The DNA was digested with NotI and NcoI enzymes (New England Biolab), according to the manufacturers instructions and subjected to an electrophoresis in 0.5% TBE and with a 0.8% agarose1% TBE gel. it is the subject of the European framework project called AntiBotABE. == Results == In this study, starting from a macaque immunized with the recombinant heavy chain of BoNT/A1(BoNT/A1-HC), an immune antibody phage-display library was generated and antibody fragments (single chain Fragment variable) ID 8 with nanomolar affinity were isolated and further characterized. The neutralization capacities of these scFvs were analyzed in the mouse phrenic nerve-hemidiaphragm assay. == Conclusions == After a three-round panning, 24 antibody fragments with affinity better than 10 nM were isolated. Three of them neutralized BoNT/A1efficiently and two cross-neutralized BoNT/A1and BoNT/A2subtypes in the mouse phrenic nerve-hemidiaphragm assay. These are the first monoclonal human-like antibodies cross-neutralizing both BoNT/A1and BoNT/A2. The antibody A1HC38 was selected for further development, and could be clinically developed for the prophylaxis and treatment of botulism. == Electronic supplementary material == The online version of this article (doi:10.1186/s12896-015-0206-0) contains supplementary material, which is available to authorized users. Keywords:Botulinum neurotoxin, Recombinant antibodies, scFv, Neutralizing antibodies, Non-human primates, Clostridium botulinum, AntiBotABE, Macaques, Biological warfare agents == Background == Botulism is a rare life-threatening disease caused by botulinum neurotoxins (BoNT), secreted by the spore-forming bacterium,Clostridium botulinum. Seven BoNTs serotypes (A to G) have been described but serotypes A, B and E are responsible for the majority of natural human intoxications [1]. Botulinum neurotoxin A (BoNT/A) is the most toxic substance known, with a human 50 % lethal dose (LD50) estimated at 1 ng.kg1(intravenous and subcutaneous routes), 10 ng.kg1(pulmonary route) or 1 g.mL1(oral route), leading the Centers for Disease Control and Prevention (CDC) to classify BoNTs among the six major biological warfare agents (CDC category A agents) [24]. BoNTs are type A-B heteromeric molecules composed of a 100 kDa heavy chain (HC) and a 50 kDa light chain (LC). The heavy chain is successively implicated in the toxin binding at the surface of the motoneurons, the internalization by dual-receptor-mediated endocytosis and the translocation of the light chain into the cytosol. The light chain is a zinc endopeptidase which cleaves proteins of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex, which is implicated in the exocytosis of the acetylcholine [5]. The SNAP25 (synaptosomal-associated protein of 25 kDa) protein is cleaved by BoNT/A-LC. This inhibition induces a flaccid muscular paralysis, affecting progressively ID 8 all the muscles and generally leading to a respiratory distress and eventually death in absence of treatment. Vaccines against botulism have been developed but vaccination is rarely used as its effectiveness has not been fully evaluated and has demonstrated adverse events [6]. Thus, vaccination is only used for individuals with a high risk of exposure, such as health care providers, researchers, first responders, and military personnel [7]. Moreover, BoNTs have been introduced as a safe and effective treatment for a wide range of disorders associated with involuntary muscle contractions and spasm disorders, and these ever-increasing medical indications prevent large scale vaccination against botulism [8]. Currently, there is no licensed small inhibitor available in the pharmacopeia. Thus, the botulism specific treatment is based on the injection of anti-toxin antibodies, which should be administered as soon as possible after the clinical diagnosis to reduce the mortality rate, complemented by several months of hospitalization in an intensive care unit with mechanical ventilation [2,9]. Infant botulism cases are treated by injections of a human-derived botulinum immunoglobulin preparation (BabyBIG), which is well tolerated, but available in very limited quantity and is expensive [10]. Adult botulism is treated by injections of a bivalent (Sanofi-Pasteur LimitedInstitut Butantan) or heptavalent (Cangene Corporation) horse-derived antitoxins, available in larger quantity. Nevertheless, because of their animal origin, these antitoxins may be poorly tolerated and could induce serious adverse effects like serum sickness or anaphylactic shock [11,12]. The development of human or human-like antibodies for passive vaccination is advised as an ID 8 alternative. In a former study, recombinant antibody fragments were isolated from an immune and a non-immune human library against botulinum toxins [13]. The immune library was built starting ID 8 from a volunteer immunized with botulinum toxoids A to E, and yielded the single chain Rabbit polyclonal to RABAC1 Fragment variables (scFvs) with best affinities and neutralization properties compared to those obtained from.
The DNA was digested with NotI and NcoI enzymes (New England Biolab), according to the manufacturers instructions and subjected to an electrophoresis in 0
