PMC:7108609 / 5340-18434 JSONTXT

{"project":"2_test","denotations":[{"id":"18818423-15582697-45167055","span":{"begin":313,"end":317},"obj":"15582697"},{"id":"18818423-15582697-45167056","span":{"begin":1474,"end":1478},"obj":"15582697"},{"id":"T77007","span":{"begin":313,"end":317},"obj":"15582697"},{"id":"T33542","span":{"begin":1474,"end":1478},"obj":"15582697"}],"text":"Results\n\nPreparation of cells coexpressing the A antigen and SARS-CoV S protein for the study of the ACE2/S protein interaction in a cell adhesion assay\nThe interaction between the SARS-CoV spike protein and its cellular receptor ACE2 can be studied using a cell-based assay, as described previously (Chou et al. 2005). In this assay, the viral S protein expressed by transfection into Chinese hamster ovary (CHO) cells mediates adhesion to Vero E6 cells that possess ACE2. CHO cells do not express ABH antigens because of the lack of an α1,2-fucosyltransferase activity and of either the A or B histo-blood group enzymes. In order to obtain cells able to express the A antigen, parental CHO cells were stably transfected successively with the rat Fut2 cDNA and a rat A enzyme cDNA. Unlike mock-transfected cells, these double transfectants strongly express cell surface A antigen as detected by flow cytometry. Transfection of the S protein–EGFP fusion construction (S–EGFP) into these cells allowed the expression of the S protein together with the histo-blood group A antigen (Figure 1A). Observation of the triple transfectants by confocal microscopy revealed that, as expected, the A antigen and the S–EGFP fusion protein partially colocalized at the cell surface (Figure 1B). In addition, western blot analysis revealed that among various A antigen positive glycoproteins, a band at the expected size of the S–EGFP fusion protein, between 210—and 230 kDa (Chou et al. 2005), was present in the extract from the triple transfectant Fut2/A/SP but absent from the double Fut2/A transfectant cell extract. It indicated that the S-protein expressed by A-positive CHO cells carried A histo-blood group epitopes. Specificity of the anti-A labeling was ensured since no band was detected in extracts from CHO Fut2 only transfectants (Figure 1C). A stable A antigen and S-EGFP expressing clone showed significantly higher adhesion to Vero cells than either mock transfectants or the A expressing clone devoid of the S protein (Figure 2A and B). Similar results were obtained after transient transfection of the S-EGFP construct (not shown). The presence of the A and/or H antigens on the S protein expressing cells did not affect adhesion since CHO cells only transfected with the S-EGFP construct, as well as CHO cells transfected with both the S-EGFP and Fut2 cDNAs, adhered to Vero cells at a similar level as the A antigen S protein triple transfectants (Figure 2A). In order to control that the cell adhesion was dependent upon the ACE2/S protein interaction, blocking experiments with either an anti-ACE2 or an anti-S protein were performed. Both antibodies significantly inhibited adhesion, although the anti-ACE2 mAb proved more efficient (Figure 2C).\nFig. 1 CHO cells coexpressing the A histo-blood group antigen and the SARS-CoV S protein. (A) Flow cytometry analysis of the expression of A antigen and the S protein–EFGP construct on CHO mock-transfected cells (mock), double transfectants either with the Fut2 and S protein constructs (Fut2/SP) or the Fut2 and A enzyme cDNAs (Fut2/A), triple transfectants with the Fut2, A enzyme and S protein constructs (Fut2/A/SP). Fluorescence of the S–EFGP molecule was directly recorded on the FL1 channel. Detection of the A antigen was performed using an anti-A mAb followed by Cy5-labeled anti-mouse IgG and recorded on the FL4 channel. (B) Confocal microscopy analysis of the A antigen (A TRITC) and the S protein–EFGP construct coexpression on CHO cells triple transfectants. Detection of the A antigen was performed using an anti-A mAb followed by TRITC-labeled anti-mouse IgG. (C) Western blot analysis of transfected CHO cells glycoproteins. Total protein extracts from CHO Fut2 simple transfectants, CHO Fut2/A double transfectants, and CHO Fut2/A/SP triple transfectants were submitted to SDS–PAGE and Western blotting. Glycoproteins carrying A histo-blood group epitopes were detected with an anti-A mAb. The arrow shows the expected molecular size of the EGFP–SP fusion protein.