Complete dmem

MB49 murine urothelial tumour cell was obtained from Millipore Corporation (Millipore, MD, USA) and B16F10 melanoma cell line was purchased from ATCC. MB49 and B16F10 cells were placed in complete DMEM (GE Healthcare Life Sciences, Pittsburgh, PA, USA) and RPMI‐1640 medium (Gibco; Thermo Fisher Science, Massachusetts, USA) medium containing 10% fetal bovine serum (FBS; Gibco; Thermo Fisher Science, Massachusetts, USA), 1% penicillin/streptomycin at 37°C in a 5% CO₂ humidified incubator. Luciferase‐expressing (MB49‐luc) was generated by transfection with lentiviral vectors (Genomeditech, Shanghai, China) encoding for firefly luciferase. All cells were tested for Mycoplasma (MycoAlert Mycoplasma Detection Kit, Lonza Group LTD) and retested prior to use.

DC2.4 cells were provided by K. Rock, University of Massachusetts Medical School, Worcester, MA, and were cultured in complete RPMI-1640 medium (GE Healthcare Life Sciences; supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin). MC-38 cells were cultured in complete DMEM (GE Healthcare Life Sciences; supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100 μg/mL streptomycin). T cells and splenocytes were cultured in complete RPMI with 20 mM Hepes, 1 mM sodium pyruvate, 0.05 mM β-mercaptoethanol, and 1× nonessential amino acids. All cells were maintained at 37 °C and 5% CO₂.

Neutralising activity of respective groups of pooled mouse sera was tested in triplicates and analysed by immunofluorescence‐based cell infection assay in HEK 293T cells. ZIKV was mixed with diluted serum to obtain an MOI of 10. This virus‐serum mix was incubated for 2 h at 37°C with gentle agitation at 350 rpm. Virus‐serum mixtures were then added to HEK 293T cells seeded in 96‐well plates and incubated for 2 h at 37°C. The mixture overlay was removed, and cells were replenished with complete DMEM (GE Healthcare Life Sciences), and incubated for 48 h at 37°C before staining with live/dead dye (Life Technologies). Cells were then fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) followed by permeabilisation with staining buffer (3% BSA, 5% FBS, 0.1% PBST, 0.1% Triton X 100). Cells were stained with ZIKV NS3 protein‐specific rabbit polyclonal antibody⁶⁸ for 1 h, followed by staining with a fluorophore‐tagged secondary goat anti‐rabbit IgG (H+L) antibody (Life Technologies) for 1 h before acquisition with MACSquant Analyser 10 (Miltenyi Biotec, Bergisch Gladbach, Germany) with MACSQuantify™ software.