Inhibition of adhesion by anti-A antibodies
The effect of anti-A antibodies on the S protein/ACE2 interaction was first tested using a monoclonal anti-A. A clear-cut inhibition of the cell adhesion was observed using the monoclonal antibody 3-3A at 2 μg/mL. Specificity of the inhibition was confirmed since a control irrelevant antibody failed to inhibit and the adhesion to Vero cells of S protein-transfected CHO cells lacking the A antigen was not inhibited by the anti-A mAb (Figure 2D). Vero cells do not express the A histo-blood group antigen. Therefore, the inhibition of adhesion mediated by the anti-A mAb can only result from a binding to CHO S protein expressing cells and not to the glycans of ACE2. In order to assay the ability of natural human anti-A to inhibit cell adhesion, plasma samples from two O blood group individuals with high anti-A titers were selected. The samples were first adsorbed on silica beads conjugated to the A type 2 tetrasaccharide in order to specifically remove the anti-A natural plasma antibodies. Efficacy of the adsorption was controlled by ELISA which showed that the reactivity to the A type 2 tetrasaccharide was almost completely abolished following adsorption (Figure 3B). The A type 2 adsorbed and mock adsorbed plasma samples from the two individuals were then added in the cell-based assay. Both mock adsorbed samples, containing the anti-A as shown in Figure 3B, strongly inhibited the adhesion of A antigen-S protein expressing cells to Vero cells. In both cases, this inhibition was almost completely lost after A type 2 adsorption, showing that it was specifically mediated by anti-A plasma antibodies (Figure 3C). Moreover, the inhibition of adhesion by blood group O plasma was dose-dependent and still detected at a plasma dilution as low as 1/32 (Figure 3D).
Fig. 3  Effect of anti-A antibodies on the interaction between the SARS-CoV S protein and ACE2. (A) The anti-A monoclonal antibody 3-3A was added at the indicated concentrations to the triple transfected CHO cells suspension prior to incubation on the Vero cell layer. An irrelevant IgG1 was used as control at 4 μg/mL. The results are presented as mean cell number per field ±SD of one representative experiment out of two. From 1.0 μg/mL to 4.0 μg/mL anti-A, values are significantly different from those for the control IgG (P < 0.05 and 0.001, respectively). (B) Adsorption of the anti-A natural antibody from human O plasmas. Plasma samples from two individuals were adsorbed on either control silica beads (mock) or A type 2 tetrasaccharide conjugated to silica beads (At2). The postadsorption plasma reactivity on A type 2 conjugated to polyacrylamide was tested by ELISA. Results are shown as O.D. 450 nm values of duplicate wells ±SD for each plasma sample diluted at 1/4. In the absence of A type 2 conjugate, mean O.D. values were 0.13. (C) Inhibition of the adhesion of CHO triple transfected cells to Vero cells by mock adsorbed (mock) or A type 2 (At2) adsorbed human blood group O plasma samples from individuals 1 and 2. Plasma samples were diluted at 1/8 in PBS. Control values were obtained in the absence of plasma. Values for the mock adsorbed plasma were significantly different from the control values (P < 0.001). (D) Inhibition of the adhesion in the cell-based assay as in C by serial dilutions of unadsorbed plasma from individual 1. All values obtained in the presence of plasma were significantly different from the control value (from P < 0.05 to P < 0.0001).