*2.7. Animal Immunizations*

A total of two batches of BALB/c mice (6 weeks old, female) were purchased from the Changchun Yisi Laboratory Animal Technology Co., Ltd. (Changchun, China) and immunized. In batch one, 24 mice were randomized into four groups (*n* = 6 per group) and were vaccinated intramuscularly. Mice in group one were vaccinated with PBS, mice in group two were vaccinated with Montanide ISA 201 VG (ISA 201) adjuvant (Seppic, Paris, France), mice in group three were vaccinated with 20 μg of SUDV VLPs-only, and mice in group four were vaccinated with 20 μg of SUDV VLPs mixed with an equal volume of Montanide ISA 201 VG (ISA 201) adjuvant, and all groups were vaccinated twice at 3-week intervals (Figure 5A). The mouse sera were collected at two-weeks after every immunization. One week after the booster immunization, splenocytes from 3 mice of each group were isolated. In batch two, nine mice were randomly distributed into three groups (*n* = 3 per group) (PBS group, ISA 201 adjuvant, 20 μg of SUDV VLPs mixed with an equal volume of ISA 201 adjuvant) and were vaccinated intramuscularly. One week after the primary immunization, the inguinal lymph nodes were collected from 3 mice.

Two healthy male horses (numbered #392 and #18), 2–6 years old, 400–500 kg in weight and without detectable antibodies against SUDV detected by indirect ELISA, were supplied by Red Hill Military horse farm. The horses were multipoint injected subcutaneously in the rear area with 1.0, 2.0, 3.0, 3.0, or 4.0 mg of purified SUDV-VLPs for a total of 5 times, primary immunization mixed with an equal volume of Freund's incomplete adjuvant (Thermo Scientific, Rockford, IL, USA) and booster immunization mixed with an equal volume of Freund's complete adjuvant, maximum immune volume does not exceed 4 mL, with boosting at two week intervals (Figure 5B). The horse sera were collected before each immunization and stored at −20 ◦C for further studies.
