Biological Characterization Of A Humanized Monoclonal Antibody Toward Staphylococcal Enterotoxin B

Background:Staphylococcus aureus is a major pathogen of hospital-and community-acquired infections in the world and presents a huge burden on the health care system.Staphylococcal enterotoxin B（SEB）is one of the main pathogenic factors of S.aureus infections.This superantigen binds directly to the major histocompatibility complex classⅡ（MHCⅡ）and specific Vβregions of T cell receptor（TCR）bringing about the activation of the immune system.Hyperactivation of monocytes/macrophages and T lymphocytes results in high expression of proinflammatory cytokines,chemokines,tissue factors,lyase and reactive oxygen species,and activation of inflammatory and blood coagulation pathways,eventually leading to endothelial cell injury,acute lung injury,acute respiratory distress syndrome and toxic shock syndrome.In addition,SEB is often associated with food poisoning with symptoms such as vomiting,abdominal pain,and diarrhea.SEB is an acterial toxins and definite biological warfare agent as a lethal and disabling agent with aerosol stability.There are no approved vaccines or specific drugs for SEB-induced diseases up to now.Current research involves a variety of prophylactic or therapeutic approaches,such as vaccines and therapeutic antibodies,inhibitors of cell receptor-toxin interaction,inhibitors of signal transduction and cytokines against SEB.High-affinity antibodies,which could neutralize toxins and exert therapeutic effect with the least delay possible,are important supports and supplements for antibiotics and vaccines,and may be the best treatment options for emergency interention-related infections and immunocompromised patients who cannot be vaccinated.The anti-SEB human monoclonal antibody assigned M0313 screened by the single B cell technique represents a potential immunotherapy for the prevention and treatment of SEB-induced diseases.Our study will evaluate the possibility of mAb M0313 as a therapeutic antibody preparation for SEB-induced diseases by detecting the neutralization effect of antibodies on SEB in vitro,the protective effect on SEB-induced toxic shock and MRSA-induced sepsis in vivo,and neutralization-based B cell epitope.Objective:In this study,to test the binding activity and neutralization effect in vitro,immunoprotection effect in vivo of mAb M0313 on SEB and MRSA strains,and to identify its immunodominant epitope,which will lay an experimental foundation for the development of therapeutic antibody preparations for SEB-induced diseases and S.aureus infection.Methods:1.Expression and identification of mAb M0313.Mab M0313 was expressed and purified by transient co-transfected of the constructed recombinant heavy and light chain plasmids of mAb M0313 in HEK 293F cells.The affinities of mAb M0313 with wild type staphylococcal enterotoxin B（wSEB）and mutant staphylococcal enterotoxin B（mSEB）were evaluated by ELISA,WB and BLI.2.In vitro neutralization effect evaluation of mAb M0313.Neutralization activities of mAb M0313 inhibiting SEB were detected by cell proliferation and cytokine release assays in mouse splenic lymphocytes and human peripheral blood mononuclear cells（PBMCs）.The interference effects of m Ab M0313 on SEB binding to MHCⅡand TCR were detected using flow cytometry.3.In vivo immunoprotective effect effect evaluation of mAb M0313.Mice challenged SEB and D-galactosamine were treated with intravenous mAb M0313,and were detected the survival rate,serum inflammatory factors and pathological conditions of mice spleen and lung to evaluate the protective effect of mAb M0313 on SEB-induced toxic shock.Mice challenged MRSA252 or clinically isolated MRSA strains JN028 or JN064 were treated with intravenous mAb M0313,and were observed the survival rate to evaluate the protective effect of mAb M0313 on MRSA-induced sepsis.Mice challenged Staphylococcus aureus Xen29（with the luciferase reporter gene）were treated with intravenous mAb M0313,and in vivo imaging was performed to observe the bacterial luminescence on day 1,3 and 5 post challenge to evaluate the inhibitory effect of mAb M0313 on the amplification of S.aureus in vivo.4.Epitope mapping of SEB binding to mAb M0313.Overlapping peptides that containing full-length of SEB,truncated peptides and mutant peptides of the immunodominant peptide were designed and synthesized.ELISA was used to test the binding activity of mAb M0313 to these peptides to identify the B cell epitope and its key amino acids binding to mAb M0313.Results:1.MAb M0313 was successfully expressed in HEK 293 eukaryotic expression system with the purity of 97.8%.MAb M0313 had good affinity with mSEB and SEB,and was proved by western blotting.It was demonstrated by BLI that mAb M0313 can bind to SEB and mSEB at the low nM level of affinity.2.MAb M0313 had a dose-dependent inhibitory effect on SEB-induced cell proliferation and production of cytokine in mouse spleen lymphocytes and human BPMCs.In addition,mAb M0313 can inhibit combination of SEB and MHCⅡas well as that of SEB and TCR,identified by flow cytometry.3.MAb M0313 had provided 100%protection in murine model of SEB-induced fatal shock,reduced the concentrations of serum inflammatory factors and pathological damage in mice spleens and lungs.Prophylactic administration of mAb M0313 had provided 100%protection against MRSA252 and its therapeutic administration had provided 25%protection in murine model of MRSA-induced sepsis.Prophylactic administration of mAb M0313 in murine model of MRSA clinical isolation-induced sepsis protected 33.3%of enterotoxin A-,C-,D-,and E-expressing JN028,and 77.8%of enterotoxin B-expressing JN064.In vivo imaging results showed that the fluorescence intensities of mice in the mAb group were significantly lower than those in the positive control group on day 1,3 and 5 after challenge in model of infected mice by peritoneal injection of Xen29（P<0.05）.4.The absorbance of SEB_85-102 among 20 overlapped peptides containing the full-length of SEB was the highest identified by ELISA.Three truncated polypeptides and six mutated polypeptides of SEB_85-102 were further identified.The combination of SEB_85-102 and SEB_88-102with mAb M0313 was positive.The combination of the three polypeptides which mutated mutated Y90,Y91 or Y92 with mAb M0313 was negative.Conclusions:1.High purity anti-SEB mAb M0313 can be obtained.mAb M0313 has a good binding affinity with SEB and mSEB,and the equilibrium dissociation constant of those was measured at the low nM level.2.MAb M0313 can inhibit the stimulating effect of SEB on cell proliferation and cytokine release in mouse spleen lymphocytes and human PBMCs,and can interfere with SEB binding to MHCⅡand TCR.These proved a good effect of in vivo neutralization of mAb M0313 on SEB.3.MAb M0313 immunotherapy can reduce serum inflammatory factors,alleviate pathological damage of viscera,and improve survival rate to protect mice against SEB-induced toxic shock.It can protect mice against MRSA-induced sepsis,reduce in vivo amplification of S.aureus,and also have cross-protection effect on non-SEB-producing MRSA infection.These proved immunoprotection of mAb M0313 on SEB-induced toxic shock and MRSA-induced sepsis.4.The B cell epitope of SEB binding to mAb M0313 is SEB_85-102 which owns high conservation in its amino acid sequences.The key amino acids are Y90,Y91 and Y92.SEB_85-102 and SEB_90-92 present in the helice connected to five-strandedβ-barrel of N-terminal domain in the three-dimensional crystal structure of SEB.