Vaccine candidates PhtD and PhtE of Streptococcus pneumoniae are adhesins that elicit functional antibodies in humans.

We evaluated the role of vaccine candidate surface proteins, PhtD and PhtE as antigens with functional importance for Streptococcus pneumoniae (pneumococci) in adherence to nasopharyngeal (D562) and lung (A549) epithelial cell lines. Comparing TIGR4 to PhtD and PhtE- isogenic mutants, a 40% (p=0.001) and 42% (p=0.002) drop in the number of epithelial cells with adherent pneumococci was observed to both cells lines with the mutants, as quantitated using flow cytometry. We expressed PhtD and PhtE on the surface of Escherichia coli and demonstrated that when PhtD and PhtE were surface expressed on E. coli, adherence increased to D562 and A549 cells, compared with the E. coli parent strain (p=0.005, 0.013 for D562 and p=0.034, p=0.035 for A549). Using flow cytometry and confocal microscopy we found that pneumococci aggregated in the presence of human serum IgG, leading to a non-specific drop in adherence. Therefore IgG Fab fragments were prepared to study the functional role of PhtD and PhtE-specific Fabs in blocking adherence. The addition of 1μg of IgG Fab from adult sera led to a 34% reduction (p=0.002) and from children a 20% (p=0.023) reduction in D562 epithelial cells with adherent pneumococci. In purified IgG from adult sera, the depletion of PhtD and PhtE specific Fab from total IgG Fab resulted in a significant increase in the number of D562 epithelial cells with adherent pneumococci (p=0.005 for PhtD and p=0.024 for PhtE). We conclude that antibody directed to PhtD and PhtE adhesins of pneumococci, if raised by vaccination, may function to prevent pneumococcal adherence to human airway epithelial cells.