Anti-human ACE2 antibody neutralizes and inhibits virus production of SARS-CoV-2 variants of concern

Figures

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  Figure 1. Generation of 293T cells expressing ACE2 (A) GFP expression in 293T-ACE2 cells. (B and C) 293T parental cells (light blue) and 293T-ACE2 cells (dark blue) were stained with either anti-Flag tag antibody (B) or with an anti-human ACE2 commercial antibody (C). Black histograms represent the background staining of 293T parental cells and gray-filled histograms represents background staining of 293T-ACE2 cells. (D) RBD-Ig staining following incubation with commercial antibody (light blue) or without antibody (dark blue). Representative of 3 experiments.

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  Figure 2. Anti-hACE2.16 antibody recognizes ACE2 and blocks RBD-Ig binding (A and B) Various anti-ACE2 antibodies (antibody number indicated in each histogram) were analyzed by flow cytometry. Staining was performed on 293T-ACE2 cells (dark blue histogram) and 293T-Parental cells (light blue). Black histograms represent the background staining of 293T parental cells and gray-filled histograms represent the background staining of 293T-ACE2 cells. (B) 293T-ACE2 cells were incubated with hybridoma supernatants (dark blue) or without antibody (light blue) and then stained with RBD-Ig. (C) 293T-ACE2 cells were incubated with increasing concentration of hACE2.16 (2-100 ug/mL), or without antibody (w\o ab), followed by RBD-Ig staining. The geometric mean of the fluorescence intensity of the staining is shown on the Y axis. Representative of 3 experiments.

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  Figure 3. hACE2.16 does not affect ACE2 enzymatic and does not leads to ACE2 internalization (A and B) Enzymatic activity of 293T-ACE2 lysates incubated with various hybridoma supernatants (A) or purified hACE2 antibodies (B). The fluorescence levels which indicate ACE2 activity are represented as mean RFU (Relative Fluorescence Unit). Necl.01 (A), anti-NKp46, commercial anti-ACE2 antibody and lysate only (B) were used as controls. Fluorescence (Ex/Em 320/420 nm) was measured in a kinetic mode (0-20 min). (C) FACS analysis of 293T-ACE2 cells incubated with hACE2.16 time 0 on ice (light blue histogram) or after 1/2/4/8/24 h (dark blue histogram). Gray histogram represent the background staining of 293T-ACE2 cells incubated with secondary antibody only. Representative of 3 experiments.

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  Figure 4. hACE2.16 inhibits SARS-CoV-2 VOCs infection and virus production (A) FACS analysis of VERO E6 cells stained with hACE2.16 (purple). Gray histogram represent the background staining of 293T-ACE2 cells. Representative of 3 experiments. (B) RT-qPCR analysis of supernatants obtained from the infected Vero E6 cells incubated with or without increasing concentrations of hACE2.16 antibody. (C) %Neutralization was calculated using the results from B and IC50 (in ug/mL) was calculated from %Neutralization. (D) FACS analysis of Calu-3 cells stained with hACE2.16 (light blue) or with commercial anti-ACE2 antibody (dark blue). Gray histogram represent the background staining of 293T-ACE2 cells incubated with secondary antibody only. Representative of 3 experiments. (E) RT-qPCR analysis of supernatants obtained from the infected Calu-3 cells incubated with or without increasing concentrations of hACE2.16 antibody. (F) % Neutralization was calculated using the results from E and IC50 (in ug/mL) was calculated from %Neutralization. (B and E). Copies/mL were calculated following the subtraction of the initial virus applied. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.0001, Student’s t test as compared to…