ectodomain and the cell-surface spike was measured using flow cytometry. Nanosota-2, which, like ACE2, only binds to the standing up RBD, replaced the ACE2 ectodomain in (C). PBS buffer was used as a negative control. Comparisons between the negative control and Nanosota-5-Fc for their impact on spike/ACE2 binding were performed using an unpaired two-tailed Student’s t-test. Error bars represent SEM (n = 3). ***p<0.001, **p<0.01, *p<0.05. (D) Post-attachment pseudovirus entry. Pseudoviruses were incubated with cells before adding Nanosota-5-Fc and allowing pseudovirus entry to occur.