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Generation, characterization, and epitope mapping of neutralizing and protective monoclonal antibodies against staphylococcal enterotoxin B-induced lethal shock - PubMed

  • ️Sat Jan 01 2011

Generation, characterization, and epitope mapping of neutralizing and protective monoclonal antibodies against staphylococcal enterotoxin B-induced lethal shock

Avanish K Varshney et al. J Biol Chem. 2011.

Erratum in

  • J Biol Chem. 2014 May 16;289(20):13706

Abstract

T-cell stimulating activity of Staphylococcal enterotoxin B (SEB) is an important factor in the pathogenesis of certain staphylococcal diseases including SEB mediated shock. SEB is one of the most potent superantigens known and treatment of SEB induced shock remains a challenge. We generated and characterized murine monoclonal antibodies (mAbs) to SEB in mice. We tested mAbs neutralize mitogenic effects of SEB in vitro and in vivo with T-cell proliferation assays and 2 murine models for SEB induced lethal shock (SEBILS). Epitope mapping suggests that all these mAbs recognize conformational epitopes that are destroyed by deleting the C terminus of the protein. Further site-directed mutagenesis identified potential residues involved in binding to SEB that differ between Methicillin resistant and sensitive Staphylococcus aureus strains. Only mAb 20B1 was effective as a monotherapy in treating SEBILS in HLA DR3 transgenic mice, which exhibit enhanced sensitivity to SEB. It is noteworthy that mAbs, 14G8 and 6D3 were not protective when given alone in the HLA DR3 mice but their efficacy of protection could be greatly enhanced when mAbs were co-administered simultaneously. Our data suggest combinations of defined mAbs may constitute a better treatment strategy and provide a new insight for the development of passive immunotherapy.

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Figures

FIGURE 1.
FIGURE 1.

Western blot analysis of mAbs 20B1, 14G8, 6D3, and 4C7 shows specificity of mAbs for SEB and not for SEA and TSST.

FIGURE 2.
FIGURE 2.

Schematic of SEB sequence in MRSA and MSSA strains demonstrate the additional nucleotide thymidine found in all MRSA strains at position 703 which results in 3-aa residues change in the C-terminal part of the protein.

FIGURE 3.
FIGURE 3.

Inhibition of T-cell proliferation and cytokine production by treatment with SEB specific mAb 20B1, 14G8, 6D3, and 4C7 individually or in different combinations. A, SEB-induced T-cell proliferation was measured by ViaLight HS Cell Proliferation kit after 48 h (A) and 96 h (B) and inhibited in the presence of all three mAb except 4C7. IFNγ (C) and IL-2 (D) were measured by ELISA in the supernatant of SEB stimulated T-cells (n = 3 wells per condition). Cytokines were significantly (p < 0.05 by t test) lower in the presence of mAbs relative to conditions with no specific antibody. The bars represent the S.D. derived from triplicate wells from same experiment.

FIGURE 4.
FIGURE 4.

Protection against SEBILS was tested in BALB/c and HLA-DR3 mice (n = 10 per group) that were injected intraperitoneal with 20 μg of SEB for BALB/c (0 h) (A and B), or 50 μg of SEB for HLA-DR3 mice (0 and 48 h) (C and D). Analysis of survival data were performed using Mantel-Cox Test. In the BALB/c model mAb 20B1 was protective at doses of 500 μg (p = <0.0001) as well as 100 μg (p = 0.0003). HLA DR-3 mice that were treated intraperitoneal with 500 μg 20B1 at the same time were 100% protected whereas all SEB-injected mice treated with PBS or up to 1000 μg of mAbs 14G8 or 6D3 (HLA/DR3) died within 6 days (p = <0.0001). In contrast, mice treated with combination of mAbs 6D3 and 14G8 survived although monotherapy with the individual mAb was not protective. Similar enhanced protection was observed in the BALB/c mouse model when 20B1 was combined either with 6D3 or 14G8. No enhanced protection was found when 4C7 was administered.

