Selection and Characterization of Murine Monoclonal Antibodies toStaphylococcus aureusIron-Regulated Surface Determinant B with Functional Activity In Vitro and In Vivo

Abstract

In an effort to characterize important epitopes of Staphylococcus aureus iron-regulated surface determinant B (IsdB), murine IsdB-specific monoclonal antibodies (MAbs) were isolated and characterized. A panel of 12 MAbs was isolated. All 12 MAbs recognized IsdB in enzyme-linked immunosorbent assays and Western blots; 10 recognized native IsdB expressed by S. aureus. The antigen epitope binding of eight of the MAbs was examined further. Three methods were used to assess binding diversity: MAb binding to IsdB muteins, pairwise binding to recombinant IsdB, and pairwise binding to IsdB-expressing bacteria. Data from these analyses indicated that MAbs could be grouped based on distinct or nonoverlapping epitope recognition. Also, MAb binding to recombinant IsdB required a significant portion of intact antigen, implying conformational epitope recognition. Four MAbs with nonoverlapping epitopes were evaluated for in vitro opsonophagocytic killing (OPK) activity and efficacy in murine challenge models. These were isotype switched from immunoglobulin G1 (IgG1) to IgG2b to potentially enhance activity; however, this isotype switch did not appear to enhance functional activity. MAb 2H2 exhibited OPK activity (>50% killing in the in vitro OPK assay) and was protective in two lethal challenge models and a sublethal indwelling catheter model. MAb 13C7 did not exhibit OPK (<50% killing in the in vitro assay) and was protective in one lethal challenge model. Neither MAb 13G11 nor MAb 1G3 exhibited OPK activity in vitro or was active in a lethal challenge model. The data suggest that several nonoverlapping epitopes are recognized by the IsdB-specific MAbs, but not all of these epitopes induce protective antibodies.

Four-Component Staphylococcus aureus Vaccine 4C-Staph Enhances Fcγ Receptor Expression in Neutrophils and Monocytes and Mitigates S. aureus Infection in Neutropenic Mice

“…Evidence is available suggesting the importance of antibodies in controlling S. aureus infections: in vitro and in vivo antibodydependent protection have been reported for S. aureus antigens (20)(21)(22)(23), antibody titers against S. aureus have been shown to rise during infections (24), and immunoglobulin deficiencies were related to an increase in susceptibility to the pathogen (25). Nevertheless, limited correlations have been established between antibody levels and disease severity or protection (22,26).…”

Naturally occurring IgG antibody levels to theStaphylococcus aureusprotein IsdB in humans

“…The nitrocellulose blots were probed with an IsdB specific monoclonal antibody, 2H2, previously described. 17 This monoclonal was demonstrated to have protective efficacy in rodent challenge models.…”

“…Importantly, enhanced protection from lethal sepsis in rodent models was mediated by both IsdB-specific CD4+ T cells, 16 and IsdB-specific mAb. 10,13,[17][18][19] The protection afforded by vaccination with IsdB in rodents was lost if the animals were challenged with an IsdB fluid (9), surgical tissue (22), synovial fluid (17), sputum (9), or other sites (5) during routine clinical care. Of the 52 S. aureus isolates, 51 were spa typed; a single isolate failed to grow sufficiently in culture for spa determination.…”

Finding Protective Bacterial Antigens

“…V710 successfully completed safety and immunogenicity studies performed in several populations (healthy adults, elderly, hemodialysis patients) with very promising results. Murine and human monoclonal antibodies generated with IsdB were shown to have in vitro opsonophagocytic activity and efficacy in S. aureus infectious models (Brown et al 2009;Ebert et al 2010). Yet this single-component vaccine failed to reach predefined efficacy endpoint in a phase IIb study to prevent S. aureus infections in patients undergoing cardiac surgery.…”