Balkiss Bouhaouala-Zahar

A bispecific nanobody to provide full protection against lethal scorpion envenoming

Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100‐kDa horse plasma antivenom serum (PAS)‐derived Fab′2 to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single‐domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI′ toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI′ ‐toxin neutralization was monitored in mice. Remarkably, the NbAahI′ F12 fully protected mice against 100 LD50 of AahI′ administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD50 of crude venom injected subcutaneously, the designed bispecific NbF12‐10 against AahI′/AahII toxins succeeded in neutralizing 5 LD50. Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 μg of NbF12‐10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12‐10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.—Hmila, I., Saerens, D., Ben Abderrazek, R., Vincke, C., Abidi, N., Benlasfar, Z., Govaert, J., El Ayeb, M., Bouhaouala‐Zahar, B., Muyldermans, S. A bispecific nanobody to provide full protection against lethal scorpion envenoming. FASEB J. 24, 3479–3489 (2010). www.fasebj.org

Identification of potent nanobodies to neutralize the most poisonous polypeptide from scorpion venom

Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary heavy-chain antibodies, have a size that closely matches that of scorpion toxins. Therefore these nanobodies might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants kon or koff, or the equilibrium dissociation constant, KD. One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37000 LD50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.

Development of Cys38 knock-out and humanized version of NbAahII10 nanobody with improved neutralization of AahII Scorpion toxin

During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size, matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However, this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage. In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr. Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections of seven LD(50) and 50% of mice survived nine LD(50) of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic venom compound of one of the most dangerous scorpions.

Efficient expression of the anti-AahI' scorpion toxin nanobody under a new functional form in a Pichia pastoris system

Most large‐scale microbial production of recombinant proteins are based on Escherichia coli, yeasts, or filamentous fungi systems. Using eukaryotic hosts, antibody fragments are generally expressed by targeting to the secretory pathway. This enables not only efficient disulfide bond formation but also secretion of soluble and correctly folded product. For this goal, a recombinant vector was constructed to produce a single‐domain antibody (NbAahI′22) directed against AahI′ scorpion toxin using the methylotrophic yeast Pichia pastoris. The corresponding complementary DNA was cloned under control of the alcohol oxidase promoter in frame with the Saccharomyces α‐factor secretion signal and then transferred to P. pastoris cell strain X‐33. Using Western blot, we detected the expression of the recombinant NbAahI′22 exclusively in the culture medium. Targeting to the histidine label, the secreted nanobody was easily purified on nickel–nitrilotriacetic acid resin and then tested in enzyme‐linked immunosorbent assay. Interestingly, the production level of the NbAahI′22 in its new glycosylated form reached more than sixfold that obtained in E. coli. These findings give more evidence for the utilization of P. pastoris as a heterologous expression system.

Venom conjugated polylactide applied as biocompatible material for passive and active immunotherapy against scorpion envenomation.

Scorpion envenoming represents a public health issue in subtropical regions of the world. Treatment and prevention need to promote antitoxin immunity. Preserving antigenic presentation while removing toxin effect remains a major challenge in toxin vaccine development. Among particulate adjuvant, particles prepared with poly (D,L-lactide) polymer are the most extensively investigated due to their excellent biocompatibility and biodegradability. The aim of this study is to develop surfactant-free PLA nanoparticles that safely deliver venom toxic fraction to enhance specific immune response. PLA nanoparticles are coated with AahG50 (AahG50/PLA) and BotG50 (BotG50/PLA): a toxic fraction purified from Androctonus australis hector and Buthus occitanus tunetanus venoms, respectively. Residual toxicities are evaluated following injections of PLA-containing high doses of AahG50 (or BotG50). Immunization trials are performed with the detoxified fraction administered alone without adjuvant. A comparative study of the effect of Freund is also included. The neutralizing capacity of sera is determined in naive mice. Six months later, immunized mice are challenged subcutaneously with increased doses of AahG50. Subcutaneous lethal dose 50 (LD50) of AahG50 and BotG50 is of 575 μg/kg and 1300 μg/kg respectively. By comparison, BotG50/PLA is totally innocuous while 50% of tested mice survive 2875 μg AahG50/kg. Alhydrogel and Freund are not able to detoxify such a high dose. Cross-antigenicity between particulate and soluble fraction is also, ensured. AahG50/PLA and BotG50/PLA induce high antibody levels in mice serum. The neutralizing capacity per mL of anti-venom was 258 μg/mL and 186 μg/mL calculated for anti-AahG50/PLA and anti-BotG50/PLA sera, respectively. Animals immunized with AahG50/PLA are protected against AahG50 injected dose of 3162 μg/kg as opposed all non-immunized mice died at this dose. We find that the detoxification approach based PLA nanoparticles, benefit the immunogenicity and protective efficacy of venom immunogen.

