Passive therapy with humanized anti-staphylococcal enterotoxin B antibodies attenuates systemic inflammatory response and protects from lethal pneumonia caused by staphylococcal enterotoxin B-producing Staphylococcus aureus

Abstract

Drugs such as linezolid that inhibit bacterial protein synthesis may be beneficial in treating infections caused by toxigenic Staphylococcus aureus. As protein synthesis inhibitors have no effect on preformed toxins, neutralization of pathogenic exotoxins with anti-toxin antibodies may be beneficial in conjunction with antibacterial therapy. Herein, we evaluated the efficacy of human-mouse chimeric high-affinity neutralizing anti-staphylococcal enterotoxin B (SEB) antibodies in the treatment of experimental pneumonia caused by SEB-producing S. aureus. Since HLA class II transgenic mice mount a stronger systemic immune response following challenge with SEB and are more susceptible to SEB-induced lethal toxic shock than conventional mice strains, HLA-DR3 transgenic mice were used. Lethal pneumonia caused by SEB-producing S. aureus in HLA-DR3 transgenic mice was characterized by robust T cell activation and elevated systemic levels of several pro-inflammatory cytokines and chemokines. Prophylactic administration of a single dose of linezolid 30 min prior to the onset of infection attenuated the systemic inflammatory response and protected from mortality whereas linezolid administered 60 min after the onset of infection failed to confer significant protection. Human-mouse chimeric high-affinity neutralizing anti-SEB antibodies alone, but not polyclonal human IgG, mitigated this response and protected from death when administered immediately after initiation of infection. Further, anti-SEB antibodies as well as intact polyclonal human IgG, but not its Fab or Fc fragments, protected from lethal pneumonia when followed with linezolid therapy 60 min later. In conclusion, neutralization of superantigens with high-affinity antibodies may have beneficial effects in pneumonia.

Keywords: HLA class II transgenic mice; T lymphocytes; pneumonia; staphylococcus aureus; superantigen.

Figure 1.

Superantigens rapidly produced in vivo during staphylococcal pneumonia cause robust T cell activation. HLA-DR3.Nur77-eGFP transgenic mice were intra-tracheally challenged with SEB-producing S. aureus strain IDRL-7419 (SEB+SA) or the non-SEB-producing isogenic strain, IDRL-7420 (SEB-SA). Mice were sacrificed 4 and 24 hours later, spleens harvested and stained with anti-CD4, anti-CD8, anti-Vβ8 and anti-CD69 antibodies. The expression of eGFP within the TCR Vβ8⁺ (left half) and CD69⁺ (right half) CD4⁺ and CD8⁺ T cell subsets was analyzed by flow cytometry. (A) Representative dot plots at 24 hours showing the gating strategy. Histogram plots show the expression of GFP within CD4⁺ TCR Vβ8⁺ and CD8⁺ TCR Vβ8⁺ gated cells or GFP expression within CD4⁺ CD69⁺ and CD8⁺ CD69⁺ gated cells. Gray filled dotted lines represent GFP expression in cells from mice challenged with IDRL-7420 (SEB-SA) and solid black lines represent GFP expression in cells from mice challenged with IDRL-7419 (SEB+SA). Plots from one representative set of experiment is shown. (B) Table shows the percentage (mean ± SE) of GFP positive cells within the gated cells at indicated time points in the histogram plots from panel A, from two mice in each group.

Figure 2.

Prophylactic administration of linezolid as well as humanized anti-SEB antibodies administered immediately after infection followed by linezolid, attenuate systemic inflammatory response in pneumonia. Experimental pneumonia was induced with S. aureus IDRL-7419 in age-matched HLA-DR3 transgenic mice. Mice were left untreated, treated prophylactically with linezolid alone 30 min prior to infection (Lin-30 min), treated with linezolid alone 60 min after infection (Lin+60 min), treated with a mixture of chimeric anti-SEB antibody clones Ch 63 and Ch 82 M (250 μg each) administered immediately following infection and linezolid 60 min after infection (αSEB&Lin + 60 min), or treated with polyclonal human IgG (500 μg) immediately following infection and linezolid 60 min after infection (IgG&Lin + 60 min). Six hours after infection, mice were sacrificed, sera collected and levels of indicated cytokines/chemokine were determined by multiplex assay (EMD Millipore). If shown, the dotted horizontal lines represent the serum concentration of respective analyte in naïve mice. Each bar represents data from 4 to 6 mice/group. @ p < 0.05 by FDR when compared with all other groups. ^#p < 0.05 by FDR when compared with all other groups except the group with #, *p < 0.05 by FDR when compared with indicated group.

Figure 3.

Impact of combined administration of linezolid and humanized anti-SEB antibodies on bacterial load. Lungs and spleens aseptically harvested from mice sacrificed in Fig 2 were quantitatively cultured to determine bacterial load. Each bar represents mean ± SE from 3–5 mice.

Figure 4.

Efficacy of administration of linezolid and humanized anti-SEB antibodies on the outcome of lethal pneumonia caused by SEB-producing S. aureus. Experimental pneumonia was induced in age-matched HLA-DR3 mice by intratracheal inoculation of S. aureus IDRL-7419 (1 × 10⁸ CFU/mouse) in a final volume of 50 μl. Mice were left untreated (n = 31) or treated as follows. Linezolid administered at 200 mg/kg of body weight either 30 min before (Lin-30 min, n = 17) or 60 min after (Lin + 60 min, n = 17) establishment of infection; treated with a mixture of 250 μg each of chimeric anti-SEB antibody clones Ch 63 and Ch 82 M (αSEB mix alone) or 500 μg of polyclonal human IgG alone (IgG alone) immediately following infection; αSEB mix, followed by linezolid 60 min after infection (αSEB&Lin + 60 min, n = 8); polyclonal human IgG followed by linezolid 60 min after infection (IgG&Lin + 60 min, n = 8); intact human IgG derived from a multiple myeloma patient (mmIgG) (n = 6) or Fab (n = 5) or Fc (n = 5) fragments derived from polyclonal human IgG, all at 500 μg/mouse given immediately following infection. Mice were closely monitored; moribund animals were removed and euthanized as per IACUC recommendations. P values of each treatment arm compared with untreated control group by Log-rank (Mantel-Cox) test. Lin-30 min, P = 0.0001; Lin + 60 min, P = 0.60; αSEB&Lin + 60 min, P = 0.012; IgG&Lin + 60 min, P = 0.0005; αSEB alone, P = 0.0040; IgG alone, P = 0.646; mmIgG&Lin + 60 min, P = 0.97, Fab &Lin + 60 min, P = 0.54, Fc &Lin + 60 min P = 0.70.