Serum anti-type 2 capsular polysaccharide IgG was measured by ELISA ( Fig. 4A). Whilst nearly all mice colonised with WT D39 developed an IgG response

as measured in whole cell ELISA ( Fig. 2A), only an occasional mouse developed a capsule-specific IgG response ( Fig. 4A). Anti-CPS IgG made a negligible contribution to total IgG binding as assayed by whole cell ELISA since pre-incubation of sera with excess purified capsular polysaccharide antigen did not inhibit IgG binding in sera from mice colonised with WT D39 ( Fig. 4B). To further confirm that colonisation with WT D39 induced antibody against non-capsular antigens, levels of IgG that bound to pneumolysin and 15 surface-accessible AZD6738 mw protein antigens was measured in the serum of 3 randomly selected WT D39 colonised mice ( Fig. 5). Antibody to pneumococcal surface protein A (PspA) and the lipoprotein pneumococcal surface adhesin A (PsaA) were detected in 3 out of 3 mice, and IgG to the lipoprotein putative proteinase maturation protein (PpmA) in 2 of 3 mice.

Thus, colonisation with the encapsulated IPI-145 price WT strain induced antibody to bacterial proteins including lipoproteins, but not to capsular polysaccharide. Colonisation with either D39-DΔ or D39Δlgt was less immunogenic, correlating with their lack of protection. Since neither D39-DΔ and D39Δpab lacked the potentially protective antigens present in WT D39, we generated the alternative hypothesis that lack of protection reflected insufficient antigen exposure during the colonisation process. To explore this, we compared the density and duration of nasopharyngeal colonisation with Megestrol Acetate these strains ( Fig. 6). D39 colonisation persisted until at least day 10 following inoculation, but no bacteria were recovered by day 17. The ability of D39-DΔ to colonise was impaired. Compared to WT, there were approximately 1-log fewer unencapsulated D39-DΔ recovered at both day 1 and day 2 post-inoculation, with colonisation cleared in nearly all mice by day 5. As seen previously with TIGR4Δpab [11], D39Δpab bacteria were rapidly cleared

within 48 h of attempted colonisation. We also found that D39Δlgt has a shorter duration of colonisation (cleared by day 10) and lower colonisation density (approximately 1–1.5 log10 fewer) compared to WT D39 (data from Chimalapati et al., under review) ( Fig. 6). Thus, the immunogenicity of the protective WT strain may reflect contributions by both capsule and surface lipoproteins to maintaining the degree of bacterial nasopharyngeal exposure required to induce protective immunity. To assess whether the duration of bacterial colonisation could be controlled using PABA supplementation of this mutant, we attempted to colonise mice with D39Δpab in the presence of PABA supplementation. PABA supplementation was commenced the day prior to colonisation, and abruptly withdrawn after 5 days ( Fig. 7A).