\nFig. 2 S Protein/ACE2-dependent adhesion of CHO cells to Vero cells. The binding assay between CHO cells and Vero E6 cells was performed as described in Material and methods. Adherent cells were counted under a fluorescence microscope. Cells from a total of 36 fields from 6 wells were counted. (A) The results shown correspond to the mean ± SD of one representative experiment out of four obtained with CHO cells mock transfectants (mock), double transfectants with the Fut2 and A histo-blood group glycosyltransferases (Fut2/A), simple transfectants with the SARS-CoV S protein construct (SP), double transfectants with the Fut2 enzyme and the SARS-CoV S protein construct (Fut2/SP), and triple transfectants with the Fut2, A glycosyltransferases and the SARS-CoV S protein cDNAs (Fut2/A/SP). Adhesion of SP, Fut2/SP, and Fut2/A/SP cells is significantly higher than that of either mock or Fut2/A cells (P \u003c 0.001, Student's t-test). (B) Representative fields illustrating the adhesion of either mock-transfected CHO cells or triple transfectants. (C) Inhibition of the adhesion of triple CHO transfectants to Vero cells by anti-ACE2 or anti-S protein mAbs. The mAbs were added to the CHO cells suspension at 20 and 25 μg/mL, respectively prior to incubation on the Vero cell layer. Adhesion in the presence of the anti-ACE2 and anti-SP are significantly lower than that of control cells (P \u003c 0.001 and P \u003c 0.01, respectively). (D) Inhibition of the adhesion to Vero cells of S protein-transfected CHO cells coexpressing either the H (Fut2/SP) or the A antigen (Fut2/A/SP) by an anti-A mAb or a control isotype matched antibody used at 4 μg/mL. Only the adhesion of the triple transfectants in the presence of the anti-A differs significantly from other conditions (P \u003c 0.01).\n\nInhibition of adhesion by anti-A antibodies\nThe 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).\nFig. 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 \u003c 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 \u003c 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 \u003c 0.05 to P \u003c 0.0001).\n\nModeling the effect of protection by natural anti-A or -B antibodies on the virus transmission in populations\nA total population NTOT = NA + NB + NO + NAB of 106 individuals including one blood group O-infected individual was simulated.\nThree different patterns of transmission of the virus, with different probabilities of transmission, were assumed according to whether some protection by anti-histo-blood group natural antibodies was present (Figure 4). A strong or moderate group effect denotes a strong or moderate protection, respectively, whereas no group effect corresponds to an absence of protection. The impact of group effect was assessed on the number of infected individuals over time in four different populations with very different frequencies of ABO phenotypes (Figure 5). A strong group effect delayed the initiation of the epidemic as well as it decreased importantly the total number of infected individuals whatever the population considered as compared to no group effect and, to a lesser extent, a moderate group effect. Moreover, a strong group effect evidenced different starting times of the epidemic according to the different populations (Figure 5B). The impact of group effect was assessed on the number of infected individuals over time according to their blood groups in the Chinese (Hong Kong) population (Figure 6). The number of blood group O-infected individuals was always the most important one, closely followed by blood group B-, A-, and AB-infected individuals whatever the transmission pattern considered. A strong group effect also delayed the initiation of the epidemic as well as it markedly decreased the total number of infected individuals. Furthermore, a strong group effect also postponed the starting time of the epidemic in blood group O individuals as compared with the other blood groups. The blood group type of the index case in the Hong Kong outbreak was not provided (Cheng et al. 2005). The modeling was first performed using a blood group O index case, as described above. In order to assess whether the results could be affected by the blood group type of this first case, further simulations were performed with different index cases of either the A, B, or AB blood group. The main results were not modified since a strong group effect always delayed the initiation of the epidemic as compared to no group effect and to a moderate group effect. Likewise, it markedly decreased the total number of infected individuals irrespective of the index case's blood group (data not shown).\nFig. 4 Transmission patterns used to model the effect of the ABO polymorphism. In the absence of ABO effect, transmission can occur irrespective of the ABO type (full arrows in all directions). In the presence of a strong ABO effect, transmission occurs strictly according to the rules of transfusion, whereas in the case of a moderate ABO effect, some incompatible transmission can occur (dashed arrows). Determination of the values of the transmission coefficients β, β1, β2 has been done based on the Hong Kong hospital outbreak data, as described in the supplemental material. Transmission coefficients correspond to the transmission rates of the disease for each contact.\nFig. 5 Influence of the ABO polymorphism, with either a moderate group effect (A) or a strong group effect (B) as compared with no group effect, on the number of infected individuals over time in four different populations presenting large differences in the frequencies of ABO phenotypes.\nFig. 6 Influence of different transmission patterns, no group effect (A), moderate group effect (B) or strong group effect (C), on the number of infected individuals over time according to their blood groups in the Chinese (Hong Kong) population."}