Fig. 2: Immunogenicity and protective effects of intranasal CCD/RBD-HR vaccine.

a Immunization scheme. Mice/rabbits were immunized intranasally with PBS, RBD-HR, or CCD/RBD-HR on days 0, 14, and 28. b, c Serum anti-RBD-HR IgG titers in immunized mice (b) and rabbits (c) on day 35. d, e Neutralizing of authentic viruses (d) and pseudoviruses (e) of SARS-CoV-2 by the immune sera on day 35. f RBD-HR-specific IgA levels in BAL fluid on day 35. Immunized mice were challenged with 5 × 10⁵ TCID₅₀ of Omicron viruses and sacrificed at 4 dpi. g Viral RNA levels in the nasal turbinates, trachea, and lungs at 4 dpi. h Histopathological changes (left) and pathological scores (right) of the lung tissues at 4 dpi. Scale bar: 100 μm. Similar results were obtained in five independent experiments. i, j Serum RBD-HR-specific IgG levels (i) and their function in neutralizing SARS-CoV-2 variant pseudoviruses (j) on day 260. On day 56, blood (k), bone marrow (l), and spleen (m) were harvested to assess SARS-CoV-2 RBD-specific IgG ASCs with ELISpot. Images (left) and quantification (right) of IgG spot-forming cells among lymphocytes were displayed. RBD-specific IgG-producing CD138⁺ plasma cells in the blood and bone marrow (n), memory B cells (RBD⁺CD38⁺CD80⁺) in the blood, bone marrow, and spleen (o) were assayed with FCM. p, q Antigen-experienced (CD44⁺) and activated (CD69⁺) CD4⁺/CD8⁺ T cells in the spleen were determined with FCM after re-stimulation with RBD protein peptide pools. r Splenic CD4⁺ and CD8⁺ T cells were assessed for IL-4 and IFN-γ expression with intracellular cytokine staining after restimulation with RBD protein peptide pools or irrelevant antigens. Data were displayed with floating bars in (k–r). The middle line indicates the median and the box shows the data range. Data were presented as mean values ± SEM. n = 4–5 mice in (b, d–r). n = 3 rabbits in (c). P values were calculated with one-way ANOVA followed by Dunnett’s multiple comparisons test in (b, c, f–i, k–q). Two-way ANOVA followed by Sidak’s multiple comparisons test in (d, e, and j). Two-way ANOVA followed by Tukey’s multiple comparisons test in (r). Source data are provided as a Source Data file.