Article Figures & Data
Figures
- FIG. 1.
Anti-Hla MAbs protect human alveolar epithelial cells from S. aureus injury. (A to F) Live (green)/dead (red) staining of A549 alveolar epithelial cells as imaged by fluorescence microscopy 4 h after infection. Cells were not infected (A) or cocultured with S. aureus Newman in medium treated with PBS (B) or with 2 μg/ml IgG2a (C), 7B8 (D), IgG2b (E), or 1A9 (F). Bars, 20 μm. The percentage of dead cells for each experimental condition was calculated by scoring live and dead cells in four independent fields (each field contained a minimum of 60 cells) and expressing the number of dead cells as a fraction of the total number of cells; the resulting values are indicated at the bottom right in each panel. The statistical significance of MAb-induced protection was calculated by using Student's t test (P = 0.004 for IgG2a versus 7B8; P = 0.002 for IgG2b versus 1A9). No statistically signifi-cant differences were observed between PBS and control IgG-treated cells. (G and H) LDH release by A549 cells was observed by using cells cocultured with S. aureus Newman in media treated with the indicated concentrations of 7B8 (G) or 1A9 (H), revealing that there was a significant reduction in LDH release for all MAb concentrations examined (P < 0.02). The error bars indicate the standard deviations.
- FIG. 2.
Passive transfer of anti-Hla MAbs protects against S. aureus pneumonia. Mice were vaccinated via i.p. injection with the indicated doses of IgG2a or 7B8 (A) or IgG2b or 1A9 (B). They were challenged 24 h later with S. aureus Newman via the i.n. route, and mortality was recorded at 24, 48, and 72 h postinfection (P < 0.039; 15 animals per group). The CFU recovered from the right lung were enumerated (C), demonstrating that there was a significant decrease in the bacterial burden in mice vaccinated with the 7B8 or 1A9 MAb (P = 0.027 and P = 0.02, respectively; 15 animals per group). The horizontal bars indicate the mean bacterial load. (D and E) Decreases in the gross pathological (D) and histopathologic (E) hallmarks of disease in animals immunized with 7B8 and 1A9 were evident when these animals were compared to control animals.
- FIG. 3.
Anti-Hla MAbs protect against highly virulent S. aureus LAC/USA300 and have therapeutic value. (A) Immunization with 7B8 and 1A9 conferred protection against 48- and 72-h mortality in mice infected via the i.n. route with S. aureus LAC/USA300 (P = 0.013 and P = 0.002, respectively; 15 animals per group). (B) Mice were passively immunized with 7B8 24 h prior to i.n. challenge with S. aureus Newman or 4, 8, or 12 h postchallenge. A significant decrease in 24-h mortality was observed for animals treated up to 8 h postinfection (P ≤ 0.021; 15 animals per group). Statistical significance (P < 0.05) is indicated by an asterisk.
- FIG. 4.
Anti-Hla MAbs bind to epitopes within the first 50 amino acids of Hla. (A) SPR was performed by amino coupling 7B8 and 1A9 to a CM5 chip. Binding of purified Hla revealed that the affinities of each MAb for Hla were in the low-nanomolar range. ka, kinetic rate constant for association; kd, kinetic rate constant for dissociation; KD, affinity constant. (B) Overlapping, serial 50-amino-acid segments of Hla were purified as GST fusion proteins and used in a dot blot analysis to map the epitopes recognized by 7B8 and 1A9, which revealed that both MAbs bind within the first 50 amino acids. The Coomassie blue-stained SDS-PAGE gel shows the integrity of each fusion protein. (C) Structure of Hla as a monomer (left structure) and as a heptamer (middle and right structures), in which the first 50 amino acids recognized by the MAbs are indicated by black lines.
- FIG. 5.
Anti-Hla MAbs prevent oligomerization of the toxin on host cells. (A) Hemolysis assays were performed with 100 nM purified Hla and 12.5% RRBC in PBS. Addition of 0.5 to 5 μM 7B8 and addition of 0.1 to 5 μM 1A9 significantly reduced hemolysis, as measured by the A450 of assay supernatants (P ≤ 0.005 and P ≤ 0.033, respectively). (B) Active [35S]methionine-labeled toxin was synthesized in vitro and added to RRBC in the presence of IgG2a, IgG2b, or the indicated concentrations of 7B8 or 1A9. Concentration-dependent inhibition of toxin oligomerization (designated Hla7) was evident with both 7B8 and 1A9. Neither MAb disrupted binding of the labeled toxin to RRBC, as indicated by the presence of the Hla monomer in all lanes.
- FIG. 6.
Active immunization with the first 50 amino acids of Hla prevents S. aureus pneumonia. C57BL/6J mice were vaccinated by i.m. injection with GST, GST-HlaH35L, or GST-Hla1-50 and challenged with S. aureus Newman via the i.n route. Mortality was recorded 24, 48, and 72 h after infection (P < 0.022; 15 animals per group). Statistical significance is indicated by an asterisk. (B) ELISA was performed using the indicated dilutions of serum from 25 human volunteers to determine the titer of anti-Hla1-50 antibodies, expressed as raw units of optical density at 450 nm (OD450). The bottom and top of each box indicate the lower and upper quartiles, respectively, and the horizontal bar indicates the median for the group of samples. The whiskers indicate the lowest and highest values within a distance of 1.5 times the interquartile range measured from the lower and upper quartiles, respectively; circles indicate outliers in each set.
