Abstract
ABSTRACTThe multidrug-resistant H30 subclone of extraintestinal pathogenic Escherichia coli sequence type 131 (ST131-H30) has spread worldwide. This clone expresses a conserved lipopolysaccharide (LPS) O antigen, O25b. Previously, we described monoclonal antibodies (MAbs) specific to the O25b antigen and characterized them as diagnostic and therapeutic tools. In this study, evidence is provided that besides the previously shown complement-mediated bactericidal effect, an O25b-specific humanized MAb, A1124, also enhances opsonophagocytic uptake by the murine macrophage cell line RAW 264.7. Both phagocyte-dependent killing and phagocyte-independent killing, triggered by A1124, were confirmed in human whole blood. Furthermore, A1124 was shown to neutralize endotoxin activity of purified LPS of clinical isolates. This activity was demonstrated in vitro using both RAW 264.7 cells and a human Toll-like receptor 4 (TLR4) reporter cell line, as well as in a murine model of endotoxemia using purified LPS for challenge. Significant protective efficacy of A1124 at low doses (<1 mg/kg of body weight) was shown in murine and rat models of bacteremia. The contribution of the bactericidal and anti-inflammatory effects was dissected in the mouse bacteremia model through depletion of complement with cobra venom factor (CVF). Protective efficacy was lost in complement-depleted mice, suggesting the essential role of complement-mediated activities for protection in this model. These data suggest that A1124 exhibits different mechanisms of action, namely, direct complement-mediated and opsonophagocytic killing as well as endotoxin neutralization in various challenge models. Which of these activities are the most relevant in a clinical setting will need to be addressed by future translational studies.
Highlights
The multidrug-resistant H30 subclone of extraintestinal pathogenic Escherichia coli sequence type 131 (ST131-H30) has spread worldwide
Bacteria were incubated in human serum samples that had been depleted of ST131specific antibodies, in the presence of different concentrations of A1124 or an isotype control IgG with irrelevant specificity
As therapeutic monoclonal antibodies (MAbs) are intended for parenteral application and LPS is known to interact with serum proteins such as lipopolysaccharide-binding protein (LBP), albumin, bactericidal permeability-increasing protein (BPI), etc., we aimed to clarify whether human serum would interfere with the endotoxin-neutralizing activity of A1124
Summary
The multidrug-resistant H30 subclone of extraintestinal pathogenic Escherichia coli sequence type 131 (ST131-H30) has spread worldwide. This clone expresses a conserved lipopolysaccharide (LPS) O antigen, O25b. Evidence is provided that besides the previously shown complement-mediated bactericidal effect, an O25b-specific humanized MAb, A1124, enhances opsonophagocytic uptake by the murine macrophage cell line RAW 264.7. Both phagocyte-dependent killing and phagocyte-independent killing, triggered by A1124, were confirmed in human whole blood. We describe three distinct mechanisms of action for this single MAb that were distinguished by various in vitro assays and in murine models
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