All strains were clinical isolates obtained from nasopharyngeal culture of children or from invasive infection in adults and children and were previously serotyped and assigned an ST by MLST as described. Bacteria were cultured at 37uC in 5% CO2 on blood agar plates or in Todd-Hewitt broth supplemented with 0.5% yeast extract to an optical density at 580 nm of 0.4 and stored at 270uC in 10% glycerol as single-use aliquots. Bacterial phase was determined using transparent medium under magnification and oblique, transmitted illumination as previously described. Capsule quantity was determined using the All-Stains assay for acidic polysaccharides. Bacteria grown to an optical density at 580 nm of 0.4 were washed and resuspended in water and chloroform added. After shaking the aqueous layer was added to All-Stains solution and the optical density measured. Pooled human serum was obtained from unvaccinated normal human volunteers. Sera were stored in single use aliquots at 270uC. To remove active IgG, sera were treated as previously described with 1% Immunoglobulin G-degrading enzyme which cleaves IgG at the hinge region, for 45 minutes at 37uC before use. Serum levels of capsule-specific antibody titres were measured using standardized ELISAs. Briefly, serum was mixed with an absorbent containing C-polysaccharide and 22F capsular PS to neutralize antibody binding to C-PS and other common contaminants before addition in serial dilutions to ELISA plates previously absorbed with individual capsular serotype antigens. Serotype specific antibody bound to the ELISA plate was detected with anti-human IgG antibody purchase Perifosine conjugated with alkaline phosphatase, followed by addition of the substrate, p-nitrophenyl phosphate and reading the optical density at 405 nm. Serum total IgG binding to S. pneumoniae using flow cytometry and a R-phycoerythrin goat anti-human IgG as described. C3b/iC3b deposition and C1q binding to S. pneumoniae were measured using previously described flow cytometry assays and fluorescein isothiocyanate conjugated polyclonal anti-human C3 or anti-C1q. Results of complement and IgG binding assays are presented as a fluorescence index in arbitrary units.