Enhanced sACE2-Fc with two single mutations exhibited broad-spectrum neutralization of SARS-CoV-2 variants.

a Schematic representation of sACE2-Fc structure (upper) and neutralization assay setup (lower). Key amino acid positions (90-92 and 374-378) involved in glycosylation and zinc binding are highlighted. Red stars mark the positions of mutations in the sACE2-Fc mutant B5-D3. SP, signal peptide; CLD, collectrin-like domain; hIgG1, human IgG1. b Comparative bar graph showing the half-maximal inhibitory concentration (IC50) values for neutralization of Wuhan-Hu-1 and D614G pseudovirues by WT sACE2-Fc and mutants (B2 to B6, A2, A3, D1 to D5, and B5-derivatives). The red arrow emphasizes the superior performance of the B5-D3 mutant. Enzymatic activity of each construct is plotted on the right axis. c List of pseudoviruses carrying spikes from different SARS-CoV-2 variants tested, categorized by the World Health Organization (WHO) into VOCs and VOIs. d Graph displaying IC50 values of WT sACE2-Fc, B5, and B5-D3/4/5 mutants against various SARS-CoV-2 VOCs and VOIs in neutralization assays. e Schematics of the plaque-reduction neutralization tests (PRNTs) process (upper) and the resulting IC50 values for B5-D3, Casirivimab, and hIgG1 against authentic SARS-CoV-2 (lower). f, g Dose-response curves depicting the neutralization efficacy of B5-D3 (orange), Casirivimab (purple), and hIgG1 (grey) in PRNTs against authentic SARS-CoV-2 Wuhan-Hu-1 and Delta strains (f), and Omicron sub-lineages (g). Data are presented as mean ± standard deviation (SD) from duplicate experiments.

Enhanced survival and reduced infection in K18-hACE2 mice through intranasal prophylaxis with B5-D3 against SARS-CoV-2.

a–e Female K18-hACE2 mice, aged 10 to 12 months, were inoculated with 1 × 104 PFU of SARS-CoV-2 (Wuhan-Hu-1 strain). Mice were treated with B5-D3 6 h prior (–6 h) via intranasal (IN, red) or intravenous (IV, green) routes, or 24 h post-infection (+24 h, blue) via IV (n = 5 each). IN PBS administered 6 h prior to viral challenge served as the vehicle control (black; n = 5), and PBS alone was used for mock control (grey; n = 4) (a). Body weight and survival were monitored over 14 days (b, c). One mouse from each group was sacrificed at 4 dpi for analysis of viral titers in lung homogenates using a median tissue culture infectious dose (TCID50) assay (d). IHC staining for N protein and H&E staining were performed on lung tissues collected at 4 dpi (e). Black arrows indicate alveolar thickening, and yellow arrows show leukocyte infiltration. Scale bar = 100 μm. ND, not detected; LOD, limit of detection. f–i Young female K18-hACE2 mice, aged 2 to 3 months, were inoculated similarly and treated with B5-D3 via IN route at 24 h before (–24 h, pink), 6 h before (–6 h, red), or 24 h after (+24 h, orange) the viral challenge (n = 5). Mice receiving IN PBS 6 h before infection served as the vehicle control (black), with mock control mice receiving PBS alone (grey) (f). Body weight (g) and survival (h) were recorded for 14 days. Neutralizing antibody titers against Wuhan-Hu-1 in serum samples from surviving mice at 14 dpi were determined using Vero E6 cells (i). nAb, neutralizing antibody. Data are presented as the geometric mean ± geometric SD. Statistical significance was determined using Dunn’s multiple comparisons test.

Efficient viral clearance at early stages through intranasal prophylaxis with B5-D3 against SARS-CoV-2 challenge in K18-hACE2 mice.

a Workflow diagram showing timelines and treatments for different mouse groups. Young female K18-hACE2 mice aged 2 to 3 months received prophylactic administration of PBS (black), B5-D3 (red), or B5-D3-LALA (purple) via the IN route 6 h prior to inoculation with 1 × 104 PFU of Wuhan-Hu-1. Mice inoculated with PBS instead of the virus served as mock controls (grey). Mice from each treatment group were sacrificed for tissue collection at 1, 2, and 4 dpi (n = 3 per time point). b Quantitative PCR results showing relative amounts of S (upper) and N (lower) viral RNA in lung tissues collected from different groups at 1, 2, and 4 dpi, normalized to mouse Gapdh (a). c The titers of infectious viruses detected in lung homogenates, measured by TCID50 assays at 1, 2, and 4 dpi. d, e Fixed lung tissues were sectioned and stained; IHC for viral N protein (d) and H&E staining for tissue damage (e) are shown (scale bar = 100 μm). Data presented as mean ± standard error of the mean (SEM). Statistical significance was determined by Tukey’s multiple comparisons test.

