Fig. 5: Long-term follow-up after vaccination with CPS14⁺ MVs or licensed vaccines.

A Timeline of immunization: CPS14⁺ MVs vs. two currently available pneumococcal vaccines, PPSV23 and PCV13. Six-week-old female BALB/c mice (n = 27) were subcutaneously immunized with PBS, PPSV23, PCV13, or CPS14⁺MVs at weeks 0, 3, and 6 (PBS: n = 3, PPSV23: n = 8, PCV13: n = 8, CPS14⁺ MVs: n = 8). In addition, a booster (4th immunization) was administered at week 30. Serum samples were collected every 4 or 5 weeks starting from week 8. Pre-immune serum samples were also obtained 1 week before first immunization (week-1). At week 56 or 59, all mice were euthanized, and nasal wash and BALF samples were collected. Among the 27 mice used in this experiment, only two mice in the PPSV23 group died accidentally at 45 and 56 weeks, respectively. B Transition of CPS14-specific serum IgM and IgG. The OD₄₀₅ values (mean ± SD) at each time point are connected by a colored line presenting each group. Arrows indicate the timing of immunization. C CPS-specific IgG subclass responses in serum at different time points (at weeks 8, 32, and 53). Serum IgG subclasses (IgG1, IgG2a, IgG2b, IgG3) were examined. Data are expressed as the mean ± SD. ND: no statistically significant difference. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. ****p ≤ 0.0001. Shown are the results of statistical analysis using one-way ANOVA followed by Tukey’s multiple comparison test, when compared to Grp. 1 (PBS) of the same age. D CPS-specific IgG and IgG subclass responses in BALF at week 56 or 59. ND: no statistically significant difference. *p ≤ 0.05. **p ≤ 0.01. ***p ≤ 0.001. Shown are the results of statistical analysis using one-way ANOVA followed by Tukey’s multiple comparison test, when compared to Grp. 1 (PBS) of the same age. In all ELISA except serum IgG, samples were used at 1:100 dilution. In ELISA for serum IgG, the samples were used at 1:1000 dilution. The results are expressed as OD₄₀₅ values (mean ± SD) after a 30-min incubation with AP substrate. E Timeline of immunization, T-cell response. CPS14⁺ MVs vs. PCV13. Six-week-old female BALB/c mice were subcutaneously immunized with PBS, PCV13, or CPS14⁺MVs at weeks 0 and 3 (n = 4 per group). Spleens were harvested at week 8, and isolated splenocytes were used for intracellular cytokine assays. F After stimulation of splenocytes with PMA and ionomycin in the presence of GolgiPlug for 6 h, intracellular IFN -γ and IL-4 in CD4⁺T cells were examined. (Top panels) The percentages of IFN-γ⁺IL-4^-CD4⁺T cells/Total CD4⁺T cells (IFN-γ^-+cells) and IFN-γ^-IL-4⁺CD4⁺T cells /Total CD4⁺T cells (IL-4⁺cells) are indicated in the lower right and upper left of each quadrant of flow cytometry findings. (Bottom panels) The percentages of IFN- γ^-+cells and IL-4⁺cells, and ratio of IL-4/IFN-γ were compared between groups using one-way ANOVA followed by Tukey’s multiple comparison test. Data are expressed as the mean ± SD of the values.