Children with invasive Staphylococcus aureus disease exhibit a potently neutralizing antibody response to the cytotoxin LukAB

Abstract

Despite the importance of Staphylococcus aureus as a common invasive bacterial pathogen, the humoral response to infection remains inadequately defined, particularly in children. The purpose of this study was to assess the humoral response to extracellular staphylococcal virulence factors, including the bicomponent leukotoxins, which are critical for the cytotoxicity of S. aureus toward human neutrophils. Children with culture-proven S. aureus infection were prospectively enrolled and stratified by disease type. Fifty-three children were enrolled in the study, of which 90% had invasive disease. Serum samples were obtained during the acute (within 48 h) and convalescent (4 to 6 weeks postinfection) phases, at which point both IgG titers against S. aureus exotoxins were determined, and the functionality of the generated antibodies was evaluated. Molecular characterization of clinical isolates was also performed. We observed a marked rise in antibody titer from acute-phase to convalescent-phase sera for LukAB, the most recently described S. aureus bicomponent leukotoxin. LukAB production by the isolates was strongly correlated with cytotoxicity in vitro, and sera containing anti-LukAB antibodies potently neutralized cytotoxicity. Antibodies to S. aureus antigens were detectable in healthy pediatric controls but at much lower titers than in sera from infected subjects. The discovery of a high-titer, neutralizing antibody response to LukAB during invasive infections suggests that this toxin is produced in vivo and that it elicits a functional humoral response.

FIG 1

Study design and subject enrollment. Healthy controls were enrolled in parallel during the same study period.

FIG 2

Geometric mean IgG titers in serum from healthy pediatric controls and acute- and convalescent-phase sera from children with invasive S. aureus disease. These target the two subunits of LukAB (LukA and LukB), alpha hemolysin (Hla), V8 protease (V8P), the two subunits of PVL (LukF-PV and LukS-PV), and the two subunits of LukED (LukE and LukD). Samples were run in duplicate, independently on separate days, and a third run was performed if the first two titer values differed by one dilution. *, the convalescent IgG response was significantly higher than the titer in the acute phase for LukA, LukB, Hla, LukF-PV, LukD, and HlgA. The paired t test was used to determine statistical significance of the rise from acute-phase to convalescent-phase titers for each antigen. Subjects for which a paired sample was not obtained (e.g., no convalescent-phase sample was available) are included in the overall GMTs displayed in the figure but were censored from the acute:convalescent statistical analysis shown in Table 1.

FIG 3

Anti-LukAB and anti-PVL responses when restricting to PVL-positive isolates, by geometric mean titer. *, the convalescent-phase IgG response against each subunit of PVL (LukF-PV and LukS-PV) was significantly higher than the acute titer, when restricting the analysis to PVL-positive isolates (paired t test, P < 0.05). Further, PVL-F and PVL-S antibody responses were significantly higher when restricting the analysis to PVL-positive isolates alone (P < 0.01 for the difference in GMT, Wilcoxon signed-rank test), a difference that was not present for anti-LukA and LukB responses. Samples were run in duplicate, independently on separate days, and a third run was performed if the first two titer values differed by one dilution.

FIG 4

Expression of LukA in clinical isolates from children with invasive S. aureus disease is correlated with cytotoxicity toward human neutrophils. (A) The majority of isolates showed cytotoxicity at 10% and 5% culture filtrate (vol/vol) after a 1-h incubation, as measured by LDH release from primary human neutrophils. Independent colonies from each isolate were used to start broth cultures, where culture filtrates were individually tested in duplicate. Data represent the means ± standard errors of the means (SEM) of LDH release of neutrophils from 6 human donors. (B) The majority of clinical isolates produced LukA and alpha toxin in vitro, as evaluated by SDS-PAGE separation and immunoblotting. Immunoblots were repeated with independent culture filtrates that showed the same results. Expression of LukA in vitro was significantly correlated with cytotoxicity, P < 0.001. Spearman correlation coefficient = 0.76.

FIG 5

Reverse cumulative distribution plot of the neutralization of LukAB-mediated PMN-HL60 cytotoxicity in the presence of sera from infected subjects or healthy controls. Convalescent-phase sera were significantly more potent than acute-phase sera (P < 0.01, paired t test) or healthy control sera (P < 0.001, independent t test) as measured by geometric mean neutralization titer. Samples were tested in duplicate, independently on separate days, and a third run was performed if the first two titer values differed by one dilution.

FIG 6

A comparison of acute-phase, convalescent-phase, and healthy control sera in neutralizing S. aureus USA300-mediated killing of human neutrophils. Human neutrophils were infected at an MOI of 10 with USA300 strain LAC in the presence of acute-phase, convalescence-phase, and healthy control sera. Convalescent-phase and acute-phase sera were significantly more potent than healthy control sera at neutralizing S. aureus USA300-mediated cytotoxicity toward human neutrophils, as assessed by SYTOX green incorporation. Assays were carried out over two separate days, on four PMN donors in total. Data from all donors were pooled, and error bars represent means ± SEM.