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      Potent Neutralization of Botulinum Neurotoxin/B by Synergistic Action of Antibodies Recognizing Protein and Ganglioside Receptor Binding Domain

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          Abstract

          Background

          Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents. Monoclonal antibody (mAb) therapeutics hold considerable promise as BoNT therapeutics, but the potencies of mAbs against BoNTs are usually less than that of polyclonal antibodies (or oligoclonal antibodies). The confirmation of key epitopes with development of effective mAb is urgently needed.

          Methods and Findings

          We selected 3 neutralizing mAbs which recognize different non-overlapping epitopes of BoNT/B from a panel of neutralizing antibodies against BoNT/B. By comparing the neutralizing effects among different combination groups, we found that 8E10, response to ganglioside receptor binding site, could synergy with 5G10 and 2F4, recognizing non-overlapping epitopes within Syt II binding sites. However, the combination of 5G10 with 2F4 blocking protein receptor binding sites did not achieve synergistical effects. Moreover, we found that the binding epitope of 8E10 was conserved among BoNT A, B, E, and F, which might cross-protect the challenge of different serotypes of BoNTs in vivo.

          Conclusions

          The combination of two mAbs recognizing different receptors' binding domain in BoNTs has a synergistic effect. 8E10 is a potential universal partner for the synergistical combination with other mAb against protein receptor binding domain in BoNTs of other serotypes.

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          Most cited references22

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          SV2 is the protein receptor for botulinum neurotoxin A.

          How the widely used botulinum neurotoxin A (BoNT/A) recognizes and enters neurons is poorly understood. We found that BoNT/A enters neurons by binding to the synaptic vesicle protein SV2 (isoforms A, B, and C). Fragments of SV2 that harbor the toxin interaction domain inhibited BoNT/A from binding to neurons. BoNT/A binding to SV2A and SV2B knockout hippocampal neurons was abolished and was restored by expressing SV2A, SV2B, or SV2C. Reduction of SV2 expression in PC12 and Neuro-2a cells also inhibited entry of BoNT/A, which could be restored by expressing SV2 isoforms. Finally, mice that lacked an SV2 isoform (SV2B) displayed reduced sensitivity to BoNT/A. Thus, SV2 acts as the protein receptor for BoNT/A.
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            Botulinum neurotoxin A selectively cleaves the synaptic protein SNAP-25.

            Neurotransmitter release is potently blocked by a group of structurally related toxin proteins produced by Clostridium botulinum. Botulinum neurotoxin type B (BoNT/B) and tetanus toxin (TeTx) are zinc-dependent proteases that specifically cleave synaptobrevin (VAMP), a membrane protein of synaptic vesicles. Here we report that inhibition of transmitter release from synaptosomes caused by botulinum neurotoxin A (BoNT/A) is associated with the selective proteolysis of the synaptic protein SNAP-25. Furthermore, isolated or recombinant L chain of BoNT/A cleaves SNAP-25 in vitro. Cleavage occurred near the carboxyterminus and was sensitive to divalent cation chelators. In addition, a glutamate residue in the BoNT/A L chain, presumably required to stabilize a water molecule in the zinc-containing catalytic centre, was required for proteolytic activity. These findings demonstrate that BoNT/A acts as a zinc-dependent protease that selectively cleaves SNAP-25. Thus, a second component of the putative fusion complex mediating synaptic vesicle exocytosis is targeted by a clostridial neurotoxin.
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              Botulinum neurotoxins serotypes A and E cleave SNAP-25 at distinct COOH-terminal peptide bonds.

              SNAP-25, a membrane-associated protein of the nerve terminal, is specifically cleaved by botulinum neurotoxins serotypes A and E, which cause human and animal botulism by blocking neurotransmitter release at the neuromuscular junction. Here we show that these two metallo-endopeptidase toxins cleave SNAP-25 at two distinct carboxyl-terminal sites. Serotype A catalyses the hydrolysis of the Gln197-Arg198 peptide bond, while serotype E cleaves the Arg180-Ile181 peptide lineage. These results indicate that the carboxyl-terminal region of SNAP-25 plays a crucial role in the multi-protein complex that mediates vesicle docking and fusion at the nerve terminal.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                29 August 2012
                : 7
                : 8
                : e43845
                Affiliations
                [1 ]School of Pharmacy, The Center for Antibody Medicine of Ministry of Education, Shanghai Jiao Tong University, Shanghai, People's Republic of China
                [2 ]International Joint Cancer Institute, The Second Military Medical University, Shanghai, People's Republic of China
                [3 ]National Engineering Research Center for Antibody Medicine, State Key Laboratory of Antibody Medicine and Targeting Therapy and Shanghai Key Laboratory of Cell Engineering, Shanghai, People's Republic of China
                [4 ]Lanzhou Institute of Biological Products, Lanzhou, Gansu, People's Republic of China
                [5 ]PLA General Hospital Cancer Center, Beijing, People's Republic of China
                Institute Pasteur, France
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: JXD YJG CCC HJW SHW. Performed the experiments: CCC SHW HJW XYM GHJ. Analyzed the data: JXD CCC HJW SHW YJG TCZ. Contributed reagents/materials/analysis tools: CCC SHW XYM TCZ GHJ XS TX WJL DPZ. Wrote the paper: JXD YJG CCC.

                Article
                PONE-D-12-10471
                10.1371/journal.pone.0043845
                3430616
                22952786
                6a689cd6-2030-430a-b323-ff544fd5e12e
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 9 April 2012
                : 26 July 2012
                Page count
                Pages: 12
                Funding
                This work was supported by grants from National Nature Science Foundation for China, Ministry of Science & Technology of China (973 and 863 Program Projects), Shanghai Commission of Science & Technology (Key Laboratory and projects) as well as special program projects for infectious diseases and new drug development from Ministry of Science & Technology of China (2010ZX09401-407). Dr. Jianxin Dai is recipient of Pujiang Scholar Award from Shanghai Commission of Education. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Biology
                Immunology
                Immunoglobulins
                Microbiology
                Bacterial Pathogens
                Gram Positive
                Toxicology
                Neurotoxicology
                Toxic Agents
                Toxin Binding
                Medicine
                Anatomy and Physiology
                Immune Physiology
                Antibodies
                Gastroenterology and Hepatology
                Bacterial and Foodborne Illness

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                Uncategorized

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