Fig. 2: Long-term animal study overview.

A Schematic representation of the types of vaccines and routes used in this study. Six-week-old female BALB/c mice (n = 43) were grouped into six different regimens. The number of mice in all groups except Grp. F was 8. The number of mice in Grp. F was 3. Vaccination groupings of mice are described in the box table in the upper right, which shows the vaccine used, the route, the amount of CPS14 per dose, and the number of mice in each group. Grps. A and B mice were intranasally immunized with MVs (vector control) and CPS14⁺MVs, respectively. The results of ELISA in mice of Grps C–F have been previously published²⁴. These samples were re-analyzed in comparison to those of Grps A (MVs, i.n.) and B (CPS14⁺MVs, i.n.) in the present study. The experiment including i.n. groups (Grps A and B) was performed at the same moment as the previously published experiment, including s.c. groups (Grps C–F)²⁴. Representative data of the long-term animal study were obtained from the 43 mice in one independent animal experiment. Created in BioRender. Konig, S. (2025) https://BioRender.com/z8u1cwz. B Timeline of vaccine regimen, sample collection, and challenge with pneumococci. As shown in Fig. 2A, BALB/c mice were intranasally immunized with MVs (vector control) and CPS14⁺MVs, and subcutaneously immunized with three different vaccines (CPS14⁺MVs, PCV, and PPSV) and PBS four times (at weeks 0, 3, 6, and 30). Serum and saliva samples were collected from the mice at seven different time points (weeks −1, 8, 16, 24, 32, 40, and 49). See the time-course data of ELISA at the seven different time points in Fig. 3. Eye wash was collected at week 53. See the data of ELISA using eye wash samples at week 53 in Fig. S2. All mice that survived until the end of the long-term animal experiment were subjected to the upper respiratory tract infection assay at week 56 or 59 (n = 40). The mice (n = 40) were divided into Class I (n = 18) and Class II (n = 20), which were assayed at weeks 56 and 59, respectively. See details of the grouping and randomization of mice in the “Materials and methods” section. See the bacterial clearance assay results in Fig. 7. Whole blood, nasal wash, and BALF were obtained after the bacterial clearance assay. See the results of ELISA and opsonophagocytic killing assays using the samples from mice at euthanasia in Figs. 4 and 6, respectively. Created in BioRender. Konig, S. (2025) https://BioRender.com/z8u1cwz.