Research On The Receptor And Neutralizing Antibodies Of Botulinum Neurotoxin Type A

Botulinum neurotoxins, a kind of protein produced by Clostridium botulinum and cause relaxation paralysis by blocking nerve-muscle synaptic transmission, are the most poisonous substance as known. There are seven serotypes designated A-G during which BoNT/A, B, E and F cause human natural intoxication. No small-molecule drug exists as therapeutic agent for the treatment of BoNTs intoxication until now. Equine antitoxin is currently used to treat botulism, but this product is involved in many disadvantages, including potent serum sickness, anaphylactic shock, high cost and a long period. Thus therapeutics by humanized neutralizing antibodies was regarded. However, there are difficulties on the research of neutralizing antibodies: (1) a neutralizing epitope is less immunogenic than other epitopes; (2) the toxoid immunogen poorly mimics the conformation of the neutralizing epitope(s); and (3) several epitopes are blocked is necessary for ef?cient neutralization.Hc (868-1296), where the receptor binding sites localize, is the antigenic determinant of BoNT/A. It is known that neutralizing antibodies inhibit the toxicity by blocking the receptor binding region so that BoNT/A cannot recognize and bind to its receptors. Therefore, the characterization of the receptor binding region in detail will provide direct evidence for neutralizing antibodies study. The receptors of BoNT/A are gangliosie GT1b and synaptic vesicle protein SV2. GT1b binds with H1253, S1264, W1266 and Y1267 of BoNT/A. There are A, B and C three isoforms of SV2, in which SV2C bind to BoNT/A most robustly. The protein receptor of BoNT/A had puzzled scientists for a long time until 2006, when Dong et al discovered that BoNT/A interacts with SV2 in vitro and in vivo. Recently, protein receptors of BoNTs have been characterized one after another except BoNT/C and BoNT/D. However, the binding detail of BoNTs with their protein receptors remains unknown. This study focused on identifying the SV2C binding site of BoNT/A and screening new neutralizing antibodies blocking receptor binding site from the fully synthetic human antibody libraries.Heavy chain (HC) expression of BoNT/A was carried out. High performance expression of HC has not yet been published. Expressing HC in E.coli will set a background or even be dispensable for futher research about BoNT/A. Coding sequence of Hn was designed and synthesized according to E.coli codon usage preference and ligated to Hc coding sequence by overlap extension PCR. HC coding sequence then was constructed into vector pTIG-TrxA and expressed in BL21(DE3). Investigation on the expressing condition showed that reducing environment is very important for the expression of HC by E.coli: expression level under the condition of culturing standingly is much higher than that of culturing agitatedly;"S"curve was observed when inspecting the relationship between expression level and the media volume. The expression level reached 64.7mg/L under optimizing condition. TrxA is a hydrogen donor in redox reaction. Now that reducing environment is in favor of HC expression, a trxA gene was added in the expressing plasmid to increase the reducibility. As new vector used, the expression of HC depended less on the reducing environment and the quantity was increased respectively. Unstability of the expression plasmid was observed and affinity purification of HC failed, so Hc which is highly expressed and purified was used in the following experiments.Coding sequence for SV2C acquired from rat brain tissue was cloned into the vector pGEX-KG and expressed as GST fusion protein. Interaction of different BoNT/A-Hc truncations with constructed SV2C tested by GST pull-down preliminarily showed that the protein receptor binding site located within the amino acid residues 1150 and 1219. Based on this result, deletion mutants were constructed for interacting detection and the probably binding site was restricted to 1173-1213 residues. At last, Ala mutants during 1173-1213 were constructed and the interaction between these mutants and SV2C was detected, demonstrating that 1179-1181(RVY) and 1191-1193(YRL) residues might be responsible for BoNT/A binding to SV2C. 1179-1181 and 1191-1193 residues are two spatially neighboring linear epitopes situated on the bottom of a pocket against the GT1b binding domain. Recent study indicated that SV2 is also the receptor for BoNT/E and F, in which V1180(BoNT/E) or Y1191-L1193(BoNT/F) conserved, showing some correlation to our results.BoNT/A-Hc was purified and coated as antigen to screen specific antibodies from fully synthetic human antibody libraries with the volume of 1.35×1010, and about 1000 clones were identified of which 30% are positive clones. A fusion peptide including GT1b binding region of BoNT/A and SV2C binding region linked with (GGGG) designated as Hc-sg was constructed and purified. These 30% positive clones were screened by Hc-sg. Except 90% that bind unspecifically with E.coli, 14 clones reacting with both Hc and Hc-sg were acquired. DNA sequencing demonstrated that there are 13 unique scFv antibodies, of which the heavy chain frameworks are all H3. The light chain frameworks of 3 clones areλ3 and the left are allλ1. Because more than one epitopes need to be blocked, 7 scFv antibodies were selected to be expressed as full antibodies. Coding sequences of VH and Vλwere constructed into vectors H293 and L293-CL with c region, then the heavy chain and the light chain expressing plasmids were transfected into human embryonic kidney cell 293-F and expressed transiently. Protein A affinity chromatography was used for purifying and concentrating the full antibodies. Preliminary experiment demonstrated that 2F3 possessed the potency of high neutralizing activity.C25, S25 and 3D12 are efficient monoclonal neutralizing antibodies as reported, which can protect the mouse from the challenge of 450,000 LD50 of BoNT/A when used combined. In our study we tried to express these antibodies as a tool to estimate other antibodies in the future. C25 has been expressed. The purpose of this study is to express S25 and 3D12. We reformed the coding sequences of the VH and Vκpublished on GenBank according to the mammalian codon usage preference, and constructed into vectors H293 and L293-CK with c region. The antibodies were expressed by the same method as the 7 antibodies mentioned above. ELISA of the product with Hc and Hc with point mutation on the reported binding sites showed that S25 was rightly expressed while 3D12 not.In this study, BoNT/A were analyzed by homology modeling to determine the interacting sites of BoNT/A to antibody, using the crystal structure of BoNT/A with antibody AR2 and CR1 as template. C25 is one of the most efficient monoclonal neutralizing antibody of which the binding epitope has been characterized. The probably binding sites acquired by Modeler covered all sites reported, and was certified in experiment, so it's regarded that the modeling result is credible, which set a background for the neutralizing epitopes research of the antibodies screened above.In summery, the heavy chain of BoNT/A was expressed at high level. The binding site of BoNT/A to its protein receptor SV2C was characterized, which would be a progress in the research on BoNTs. The peptide just contains GT1b and SV2 binding region of BoNT/A was constructed and antibodies against the peptide was screened and expressed as full antibodies. Probably high neutralizing activity was obtained. The structure of BoNT/A interacting with C25 was predicted by homology modeling, and the result was confirmed by experiment.