Papers by zakaria benlasfar
Molecular Immunology, 2008
Many efforts aim at solving the serious problems encountered with immunotherapy against scorpion ... more Many efforts aim at solving the serious problems encountered with immunotherapy against scorpion envenoming. The most attractive approach consists in generating single-chain antibody fragments (scFv) as their pharmaco-kinetic properties should match closely those of the scorpion toxins. Although high affinity scFv reagents have been generated in the past, their production level, stability, and toxin neutralizing capacity remain disappointingly poor. In the current study, we identified one Nanobody (Nb), a single-domain antigen-binding fragment of a dromedary Heavy-chain antibody (HCAb) that recognizes specifically the Androctonus australis hector AahI toxin. This Nb has excellent production, stability and solubility characteristics. With this Nb we further manufactured a tandem linked bivalent construct and assembled a HCAb with improved antigen binding due to avidity effects. All these constructs were shown in mouse models to possess a scorpion toxin neutralization capacity that exceeds by far all previous attempts with scFv-based materials, even when used at lower doses. It is therefore clear that in the near future Nanobodies will be at the core of novel serotherapeutics as they combine multiple benefits over other reagents to treat scorpion envenomed patients.
Faseb Journal, 2010
Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was... more 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 NbAahIF12 fully protected mice against 100 LD 50 of AahI administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD 50 of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI/AahII toxins succeeded in neutralizing 5 LD 50 . Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 g 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 Scorpion envenoming is a frequent medical emergency in several countries, including Tunisia, where the Androctonus australis hector (Aah) scorpion is responsible for the majority of the fatal stings. The toxicity of the Aah scorpion venom is essentially due to the presence of three small basic toxins (7 kDa) that act on the voltage-gated sodium channels of excitable cells. These toxins belong to two distinct structural and antigenically unrelated groups: group I (polypeptides AahI, AahIЈ, and AahIII) and group II (AahII) (1, 2). 3479 0892-6638/10/0024-3479 © FASEB
Toxicon, 2003
Scorpion envenoming is a real health problem. The only specific treatment is immunotherapy with a... more Scorpion envenoming is a real health problem. The only specific treatment is immunotherapy with antibodies from immunized horses. The severity of scorpion envenoming and the rapid diffusion of the toxins into the blood compartment require an improvement of the present antivenom therapy. In this study, we report successful immunization of dromedaries (Camelus dromedarius) against the small weakly antigenic neurotoxins of Androctonus australis hector scorpion. Camel immune sera was tested for its specific antigenic reactivity and neutralizing capacity against Aah toxic fraction and AahII toxin. We demonstrate that a substantial proportion of polyclonal heavy chain antibodies bind to Aah toxins and in particular to AahII, the most toxic one scorpion venom component. Furthermore, we show that both dromedary sera and heavy chain antibody subclasses are capable of neutralizing the toxicity of Aah toxins in mice.
Biochemical Journal, 2009
Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patie... more 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 subnanomolar 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 k on or k off , or the equilibrium dissociation constant, K D . One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD 50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37 000 LD 50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.
Biochemical Journal, 2009
Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patie... more 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 subnanomolar 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 k on or k off , or the equilibrium dissociation constant, K D . One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD 50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37 000 LD 50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.
Archives de l'Institut Pasteur de Tunis
The scorpionic and ophidian envenomations are a serious public health problem in Tunisia especial... more The scorpionic and ophidian envenomations are a serious public health problem in Tunisia especially in Southeastern regions. In these regions Artemisia campestris L is a plant well known which has a very important place in traditional medicine for its effectiveness against alleged venom of scorpions and snakes. In this work, we tested for the first time, the anti-venomous activity of Artemisia campestris L against the scorpion Androctonus australis garzonii and the viper Macrovipera lebetina venoms. Assays were conducted by fixing the dose of extract to3 mg/mouse while doses of venom are variable. The leaves of Artemisia campestris L were extracted by various organic solvents (Ether of oil, ethyl acetate, methanol and ethanol) and each extract was tested for its venom neutralizing capacity. For the ethanolic extract, a significant activity with respect to the venoms of scorpion Androctonus australis garzonii (Aag), was detected. Similarly, a significant neutralizing activity against...
Archives de l'Institut Pasteur de Tunis
Archives de l'Institut Pasteur de Tunis
Archives de l'Institut Pasteur de Tunis
Phytomedicine, 2011
The present study reports for the first time, the in vivo wound healing potential of Punica grana... more The present study reports for the first time, the in vivo wound healing potential of Punica granatum L. peels. A 5% (w/w) methanolic extract based-ointment was formulated and evaluated for its wound healing in guinea pigs. The ointment was applied in vivo on the paravertebral area of twelve excised wounded models once a day for 10 consecutive days. The ointment significantly enhanced the wound contraction and the period of epithelialization as assessed by the mechanical (contraction rate, tensile strength), the biochemical (increasing of collagen, DNA and proteins synthesis) and the histopathological characteristics. Such investigation was encouraged by the efficiency of the methanolic extract as antimicrobial and antioxidant. Indeed, the extract showed antioxidant activity as strong as natural and synthetic compounds (Trolox, BHA, Quercetin). Furthermore, the extract exhibited significant antibacterial and antifungal activity against almost all tested bacteria: Pseudomonas aeruginosa ATCC 9027, Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Klebsiella pneumoniae, Salmonella anatum, Salmonella typhimurium, Streptococcus pneumoniae, and fungi Candida albicans, Candida glabrata, Trichopyton rubrum and Aspergillus niger. The formulated ointment might well find use as skin repair agent without hazard to human health based on these results and on the fact that it has been well established that the extracts of pomegranate used in conditions similar to those applied by traditional medicine, showed no toxic effects.
