Production and characterization of a recombinant chimeric antigen consisting botulinum neurotoxin serotypes A, B and E binding subdomains (original) (raw)
Related papers
Toxicon, 2013
Sequences mimicking epitopes of pathogens can be used as effective tools for fast development and characterization of pathogen-specific antibodies. To demonstrate this, we used phage displays and isolated a number of short peptides mimicking epitopes of botulinum neurotoxin serotype A (BoNT/A). Presented data suggest that some of these peptides mimic linear epitopes while others mimic structural epitopes of BoNT/A. All tested peptides retained their ability to be recognized by BoNT/A-specific antibodies even when transferred into new carrier proteins. We demonstrated how such new hybrids can be used for fast conversion of pathogen-specific serums into panels of mono-epitope specific antibodies. We also demonstrated that hybrid proteins carrying multiple isolated peptides can be used as a substitute for BoNT/A in immunization studies and triggers production of BoNT/A-specific antibodies.
Generation of a Recombinant Full-Length Human Antibody Binding to Botulinum Neurotoxin A
Applied Biochemistry and Biotechnology, 2010
In order to develop a recombinant full-length human anti-botulinum neurotoxin A (BoNT/A) antibody, human peripheral blood mononuclear cells (PBMC) were collected from three healthy volunteers and induced for BoNT/A-specific immune response by in vitro immunization. The genes encoding human Fd fragment, consisting of antibody heavy chain variable region and constant region 1 with the genes encoding antibody light chain, were cloned from the immunized PBMC. Afterwards, one combinatory human antigenbinding fragment (Fab) library was constructed using a lambda phage vector system. The size of the constructed library was approximately 10 5 Escherichia coli transformants. After screening the library by BoNT/A antigen using a plaque lifting with immunostaining approach, 55 clones were identified as positive. The Fab gene of the most reactive clone exhibiting particularly strong BoNT/A binding signal was further subcloned into a fulllength human IgG1 antibody gene template in an adenoviral expression vector, in which the heavy and light chains were linked by a foot-and-mouth-disease virus-derived 2A selfcleavage peptide under a single promoter. After the full-length human IgG1 was expressed in mammalian cells and purified with protein L column, sodium dodecyl sulfatepolyacrylamide gel electrophoresis showed that the heavy and light chains of the antibody were cleaved completely. The affinity expressed as the dissociation constant (K d ) for the recombinant human antibody to bind to BoNT/A was determined by indirect enzyme-linked immunosorbent assay and results confirmed that the recombinant full-length human antibody retained BoNT/A-binding specificity with K d value of 10 −7 M.
Botulinum neurotoxin (BoNT) serotypes A, B and E are most frequently associated with human botulism. Recombinant vaccines against BoNTs are usually based on one or more domain(s) of the toxin molecule. In this study, we investigate a new-designed multiple antigenic polypeptide for serotypes A and E (MAP/AE), containing two linear epitopes from each serotype. A synthetic gene was used to express the recombinant MAP/AE, in E. coli. Anti-MAP/AE antibodies were produced by injecting the purified MAP/AE to Balb/C mice. The interactivity of these antibodies and BoNT/A and E has been shown by ELISA. High titers of anti-MAP/AE antibodies were detected in mice sera. The anti-MAP/AE antibody titer is clearly detectable even at 25,600 dilution level. The anti-MAP/AE antibodies bound to both BoNT/A and BoNT/E holotoxin molecules. Neutralization ability of the antibodies for both toxin serotypes was determined, by an inhibitory ELISA assay. Results are suggestive of the feasibility of this epitope targeting strategy to develop novel multivalent recombinant vaccines against BoNTs.
Infection and Immunity, 1997
The domain organization of the botulinum neurotoxin serotype A was studied by using antibody mapping of 44 monoclonal single-chain variable fragments. The analysis was carried out on (i) the individual domains of botulinum neurotoxin holotoxin (binding, translocation, and catalytic), (ii) botulinum neurotoxin holotoxin, (iii) the botulinum neurotoxin holotoxin in complex with the nontoxic portion, and (iv) botulinum neurotoxin holotoxin and nontoxic portion of the complex recombined in vitro. All 44 antibodies mapped to individual domains of botulinum neurotoxin. Forty of the 44 single-chain variable fragments bound the botulinum neurotoxin holotoxin relative to the isolated domains, suggesting that 4 epitopes are covered when the individual domains are in the holotoxin form. Only 20 of the antibodies showed a positive reaction to the toxin while in complex with the nontoxic portion. All of the covered epitopes were mapped to the binding domain of botulinum neurotoxin, which suggest...
