SpA_KKAA-specific monoclonal antibodies (MAbs) protect mice against MRSA infection. Cohorts of animals (n = 10) were passively immunized by intraperitoneal injection with either isotype control (IgG_2a) or SpA_KKAA MAb (3F6) at 20 mg · kg⁻¹. After 24 h, animals were challenged with 5 × 10⁶ CFU of S. aureus MW2. (A) At 15 days postchallenge, animals were euthanized to enumerate the staphylococcal load in kidneys. (B) Serum samples of mice infected for 15 days were analyzed for antibodies against the staphylococcal antigen matrix. ClfA, clumping factor A; ClfB, clumping factor B; FnBPA, fibronectin binding protein A; FnBPB, fibronectin binding protein B; IsdA, iron surface determinant A; IsdB, iron surface determinant B; SdrD, serine-aspartic acid repeat protein D; SpA_KKAA, nontoxigenic staphylococcal protein A; Coa, coagulase; EsxA, Ess (early secreted antigen target 6 kDa [ESAT-6] secretion system) extracellular A; EsxB, Ess (ESAT-6 secretion system) extracellular B; Hla, alpha-hemolysin; LukD, leukocidin D; vWbp, von Willebrand binding protein. The values represent the fold increases of samples from MAb 3F6-treated animals over the isotype control animal serum samples (n = 7 for IgG_2a, n = 8 for 3F6). Data are the means, and error bars represent ± standard errors of the means (SEM). Results in panels A and B are representative of two independent analyses.

SpA monoclonal antibody (Spa27) fails to elicit protective immunity in mice. (A) ELISA examining the association of SpA MAb (Spa27) and SpA_KKAA MAb (3F6) with immobilized wild-type protein A (SpA) and variants lacking immunoglobulin binding via Fcγ (SpA_KK), Fab (SpA_AA), or Fcγ and Fab (SpA_KKAA) (n = 3). (B) Cohorts of animals (n = 9 to 15) were passively immunized by intraperitoneal injection with either mock (PBS), Spa27 at 5 mg · kg⁻¹, or 3F6 at 5 or 50 mg · kg⁻¹. Twenty-four hours postimmunization, animals were challenged with 5 × 10⁶ CFU of S. aureus USA300. Four days postchallenge, animals were euthanized to enumerate the staphylococcal load in kidneys.

Avidity of protein A-specific monoclonal antibodies. Monoclonal antibodies 5A10, 3F6, and 3D11 were incubated with increasing concentrations (0 to 4 M) of ammonium thiocyanate to perturb antigen-antibody-specific interactions. Data are the means, and error bars represent ± SEM. Results are representative of three independent analyses.

SpA_KKAA MAb 3F6 binds to Sbi (staphylococcal binder of immunoglobulin). (A) Coomassie blue-stained SDS-PAGE gel revealing the electrophoretic mobility of human immunoglobulin (hIgG, lane 1), purified recombinant Sbi encompassing its immunoglobulin binding domains (Sbi_1-4, lane 2), and variant Sbi, which cannot bind hIgG (Sbi_1-4/KKAA, lane 3). Using affinity chromatography, His-tagged Sbi_1-4 and Sbi_1-4/KKAA were eluted from nickel-nitriloacetic acid Sepharose without (−hIgG; lanes 1 to 3) or with (+hIgG; lanes 4 and 5) incubation with human immunoglobulin. (B) ELISA examining the association of immobilized Sbi_1-4/KKAA with protein A-specific MAbs (n = 3). Data are the means, and error bars represent ± SEM. Results in panels A and B are representative of three independent analyses.

SpA_KKAA-specific MAbs bind wild-type protein A. (A) ELISA examining the binding of immobilized wild-type protein A (SpA) to isotype control antibodies (IgG1, IgG_2a, or IgG_2b) or SpA_KKAA-specific MAbs (5A10, 3F6, and 3D11). (B) Association of horseradish peroxidase (HRP)-conjugated SpA_KKAA-specific MAbs (5A10-HRP, 3F6-HRP, and 3D11-HRP) to immobilized SpA_KKAA was examined in a plate reader experiment where SpA_KKAA was first incubated with isotype control antibodies (IgG1, IgG_2a, or IgG_2b) or three different SpA_KKAA-specific MAbs (5A10, 3F6, and 3D11) to assess the possibility of competitive inhibition for antibodies that bind the same or closely related sites (n = 3). The values at an optical density at 405 nm (OD₄₀₅) were measured and normalized to the interaction of SpA_KKAA and HRP-conjugated SpA-specific MAbs. Data are the means, and error bars represent ± SEM. Data in panels A and B are representative of three independent analyses. Asterisks denote statistical significance (P < 0.05), which was calculated using the two-tailed Student's t test: 5A10-HRP versus 5A10, P = 0.0017; 5A10-HRP versus 3F6, P = 0.0343; 5A10-HRP versus 3D11, P = 0.001; 3F6-HRP versus 5A10, P = 0.0279; 3F6-HRP versus 3F6, P = 0.0001; 3F6-HRP versus 3D11, P = 0.0584; 3D11-HRP versus 5A10, P = 0.0001; 3D11-HRP versus 3F6, P = 0.0005; 3D11-HRP versus 3D11, P = 0.0053.

SpA_KKAA MAbs prevent the association of staphylococcal protein A with immunoglobulin. (A) Isotype control antibodies or SpA_KKAA MAbs were used to perturb the binding of human IgG to wild-type protein A (SpA) or variants that lack the ability to bind Fcγ (SpA_KK) or Fab (SpA_AA) immobilized on ELISA plates. The values were normalized to the protein A interaction with human IgG without antibodies (n = 4). (B) Staphylococci were grown to mid-log phase and incubated with either isotype control antibody or MAb 3F6 and followed by the addition of 2 μg wild-type Sbi_1-4. Upon incubation, Sbi_1-4 consumption was measured by immunoblot using affinity-purified α-SpA_KKAA rabbit antibody. The values were normalized to Sbi_1-4 sedimentation without antibody (No Ab). (C) Affinity-purified SpA (200 μg) was injected into the peritoneal cavity of mice pretreated with 85 μg (5 mg · kg⁻¹) of either isotype control antibody or MAb 3F6. Animals were euthanized at indicated time points to measure the amount of SpA in circulating blood by immunoblotting with affinity-purified α-SpA_KKAA rabbit antibody (n = 3 per time point). The values were normalized to the total amount of SpA injected at 0 min. Data are the means, and error bars represent ±SEM. Results in panels A to C are representative of two independent analyses. The asterisks denotes statistical significance (P < 0.05).

SpA_KKAA MAbs promote opsonophagocytic killing of S. aureus in mouse and human blood. (A) Anticoagulated mouse blood was incubated with 5 × 10⁵ CFU S. aureus Newman in the presence of isotype mouse antibody controls or SpA_KKAA MAbs (2 μg · ml⁻¹) for 30 min, and survival was measured (n = 3). (B) Anticoagulated human whole blood was incubated with 5 × 10⁶ CFU S. aureus MW2 in the presence of isotype mouse antibody controls or SpA_KKAA MAbs (10 μg · ml⁻¹) for 120 min, and survival was measured (n = 3). (C to H) At 60 min of incubation of staphylococci in anticoagulated human blood, clusters of extracellular staphylococci were detected in samples incubated with mouse isotype antibody controls (red arrowheads), whereas staphylococci were found in close proximity to neutrophils (blue arrowheads) in samples with SpA_KKAA MAbs. Data are the means, and error bars represent ±SEM. Results in panels A to H are representative of three independent analyses. The asterisks denotes statistical significance (P < 0.05).