Characterization of a protective monoclonal antibody recognizing Staphylococcus aureus MSCRAMM protein clumping factor A

Abstract

The Staphylococcus aureus MSCRAMM (microbial surface components recognizing adhesive matrix molecules) protein clumping factor A (ClfA) has been shown to be a critical virulence factor in several experimental models of infection. This report describes the generation, characterization, and in vivo evaluation of a murine monoclonal antibody (MAb) against ClfA. Flow cytometric analysis revealed that MAb 12-9 recognized ClfA protein expressed by all of the clinical S. aureus strains obtained from a variety of sources. In assays measuring whole-cell S. aureus binding to human fibrinogen, MAb 12-9 inhibited S. aureus binding by over 90% and displaced up to 35% of the previously adherent S. aureus bacteria. Furthermore, a single infusion of MAb 12-9 was protective against an intravenous challenge with a methicillin-resistant strain of S. aureus in a murine sepsis model (P < 0.0001). These data suggest that anti-ClfA MAb 12-9 should be further investigated as a novel immunotherapy for the treatment and prevention of life-threatening S. aureus infections.

FIG. 1.

Characterization of protein interactions by BIAcore. The binding characteristics of anti-ClfA MAbs 12-9 (solid line) and 15EC6 (broken line) compared to those of buffer alone (dotted line) were studied by BIAcore. The progressive real-time analysis begins at time point A, when the MAb of interest was adsorbed to the chip via rabbit anti-mouse Fcγ antibody binding. At time point B, rClfA(40-559) was injected over the chip. Point C represents the termination of the ClfA injection over the chip. Point D represents the initial injection of fibrinogen over the MAb-ClfA complex, while point E represents the termination of the fibrinogen injection over the chip.

FIG. 2.

Antibody-dependent inhibition of fibrinogen binding to rClfA(40-559). Anti-ClfA MAbs 12-9 (filled triangles) and 15EC6 (filled circles) were tested at various concentrations (0 to 10 μg/ml) for the ability to inhibit human fibrinogen binding to rClfA(40-559) compared to that of isotype control CRL-1771 (open circles). Data are representative of at least three independent experiments.

FIG. 3.

Inhibition of adhesion by preincubation of the bacterial suspension with a MAb at a concentration of 5 μg/ml and shear rates of 100, 300, and 1,000 s⁻¹. Data are representative of at least three independent experiments.

FIG. 4.

Newman destabilization experiments at an antibody concentration of 5 μg/ml and shear rates of 100, 300, and 1,000 s⁻¹. S. aureus Newman WT alone was below the limit of detection. Data are representative of at least three independent experiments.

FIG. 5.

Prophylaxis in a murine model of S. aureus-induced sepsis. (A) The biological activity of MAbs 12-9 and 35-052 was studied in a mouse septicemia model with MRSA strain 67-0. Percent survival of mice (n = 30) after treatment with anti-ClfA MAb 12-9 (dotted line) or 35-052 (solid line) is shown. *, P < 0.0001. (B) The biological impact of inhibiting fibrinogen binding to S. aureus was evaluated with MAbs 12-9 (n = 29) and 15EC6 (n = 30). Mice were challenged i.v. with S. aureus strain Newman. Percent survival of mice after treatment with anti-ClfA MAbs 12-9 (dotted line) and 15EC6 (solid line) is shown. *, P < 0.006.