*3.6. SUDV VLPs-Enhanced Splenocytes Cytokine Secretion*

To further investigate the antigen-specific cellular immune responses induced by SUDV VLPs, cytokines secreted by splenocytes were measured by commercial ELISA kits. Splenocytes were harvested from mice one week after the booster immunization and stimulated with Prokaryotic-expressed SUDV GP (158~368 aa). Cytokines secreted into the supernatant by splenocytes were assessed. Type 1 cytokines such as IL-2 (Figure 6A), IFN-γ (Figure 6B), and TNF-α (Figure 6C) were significantly higher in the group immunized with 20 μg SUDV VLPs mixed with an ISA 201 adjuvant than they were in the PBS and ISA 201 adjuvant group. Type 2 cytokines, such as IL-4 (Figure 6D) and IL-10 (Figure 6E), showed the same trend. These results indicate that 20 μg SUDV VLPs mixed with an ISA 201 adjuvant potently enhance cellular immune response in mice.

**Figure 6.** Detection of cytokine secretion in splenocytes. Splenocytes were collected from mice one week after following the booster immunization and were stimulated with purified-prokaryotic expressed SUDV GP protein for 48 h. The level of IL-2 (**A**), IFN-γ (**B**), TNF-α (**C**), IL-4 (**D**), and IL-10 (**E**) in the supernatant were measured with commercial ELISA kits (*n* = 3). Data are shown as the means ± SDs and were analyzed by using Tukey's multiple comparison test (\* *p* < 0.05, \*\* *p* < 0.01, \*\*\* *p* < 0.001).

#### *3.7. Enhancing E*ff*ects of SUDV VLP on B Cell Activation*

To study whether the vaccine can induce B cell activation, inguinal lymph nodes were collected from the mice vaccinated with PBS, ISA 201 adjuvant and 20 μg SUDV VLPs mixed with ISA 201 adjuvant. The result showed (Figure 7) that the percentage of activated B cells (CD19+CD40+) was significantly higher in the 20 μg SUDV VLPs mixed with ISA 201 adjuvant group than in the PBS group and ISA 201 adjuvant group.

**Figure 7.** Activation of B cell in BALB/c mice. At seven days after the primary immunization, inguinal lymph nodes were collected from the mice treated with PBS, ISA 201 adjuvant and 20 μg SUDV VLPs mixed with ISA 201 adjuvant, and B cell activation was analyzed by staining with anti-CD19-APC and anti-CD40-FITC antibodies. Data are shown as the means ± SDs and were analyzed by using Tukey's multiple comparison test (\*\*\* *p* < 0.001).
