FIGURE 7.

Only group W-135 and Y capsular polysaccharides bound to bacteria, but not free soluble polysaccharide, enhance alternative pathway activation. A, purified capsular polysaccharides in solution do not generate C3a. C2-depleted serum (25% (v/v)) was incubated with soluble purified capsular polysaccharides (each at a concentration of 125 μg/ml) for 10 min at 37 °C and C3a generated was measured by ELISA. Each bar represents the mean ± S.D. of 2 separate experiments. B, binding of purified capsular polysaccharides to Cap− N. meningitidis. W171 Cap−/fHbp−/NspA− (referred to as W Cap−) was incubated separately with purified polysaccharides (125 μg/ml) representing each of the 5 major meningococcal serogroups. Bound polysaccharide was detected using a mAb specific for each of the serogroups (gray shaded histograms). Binding of the mAb to the corresponding Cap+ mutant strain used in this study (i.e. A2594, H44/76, C2120, W171, and Y2220) is shown by the solid black histogram. Controls where the Cap− W171 mutant without any added polysaccharide was incubated with the anti-capsule mAb and secondary conjugate are shown by the broken lines. One of two reproducible experiments is shown. C, group W-135 and Y polysaccharides induce rapid C3a generation when bound to bacteria. Strain W171 Cap− was incubated with soluble purified capsular polysaccharides (each at a concentration of 125 μg/ml), followed by the addition of C2-depleted serum (25% (v/v)) for 10 min at 37 °C; C3a generated in the reaction mixture was measured by ELISA. Encapsulated strains W171 and Y2220 served as positive controls, whereas W171 Cap− plus C2-depleted serum (no added capsular polysaccharide) and C2-depleted serum alone were used as negative controls. Each bar represents the mean ± S.D. of 3 separate experiments. D, deposition of iC3b on W171 Cap− coated with purified capsular polysaccharides. The bacterial strains incubated with C2-depleted serum as described in C were washed, lysed, and Western blotted to analyze iC3b binding.