FIG 3.

Immunization with 5CP through the i.n. route provided cross-serotype protection against GAS mucosal and systemic infection. Mice were immunized as described in the legend to Fig. 1. (a) Two weeks after the last immunization, mice were i.n. challenged with GAS M1. Colony-forming units (CFU) in NALT were determined 24 h after challenge. Data are from two independent experiments and presented as means ± SEM (n = 5 to 10). Statistical significance was determined by one-way ANOVA with Tukey’s posttest and indicated as follows: ***, P < 0.001; ns, not significant. (b) Immunized mice were challenged with GAS M1 or GAS M12. CFU in NALT were determined 24 h after challenge (n = 5). Statistical significance was determined by unpaired Mann-Whitney U nonparametric t tests and indicated as follows: *, P < 0.05. (c) Bacteria (GAS M1, M12, or M49) were coincubated with whole blood from immunized (5CP) or GAS-infected mice for 2 h, and bacterial growth rate was determined. (d) Bacteria (GAS M1 or M12) were coincubated with differentiated HL-60 cells in the presence of serum from immunized (5CP) or infected (GAS) mice for 1.5 h, and the bacterial growth rate was determined. Data are from two independent experiments and presented as means plus SEM (n = 8). Statistical significance was determined by one-way ANOVA with Tukey’s posttest and indicated as follows: ****, P < 0.0001; ***, P < 0.001. (e and f) Two weeks after the last immunization, mice were i.n. challenged with a lethal dose of GAS M1 (e) or M49 (f). Data are from two independent experiments (n = 20 or 21). Curves were compared using the log rank test for significance, and statistical significance was indicated as follows: ****, P < 0.0001; **, P < 0.01; *, P < 0.05; ns, not significant.