Fig. 5

Neutralizing activity of the Ab induced by HBD 2-conjugated Ag injection against the binding of S RBD to MERS-CoV receptor-expressing cells. (a, b) Serum neutralizing activity and (c) in vitro inhibitory activity against MERS-CoV infection were determined by Ab-mediated receptor-binding inhibition assay. (a) Huh-7 cells were treated with S RBD that had been incubated with 50-fold diluted sera drawn from mice immunized with the indicated Ag. A specific monoclonal Ab against S RBD was used for indirect immunofluorescence, and slides were analyzed by confocal microscopy. Nuclei were stained with DAPI (blue), whereas the S RBD signal was stained with an Alexa Fluor 488-conjugated secondary Ab (green, arrows). Representative fields were observed at 200× magnitude, and the scale bar represents 10 μm. (b) Vero E6 cells were treated with S RBD alone (red line in upper left panel) or with S RBD that had been incubated with 50-fold-diluted sera drawn from mice immunized with either PBS (green line in upper right panel), S RBD alone (sky-blue line in lower left panel), or HBD 2-conjugated S RBD (blue line in lower right panel). Grey line in upper left panel represents the control Ab treatment result. (c) Viral upE gene transcript level in Vero E6 cells 24 h after treatment with MERS-CoV (10³ PFU) that had been incubated with 100-fold diluted sera drawn from mice immunized with the indicated Ag. qRT-PCR was performed as described in Materials and Methods. Values are expressed as relative levels using the value normalized to the expression level of the internal control gene (Vero E6 β-actin). The relative quantification level using the value of PBS-treated control cells as a basal reference level for comparison is shown as the mean ± SD. * p < 0.05 and ** p < 0.01 indicate significant differences between groups