Immunofluorescence Assay for ZIKV Vaccine Evaluation

For the immunofluorescence assay, the cells were grown on coverslips and transfected with 5 μg of ZIKV-prME vaccine. Two days after transfection, the cells were fixed with 4% paraformaldehyde for 15 min. Nonspecific binding was then blocked with normal goat serum diluted in PBS at room temperature for 1 h. The slides were then washed in PBS for 5 min and subsequently incubated with sera from immunised mice or RM at a 1:100 dilutions overnight at 4 °C. The slides were washed as described above and incubated with appropriate secondary antibody (goat anti-mouse IgG-AF488; for mouse serum and goat anti-human IgG-AF488 for RM serum; Sigma, St Louis, MO, USA) at 1:200 dilutions at room temperature for 1 h. After washing, Flouroshield mounting media with DAPI (Abcam, Cambridge, MA, USA) was added to stain the nuclei of all cells. After which, coverslips were mounted and the slides were observed under a microscope (EVOS Cell Imaging Systems; Life Technologies).²⁵ . In addition, Vero, SK-N-SH or U87-MB cells were grown on four-chamber tissue culture treated glass slides and infected at MOI of 0.01 with ZIKV-MR766 or PR209 that were preincubated with/without RM immune sera (1:200), and stained at 4 days post ZIKV infection using pan-flavivirus antibody as described.^{17 (link)}