Fig. 5: Role of humoral responses in J8-Lipo-DT-PHAD-mediated protection.

a, b Role of mucosal antibodies. pIgR^−/− mice (n = 10/group; male and female, 4–6 weeks old) were immunized i.n. with J8-Lipo-DT-PHAD or PBS. One-week post-last boost serum samples were analyzed for J8-specific serum IgA and IgG antibody responses by ELISA (a), represented as mean ± SEM. Two weeks after last boost, the mice were infected via the URT with S. pyogenes 2031 (5 × 10⁶ cfu/mouse). On day 3 post-infection all surviving mice were euthanized and NALT and lungs collected to determine S. pyogenes burden (b), data represent the geomean ± geometric SD (cfu/tissue) on a Log 10 scale. c Role of serum antibodies. μMT (MuMT) mice (n = 10/group; male and female, 4–6 weeks old) were immunized i.n. with J8-Lipo-DT-PHAD or PBS. Two weeks after last boost, the mice were infected via the URT with S. pyogenes 2031 (5 × 10⁶ cfu/mouse). On day 3 post-infection all surviving mice were euthanized and NALT and lungs collected to determine S. pyogenes burden, data represented as the geomean ± geometric SD (cfu/tissue) on a Log 10 scale. Statistical analysis was performed using a nonparametric, unpaired Mann–Whitney U test on the raw CFU values (one-tailed) for both pIgR^−/− and μMT experiments (**p < 0.01; ****p < 0.0001). The percent reduction in bacterial burden was calculated by comparing the geomean (cfu/tissue) of the PBS group with the geomean (cfu/tissue) of the vaccine group. d–f Cytokine expression in pIgR^−/− mice vaccinated intranasally with J8-Lipo-DT-PHAD. pIgR^−/− and C57BL/6 mice (n = 3/group; male/female, 4–6 weeks old) were immunized i.n. with J8-Lipo-DT-PHAD or PBS. Two weeks after last boost, mice were euthanized, and spleens were excised. The splenocytes were stimulated ex-vivo either with J8-DT (50 μg/well) or media (left unstimulated). At 72 h post-stimulation, supernatants were isolated and levels of (d) IFN-γ, (e) IL-2, and (f) IL-17A were assessed using cytometric bead array as per manufacturer’s instructions. Data are represented as mean ± SEM. Statistical analysis was performed using two-way ANOVA, corrected for multiple comparisons using Sidak’s multiple comparison test (****p < 0.0001). All experiments were repeated once to confirm the results. Source data are provided as a Source Data file.