Fig. 5: Humoral immune responses of young and old mice.

A Timeline of vaccine regimen to young (7 weeks old) and old (54 weeks old) mice. Female BALB/c mice aged at 7 weeks old (n = 8) and 54 weeks old (n = 8) were used in this study. The young mice (n = 8) and old mice (n = 8) were equally distributed into two groups, and were intranasally (i.n., n = 4) or subcutaneously (s.c., n = 4) immunized with 1 µg of CPS14⁺MVs, respectively. One µg of CPS14⁺MVs contained 0.013 µg of CPS14. Vaccination was performed three times at 3-week intervals (weeks 0, 3, and 6). Serum and saliva samples were collected at week 8. The results of ELISA in young and old mice subcutaneously immunized with CPS14⁺MVs have been previously published²⁴. These samples were re-analyzed in comparison to those of intranasally immunized with CPS14⁺MVs in the present study. The experiments of young and old mice intranasally immunized with CPS14⁺MVs were performed at the same time as the previously published experiments of young and old mice subcutaneously immunized with CPS14⁺MVs. Representative data shown in B and C were obtained from the young mice (n = 8) and old mice (n = 8) in one independent animal experiment. Created in BioRender. Konig, S. (2025) https://BioRender.com/z8u1cwz. B CPS14-specific antibodies in serum samples. In all ELISAs except for IgG, serum samples were used at a 1:100 dilution. In the ELISA for serum IgG, the samples were used at a 1:1000 dilution. The results are expressed as OD₄₀₅ values (mean ± SD). Incubation time after AP substrate addition was as follows: Serum IgM (90 min), Serum IgG (45 min), Serum IgA (120 min), Serum IgE (120 min), Serum IgG1 (90 min), Serum IgG2a (90 min), and Serum IgG2b (45 min), Serum IgG3 (60 min). Asterisks denote a statistically significant difference (p < 0.05) between intranasal and subcutaneous administrations. A dagger in the panel of serum IgG denotes a statistically significant difference (p < 0.05) between young and old mice when intranasally vaccinated with CPS14⁺MVs. Shown are the results of the statistical analysis using the Mann–Whitney U-test. C CPS14-specific IgA in saliva, nasal wash, and BALF samples. Saliva, nasal wash, and BALF samples were used at a 1:100 dilution. The results are expressed as OD₄₀₅ values (mean ± SD). Incubation time after AP substrate addition was as follows: Salivary IgA (120 min), Nasal wash IgA (120 min), and BALF IgA (120 min). Asterisks denote statistically significant difference (p < 0.05) between intranasal and subcutaneous administrations. Shown are the results of statistical analysis using the Mann–Whitney U-test.