Virtually all uropathogenic strains of Escherichia coli, the primary cause of cystitis, assemble adhesive surface organelles called type 1 pili that contain the FimH adhesin. Sera from animals vaccinated with candidate FimH vaccines inhibited uropathogenic E. coli from binding to human bladder cells in vitro. Immunization with FimH reduced in vivo colonization of the bladder mucosa by more than 99 percent in a murine cystitis model, and immunoglobulin G to FimH was detected in urinary samples from protected mice. Furthermore, passive systemic administration of immune sera to FimH also resulted in reduced bladder colonization by uropathogenic E. coli. This approach may represent a means of preventing recurrent and acute infections of the urogenital mucosa.

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.

CD2 is a 50-kDa transmembrane glycoprotein that plays an important role in T and natural killer (NT) lymphocyte functions. CD2 serves as both an adhesion molecule and as a costimulatory molecule through interactions with its ligand, CD58, on antigen presenting or target cells. Consistent with earlier studies using a rat anti-CD2 mAb, we have shown that treatment of alloantigen stimulated T lymphocytes with a humanized mAb, MEDI-507 (IgG1, kappa), induced hyporesponsiveness to subsequent stimulation with alloantigen but not to mitogen (phytohemagglutinin). Fluorescence-activated cell sorting analysis of cells from mixed lymphocyte reaction (MLR) treated with MEDI-507 revealed pronounced deletion of T and NK cells, consistent with lack of proliferation in the MLR. MEDI-507 F(ab')2 fragments did not have inhibitory activity or induce deletion of lymphocytes in the MLR. Removal of the NK cell subset by magnetic bead depletion using anti-CD16 and anti-CD56 mAbs eliminated both the T cell deletion and the inhibitory effect. Reconstitution of NK depleted responder populations using autologous NK cells restored the MEDI-507-mediated deletion activity to levels measured in the original MLR. Formaldehyde-fixed NK cells failed to mediate the MEDI-507-induced deletion effect. Altogether, our studies indicate that activated T cells with MEDI-507 bound to CD2 are preferential targets for autologous NK cells through a nonapoptotic cytotoxic mechanism.

Pneumococcal adherence to alveolar epithelial cells and nasopharyngeal epithelial cells has been well characterized. However, the interaction of Streptococcus pneumoniae with bronchial epithelial cells has not been studied. We have now shown that pneumococci bind specifically to a human bronchial epithelial cell line (BEAS-2B cells). Pneumococci adhered to BEAS-2B cells in a time- and dose-dependent manner. These results suggest that the bronchial epithelium may serve as an additional site of attachment for pneumococci and demonstrate the utility of the BEAS-2B cell line for studying mechanisms of pneumococcal infection.

Splenectomy is associated with an increased risk of serious infection with encapsulated micro-organisms in a small number of individuals. The immunological defects in this state are unclear. We have investigated in rabbits the effect of splenectomy, before and after primary immunization with a specific antigen (sheep erythrocytes) similar in structure to the pneumococcal capsular antigen, on the organ localization of antibody forming cells and the long-lived memory cells. Antibody forming cell and memory cell assays were performed at intervals up to day 270 after primary immunization. Antibody titres following secondary immunization were also measured. The results show that the major site of antibody forming cells is in the spleen following primary immunization of the untreated rabbit and that antibody forming cells subsequently migrate to the lymph nodes and thymus. On the other hand, in rabbits splenectomized before primary immunization the major site of the antibody forming cells is the peripheral lymph nodes. These rabbits also gave good secondary immune responses. However, splenectomy 40 days after primary immunization results in a poor secondary immune response, indicating that essentially all the antibody forming cells following primary immunization were localized in the spleen at the time of splenectomy.