In this regard, it would

be interesting to directly compare the immunogenicity and protective efficacy of colonisation with unencapsulated strains that are known to protect [6] with those of their WT parent strains. It is possible that WT strains in general would emerge as more immunogenic than unencapsulated isogenic mutants. The reduced immunogenicity of the Δlgt mutant is likely to reflect a combination of factors. Most important of these may be the reduced Imatinib in vivo colonisation density and duration. In addition, colonisation with WT D39 induced serum IgG to only 3 of 16 proteins antigens tested and two of these three were lipoproteins. Thus if the antibodies binding these antigens makes a critical

contribution to protection of the WT strain, the absence of the antigens in D39Δlgt would find more significantly impair its ability to protect. TLR2 signalling is important in the induction of Th17-cell responses through S. pneumoniae colonisation. Thus, mice lacking TLR2 have delayed clearance of S. pneumoniae [22] and [23]. Reduced TLR2 signalling from D39Δlgt may therefore impair the induction of the Th17 response and could reduce the immunogenicity of the Δlgt strain. However, data from TLR2 deficient mice suggest that this pathway may be redundant in the induction of robust serum IgG responses to colonisation [24], perhaps due to other compensating pathogen recognition pathways. Similarly, TLR4 [25] and inflammasome [26] and [27] activation by

pneumolysin may also be redundant in this regard, since pneumolysin-deficiency bacteria are also capable of inducing protection [7], perhaps due to intact TLR2 signalling. Prior colonisation protects against re-colonisation through Th17-mediated rapid neutrophil recruitment [23]. Hence, although we did not measure the bacterial load in the nasopharynx after the second dose, we would anticipate it is cleared more rapidly than the original inoculum. The ability of repeated doses of nasopharyngeal inoculation to induce stronger immune Carnitine palmitoyltransferase II responses has been previously reported and can be protective even with mutant strains [6] and [28]. Hence once sufficient bacterial exposure has occurred to induce a primary immune response, further exposure with a second inoculation probably acts as an immunological booster even without prolonged duration of dense colonisation. It is thus possible that administering repeated doses of any of the non-protective mutant strains reported in this work may enhance immunity sufficient to cause protection. The data presented here directly comparing the several non-protective mutant bacterial strains with their protective parent WT strain aid our understanding of why certain live attenuated strains are able to function as effective vaccines.