*2.10. Cell-Mediated Immune Responses in Mice*

One week after the booster immunization in batch one, three mice from each group were randomly selected and euthanized. Their spleens were harvested into a tissue culture dish, and each spleen was roughly minced and pressed through a 5-mL syringe. The cells were filtered through a 40-μm filter (BD Falcon 40-μm strainer) and centrifuged at 2000 rpm for 10 min at the room temperature. The cells were processed by resuspension in a red blood cell lysis buffer and centrifuged at 2000 rpm for 10 min at twice the room temperature. The splenocytes were harvested and washed with RPMI 1640 medium (Gibco, San Diego, CA, USA) containing 10% FBS (Gibco, San Diego, CA, USA). Then, the splenocytes were cultured in RPMI 1640 medium containing 10% FBS and stimulated with or without the purified SUDV-GP antigen (10 μg/mL). Following incubation at 37 ◦C in 5% CO2 for 48 h, the frequencies of splenocytes producing IFN-γ or IL-4 were measured using mouse ELISpot kits (Mabtech AB, Stockholm, Sweden) according to the manufacturer's instructions. Spot-forming cells (SFCs) were enumerated by an automated ELISpot reader (AID ELISPOT reader-iSpot, Germany).

The levels of cytokines in the supernatant of stimulated splenocytes were detected by commercial ELISA. Splenocytes were stimulated as described above and were then incubated for 48 h. The cell culture supernatant was collected by centrifugation at 3000 rpm for 15 min, and the manufacturer directions were followed to detect IL-2, IL-4, IL-10, IFN-γ, or TNF-α by ELISA kits (Mabtech AB, Stockholm, Sweden).

#### *2.11. SUDV VLPs Induce Activation of B cells*

Inguinal lymph node samples were harvested from batch two vaccinated mice 7 days after the primary immunization. Lymphoid cells were harvested into a tissue culture dish, and were roughly minced and pressed through a 5 mL syringe. The cells were filtered through a 40-μm filter (BD Falcon 40-μm strainer). Then, prepared in PBS with 2% FBS and were stained with equal volumes of 1:250 dilutions of anti-CD19-APC and anti-CD40-FITC antibodies (BD Biosciences, Franklin, VA, USA) for 30 min at 4 ◦C; the labeled B cells were then washed twice with PBS with 2% FBS and analyzed using a FACSAria TM Cell Sorter (BD Biosciences, Franklin, VA, USA).
