Human antibodies to PhtD, PcpA, and Ply reduce adherence to human lung epithelial cells and murine nasopharyngeal colonization by Streptococcus pneumoniae

Abstract

Streptococcus pneumoniae adherence to human epithelial cells (HECs) is the first step in pathogenesis leading to infections. We sought to determine the role of human antibodies against S. pneumoniae protein vaccine candidates PhtD, PcpA, and Ply in preventing adherence to lung HECs in vitro and mouse nasopharyngeal (NP) colonization in vivo. Human anti-PhtD, -PcpA, and -Ply antibodies were purified and Fab fragments generated. Fabs were used to test inhibition of adherence of TIGR4 and nonencapsulated strain RX1 to A549 lung HECs. The roles of individual proteins in adherence were tested using isogenic mutants of strain TIGR4. Anti-PhtD, -PcpA, and -Ply human antibodies were assessed for their ability to inhibit NP colonization in vivo by passive transfer of human antibody in a murine model. Human antibodies generated against PhtD and PcpA caused a decrease in adherence to A549 cells (P < 0.05). Anti-PhtD but not anti-PcpA antibodies showed a protective role against mouse NP colonization. To our surprise, anti-Ply antibodies also caused a significant (P < 0.05) reduction in S. pneumoniae colonization. Our results support the potential of PhtD, PcpA, and Ply protein vaccine candidates as alternatives to conjugate vaccines to prevent non-serotype-specific S. pneumoniae colonization and invasive infection.

FIG 1

Adherence of TIGR4 wild type (TIGR4-WT) and its isogenic PcpA, PhtD, and PhtA, -B, and -D mutants on A549 cell line (lung epithelial cells). The mean adherence of TIGR4-WT on A549 cells was normalized, and the percent adherence of TIGR4 and its mutants was calculated compared to mean adherence of TIGR4-WT. Data are means with standard errors from two experiments done in triplicate. **, mean P value < 0.01; *, mean P value < 0.05 (adherence of TIGR4-WT versus that of its isogenic mutants).

FIG 2

Effect of Anti-PhtD, -PcpA, and -Ply specific Fabs on pneumococcal adherence to A549. (A) Flow cytometry data showing the actual percent adherence of labeled bacteria to A549 cells. Gated boxes represent concatenated data of triplicates with labeled TIGR4 as the control and pretreated TIGR4 with anti-PhtD, -PcpA, and -Ply Fabs. (B) Decrease in adherence of TIGR4 to A549 cells with treatment of 1 μg of anti-PhtD, -PcpA, and -Ply Fabs. The mean adherence of TIGR4 on A549 cells was normalized, and the percent adherence of TIGR4 with the Fabs was compared to that with TIGR4 alone. Data are means with standard deviations from two experiments done in triplicate. **, P < 0.01; ns, not significant.

FIG 3

Decrease in adherence of strains TIGR4 and RX1 to A549 cells with treatment of 1.5 μg of mixed Fabs (0.5 μg of each anti-PhtD, -PcpA. and -Ply Fabs). The mean adherence of TIGR4 and RX1 alone on A549 cells was normalized, and the percent adherence of TIGR4 and RX1 with mixed Fab treatment compared to that with no treatment is shown. Data are means with standard deviations from triplicates.

FIG 4

Effect of passive transfer of human anti-PhtD, -PcpA, and -Ply IgG antibodies in a mouse pneumococcal NP colonization model. A significant decrease in S. pneumoniae colonization was observed with 25 μg of anti-PhtD and -Ply antibodies compared to nonspecific control antibodies against P6 of NTHI.