Neutralization Of Staphylococcal Enterotoxin B (seb) By Combined Murine Monoclonal Antibodies And Molecular Mechanism

Background: Staphylococcal enterotoxin B is a kind of Superantigen (SAG) and acts by binding to Vβregion of the T cell receptor (TCR) and to MHC classⅡmolecular, therefore activating T cells. The activated T cells could release a large amount of cytokins including IFN-γand TNF-αmainly, lead to toxic shock syndrome (TSS) and even death. Owing to its toxixity, SEB is considered to be a biological weapon, and lethal dose of SEB is about ng/kg bodyweight. In general, therapy of diseases caused by toxins depends on neutralizing antibodies mainly at present. For example, antiserum specific to botulinum neurotoxin is used for treatment of botulism and intravenous immunoglobulin (IVIG) for treatment of TSS induced by SEB. According to the research work of neutralization mechanism of monoclonal antibidy (mAb) specific to botulinum neurotoxin and tetanus toxin, results showed that there was significant synergy effect produced by combined mAbs recognizing different epitopes. So, mAbs combination provides a new way for potent neutralization, avoids using antiserum and makes it easy for antibody humanization. It has been reported that mechanism of synergy relating to increased apparent affinity after mAbs combination and improved area that mAbs occupied, but it is not known in details.Resear aim: To prepare amount of mAbs specific to SEB and identify most potent neutralizing mAbs combination by in vitro and in vivo experiments, map epitope recognized by mAb and analyze relationship among epitope, toxin structure and mAbs potent neutralization.Methods and results: We prepared 22 hybridoma clones secreting mAbs to SEB with high specificity and ascties titers. A group of combined mAbs composed of FMU-SEB-1, 2 and 10 were identified with most potent neutralization by testing randomly combined mAbs inhibition efficiency to T cell activation induced by SEB in vitro, and potent neutralization was further tested in vivo. Epitopes were mapped by site-directed mutagenesis with amino acid situated in SEB-TCR and SEB-MHC classⅡmolecule binding site and by the reactivity of hybrid molecules constructed by SEB and SEC1 molecules. mAb FMU-SEB-1 binds amino acids E138 and L143, but SEB structure around these amino acids is far away from binding site of SEB-TCR or SEB-MHC classⅡmolecule, this means that the mAb functioned by changing SEB conformation; FMU-SEB-2 binds amino acids D55, T56, Y61, Y81 and N88, these amino acids cover the core region of binding site between SEB and TCR Vβ; Amino acids Y91 and F177 form the epitope of FMU-SEB-10, the 2 amino acids lie in the edge of the SEB-TCR binding site and near SEB-MHC classⅡmolecule binding site, so the mAb could block the interaction of SEB-TCR and SEB-MHC classⅡmolecule simultaneously.Conclusion: We mapped the epitopes of a group of mAbs with most potent neutralization agains SEB, and by resolving the epitope location, we further analyzed the relationships contained by toxin structure, epitope, mAb affinity and neutralization. The results help to understand SAG function better and elucidate mechanism of combined mAbs synergy for their neutralization of SEB.