Transcriptomic analysis revealed early immune activation in IN B5-D3-prophylaxis mouse group after SARS-CoV-2 challenge.

a–d DGE analysis comparing PBS (a), B5-D3 (c), and B5-D3-LALA (d) against the mock control at specific time points (n = 3). Volcano plots illustrate the gene expression changes (a, c, d), while red and blue dots represent significantly upregulated and downregulated genes, respectively, with |log2 fold change (log2FC)| ≥ 1 and a false discovery rate (FDR) < 0.05. Bar chart in b shows the enrichment of GOBP “response to virus” observed in PBS groups at 1, 2, 4 dpi, in which adjusted p values are indicated for individual comparisons. e–g Comparison between IN B5-D3 and PBS group at 1 dpi. Volcano plot illustrates the DGE analysis between IN B5-D3 to PBS group at 1 dpi (e), with red and blue dots representing significantly upregulated and downregulated genes, respectively, with |log2FC| ≥ 1 and FDR < 0.05. GSEA shows top 15 significantly activated GOBPs (f) and KEGG pathways (g) in IN B5-D3 compared to PBS group at 1 dpi. NES, normalized enrichment score; p.adj, adjusted p value. h–j GSEA plots of chemotaxis (h), Rap1 signaling pathway (i), and Th1 and Th2 cell differentiation in B5-D3 vs PBS comparison at 1 dpi. k, l Heatmaps show NES of GSEA comparing various treatments to the mock control (k) and between B5-D3 to PBS (l), focusing on top 10 GOBPs highlighted in f and Supplementary Figure 9c, d, respectively, and those related to immune cell chemotaxis. Significant NES values (p < 0.05, FDR < 0.25) were highlighted in yellow. Benjamin– Hochberg method was used for FDR adjustment.

In vivo bio-distribution of B5-D3 after IN administration.

a Schematic workflow of in vivo and ex vivo imaging. Female K18-hACE2 mice aged 2 to 3 months received IN administration of fluorescently labeled B5-D3 (B5-D3-AF750) and was visualized at different time points. b Representative whole-body images of control and treated mice at 5 min, 1 h, and 24 h after B5-D3-AF750 administration, showing the signal captured by in vivo imaging (left). White circles indicate regions of interest (ROIs) for quantification of fluorescence signals in the nasal cavities. Average (Avg) Radiance measured at all time points are shown on the right. c Ex vivo images of tissues from control and treated mice sacrificed at indicated time points after B5-D3-AF750 administration. Blue circles indicate ROIs for signal quantification. Br, brain; NC, nasal cavity; T, trachea; Lu, lung; H, heart; Lv, liver; S, spleen; K, kidney; UB, urinary bladder; Bl, blood; Ur, urine. d Avg Radiance shows the fluorescence signals in excised tissues measured ex vivo. e Schematic workflow for BALF analysis. Female K18-hACE2 mice aged 2 to 3 months received IN administration of B5-D3-AF750 (n = 3) or PBS (n = 4) and were sacrificed at 6 h later for collection of BALF cells. f Percentage of CD45+ cells in live BALF cells. g Positive rates (left) and histograms (right) of B5-D3 binding/uptake in CD45+ BALF cells. Histograms show B5-D3-AF750 fluorescence intensities in CD45+ BALF cells from individual mice. h Frequency of individual immune cell types in CD45+B5-D3+ BALF cells. Red arrows point out AM and Mono-Mac with high abundance. AM, alveolar macrophage; Mono-Mac, monocyte-derived macrophage; cDC1/2, type 1 or 2 conventional dendritic cells. i, j Positive rates (left) and histograms (right) of B5-D3 binding/uptake in CD11c+Siglec-F+ AMs (i) and CD11b-F4/80+ mono-Macs (j). k Median fluorescence intensity (MFI) of AF750 indicate B5-D3 binding/uptake in different CD45+B5-D3+ populations. l Confocal images (scale bar = 50 μm) of BALF cells collected at 6 h and stained for sACE2-Fc (red, Abcam, ab98596), Siglec-F (green, BD #564514), and nuclei (blue, Hoechst). Magnified views are shown in white rectangles. Data are presented as mean ± SEM, and statistical significance was determined by Tukey’s multiple comparisons test or Student’s t-test.

B5-D3 enhanced phagocytosis and degradation of SARS-CoV-2 pseudovirus in THP-1-derived macrophages.

a Immunostaining of p24 (Invitrogen #PA5-81773), sACE2-Fc (Abcam #ab98596), and LAMP1 (Abcam #ab25630) in THP-1-differentiated M0 macrophages showing phagocytosis of SARS-CoV-2 pseudovirus (p24+) after 6 h of incubation with or without sACE2-Fc (scale bar = 50 µm). LAMP1 was stained to identify lysosomes. b Quantification of p24 signal intensity as shown in a. Intensity Density (IntDen) per cell number indicates the mean p24 signal per cell, calculated using ImageJ. Each dot represents one image. c Manders’ coefficient indicating the colocalization of p24 and LAMP1 in THP-1 M0 macrophages as shown in a. d Immunostaining of p24, sACE2-Fc, and LAMP1 in hACE2-Calu-3 cells after 6 h incubation with pseudovirus, with or without B5-D3 (scale bar = 50 µm). e Quantification of mean p24 signal intensity as shown in d. f Manders’ coefficient for the colocalization of p24 and LAMP1 in hACE2-Calu-3 cells, as shown in d. g Quantification of pseudovirus infection in THP-1, M0 macrophages, M1 macrophages, hACE2-Calu-3, and hACE2-293T cells, in the presence or absence of sACE2-Fc. Results shown were luciferase activities measured at 2 days post-transduction. h Immunoblot staining to detect SARS-CoV-2 spike cleavage after cell entry. M0 macrophages, M1 macrophages, and hACE2-293T cells were incubated with pseudovirus for 6 h, with or without sACE2-Fc, before protein extraction. Data are presented as mean ± SEM, and statistical significance was determined by Tukey’s multiple comparisons test.

Proposed mechanisms of action of IN sACE2-Fc decoy in preventing SARS-CoV-2 infection.

a, b Schematics illustrating the actions and outcomes of SARS-CoV-2 infection, in the absence (a) and presence (b) of IN delivered sACE2-Fc decoys. The figure was created with BioRender.com.