Fig 1: Antibody and cellular responses elicited by homologous and heterologous prime-boost vaccination.BALB/c mice were randomly assigned into eight groups (n = 17 per group). In four vaccination groups, mice were vaccinated with homologous and heterologous prime-boost regimens (permutations of 10 µg Pvs25F mRNA–LNP or 10 µg rPvs25 Protein-ISA51 VG). In the other 4 groups, 10 µg poly(C) RNA–LNP and Montanide ISA51 VG were administered as controls. Four weeks after the booster dose, eight mice from each group were terminated. Serum and spleen from each animal were collected to evaluate antibody and cellular responses. a Pvs25-specific IgG titers 4 weeks after the prime and 4 weeks after the boost vaccination were evaluated by endpoint dilution ELISA (n = 17 per group) and reported as GMT with 95% confidence intervals (CI). b Avidity indices of antibodies 4 weeks post boost assessed by ELISA (n = 8 per group). The avidity index (AI) of each mouse was calculated as the ratio of the areas under the curve obtained in the ELISA graphic (OD vs. log of dilution) with and without the denaturant. c Measurements of Pvs25-specific IgG2a and IgG1 subclasses from individual mice (n = 8 per group), expressed as the IgG2a/IgG1 ratio. d, e Splenocytes were obtained 4 weeks post boost (n = 5–6 per group). Control animals (n = 2) were randomly selected from the 4 control mice. d The Pvs25-specific memory B cell response was measured by ELISpot assay. Data are reported as spot-forming cells (SFC) per 200,000 input cells. e Splenocytes were stimulated with a Pvs25-specific peptide pool to analyze the production of IFN-γ and IL-2 by CD4+ T cells. Error bars represent SEM. a, d, e Each symbol represents one animal. Statistical analyses: a Two-tailed t-test to compare prime vs. boost values (P < 0.0001 for all comparisons); one-way ANOVA with Dunnett’s post hoc test to compare antibody titers to that of mRNA/mRNA (****P < 0.0001). b, c One-way ANOVA with Bonferroni correction (P > 0.05, n = 8 per group). d, e One-way ANOVA with Dunnett’s post hoc test to compare values to that of mRNA/mRNA (**P < 0.01, ***P < 0.001, ****P < 0.0001).
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