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Microbial targets for protective humoral immunity are typically surface-localized proteins and contain common sequence motifs related to their secretion or surface binding. Exploiting the whole genome sequence of the human bacterial pathogen Streptococcus pneumoniae, we identified 130 open reading frames encoding proteins with secretion motifs or similarity to predicted virulence factors. Mice were immunized with 108 of these proteins, and 6 conferred protection against disseminated S. pneumoniae infection. Flow cytometry confirmed the surface localization of several of these targets. Each of the six protective antigens showed broad strain distribution and immunogenicity during human infection. Our results validate the use of a genomic approach for the identification of novel microbial targets that elicit a protective immune response. These new antigens may play a role in the development of improved vaccines against S. pneumoniae.Streptococcus pneumoniae (the pneumococcus) is the leading cause of bacterial sepsis, pneumonia, meningitis, and otitis media in young children in the United States. Annually, 7,000,000 middle-ear infections are ascribed to this organism (4). The vaccines in current use are formulations of capsular carbohydrate from the 23 serotypes responsible for 85 to 90% of infections in the United States, but these vaccines are poorly efficacious in infants and the elderly, the populations that are most at risk (1). A heptavalent-capsular-carbohydrate vaccine conjugated to the protein carrier CRM197 has been shown to be well tolerated and efficacious against invasive disease caused by the seven vaccine serotype strains (3) and has recently been approved for use in young children. However, this type of vaccine has several potential limitations, including serotype replacement by strains that are not represented (14).The advent of whole-genome sequencing of microbes, including microbial pathogens, has revolutionized the methods by which these organisms are studied and has heightened expectations regarding the ability to predict potential targets for antimicrobial agents and vaccines (2,12,20). We combined sequence scanning for prediction of surface-localized proteins with an animal model which allowed us to directly screen proteins for vaccine efficacy to identify novel vaccine candidates from the genome sequence of S. pneumoniae. Here we describe the use of a clinically relevant animal model for the evaluation of the vaccine efficacy of proteins identified from the genome sequence of pneumococcus. This approach was validated by the discovery of five previously unidentified genes whose products induced immune responses that protected mice from pneumococcal infection. Similar sequence scanning methods were recently used to identify potential vaccine candidates from the genomic sequence of the gram-negative pathogen Neisseria meningitidis (21) predicted by in vitro correlates of vaccine effectiveness. Here we expand upon the use of genomics to directly demonstrate vaccine efficacy in an animal model for...

Four pneumococcal genes (phtA, phtB, phtD, and phtE) encoding a novel family of homologous proteins (32 to 87% identity) were identified from the Streptococcus pneumoniae genomic sequence. These open reading frames were selected as potential vaccine candidates based upon their possession of hydrophobic leader sequences which presumably target these proteins to the bacterial cell surface. Analysis of the deduced amino acid sequences of these gene products revealed the presence of a histidine triad motif (HxxHxH), termed Pht (pneumococcal histidine triad) that is conserved and repeated several times in each of the four proteins. The four pht genes (phtA, phtB, phtD, and a truncated version of phtE) were expressed in Escherichia coli. A flow cytometry-based assay confirmed that PhtA, PhtB, PhtD and, to a lesser extent, PhtE were detectable on the surface of intact bacteria. Recombinant PhtA, PhtB, and PhtD elicited protection against certain pneumococcal capsular types in a mouse model of systemic disease. These novel pneumococcal antigens may serve as effective vaccines against the most prevalent pneumococcal serotypes.

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