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

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.

Keywords: antibody characterization; bacterial protease; host-pathogen interaction; neutralization; passive vaccination; vaccine candidate.

Fig 1

α-SplB1 mAb binds SplB in a concentration-dependent manner. (A) Binding of purified α-SplB1 mAb to recombinant tag-free SplB was determined by ELISA. One of three similar experiments is shown. (B) Dot blot immunoassay with 1 µg of native or heat-denatured tag-free SplB (upper picture; protein stained with amido black dye) incubated with 10 ng/mL α-SplB1. The graph (lower picture) shows the signal intensities of the dot blot spots as determined using ImageJ 1.52a, presented as mean ± SD of three independent experiments. Statistics: Unpaired t-test, ns, not significant. (C) Binding affinity of α-SplB1 to tag-free SplB was determined by MicroScale Thermophoresis and is depicted as normalized fluorescence (F_Norm [‰]). α-SplB1 was titrated to a constant amount of labeled, tag-free SplB. One out of three similar experiments is shown. The depicted K_D is the mean of three replicates.

Fig 2

α-SplB1 mAb does not cross-react with other S. aureus proteins. (A) Amino acid sequence identities among the Spl proteins based on paired alignments. Sequences were derived from S. aureus NCTC 8325-4. (B) A potential cross-reactivity of α-SplB1 with other Spl proteases was evaluated by ELISA using recombinant tag-free SplA, SplD, SplE, and SplF. (C) Extracellular proteins (ECPs) from TSB culture supernatants of S. aureus NCTC 8325-4 and D29 were harvested in the stationary growth phase. Quantitative Western blot was performed with 5 µg of ECPs and purified tag-free SplB in different amounts (0.002–0.1 µg). The left panel shows total protein staining using Revert 700 total protein stain (LI-COR Biosciences, Lincoln, USA [red]); the right panel shows the Western blot signals using murine α-SplB1 (300 ng/mL) as primary antibody and an IRDye 800CW-labeled goat anti-mouse IgG Ab (1:10,000 dilution) as secondary antibody (green). Prestained PageRuler (ThermoFisher Scientific, Waltham, USA) served as a size marker.

Fig 3

α-SplB1 mAb efficiently neutralizes the enzymatic activity of SplB in vitro. (A) The neutralizing activity of α-SplB1 mAb was assessed using the synthetic peptide substrate Ac-VEID-MCA. Then, 2.5 µM tag-free SplB was pre-incubated with α-SplB1 in triplicates at the indicated molar ratios at 37°C for 1 h. After substrate addition, fluorescence was quantified over 60 min. A total of 2.5 µM SplB alone and 5 µM α-SplB1 mAb alone served as negative controls. One out of five similar experiments is depicted. (B) Comparison of fluorescence intensity at t = 60 min between SplB and SplB + αSplB1 at a 1:1 molar ratio. Mean ± SD is shown. Statistics: Unpaired t-test, *P < 0.05. (C) Culture supernatants (TSB, stationary phase) from three splB-negative and three splB-positive S. aureus isolates were concentrated 250-fold to enrich the secreted SplB, incubated with the α-SplB1 mAb for 1 h at varying concentrations or PBS as control, and subsequently screened for AMC substrate cleavage. One out of two experiments is shown. AU, arbitrary units.

Fig 4

α-SplB1 mAb blocks SplB-induced vascular leakage in a murine cremaster muscle microvascular leakage model. (A) Male 6–8-week-old C57BL/6N mice were intrascrotally challenged with 10 µg tag-free SplB, with or without prior administration of α-SplB1 mAb. Vascular injury was assessed by examining the leakage of intravenously administered FITC-dextran into the perivascular tissue through fluorescence microscopy. FITC-dextran leakage was captured using a CCD camera and is presented in false colors. SplB induced vascular leakage, as indicated by a perivascular spread of the FITC signals (upper panel). Pretreatment with α-SplB1 mAb prevented SplB-induced vascular leakage (lower panel). (B) Quantification of the perivascular FITC signal using ImageJ software (mean MFI ± SD) from 3 to 8 mice per experimental group. Statistics: Unpaired t-test, *P < 0.05.