FIG 5

CEACAM5-specific antibody blocks MERS-CoV entry and replication. (A) The antibody blocking assay was performed in Calu3 cells using MERS-S-pseudotyped virus. Antibodies were diluted to 2 μg/ml and incubated with Calu3 cells for 1 h at 37°C. The pseudotyped viruses were then added at a ratio of 100 LP per cell for 1 h. Luciferase activity was determined at 48 h postinfection and was normalized to that of the mock-treated cells. (B) The antibody blocking assay was performed in Calu3 cells using MERS-CoV. Calu3 cells were preincubated with antibodies at the indicated concentrations for 1 h at 37°C. The cells were then inoculated with MERS-CoV at an MOI of 1 for 1 h at 37°C in the presence of the antibodies. After 1 h, the cells were washed and harvested. MERS-CoV entry was assessed by qPCR, and the result was normalized to that of the mock-treated cells. (C) Calu3 cells were treated with CEACAM5 antibody for a total of 2 h during preincubation and virus inoculation (−1 to 1) or after virus inoculation (1 to 3). MERS-CoV entry was assessed by qPCR. (D) The antibody blocking assay was performed in Caco2 cells. (E and F) The impact of CEACAM5 inhibition on MERS-CoV replication was investigated in Huh7 cells. Huh7 cells were preincubated with antibodies at 2 μg/ml for 1 h at 37°C. The cells were then inoculated with MERS-CoV at an MOI of 0.0005 for 1 h at 37°C in the presence of the antibodies. At the end of the inoculation period, the inoculum was replaced with culture medium containing the indicated antibodies. Cell lysates (E) and supernatants (F) were harvested at 1, 24, and 48 h postinfection. The virus genome copy number was determined with qPCR. ND, virus was not detected. The data are represented as means and standard deviations of the results of three independent experiments. The results for the anti-DPP4-, anti-CEACAM5-, and control IgG-treated samples were compared with those for the mock-treated samples. Statistical analyses were carried out using Student's t test. Statistical significance is indicated by the asterisks (P < 0.05).