*3.4. SUDV VLPs Elicit Antibody Responses in Vaccinated Mice*

To detect a SUDV-VLP-induced humoral immune response in vaccinated BALB/c mice, indirect ELISA and virus neutralization assay were performed to evaluate the production of SUDV GP-specific antibodies and neutralizing antibodies. At the fifth week of the immunization, the sera from mice treated with 20 μg SUDV VLPs mixed with an ISA 201 adjuvant group had high titers of SUDV specific IgG antibodies with endpoint titers up to 1:81,920 (Figure 4C), virus-neutralizing antibody titers averaged 1:320 and individual mice neutralizing antibody titers were up to 1:640 (Figure 4D), showing significant divergence when compared to the PBS group and ISA 201 adjuvant group. But the SUDV specific IgG antibodies and neutralizing antibodies were not detected in the VLPs-only group mice. These results demonstrate that SUDV VLPs at 20 μg antigen dose cannot stimulate the production of antibodies in mice, but this antigen dose mixed with ISA201 adjuvant can stimulate mice to produce specific IgG antibodies and neutralizing antibodies.

**Figure 4.** Mice and horse immunization procedure, analysis of SUDV GP-specific antibody, and neutralizing antibodies in vaccinated mice. The immunizations of mice (**A**) or horses (**B**). Analysis of SUDV-VLP-induced specific IgG antibody response by indirect ELISA at two weeks after every immunization of vaccinated mice (**C**). SUDV neutralizing antibody titers are detected in immunized mice at two weeks after booster immunization (**D**). Limit of detection (LOD) means the minimum concentration or content that can be detected under the determined experimental conditions. Error bars represent the standard deviation. The *p-*values were determined according to a Tukey's multiple comparison test (\* *p* < 0.05, \*\*\* *p* < 0.001).
