Targeting Alpha Toxin and ClfA with a Multimechanistic Monoclonal-Antibody-Based Approach for Prophylaxis of Serious Staphylococcus aureus Disease

Abstract

Staphylococcus aureus produces numerous virulence factors, each contributing different mechanisms to bacterial pathogenesis in a spectrum of diseases. Alpha toxin (AT), a cytolytic pore-forming toxin, plays a key role in skin and soft tissue infections and pneumonia, and a human anti-AT monoclonal antibody (MAb), MEDI4893*, has been shown to reduce disease severity in dermonecrosis and pneumonia infection models. However, interstrain diversity and the complex pathogenesis of S. aureus bloodstream infections suggests that MEDI4893* alone may not provide adequate protection against S. aureus sepsis. Clumping factor A (ClfA), a fibrinogen binding protein, is an important virulence factor facilitating S. aureus bloodstream infections. Herein, we report on the identification of a high-affinity anti-ClfA MAb, 11H10, that inhibits ClfA binding to fibrinogen, prevents bacterial agglutination in human plasma, and promotes opsonophagocytic bacterial killing (OPK). 11H10 prophylaxis reduced disease severity in a mouse bacteremia model and was dependent on Fc effector function and OPK. Additionally, prophylaxis with 11H10 in combination with MEDI4893* provided enhanced strain coverage in this model and increased survival compared to that obtained with the individual MAbs. The MAb combination also reduced disease severity in murine dermonecrosis and pneumonia models, with activity similar to that of MEDI4893* alone. These results indicate that an MAb combination targeting multiple virulence factors provides benefit over a single MAb neutralizing one virulence mechanism by providing improved efficacy, broader strain coverage, and protection against multiple infection pathologies.

Importance: Alternative strategies to broad-spectrum antibiotics are required to combat the antibiotic resistance epidemic. Previous attempts at active or passive immunization against Staphylococcus aureus targeting single antigens have failed in clinical trials despite positive preclinical data. To provide broad disease and isolate coverage, an effective immunization strategy likely must target multiple virulence mechanisms of the pathogen. Herein, we tested a multimechanistic MAb combination targeting alpha toxin (AT) and clumping factor A (ClfA) that neutralizes AT-mediated cytotoxicity, blocks fibrinogen binding by ClfA, prevents bacterial agglutination, targets the bacteria for opsonophagocytic killing, and provides broad isolate coverage in a lethal-bacteremia model. Although each MAb alone was effective in bacteremia against some individual isolates, the MAb combination provided improved protection against other isolates. These results illustrate the importance of targeting multiple virulence mechanisms and highlight the potential for an MAb combination targeting AT and ClfA to effectively prevent S. aureus disease.

FIG 1

ClfA and AT contribute to virulence in lethal bacteremia in mice. Mice (n = 10) were i.v. infected with 6 × 10⁷ CFU of wild-type (WT) SF8300 or an isogenic ΔclfA, Δhla, or ΔclfA Δhla mutant. (A) Survival was monitored for 2 weeks postinfection. Statistical differences from wild-type SF8300 were analyzed with a log rank (Mantel Cox) test. Bacterial burdens in hearts 14 h postinfection (B) and in kidneys 48 h postinfection (C) were determined. Horizontal lines represent geometric mean numbers of CFU. Statistical differences from the SF8300 wild type were analyzed with a Mann-Whitney U test. Data were considered statistically different if P was <0.05. Results are representative of three independent experiments.

FIG 2

In vitro characterization of human anti-ClfA MAb and 11H10. (A) Antibody affinity to ClfA determined with Octet. (B) Inhibition of fibrinogen (Fg) binding. ClfA binding to fibrinogen was measured in the presence of serially diluted 11H10 (300 to 0.03 µg/ml) and c-IgG or in the absence of any MAb. Data are the mean values ± standard deviation (SD) from three independent experiments. (C) Agglutination of S. aureus clinical isolates in the presence of human plasma. The graph illustrates the minimal concentration of 11H10 required to inhibit bacterial agglutination. A human negative-control MAb, c-IgG, did not show any inhibitory effect up to 200 µg/ml. Data are representative of three independent experiments, with the same donor as a plasma source. (D) Bacterial OPK assay. S. aureus strain Newman was incubated with human HL-60 cells, human sera, and serial dilutions of 11H10 or negative-control IgG. The graph represents mean values ± SD from three independent experiments. (E) 11H10 binding (blue lines) to Δspa SF8300 grown to stationary phase in TSB (left) or ex vivo after passage in mice for 1 h (right) was measured by fluorescence-activated cell sorting (FACS). Histograms represent the typical binding of 11H10 compared to that of negative-control c-IgG (red lines).

