Functional antibodies targeting IsaA of Staphylococcus aureus augment host immune response and open new perspectives for antibacterial therapy

Abstract

Staphylococcus aureus is the most common cause of nosocomial infections. Multiple antibiotic resistance and severe clinical outcomes provide a strong rationale for development of immunoglobulin-based strategies. Traditionally, novel immunological approaches against bacterial pathogens involve antibodies directed against cell surface-exposed virulence-associated epitopes or toxins. In this study, we generated a monoclonal antibody targeting the housekeeping protein IsaA, a suggested soluble lytic transglycosylase of S. aureus, and tested its therapeutic efficacy in two experimental mouse infection models. A murine anti-IsaA antibody of the IgG1 subclass (UK-66P) showed the highest binding affinity in Biacore analysis. This antibody recognized all S. aureus strains tested, including hospital-acquired and community-acquired methicillin-resistant S. aureus strains. Therapeutic efficacy in vivo in mice was analyzed using a central venous catheter-related infection model and a sepsis survival model. In both models, anti-IsaA IgG1 conferred protection against staphylococcal infection. Ex vivo, UK-66P activates professional phagocytes and induces highly microbicidal reactive oxygen metabolites in a dose-dependent manner, resulting in bacterial killing. The study provides proof of concept that monoclonal IgG1 antibodies with high affinity to the ubiquitously expressed, single-epitope-targeting IsaA are effective in the treatment of staphylococcal infection in different mouse models. Anti-IsaA antibodies might be a useful component in an antibody-based therapeutic for prophylaxis or adjunctive treatment of human cases of S. aureus infections.

FIG. 1.

Quantitative analysis of the interaction of UK-66P with rIsaA, carried out using surface plasmon resonance (SPR). Various concentrations of rIsaA (0.8 to 400 nM) were flushed over the antibody UK-66P, immobilized on the sensor chip surface. Sensorgrams were recorded at a flow rate of 30 μl/min at 25°C. From these sensorgrams, an equilibrium dissociation constant (K_D) of 1.7 nM was determined. Rate constants for association (k_a) and dissociation (k_d) were determined to be 1.8 × 10⁵ M⁻¹ s⁻¹ and 3.1 × 10⁻⁴ s⁻¹, respectively. The figure shows one representative result from two independent experiments yielding identical kinetic constants.

FIG. 2.

Display of UK-66P on the staphylococcal surface and positive binding to representative clinical S. aureus isolates are shown. Binding of FITC-labeled anti-mouse IgG to UK-66P was analyzed by conventional microscopy of S. aureus (top) and corresponding fluorescence microscopy with the data superimposed (bottom). (A) UK-66P binds specifically to wild-type S. aureus MA12. (B) The isogenic mutant strain MA12 ΔisaA failed to bind UK-66P. (C) The S. aureus protein A knockout strain (Cowan I Δspa) binds UK-66P, indicating no antibody cross-reactivity with protein A. Magnification, ×100. (D) Reactivity of UK-66P to IsaA of representative clinical isolates strain ANS46 (SCCmec III), strain BK2464 (SCCmec II), strain HDE288 (SCCmec IV), strain MU50 (vancomycin-resistant S. aureus [VRSA]), strain MW2 (CA-MRSA), strain USA300 (CA-MRSA), and strain EMSRA-15 (epidemic MRSA) was constant, as verified by Western blotting.

FIG. 3.

UK-66P activates neutrophils. Mouse blood (100 μl) was incubated with 5 × 10⁷ CFU of UK-66P-opsonized wild-type S. aureus MA12 or IsaA mutant S. aureus MA12 ΔisaA. Controls included isotype control antibody (IC)-opsonized and unopsonized (saline) bacteria. The percentages of activated and oxidizing neutrophils were determined using a DHR123/R123 assay with flow cytometric analysis. (A) At 30 and 60 min, the fraction of oxidizing neutrophils was significantly higher for the wild-type (wt) bacteria coincubated with UK-66P than for the IC- and saline-coincubated bacteria (Mann-Whitney test, P < 0.05). (B) As a specificity control, the respective percentages of oxidizing neutrophils were similar for UK-66P-, IC-, and saline-coincubated IsaA mutant bacteria. Error bars represent SDs.

FIG. 4.

Oxidative burst of neutrophils is significantly enhanced in response to UK-66P-opsonized S. aureus. The oxidative burst activity of native mouse blood neutrophils was determined using a DHR123/R123 assay and flow cytometric analysis. (A) Oxidative burst was monitored by observing the fluorescence events (M1) in an FL1 overlay histogram. Wild-type S. aureus MA12-stimulated neutrophils with the addition of saline, isotype control antibody (IC), or UK-66P at concentrations of 0.3 mg/ml and 0.6 mg/ml. (B) As a specificity control for UK-66P, the oxidative burst was additionally monitored for IsaA mutant S. aureus MA12 ΔisaA-stimulated neutrophils with the addition of saline, IC, or UK-66P at concentrations of 0.3 mg/ml and 0.6 mg/ml. (C) Mean fluorescence intensity (MFI) of UK-66P-opsonized bacteria at concentrations of 0.3 and 0.6 mg/ml (equivalent to 15 mg/kg and 30 mg/kg body weight, respectively) compared to those for IC- or saline-opsonized wild-type and IsaA mutant bacteria. Significant differences are denoted (Mann-Whitney test). Error bars represent SDs.

FIG. 5.

Effect of UK-66P on survival of S. aureus within neutrophils in whole mouse blood. Mouse blood (100 μl) was incubated with 5 × 10⁷ CFU of UK-66P-opsonized wild-type S. aureus MA12 or IsaA mutant S. aureus MA12 ΔisaA. Controls included isotype control antibody (IC)-opsonized bacteria. The number of neutrophil-associated CFU was determined by serial dilution and plating on TSB. UK-66P-opsonized wild-type S. aureus bacteria were killed significantly better than IC-opsonized bacteria (mean CFU ± SD, 1.13 × 10⁵ ± 9.38 × 10³ and 2.99 × 10⁵ ± 3.65 × 10³, respectively; Mann-Whitney test, P = 0.0286). UK-66P- and IC-opsonized IsaA mutant S. aureus produced similar results (mean CFU ± SD, 1.8 × 10⁵ ± 1.3 × 10⁴ and 1.9 × 10⁵ ± 6.1 × 10³, respectively). Error bars represent SDs.

FIG. 6.

Bacterial burden after UK-66P and isotype control antibody treatment in lung, heart, liver, spleen, and kidneys 5 days after infection with S. aureus MA12 in the catheter-related infection model. The mice (7 or 9 per group) were inoculated via catheter with 1 × 10⁷ CFU bacteria. *, P < 0.05 compared with IC-treated mice, by Kruskal-Wallis testing with posthoc Dunn's multiple-comparison testing. Data are graphed as a box-and-whisker plot.

FIG. 7.

Immunotherapy with UK-66P generates protection against lethal S. aureus challenge. Mice (7 or 8 per group) were given UK-66P antibody preparation or isotype control antibody (IC). Animals were challenged with 5 × 10⁸ CFU of wild-type S. aureus USA300, MA12, or IsaA mutant S. aureus MA12 ΔisaA by intravenous injection, and then they were monitored for 8 days. The significance of protection compared to that for animals receiving control IgG1 was measured with the log rank Mantel-Cox test.