2.7.3. For Zika Studies

Spleens were isolated from IFNAR−/<sup>−</sup> mice 14 days post-final vaccination. Single-cell suspensions of splenocytes were made by homogenizing and processing the spleens through a 40-μm cell strainer. Cells were then re-suspended in ACK Lysing buffer (GibcoTM) for 5 min to lyse red blood cells before two washes with PBS and final re-suspension in RPMI complete media (RPMI 1640 + 10% FBS + 1% penicillin–streptomycin). Two hundred thousand splenocytes were added to each well and stimulated overnight at 37 ◦C in 5% CO2 with R10 (negative control), concanavalin A (3 μg/mL; positive control), or 15-mer Zika peptides overlapping by 9 amino acids spanning the length of the Zika prME protein. The Zika prME peptides were pooled at a concentration of 1 mg/mL/peptide into six pools as antigens for specific stimulation of IFN-γ release.

After 18 h of stimulation, the plates were washed and developed following manufacturer's protocol. The plates were then rinsed with distilled water and dried at room temperature overnight. Spots were counted by an automated ELISpot reader (Cellular Technology Ltd., Shaker Heights, OH, USA).
