Figure 5.

Immunogenicity of GAC physically mixed to CRM₁₉₇ and when conjugated to CRM₁₉₇ or GAS proteins SLO, SpyAD and SpyCEP. CD1 mice were immunized intraperitoneally (i.p.) at day 0 and 28 with 1.5 µg GAC/dose or with the corresponding dose of the carrier protein alone, all formulated with 2 mg/mL Alhydrogel. Sera were analyzed by enzyme-linked immunosorbent assay (ELISA) using as coating antigens GAC-HSA (a) or SLO, SpyAD and SpyCEP (b). Summary graphs of anti-antigen specific IgG geometric mean units (bars) and individual antibody levels (dots) are reported. Not enough sera was available for one mice of day 27 for GACox-SLO, GACox-SpyAD and GACox-CRM₁₉₇ groups. Kruskal-Wallis test was performed among the 4 groups immunized with the conjugates in graph (a) (p > 0.05), Wilcoxon test was performed between response at day 27 and day 42 in graph (a) and Mann-Whitney two-tailed test between each group immunized with protein alone or GACox-protein conjugate in graph (b) (* p < 0.05, ** p < 0.01, *** p < 0.001). Sera were tested in the hemolysis inhibition assay (c) and in the IL-8 cleavage inhibition assay (d) to evaluate their ability to block native SLO and SpyCEP activity, respectively. The amount of hemoglobin released by rabbit red blood cells (c) and of uncut IL-8 (d) observed at each serum dilution tested is reported for pre-immune serum, standard serum and one selected day 42 serum for each immunization group. Each serum dilution was tested twice and the mean value with error bar was reported in the graph (for some points the error bars are not shown as shorter than the height of the symbol). Pooled sera at day 42 were tested in Flow cytometry (FACS) (e) to evaluate their ability to bind to GAS bacterial cells. Following incubation of bacteria with the different sera, APC-conjugated anti-mouse IgG secondary antibody was used for detection. The mean fluorescence intensity (MFI) of 10,000 acquired events, measured for each serum, is reported as compared to pre-immune sera. Results obtained with sera diluted 1:500 are reported, as representatives of the three dilutions tested (1:500, 1:5000 and 1:10,000).