Fig. 5. The designed subunit vaccines induced SARS-CoV-specific immune responses and protected K18-hACE2 mice against SARS-CoV infection.
K18-hACE2 mice were immunized with S-6P, S-6P-Delta-RBD, S-6P-BA5-RBD, S-6P-WT-RBD, or S-6P-no-RBD, the cocktail (combination of S-6P-Delta-RBD and S-6P-BA5-RBD proteins) in the presence of adjuvants, or PBS plus adjuvants (control), as described in Fig. 1. The immunized mice were challenged with a lethal dose of SARS-CoV (MA15 strain) 2 weeks after the 3rd immunization. The challenged mice were observed for overall survival (a–g) and weight changes (h) for 13 days after SARS-CoV challenge. In a separate experiment, C57BL/6 mice were immunized as described above, collected for sera and splenocytes 4 months after the 3rd immunization, and detected for neutralizing antibodies against pseudotyped SARS-CoV in hACE2/293T cells (i), as well as IFN-γ or TNF-α-producing CD4+ (j, k) and CD8+ (l, m) T cells, respectively. NT50 is expressed as 50% neutralizing antibody (Ab) titers of sera against SARS-CoV pseudovirus in hACE2/293T cells, and the data (i) are presented as the mean plus s.e.m. of four wells from pooled sera of five mice in each group. The limit of detection for the neutralization assay was 1:5. The splenocytes were stimulated with a Fc-fused SARS-CoV RBD protein, and the secreted cytokines were measured by flow cytometry. The data (j–m) are presented as the mean plus s.e.m. of five mice in each group. Ordinary one-way ANOVA (Dunnett’s multiple comparison test) was used to compare the statistical differences between S-6P-Delta-RBD and other vaccinated groups (i), and Ordinary one-way ANOVA (Tukey’s multiple comparison test) was used to compare the statistical differences among different groups (l, m). *P < 0.05, **P < 0.01, and ***P < 0.001 designate significant differences among various groups. The experiments were repeated twice, with similar results.