Function And Mechanism Study Of A Potent Neutralizing Antibody Against Staphylococcal Enterotoxin B

Staphylococcal enterotoxins are produced by staphylococcal and streptococcalexotoxins. Staphylococcal enterotoxin (Staphyloccucal enterotoxins, SEs) is a family ofbacterial exotoxins, including SEA, SEB, SEC1-3, SED SEE, the SEF, the SEG et alabout20serotypes, secreted by Staphylococcus aureus. These toxins are responsible for anumber of severe human illnesses, including food poisoning, purulent infections andnosocomial infections.As a superantigen, SEs can bridge MHC class II molecules on antigen presenting cellsand TCR β chain V region on T cells leading to the formation of MHC class II molecules:SEs: TCR ternary complex, which can directly activate T cells and lead to the release of aseries of inflammatory cytokines causing high fever, nausea, diarrhea, and toxic shocksyndrome (TSS). Due to up to50%case fatality rate of TSS caused by Staphylococcusaureus enterotoxin B (SEB) and as high as90%of mortality by SEB infection concurrentwith the influenza, SEB was considered to be one of standard (“classic”) biological warfareagents with conventional storage by the U.S. Special Operations Department.Currently there are no vaccines and medicines available for treating SEB-inducedshock., however, therapeutic monoclonal antibodies become a preferred solution due to itshigh specificity and low toxicity. Several mouse-derived anti-SEB neutralizing monoclonalantibodies (mAbs) have been generated against SEB, but only partial (about63%)inhibition was achieved. This fact demonstrates that a single monoclonal antibody is notsufficient to provide the protection against diseases induced by SEB. The combination of apair of non-cross-reactive neutralizing anti-SEB monoclonal chimeric antibodies wasreported to act synergistically against SEB in vitro and in vivo. Because each antibody hasto be individually developed, tested for safety and efficacy, and then the whole thing has tobe done also for the combination, it’s really a doubling or even tripling of the effortrequired for developing a single antibody, which is already very costly. In addition,unknown epitopes and undefined neutralizing mechanisms are also the bottle neck of thedevelopment of effective therapeutic method. Therefore, epitope identification andneutralizing mechanism study play a pivotal role in the development of therapeuticantibodies against SEB.In this study, we studied the identification and characterization of3E2, a neutralizingmAb that recognizes SEB with high specificity and subnanomolar affinity. To gain a better understanding of these neutralization and specificity mechanisms against SEB, wesubsequently determined the crystal structure of the3E2Fab in complex with SEB andobtained detailed molecular insights of their inhibition mechanism. In addition, weconstructed double-variable domain (DVD) antibody3E2-4A3which can block the MHCClass II and TCR-binding sites on SEB surface simultaneously. We found that3E2-4A3DVD was significantly more effective in inhibiting SEB induced lymphocyte activation invitro and in vivo. This study includes three parts:Part I, structure basis of neutralizing antibody against SEBIn our previous study, we generated10mouse anti-SEB mAbs via classic hybridomaprotocol.4mAbs with higher affinity for SEB were selected for epitope mapping. Theepitopes of3C1and4A3located at the N terminal of SEB next to α1-helix, which fold intopart of the TCR-Vβ binding site. The epitope of3E2was located within the MHC Ⅱbinding sites, whereas the epitope of1A5was near the MHC Ⅱ binding sites.To gain a better understanding of these neutralization and specificity mechanismsagainst SEB, we subsequently determined the crystal structure of the3E2Fab in complexwith SEB. We found that3E2recognized domain44FLYF47and71KDLADK76. Todetermine the key amino acids that could be involved in the epitope structure, wegenerated a series of substitutions of SEB by replacing the interacting residues withglycine based on structural information. The binding of3E2to the mutated proteins andthat to wild-type (wt) SEB were compared by Western blot analysis and ELISA. We foundthat46Y and71K are the key residues for the interaction between3E2and SEB. They arealso the important residues for SEB’s activity. To further investigate the effect of aminoacids side chains on binding affinity between SEB and3E2, we constructed several pointmutations onSEBY46andSEBK71. These point mutants were evaluated by ELISA andsurface plasmon resonance for their binding affinities to3E2. The results indicated that thehydrophobic side chain, as well as the hydroxyl group, is essential for the function ofSEBY46in the interaction between3E2and SEB, and the positively charged characteristicsof theSEBK71side chain play a critical role in3E2binding.Part II, synergistically protective effects of anti-SEB mAbsIn order to investigate the potential synergistic action between3E2and other mAbs,we evaluated the inhibitory effect of different antibody combination for T-cell proliferationand cytokine release by3H thymidine incorporation and ELISA assays. We found that3E2+4A3and3E2+3C1combinations suppressed the T-cell proliferation by73%and68% respectively, indicating that3E2+4A3or3E2+3C1combinations with targeting againstdifferent functional domains on the SEB have the synergistic action. However, thecombination between antibodies3E2+1A5or4A3+3C1, which recognize the samefunctional domain on the SEB, could not raise the T-cell proliferation and cytokine releaseinhibition rate significantly. These results suggest that the combination of antibodiesrecognizing different functional domains will be more efficient in protection against SEBcontrast with the combination between the antibodies recognizing the same functionaldomain.Part III, construction and functional evaluation of anti-SEB DVD antibodiesUsing genes encoding the V regions of3E2and4A3fused with the C regions ofhuman IgG, we developed two DVD antibodies3E2-4A3and4A3-3E2. ELISA testingshowed that the3E2-4A3DVD antibody has a higher binding potency for SEB than4A3-3E2DVD antibody. Compared with4A3and3E2alone, or4A3and3E2together,3E2-4A3DVD was significantly more effective in inhibiting SEB induced lymphocyteproliferation and cytokines releasing in vitro and in enhancing the protective efficiency invivo. Our results suggested that the DVD-Ig of bi-specifically against2function domain ofSEB may constitute a better treatment strategy and provide a promising therapeutic agentfor treatment of SEB intoxication. Moreover,3E2-4A3DVD antibody exhibited10timeshigher binding affinity compared with4A3and3E2alone. This result indicates that theexcellent protective activity conferred by DVD antibody is not only due to the dualblockade of two different functional domains on the SEB, but also for its higher affinity toSEB.In summary, based on the previously obtained mouse anti-SEB mAbs, we decoded theinterface between3E2and SEB by crystallization analysis, identified that46Y and71K arethe key amino acid residues for the neutralizing antibody3E2recognization. Functionalanalyses reveals that the combination of antibodies recognizing different functionaldomains will be more efficient in protection against SEB contrast with the combinationbetween the antibodies recognizing the same functional domain. We further prepared anew DVD antibodies3E2-4A3targeting TCR and MHC-II-interactive domains on the SEBsimultaneously and found that3E2-4A3DVD was significantly more effective ininhibiting SEB induced lymphocyte proliferation and cytokines releasing in vitro and inenhancing the protective efficiency in vivo. Our results suggested that the DVD-Ig ofbi-specifically against2function domain of SEB may constitute a better treatment strategy and provide a promising therapeutic agent for treatment of SEB intoxication.