Fig. 2: Comparison of the immunogenicity of the RBD vaccine and a spike mRNA vaccine in mice.

BALB/c female mice (n = 5 per group) were immunized intramuscularly twice with a 4 week interval with 1 µg of saRNA-LNP expressing SARS-CoV-2 Spike variant RBD with or without TM or with 10 µg of mRNA-LNP expressing the Spike protein (S2P). All experiments were performed once. a, b Antibody titers of sera (at Day 42) from the immunized mice were evaluated by ELISA against SARS-CoV-2 RBD from Wuhan-Hu-1, Gamma, Delta, and Omicron BA.1 variants. Plots represent individual endpoint titers with medians (lines). Ratio of IgG2a to IgG1 was calculated based on the endpoint titers. One-way ANOVA was used to determine significance. c Frequencies of RBD-specific B cells in memory B cells (gated on CD3^-NK1.1^-CD19⁺B220⁺CD138^-CD38⁺) were measured by flow cytometric analysis. One-way ANOVA was used to determine significance. d Frequencies of RBD VoCs cross-binding B cells as percentages of memory B cells. Two-way ANOVA was used to determine significance. e Dot plots represent the frequencies of IFN-γ, TNF, or IL-2-secreting CD4⁺ T cells responding to SARS-CoV-2 spike variant RBD peptides in CD4⁺ total memory cells. The lines show the medians. One-way ANOVA was used to determine significance. f Frequencies of RBD-specific CD4⁺ T cell subpopulations producing IFN-γ, TNF, and/or IL-2 in cytokine secreting CD4⁺ total memory cells. The x-axis displays each response pattern, whose composition is denoted with a plus for the presence of IFN-γ, TNF and IL-2. The response patterns are grouped and color-coded by number of positive functions and summarized in pie chart form where each pie slice represents the mean proportion of the total CD4⁺ T cell response contributed by response patterns that have all three (red), any combination of two (blue) or one (green) of the measured functions from each group immunized with saRNA-LNP expressing SARS-CoV-2 Spike variant RBD with (RBD-TM) or without (Secreted RBD) HA TM or 10 µg of mRNA-LNP expressing Spike protein (mRNA S2P). Two-way ANOVA and permutation test were used to determine significance. g Dot plots represents the correlation between anti-RBD endpoint titer and frequency of Th1 (producing IFN-γ, TNF or IL-2 responding to RBD peptides). Correlations and two-sided p-values were calculated using the nonparametric Spearman’s rank test. h Dot plots represent the frequencies of CD107a, IFN-γ, TNF, or IL-2-secreting CD8⁺ T cells responding to SARS-CoV-2 spike variant RBD peptides in CD8⁺ total memory cells. The lines show the medians. One-way ANOVA was used to determine significance. i Frequencies of RBD-specific CD8⁺ T cell subpopulations producing CD107a, IFN-γ, TNF, and/or IL-2 in cytokine secreting CD8⁺ total memory cells. The x-axis displays each response pattern, whose composition is denoted with a plus for the presence of CD107a, IFN-γ, TNF and IL-2. The response patterns are grouped and color-coded by number of positive functions and summarized in pie chart form where each pie slice represents the mean proportion of the total CD8⁺ T cell response contributed by response patterns that have all four (red), any combination of three (blue), two (green) or one (gray) of the measured functions from each group immunized with saRNA-LNP expressing SARS-CoV-2 Spike variant RBD with (RBD-TM) or without (Secreted RBD) HA TM or 10 µg of mRNA-LNP expressing Spike protein (mRNA S2P). Two-way ANOVA and permutation test were used to determine significance.