Screening, Characterization And Synergistic Protection Mechanisms Of Antibodies Against Bonts

Clostrididal botulinum neurotoxins(BoNTs) are a group of proteins produced by different strains of clostridium botulinum under anaerobic circumstance, which are responsible for botulism. BoNTs are presently known as the most toxic substance. Seven stereotypes, including types A to G, are known to exist. BoNTs are produced as a 150 kD single polypeptide chain. And then the protein is proteolyzed to form a dichain in which the heavy chain(HC,~100KD) and the light chain(LC,~50KD) are linked by a disulfide bond. HC is composed of two 50 KD domains. The Nterminal half(HN) is involved in translocation and the C-terminal half(HC) is involved in binding with nerve cell and the LC is confined to catalysis.Botulism is a severe disease charactered by flaccid muscle paralysis, which is caused by Bo NTs-mediated blockage of neurotransmitter releasing. Naturally, toxin types A、B、E,and F usually cause human botulinum. Although the natural cases of botulism are rare, due to their extreme toxicity and easy production, BoNTs have become potential biowarfare agents, and create maximum fear among populations concerned with bioterror agents. The only available antidote against BoNTs is equine anti-toxin, which can only target the toxin at extracellular level, but can not reverse the paralysis caused by botulism. In addition, equine antibody can cause severe hypersensitivity reactions, and is limited to be used for prophylaxis treatment.However antibody-based therapy is an important method for treatment of botulism. With the great progress in the last few years, therapeutic monoclonal antibodies(mAbs) have more and more advantages over other therapeutics. mAbs are more effective, specific with less side-effects and have the ability of eliciting toxin by CDC and ADCC. Previous studies have shown that neutralizing m Abs could implement therapeutic effects on botulism.On the other hand, neutralizing mAbs against botulium have the potential therapeutic applications, because mAbs can be manufactured in unlimited supply, do not require a source of immune donors, are consistent batch to batch, and have no infectious risk.Collective evidence showed that much PPADS appeared on the heavy chain(HC) of BoNTs, which is a good imunogen(antigen) to imunize mice, leading to remarkable response of mice. Therefore, it is very significant to choose BoNT/Hc as immunogen for developing vaccine and therapeutic antibodies.In this study we aimed to generate neutralizing mAbs against BoNTs by using hybridoma technology, and mapped neutralizing epitopes, then identified key amino acids. Furthermore, we performed study on the action protective mechanism of neutralizing antibody against BoNTs. Five individual parts described as follows: Part I. Expression and purification of BoNT/AHc, BoNT/BHc, BoNT/EHc, BoNT/FHc proteinIn this part, we obtained DNA sequence of BoNT/AHc, BoNT/BHc, BoNT/EHc, BoNT/FHc from Genebank, and synthesized their DNA fragments, after carrying out a codon optimization. Then Reconstructed Bo NTs/HC protein was expressed in E.coli BL21(ED3) in soluble form respectively and confirmed recombinant protein by Western blot technology. Part II. Generation and screening of mAbs against BoNTs.In this part, in order to generate mAbs against BoNTs, we immunized mice with purified recombinant Bo NT/BHc protein. We obtained 16 mAbs against BoNT/B by using hybridoma technology. Among them, 7 mAbs(8D1, 8E10, 1F4, 2F4, 5G10, 2H12 and 2B2) could neutralize BoNT/B in vivo. One of them were found to be able to cross-bind to BoNT/A,BoNT/E and BoNT/F. And we also analyzied mAb’s isotype by ELISA, then measured kinetics and affinity using SPR. Part III Neutralizing potency of mAbs against BoNTs Tested in vivoIn this part, In vivo toxin neutralization was studied using a standard mouse protection assay in which toxin and mAb were premixed and injected i.p. and time to death and numbers of survival mice were recorded. Two mAbs(2F4, 5G10) could completely protect mice challenged with 40 mouse LD50 s of Bo NT/B, only prolonged the time to death but failed to protect mice challenged with 80 mouse LD50 s. 8E10 could completely protect mice challenged with 20 mouse LD50 s of BoNT/B and couldn’t completely protect mice with 20 mouse LD50 s of Bo NT/A, BoNT/E or BoNT/F, and prolonged the time to death but failed to protect mice challenged with 40 mouse LD50 s of BoNT/E or BoNT/F. Part IV Screening and identification of neutralizing epitopesFor analyzing the neutralizing epitope that 8E10, 2F4 and 5G10 mAb recognizes, we carried out bio-panning technique by screening phage-displayed peptide library and sequence alignment. Then, we identified the epitopes that antibodies recognize by the competitive ELISA assay with synthetic polypeptide. Alignment of the epitope sequence 8E10 recognize on the sequence of BoNT/B provides a best fit with 1259SKWY1262. 5G10 and 2F4 bind to SDXFY and KXSP, which correspond exactly to residues 1201SDEFY1205 and 1114KDSP1117 of BoNT/B respectively.The binding epitope of 8E10 was conserved among BoNT/A, B, E and F. It could cross-protect the challenge of different serotypes of BoNTs in vivo. Part V. Mabs recognizing different epitopes of BoNT/B synergistic protection mechanismsIn this part, we selected three neutralizing mAbs, which recognize different nonoverlapping epitopes of BoNT/B, and compare the neutralizing effects among different antibody combinations. We found that 8E10, response to ganglioside receptor binding site, could collaborate with 5G10 or 2F4, recognizing nonoverlapping epitopes within Syt II binding sites. However, the combination between two m Abs, 5G10 and 2F4, blocking protein receptor binding sites, did not achieve synergistical effects. The binding epitope of 8E10 was conserved among BoNT A, B, E and F. It could cross-protect the challenge of different serotypes of BoNTs in vivo. The results provide a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.In summary, this study generated two BoNT/B type-specific neutralizing antibody((5G10, 2F4) and one novel cross-protective neutralizing antibody 8E10 against BoNT/A、BoNT/B、BoNT/E and Bo NT/F. We mapped three neutralizing epitope that three mAbs recongnize. We found that 8E10, response to ganglioside receptor binding site, could collaborate with 5G10 or 2F4, recognizing nonoverlapping epitopes within Syt II binding sites. However, the combination between two m Abs, 5G10 and 2F4, blocking protein receptor binding sites, did not achieve synergistical effects. These findings help us to elucidate the relationship between neutralizing function of m Abs and structure basis of BoNT/Hc, providing a potential universal partner for the synergistical combination with other m Ab against protein receptor binding domain in BoNTs of other serotypes.