Streptococcus pneumoniae infections lead to high morbidity and mortality rates worldwide. Pneumococcal polysaccharide conjugate vaccines significantly reduce the burden of disease but have a limited range of protection, which encourages the development of a broadly protective protein-based alternative. We and others have shown that immunization with pneumococcal lipoproteins that lack the lipid anchor protects against colonization. Since immunity against S. pneumoniae is mediated through Toll-like receptor 2 signaling induced by lipidated proteins, we investigated the effects of a lipid modification on the induced immune responses in either intranasally or subcutaneously vaccinated mice. Here, we demonstrate that lipidation of recombinant lipoproteins DacB and PnrA strongly improves their immunogenicity. Mice immunized with lipidated proteins showed enhanced antibody concentrations and different induction kinetics. The induced humoral immune response was modulated by lipidation, indicated by increased IgG2/IgG1 subclass ratios related to Th1-type immunity. In a mouse model of colonization, immunization with lipidated antigens led to a moderate but consistent reduction of pneumococcal colonization as compared to the non-lipidated proteins, indicating that protein lipidation can improve the protective capacity of the coupled antigen. Thus, protein lipidation represents a promising approach for the development of a serotype-independent pneumococcal vaccine.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite for pneumococcal transmission and disease. Current vaccines protect only against disease and colonization caused by a limited number of serotypes, consequently allowing serotype replacement and transmission. Therefore, the development of a broadly protective vaccine against colonization, transmission and disease is desired but requires a better understanding of pneumococcal adaptation to its natural niche. Hence, we measured the levels of free and protein-bound transition metals in human nasal fluid, to determine the effect of metal concentrations on the growth and proteome of S. pneumoniae. Pneumococci cultured in medium containing metal levels comparable to nasal fluid showed a highly distinct proteomic profile compared to standard culture conditions, including the increased abundance of nine conserved, putative surface-exposed proteins. AliA, an oligopeptide binding protein, was identified as the strongest protective antigen, demonstrated by the significantly reduced bacterial load in a murine colonization and a lethal mouse pneumonia model, highlighting its potential as vaccine antigen.

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