Mucosal immunization with a killed whole-cell pneumococcal vaccine, given with enterotoxinrelated adjuvants, has been shown to confer multi-serotype protection against colonization of the nasopharynx and middle ear in mice. However, because novel mucosal immunization strategies may be difficult to implement, here we evaluated subcutaneous injection. Strain RM200 was engineered to be capsule-negative, autolysin-negative, and to express a non-toxic mutant pneumolysoid. Liter-scale and 60-L Good Manufacturing Practice (GMP) cultures were grown in bovine-free soy-based medium, killed with chloroform or beta-propiolactone, and injected into C57Bl/6 mice without or with aluminum adjuvant. The adjuvant Al(OH) 3 strongly increased responses, particularly if pre-treated with phosphate. Protection was found in several tested model infections: nasal colonization with a serotype 6B strain and fatal aspiration-sepsis with strains of serotype 3 and 5. Protection against colonization was mechanistically dependent on the presence of CD4+ T cells at the time of challenge; in contrast, in the type 3 aspiration-sepsis model, CD4+ T cells were not required for protection at the time of challenge, suggesting that antibody alone was sufficient to protect against death in this model. Rabbits receiving sequential intramuscular injections in a pilot toxicity study displayed local reactogenicity at injection sites but no clinical signs. The rabbit antiserum thus produced was active in an in vitro phagocytic killing assay and passively protected mice in the type 3 aspiration-sepsis model. Approval is being sought for human trials of this vaccine.

The pneumococcal type 1 pilus, which is present in 25 to 30% of clinical isolates, has been associated with increased adherence and inflammatory responses and is being evaluated as a potential vaccine candidate. Here we show that expression of the pilus is bistable as a result of the molecular interaction between the transcription activator RrlA and a structural component of the pilus called RrgA. Sampling various clinical pneumococcal isolates that harbor the type 1 pilus-encoding islet, we show that distinct populations of cells can be identified with either undetectable or prominent pilus expression. When these two populations are separated and regrown in liquid medium, they are phenotypically different: the nonexpressing population reverts to the previous bimodal distribution, whereas the expressing population retains the same high level of pilus expression. Controlled exogenous expression of the regulatory pilus gene rlrA in a strain from which the endogenous version has been deleted increases pilus expression steadily, suggesting that the bistable expression of the pilus observed in wild-type cells is dependent on the native rlrA promoter. Finally, we demonstrate that RrgA is a negative regulator of pilus expression and that this repression is likely mediated through direct interaction with RlrA. We conclude that type 1 pilus expression in pneumococcus exhibits a bistable phenotype, which is dependent upon the molecular interplay between the RlrA and RrgA proteins. We suggest that this flexibility in expression may assist adaptation to a range of immune conditions, such as evasion of antipilus antibodies, within potential hosts.

Background:The pneumococcal pilus is associated with increased inflammation. Results: A 49-amino acid region of the pilus protein RrgA activates TLR2 and is associated with increased inflammation and virulence. Conclusion:The pneumococcal pilus is a TLR2 agonist; RrgA is a key component. Significance: A better understanding of the pilus in bacterial pathogenesis is crucial for the development of novel strategies against this pathogen.