A complementary LC-ESI-MS and MALDI-TOF approach for screening antibacterial proteomic signature of farmed European sea bass mucus.

Antibacterial protection in the mucus is provided by antimicrobial compounds and till now few numbers of AMP and proteins were identified. Herein, mass spectral profiling of fresh mucus from farmed sea bass (Dicentrarchus labrax) using Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometer (MALDI-TOF) and liquid chromatography mass spectrometry is investigated in order to survey the infective/healthy status of the mucus. We identify AMP peptides of 2891.7, 2919.45 and 2286.6 Da molecular weight respectively and characterize Chrysophsins in the mucus of Dicentrarchus labrax. These peptides display broad-spectrum bactericidal activity against Gram-negative (Minimum Inhibitory Concentrations namely MICs < 0.5 μM) and Gram-positive bacteria (MICs < 0.5 μM) including Escherichia coli and Bacillus subtilis. Furthermore, sensitivity to yeast Candida albicans is reported for the first time and shows interesting MICs of less than 2 μM. We also demonstrate that the fish pathogen Aeromonas salmonoicida is sensitive to Chrysophsins (MICs ranging between 5 and 14 μM). Our mucus molecular mass mapping developed approach allows for fast exploration of immune status. Our data provides evidence that Chrysophsins are secreted by immune cells and are released in mucus of non-challenged farmed European sea bass. These results suggest that Chrysophsins, secreted by gills of red sea bream, are an important widespread component of Teleostei defense against disease.

Dromedary immune response and specific Kv2.1 antibody generation using a specific immunization approach

Voltage-gated potassium (Kv) channels form cells repolarizing power and are commonly expressed in excitable cells. In non-excitable cells, Kv channels such as Kv2.1 are involved in cell differentiation and growth. Due to the involvement of Kv2.1 in several physiological processes, these channels are promising therapeutic targets. To develop Kv2.1 specific antibody-based channel modulators, we applied a novel approach and immunized a dromedary with heterologous Ltk- cells that overexpress the mouse Kv2.1 channel instead of immunizing with channel protein fragments. The advantage of this approach is that the channel is presented in its native tetrameric configuration. Using a Cell-ELISA, we demonstrated the ability of the immune serum to detect Kv2.1 channels on the surface of cells that express the channel. Then, using a Patch Clamp electrophysiology assay we explored the capability of the dromedary serum in modulating Kv2.1 currents. Cells that were incubated for 3h with serum taken at Day 51 from the start of the immunization displayed a statistically significant 2-fold reduction in current density compared to control conditions as well as cells incubated with serum from Day 0. Here we show that an immunization approach with cells overexpressing the Kv2.1 channel yields immune serum with Kv2.1 specific antibodies.

Wheat germ in vitro translation to produce one of the most toxic sodium channel specific toxins

Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Aah (Androctonus australis hector)II protein that requires the proper formation of four disulphide bonds. Soluble, recombinant N-terminal GST (glutathione S-transferase)-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified rAahII (recombinant AahII) toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after i.c.v. (intracerebroventricular) injection in Swiss mice. An LD50 (median lethal dose)-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD50 of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active rAahII. In addition, NbAahII10 (Androctonus australis hector nanobody 10), a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified rAahII upon injection in mice.