The botulinum neurotoxins (BoNTs), the most toxic biological compounds, are known to cause muscle paralysis. These toxins are enzymatic properties which inhibit the release of the acetylcholine. The purposes of this study are the recombinant expression of light chain (LC) of botulinum toxin types A (rBoNT/A-LC) with high purity, the evaluation of its antibody titer yield and its immunogenicity, and finally the evaluation of in vitro reorganization of BoNT/A-holotoxin by produced antibody. BoNT/A light chain gene sequences were obtained from the NCBI genome database. After codon optimization of target gene to better express in E. coli, the target gene was ordered to be synthesized in pET-28a (+). E. coli BL21 (DE3) was transformed by the mentioned vector containing the gene. The expression process was performed in standard conditions. In order to express the recombinant protein in soluble form, optimization of host cell culture and expression process was performed. The protein was purified by affinity chromatography (Ni-NTA column) and was confirmed with specific antibodies. In this study, the highest expression of soluble protein was obtained at 0.5 mM IPTG, cell culture optical density of 0.5, and the induction time of 18 h at 18 °C. Western blot analysis confirmed the target protein. The results showed that rBoNT/A-LC was highly expressed in soluble form and highly purified using affinity chromatography. Immunology results showed that the immune mice with rBoNT/A-LC are able to endurance up to 100 LD 50 of BoNT/A. The results also indicated that the produced antibodies can recognize up to 200 ng of BoNT/A-holotoxin.
Protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D
Anaerobe, 2016
Botulinum neurotoxin (BoNT) serotypes C and D are responsible for cattle botulism, a fatal paralytic disease that results in great economic losses in livestock production. Vaccination is the main approach to prevent cattle botulism. However, production of commercially available vaccines (toxoids) involves high risk and presents variation of BoNT production between batches. Such limitations can be attenuated by the development of novel nontoxic recombinant vaccines through a simple and reproducible process. The aim of this study was to evaluate the protective potential of recombinant non-purified botulinum neurotoxin serotypes C and D. Bivalent vaccines containing 200 μg rHCC and rHCD each were formulated in three different ways: (1) purified antigens; (2) recombinant Escherichia coli bacterins; (3) recombinant E. coli cell lysates (supernatant and inclusion bodies). Guinea pigs immunized subcutaneously with recombinant formulations developed a protective immune response against the ...
Separation of the components of type A botulinum neurotoxin complex by electrophoresis
Toxicon, 2003
Clostridium botulinum neurotoxins (BoNTs) are the most toxic substances known. They exert potent neuroparalysis on vertebrates. C. botulinum produces seven serotypes of neurotoxin (A-G). BoNT/A, found in bacterial cultures of C. botulinum type A, is produced as a complex with a group of neurotoxin associated proteins (NAPs). Botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against the acidity and proteases of the stomach. Here, we used sodium dodecyl sulfate -polyacrylamide gel electrophoresis (SDS -PAGE) for separation and identification of the constituent proteins of BoNT/A complex. A range of homogenous and gradient SDS -PAGE gels was used to resolve the BoNT/A complex. These gels were run under constant voltage and constant current conditions. The molecular weight and relative amount of each protein band were determined. On a 12.5% homogenous SDS -PAGE under reducing conditions, seven protein bands were identified with average molecular weights of 118, 106, 90, 56, 36, 23 and 17 kDa. The relative amounts of these seven proteins were determined densitometrically as 10, 6, 13, 27, 22, 13 and 8%, respectively. The separation and identification of BoNT/A complex will help in understanding the molecular structure and function of BoNT/A NAPs and their interaction with the toxin, in the toxico-infection process of the botulism diseased state. In particular, the stoichiometry of the individual components is established for a typical preparation of BoNT/A complex. Furthermore, the studies reported here identify the most favorable conditions for the baseline resolution of BoNT/A NAPs proteins for other workers in this field. q
Production and Characterisation of a Neutralising Chimeric Antibody against Botulinum Neurotoxin A
PLoS ONE, 2010
Botulinum neurotoxins, produced by Clostridium botulinum bacteria, are the causative agent of botulism. This disease only affects a few hundred people each year, thus ranking it among the orphan diseases. However, botulinum toxin type A (BoNT/A) is the most potent toxin known to man. Due to their potency and ease of production, these toxins were classified by the Centers for Disease Control and Prevention (CDC) as Category A biothreat agents. For several biothreat agents, like BoNT/A, passive immunotherapy remains the only possible effective treatment allowing in vivo neutralization, despite possible major side effects. Recently, several mouse monoclonal antibodies directed against a recombinant fragment of BoNT/A were produced in our laboratory and most efficiently neutralised the neurotoxin. In the present work, the most powerful one, TA12, was selected for chimerisation. The variable regions of this antibody were thus cloned and fused with the constant counterparts of human IgG1 (kappa light and gamma 1 heavy chains). Chimeric antibody production was evaluated in mammalian myeloma cells (SP2/0-Ag14) and insect cells (Sf9). After purifying the recombinant antibody by affinity chromatography, the biochemical properties of chimeric and mouse antibody were compared. Both have the same very low affinity constant (close to 10 pM) and the chimeric antibody exhibited a similar capacity to its parent counterpart in neutralising the toxin in vivo. Its strong affinity and high neutralising potency make this chimeric antibody interesting for immunotherapy treatment in humans in cases of poisoning, particularly as there is a probable limitation of the immunological side effects observed with classical polyclonal antisera from heterologous species.