FIG 3

Activities of anti-AT and anti-ClfA in an SF8300 lethal-bacteremia model. (A) BALB/c mice (n = 30) were immunized i.p. with MEDI4893*, 11H10, or c-IgG at the indicated concentrations 24 h prior to i.v. challenge with SF8300’s LD₉₀ (5e7 CFU), and survival (n = 10) was monitored for 2 weeks. mpk, mg/kg. Ten animals were euthanized after 14 h and 48 h for bacterial enumeration in their hearts (B) and kidneys (C). Horizontal bars represent geometric mean numbers of CFU. Statistical differences from the c-IgG group were analyzed with a log rank (Mantel Cox) test, and values were considered statistically different if P was <0.05. Data are representative of three independent experiments.

FIG 4

OPK is required for 11H10 efficacy in lethal bacteremia. (A) BALB/c mice (n = 10) were injected i.p. with 15 mg/kg (mpk) of 11H10, 11H10_N297Q, or c-IgG 24 h prior to i.v. challenge with SF8300 (5e7 CFU), and survival was monitored for 2 weeks. Statistical analysis was performed with a log rank test (Mantel Cox test). (B) Bacteria were enumerated in kidneys 48 h after infection. Horizontal lines represent geometric mean numbers of CFU. Statistical analyses were conducted with a Mann-Whitney U test. Data are representative of three independent experiments.

FIG 5

An anti-AT–anti-ClfA MAb combination provides benefit over monotherapy in preventing murine sepsis. Mice (n = 10) were immunized i.p. with MEDI4893* (2 mg/kg), 11H10 (2 mg/kg), a combination of both MAbs (1 mg/kg each), or c-IgG (2 mg/kg) 24 h prior to infection, and survival was monitored for 2 weeks. (B and C) Statistical differences from the c-IgG group were analyzed with a log rank (Mantel Cox) test. Bacteria in the heart 14 h postinfection (B) or the kidneys 48 h postinfection (C) were enumerated. Horizontal lines represent geometric mean numbers of CFU. Statistical analysis versus c-IgG was performed with a Mann-Whitney U test. Statistical analyses were considered statistically different if P was <0.05. Data are representative of three independent experiments.

FIG 6

Effect of monotherapy and MAb combination on plasma biomarkers of organ damage. Mice (n = 10) were immunized i.p. with MEDI4893* (2 mg/kg), 11H10 (2 mg/kg), a combination of both MAbs (1 mg/kg each), or c-IgG (2 mg/kg) 24 h prior to infection with WT SF8300 (5e7 CFU). Plasma biomarkers of organ damage were measured 48 h after infection. Graphs represent mean values ± standard errors of the means for the biomarkers cardiac troponin 1 in nanograms per milliliter (top left) and for creatine kinase myocardial band (CK-MB) (top right), alanine aminotransferase (bottom left), and aspartate aminotransferase (bottom right) in units per liter. Background levels for each marker were measured in the plasma of mock-infected animals (200 µl PBS i.v.). Statistical analysis relative to c-IgG was performed with Student’s unpaired t test. Values were considered statistically different if P was <0.05 (P values are indicated on the graphs). Data are representative of three independent experiments.

FIG 7

Protection from dermonecrosis and pneumonia with MEDI4893* and the MEDI4893*-11H10 combination. BALB/c mice (n = 10) were immunized i.p. with MEDI4893*, a combination of both MEDI4893* and 11H10 MAbs, or c-IgG at the indicated concentrations 24 h prior to intradermal infection with SF8300 (5e7 CFU). (A) The graph represents lesion sizes at day 1 postinfection as mean values ± standard errors. (B) Bacteria in the skin lesions were enumerated 7 days postinfection. Statistical analysis for each group versus c-IgG was performed with a Student unpaired t test. (C and D) C57/Bl6 mice (n = 10) were injected i.p. with MEDI4893*, 11H10, a combination of both MAbs, or c-IgG at the indicated concentrations 24 h prior to intranasal (IN) infection with SF8300 (1.8e8 CFU). (C) Survival was monitored for 5 days. (D) Bacteria were enumerated in the lungs 24 h postinfection with SF8300 (1e8 CFU). Statistical analysis for each group versus c-IgG was performed with a Student unpaired t test. Values were considered statistically different if P was <0.05, and statistically significant differences are indicated with a number sign. Data are representative of two independent experiments.