2.3. Mouse immunization

Female BALB/c mice (aged 6 weeks; n = 6) were vaccinated three times at three-week intervals via the intranasal, intradermal, or intramuscular route, as shown in Fig. 1B. Ten μg of RBD was administered per mouse with or without 100 μg of alum via intranasal route under light isoflurane anesthesia, via microneedle-intradermal injection in the back after shaving, or through intramuscular injection in a right hindlimb. Each animal in the control groups (PBS groups and alum groups) received 100 μg of alum or the same volume of PBS. The volumes for injected (intradermal or intramuscular injection) and mucosal administration (intranasal inoculation) were adjusted to 50 μL and 25 μL per dose respectively. The study protocol was approved by the Institutional Animal Ethics Committee of Foshan Huamio Biotechnology Company Ltd.

Characterization of recombinant RBD and immunization schedule of mice. (A) Analysis of purified RBD. Rabbit anti-RBD polyclonal antibody was used for RBD detection. Left panel: SDS-PAGE of purified RBD. Right panel: Western blot of purified RBD. (B) Schematic of the vaccination schedule of mice. They were immunized three times at intervals of 3 weeks through intranasal immunization (i.n.), intradermal immunization (i.d.), or intramuscular immunization (i.m.). Blood samples were collected before each boost immunization and before sacrifice. Vaginal washes were collected for 3 consecutive days before sacrifice. Samples of spleens, nasal washes, bronchoalveolar lavage fluids, and intestines were collected at the time of sacrifice.