Rabies in the Tropics, 1985
Toxicon, 1997
Two polymeric forms of Buthus occitamus tunetanus (Bot) G-50 and Androctonus australis hector (Aa... more Two polymeric forms of Buthus occitamus tunetanus (Bot) G-50 and Androctonus australis hector (Aah) G-50 were obtained by controlled polymerization with glutaraldehyde. Their mol. wts, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and exclusion high-performance liquid chromatography using radiolabelled toxin, ranged from 14,000 to 90,000 and 90,000 to 100,000 for Aah G-50 and Bot G-50, respectively. Modification of about 20% of the lysine residues yielded completely detoxified Bot G-50 fractions which retained the antigenic cross-reactivity with the native G-50 fraction, venom and toxins. High levels of specific antibodies against venoms and toxins were generated by immunization with prepared polymeric forms because absorbance values higher than 3 units were regularly observed by enzyme-linked immunosorbent assay with immune sera dilutions of 1/10,000. In vitro protection experiments demonstrated that immune sera from mice, horse and monkeys efficiently neutralized either Aah or Bot venom. Depending on the adjuvant and animal model species used, the neutralizing titre estimated by subcutaneous injection in mice varied between 20 and 40 LD50/ml. The in vivo protection assays showed that immunized mice could resist the challenge by six times the LD50 amounts of the toxic fraction. This protection was found to be long-lived. It was concluded that G-50 polymeric forms are highly detoxified and immunogenic, and should be useful for the production of potent polyvalent antivenom against scorpion toxins. They could also be considered for further studies towards the development of vaccine candidates.
The FASEB Journal, 2010
Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was... more 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 NbAahIF12 fully protected mice against 100 LD 50 of AahI administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD 50 of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI/AahII toxins succeeded in neutralizing 5 LD 50 . Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 g 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 Scorpion envenoming is a frequent medical emergency in several countries, including Tunisia, where the Androctonus australis hector (Aah) scorpion is responsible for the majority of the fatal stings. The toxicity of the Aah scorpion venom is essentially due to the presence of three small basic toxins (7 kDa) that act on the voltage-gated sodium channels of excitable cells. These toxins belong to two distinct structural and antigenically unrelated groups: group I (polypeptides AahI, AahIЈ, and AahIII) and group II (AahII) (1, 2). 3479 0892-6638/10/0024-3479 © FASEB
Molecular Immunology, 2008
Many efforts aim at solving the serious problems encountered with immunotherapy against scorpion ... more Many efforts aim at solving the serious problems encountered with immunotherapy against scorpion envenoming. The most attractive approach consists in generating single-chain antibody fragments (scFv) as their pharmaco-kinetic properties should match closely those of the scorpion toxins. Although high affinity scFv reagents have been generated in the past, their production level, stability, and toxin neutralizing capacity remain disappointingly poor. In the current study, we identified one Nanobody (Nb), a single-domain antigen-binding fragment of a dromedary Heavy-chain antibody (HCAb) that recognizes specifically the Androctonus australis hector AahI toxin. This Nb has excellent production, stability and solubility characteristics. With this Nb we further manufactured a tandem linked bivalent construct and assembled a HCAb with improved antigen binding due to avidity effects. All these constructs were shown in mouse models to possess a scorpion toxin neutralization capacity that exceeds by far all previous attempts with scFv-based materials, even when used at lower doses. It is therefore clear that in the near future Nanobodies will be at the core of novel serotherapeutics as they combine multiple benefits over other reagents to treat scorpion envenomed patients.
Bioscience, Biotechnology, and Biochemistry, 2007
Biochemical Journal, 2009
Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patie... more 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 subnanomolar 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 k on or k off , or the equilibrium dissociation constant, K D . One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD 50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37 000 LD 50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.
Toxicon, 2013
We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein... more We have previously identified Heminecrolysin, a sphingomyelinase D (SMaseD), as the major protein responsible for the main pathological effects observed following Hemiscorpius (H.) lepturus scorpion envenomation. We aimed herein to further investigate the kinetics and molecular mechanisms triggered by Heminecrolysin to initiate hematological disorders and inflammatory reaction. We show that Heminecrolysin highly hydrolyzes lysophosphatidylcholine (LPC) into lysophosphatidic acid (LPA) and choline, with a Vmax = 1481 ± 51 μmol/min/mg and a Km = 97 ± 16.78 μM, at a much lesser extend sphingomyelin but not phosphatidylcholine substrates. Its lysophospholipase D (lysoPLD) catalytic efficiency, up to three orders of magnitude higher, comparatively to spider's SMaseDs (newly referred as phospholipases D; PLDs), could explain its strong hemolytic capacity. Chelating agents such as EDTA, EGTA, and 1, 10-phenantroline blocked Heminecrolysin-induced LPC hydrolysis at 98, 48, and 70% respectively. Hemolysis blockade occurs only when the toxin is added to erythrocytes in the presence of serum, source of LPC and complement, indicating that the production of LPA and the presence of complement are mandatory for hemolysis. Moreover, we show that Heminecrolysin efficiently binds to erythrocyte's membrane and provokes phosphatidylserine (PS) translocation without cleavage of glycophorin A, suggesting that, unlike spider's PLDs, complement was activated only via the classical pathway. Interestingly, Heminecrolysin was found to induce PS exposure on human nucleated Jurkat T cells, to stimulate secretion of the pro-inflammatory (TNF-α, IL-6), and anti-inflammatory (IL-10) cytokines by human monocytes, and to provoke a disseminated intravascular coagulation on chick embryo chorioallantoic membrane model system. Taken together, our results indicate that Heminecrolysin evokes the major characteristic clinical features of H. lepturus envenomation by using mainly its lysoPLD, rather than its SMaseD's, activity.
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Papers by zakaria benlasfar