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

Abstract

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-deleted 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 E 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 1 μg 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 are adhesins of pneumococci, if raised by vaccination, may function to prevent pneumococcal adherence to human airway epithelial cells.

Human Antibodies to PhtD, PcpA, and Ply Reduce Adherence to Human Lung Epithelial Cells and Murine Nasopharyngeal Colonization by Streptococcus pneumoniae

“…Our previous work demonstrated the role of PhtD, PhtE, and PcpA in adherence of S. pneumoniae to mucosal epithelium in vitro (9,33). In this report, we show a direct role of human antibodies directed against PhtD and PcpA in blocking S. pneumoniae adherence to human lung epithelial cells, and we demonstrate a significant reduction in NP colonization of mice after passive transfer of natural human anti-PhtD and anti-Ply specific antibodies.…”

“…Streptococcus pneumoniae wild-type strain TIGR4 (serotype 4), used in this study, and its isogenic PhtD, PcpA, and PhtABD mutants were received from Sanofi Pasteur. Methods for generating these mutants were described previously (9,33). A noncapsular strain (RX1) was also used in the study.…”

“…Our group is working with three of these vaccine candidate proteins, PhtD, PcpA, and PlyD1. We know that children, who are naturally exposed to S. pneumoniae during NP colonization and during AOM, generate antibody responses against PhtD, PcpA, and PlyD1 (9,10). In a recent study, we demonstrated that both infant and adult mice vaccinated with a trivalent formulation of PhtD, PcpA, and PlyD1 are protected against lethal pneumonia infection (11).…”

Discovery of Immunodominant B Cell Epitopes within Surface Pneumococcal Virulence Proteins in Pediatric Patients with Invasive Pneumococcal Disease

“…This finding suggests that binding of anti-peptide antibodies to this exact region may block the Pht protein-zinc interaction and, therefore, hamper Pht proteins function, including zinc concentration homeostasis in the bacterial environment, adherence to epithelial cells, and C3b deposition inhibition, leading to impaired opsonophagocytosis and, therefore, attenuated bacterial virulence (14 -16, 24). In this regard, previous studies have shown that a quadruple S. pneumoniae mutant in which all Pht genes are deleted is completely avirulent (25), whereas antibodies against PhtD and PhtE prevent bacterial adhesion to the human respiratory epithelium (24). We are currently undertaking further studies to investigate how antibody binding may affect the zinc binding potential of zinc-finger B cell epitopes as well as their changes in conformation and function, as we have described previously in a different clinical setting (26).…”

Review: Current and new generation pneumococcal vaccines

“…Two of these, PhtD and PhtE, are adhesins which promote attachment of the pneumococcus to airway epithelial cells via interactions that remain to be established. 98,99 Because of their broadly serotype-independent expression, immunogenicity and involvement in pneumococcal colonisation, these proteins are considered to be priority vaccine candidates.…”