Monoclonal antibody neutralizes Staphylococcus aureus serine protease-like protein B (SplB)-induced pathology

Abstract

ABSTRACT Staphylococcus aureus is a versatile pathogen, renowned for its arsenal of virulence and immune evasion factors. Several S. aureus virulence factors have been targeted in vaccination trials; however, so far, without success. Promising new vaccine candidates are the staphylococcal serine protease-like proteins (Spl A–F), which are involved in the pathogenesis and immune evasion of S. aureus . SplB, for instance, promotes type 2 immune responses and inactivates human complement factors. In this study, we report on the production and characterization of a murine monoclonal antibody (mAb) against SplB. The murine anti-SplB mAb α-SplB1 was produced by hybridoma technology, and its binding characteristics were investigated using enzyme-linked immunosorbent assay (ELISA), Western blot, and MicroScale Thermophoresis. Its neutralizing capacity was determined in a fluorogenic substrate assay, Western blot, and a murine vascular leakage model. α-SplB1 bound to recombinant SplB with high specificity, showing no cross-reactivity to other Spls or secreted proteins of S. aureus . MicroScale Thermophoresis revealed a K D value of 37.9 nM for the α-SplB1:SplB interaction. α-SplB1 neutralized the enzymatic activity of SplB in vitro in a dose-dependent manner, yielding complete neutralization at a twofold molar excess of the antibody. In a murine vascular leakage model, the antibody completely abolished SplB-mediated endothelial damage. In summary, we produced a neutralizing mAb against the staphylococcal protease SplB, which merits further investigation as a candidate for the immunotherapy of SplB-induced pathologies.