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Cooperative Immunological And Pharmacological Control Of SEB-Induced T Cell Activation And Subsequent Pathology

Abstract
Staphylococcal Enterotoxin B (SEB) is one of the potent exotoxins synthesized by Staphylococcus aureus that causes toxic shock, is a primary cause of food poisoning and is a Class B bioterrorism agent. SEB, a superantigen, mediates antigen-independent activation of a major subset of the T-cell population by crosslinking TCRs of T-cells with MHC class II molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. This excessive secretion of cytokines, some of which are inflammatory, causes immune dysregulation, systemic inflammation and disease. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC class II and provide protection against the debilitating effects of this superantigen. In a series of experiments, we derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of noncrossreactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically-acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human PBMCs, or splenocytes derived from BALB/c or HLA- DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T cell activation and cytokine production. When tested in vivo in HLA-DR3 transgenic mouse TSS model, the two chimeric antibodies acted synergistically and provided full protection against SEB-mediated TSS symptoms and lethality of SEB. Furthermore, combination of chimeric anti-SEB, an extracellular inhibitor of SEB, and pharmacological agents (γ-secretase inhibitors, rapamycin, or lovastatin), an inhibitor of intracellular pathways recruited by SEB, provided significant reduction of SEB-induced T cell activation in cultures of mouse splenocytes and human PBMCs. Combination of chimeric anti-SEB antibody and lovastatin also provided in vivo protection against lethal doses of SEB in HLA-DR3 transgenic TSS model. In this study, we have developed a pair of chimeric anti-SEB antibodies (for the first time) that neutralize SEB efficiently in vitro as well as in vivo . In addition, we demonstrated that in vivo protection against lethal doses of SEB can be achieved by a statin of proven safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts. Both these findings have provided potential treatment options for diseases mediated by SEB, as there is no prophylaxis, or therapy against accidental or malicious exposure.
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campus
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dissertation
Date
2010-02-01
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