Fig 2: FXa cleavage reduces the binding between S protein and ACE2.a The binding between ACE2 with S protein or FXa-pre-treated S protein was measured by ELISA (n = 9 biologically independent samples). b The binding between S protein or FXa-pre-treated S protein and ACE2 expressed on A549 human lung cancer cells was measured by flow cytometry (left, representative flow cytometry histogram; right, summary data) (n = 4 independent experiments). c The binding of FXa with S protein, S protein–ACE2 complex, or PBS control was measured by ELISA (n = 9 biologically independent samples). d The binding of membrane-bound (mb) FXa with S protein or the binding of mb FXa with S protein–ACE2 complex on 293 T cells was measured by flow cytometry (left, representative flow cytometry histogram; right, summary data) (n = 3 independent experiments). PBS served as control for S protein and the S protein–ACE2 complex. e The effect of RIVA or FONDA on the binding of FXa with S protein was measured by ELISA (n = 3 independent experiments). f, g Infectivity of VSV-SARS-CoV-2 in MA104 cells that were pre-treated or not treated with FXa in the presence or absence of RIVA or FONDA was examined with fluorescent microscopy (f) and flow cytometry (g, n = 3 biologically independent samples). h S protein cleavage by FXa was examined by immunoblotting 3 hours after their incubation in the presence or absence of RIVA or FONDA, using an anti-S protein antibody (40591-T62, Sino Biological). i, j FXa pre-treated or not treated with RIVA or FONDA was incubated with S protein; then the binding capability of those S proteins with ACE2 coated on a plate (i, n = 9 biologically independent samples) or ACE2 expressed on 293 T cells (j) was assessed. k Infectivity of live SARS-CoV-2 in A549-ACE2 cells pre-treated or not treated with FXa in the presence or absence of RIVA or FONDA was examined using an immuno-plaque assay (n = 3 independent experiments). a, c, i, the top and the bottom bound of box are 75th percentile and 25th percentile, respectively; horizontal bar indicates median; and whiskers indicate data ranges. Experiments in h and j are representative of three independent experiments with similar data. For all panels, data are presented as mean values ± SD, and statistical analyses were performed by one-way ANOVA models. MFI data were log2 transformed before running the statistical models. Source data are provided as a Source Data file.

Fig 3: FXa suppresses viral entry by binding to and cleaving the SARS-CoV-2 S protein.a The binding affinity of FXa with full-length wild-type S protein, subunit S1, subunit S2, or RBD was quantified by ELISA (n = 9 biologically independent samples). Bounds of box is from 25% percentile to 75% percentile, horizontal bar indicates median, and whiskers indicate data ranges. b The binding affinity of FXa to VSV-SARS-CoV-2 viral particles was quantified by ELISA (n = 3 biologically independent samples). c The interaction between FXa protein and full-length S protein was examined with a pull-down assay. d The binding affinity at indicated concentrations of FXa was measured by ELISA (n = 3 biologically independent samples). e The cleavage of S protein by furin, TMPRSS2, and FXa after 3-hour incubation was analyzed by immunoblotting using an anti-S protein antibody (40591-T62, Sino Biological). f S protein was cleaved by FXa, followed by immunoblotting with an anti-RBD antibody (MAB10540-100, R&D) (left) and an anti-S2 antibody (MA5-35946, Invitrogen) (right). g The cleavage of VSV-SARS-CoV-2 by FXa or furin was analyzed by immunoblotting using an anti-S protein antibody (40591-T62, Sino Biological). The larger size of S protein from VSV-SARS-CoV-2 (~200 kD) compared to recombinant S protein (~150 kD) may be due to the glycosylation of the former. All data are representative of at least three independent experiments. a, b, d, data are presented as mean values ± SD. Statistical analyses were performed by two-sided Student’s t test (b) or one-way ANOVA models (a). Source data are provided as a Source Data file.

Fig 4: Experimental validation of codon optimization of the S gene. a) The distribution of CAI values of human genes and the partially or fully optimized codons for the S gene of SARS-CoV-2. For the S gene of BA.2.12.1, BA.4/5, and XBB.1.5 variants in the Omicron lineage, s1 represents the native viral CDS sequence, s2, s3, s4 and BA.4/5-s5 represent the sequences with different CAI optimization levels, respectively, BA.2.12.1-s5, BA.4/5-s6, and XBB.1.5-s5 represent the fully optimized CDS sequence that had a CAI value of 1. b) Western blotting analysis of S protein expression in HEK293T cells. The bands of full-length and cleaved S protein are labeled. All the samples were probed using polyclonal rabbit anti-SARS-CoV-2 S antibody (40591-T62, Sino Biological) at a dilution of 1:2000. c) Correlation analysis between CAI value and S protein expression level. Experiments were repeated three times, error bars indicate stand error of the mean.