FIGURE 5.
FIGURE 5.

Protection against MRSA-derived SEB protein induced lethal shock was also determined in BALB/c mice by treatment with mAb 20B1 (p = 0. 0109). n = 10 each group. Analysis of survival data were performed using Mantel-Cox Test.

FIGURE 6.
FIGURE 6.

SEB level in the serum of (A) BALB/c and (B) HLA-DR3 mice (n = 10 per group) was measured by ELISA. Note that mice injected with SEB and mAb 20B1 exhibited the highest SEB serum levels both in BALB/c and HLA/DR3 mice. Bars are averages of SEB measurements in the serum of five mice in each group and brackets denote intra-assay S.D. The experiment was repeated and yielded similar differences. Gala, galactosamine.

FIGURE 7.
FIGURE 7.

Capture ELISA with mAbs shows that two different SEB-specific mAbs can bind to SEB at the same time. Bars represent the average of three absorbance units at wavelength 405 nm and brackets denote intra-assay S.D. Inset, schematic diagram of ELISA, which applies to this experiment.

FIGURE 8.
FIGURE 8.

A, schematic diagram of SEB deletion mutants. B, SDS-PAGE shows the expression of SEB and deletion mutants (M, marker, 1, uninduced cells, 2, induced SEB, 3, induced mutant-1 (5del SEB), 4, induced mutant-2 (11 del SEB), 5, induced mutant-3 (15 del SEB). C, Western blot with mAbs and SEB deletion mutants shows that all three mAbs fail to bind to mutant 2 (11 residue deletion) and 3 (15 residue deletion). Not shown is that these mAbs also do not bind to the shorter SEB fragments. D, dot blot analysis shows binding of 10-mer peptide with all three mAbs with SEB and mutant-1 and no binding with mutant-2. The binding affinity for the 10-mer peptide was low. E, ELISA with purified SEB mutants protein (1 and 2) confirmed no binding of mutant 2 by mAbs 20B1, 14G8, and 6D3. FL, full-length.

FIGURE 9.
FIGURE 9.

ELISA shows the effect of binding using different site directed mutagenesis proteins. Mutant proteins were coated in polystyrene plates at a concentration of 0.5 μg/ml. Further mAb 20B1 or 14G8 or 6D3 or 4C7 was added, detected by alkaline phosphatase (AP)-conjugated goat anti-mouse IgG1 and developed by PNPP tablets. The x-axis represents absorbance at 405 nm and y-axis represents the log of antibody concentration (in μg). Results identify different critical residues, which could interact with the individual SEB specific mAbs. For mAb 20B1 mutation of residue 135-R, 137-F, 186-Y, 235 & 236-T affected binding. The residues 135-R, 186-Y were required for the interaction with mAb 6D3. mAb 14G8 bind to residues 135-R, 137-F, 186-Y, 188-K, 231-E, 233-Y, and 235, 236-T, whereas mAb 4C7 interact with 135-R, 137-F, 186-Y, 188-K, and 235, 236-T.

FIGURE 10.
FIGURE 10.

Schematic representation of the potential residues recognized by SEB specific mAbs 20B1, 14G8, 6D3, and 4C7. All mAbs recognize non-continuous residues that are likely to contribute to conformational epitopes. A, schematic illustration of the three-dimensional structure of SEB recognizing potential residues of mAbs. B, schematic diagram of expanded view of the β-sheet formed by the three strands, which could disrupt by deleting C-terminal residues. C, surface plot of SEB shows mutated residues (red color) which are distinct from D the MHC surface (rotating 180 degrees around vertical axis) shows in cyan (residues 43, 44, 45, 46, 47, 65, 67, 89, 92, 94, 96, 98, 115, 209, 211, 215) and TCR surface in green (residues 18, 19, 20, 22, 23, 26, 60, 90, 91, 177, 178, and 210).

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