The venom of the scorpion Centruroides limpidus, which causes the highest number of stings in Mexico, is neutralized by two recombinant antibody fragments.

Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.

Antigenic and Substrate Preference Differences between Scorpion and Spider Dermonecrotic Toxins, a Comparative Investigation.

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.

Specificity of the female's local cellular immune response in genital plug producing scorpion species.

Immune defense is a key feature in the life history of organisms, expensive to maintain, highly regulated by individuals and exposed to physiological and evolutionary trade-offs. In chelicerates, relatively scarce are the studies that relate postcopulatory mechanisms and immune response parameters. This work makes an approximation to the female's immunological consequences produced after the placement of a foreign body in the genitalia of three scorpions species, two species that normally receive genital plugs during mating (Urophonius brachycentrus and U. achalensis) and one that does not (Zabius fuscus). Here we performed the first morphological description of the natural plugs of the two Urophonius species. We described complex three zoned structure anchored to the female genital atrium and based on this information we placed implants in the genitalia (for eliciting the local immune response) of virgin females of the three species and measured the immune encapsulation response to this foreign body. We found a greater and heterogeneous response in different zones of the implants in the plug producing species. To corroborate the specificity of this immune response, we compared the local genital reaction with the triggered response at a systemic level by inserting implants into the female body cavity of U. brachycentrus and Zabius fuscus. We found that the systemic response did not differ between species and that only in the plug producing species the local response in the genitalia was higher than the systemic one. We also compared the total hemocyte load before and after the genital implantation to see if this parameter was compromised by the immunological challenge. We confirmed that in Urophonius species the presence of a strange body in the genitalia caused a decrease in the hemocyte load. Besides, we find correlations between the body weight and the immunological parameters, as well as between different immunological parameters with each other. Complementarily, we characterized the hemocytes of the three scorpion species for the first time. This comparative study can help to provide a wider framework of the immunological characteristics of the species, their differences and their relationship with the particular postcopulatory mechanism such as the genital plugs.

CD36 Shunts Eicosanoid Metabolism to Repress CD14 Licensed Interleukin-1ß Release and Inflammation.

Interleukin (IL)-1ß is a potential target for treatment of several inflammatory diseases, including envenomation by the scorpion Tityus serrulatus. In this context, bioactive lipids such as prostaglandin (PG)E2 and leukotriene (LT)B4 modulate the production of IL-1ß by innate immune cells. Pattern recognition receptors (PRRs) that perceive T. serrulatus venom (TsV), and orchestrate LTB4, PGE2, and cyclic adenosine monophosphate (cAMP) production to regulate IL-1ß release are unknown. Furthermore, molecular mechanisms driving human cell responses to TsV remain uncharacterized. Here, we identified that both CD14 and CD36 control the synthesis of bioactive lipids, inflammatory cytokines, and mortality mediated by TsV. CD14 induces PGE2/cAMP/IL-1ß release and inflammation. By contrast, CD36 shunts eicosanoid metabolism toward production of LTB4, which represses the PGE2/cAMP/IL-1ß axis and mortality. Of importance, the molecular mechanisms observed in mice strongly correlate with those of human cell responses to TsV. Overall, this study provides major insights into molecular mechanisms connecting CD14 and CD36 with differential eicosanoid metabolism and inflammation mediated by IL-1ß.

New and safe formulation for scorpion immunotherapy: Comparative study between saponin and FCA adjuvants associated to attenuated venom.

Envenoming by scorpion is a major health problem in Maghreb regions as well as in several regions of the world. Immunotherapy is the only effective treatment for scorpion stings. The immune sera are obtained from hyper-immunized animals with a formulation of venom associated to Freund's Complete Adjuvant (FCA). This formulation seems to protect against several alterations in immunized animals leading to worsening of their health due to added toxicity of native venom and FCA adjuvant. This study aims to provide a more efficient and non-toxic alternative to this formulation. Two formulations of saponin or FCA associated to irradiated venom of Androctonus australis hector (Aah) were used to compare their safety and their efficiency to better enhance the antibody titers against toxic antigens. Both of these formulations were used in immunization schedule of three months. Blood samples were collected every week, cell count, myeloperoxydase (MPO) and eosinophil peroxidase (EPO) activities and specific antibody titers were evaluated. Four months after the last immunization, rabbits were challenged with increased doses of native Aah venom. Results showed that immunization with saponin formulation induced lower inflammatory cell activation as well as reduced MPO and EPO activities compared to that using FCA. The formulation of irradiated venom with saponin seems also to be more efficient in the activation of lymphocytes resulting in higher titers of specific IgG. The immunoprotective effect evaluation showed that the formulation using saponin seems to protected animals until 3 LD50 of native venom compared to that using FCA which protected only until 2 LD50. These results indicate that saponin formulation with irradiated antigen could be more efficient and safe immunizing preparation for the production of sera against scorpion envenomation.

Functional and immuno-reactive characterization of a previously undescribed peptide from the venom of the scorpion Centruroides limpidus.

A previously undescribed toxic peptide named Cl13 was purified from the venom of the Mexican scorpion Centruroides limpidus. It contains 66 amino acid residues, including four disulfide bonds. The physiological effects assayed in 7 different subtypes of voltage gated Na+-channels, showed that it belongs to the ß-scorpion toxin type. The most notorious effects were observed in subtypes Nav1.4, Nav1.5 and Nav1.6. Although having important sequence similarities with two other lethal toxins from this scorpion species (Cll1m and Cll2), the recently developed single chain antibody fragments (scFv) of human origin were not capable of protecting against Cl13. At the amino acid sequence level, in 3 stretches of peptide Cl13 (positions 7-9, 30-38 and 62-66) some differences with respect to other similar toxins are observed. Some of these differences coincide with contact points with the human antibody fragments.

Scorpion venom activates natural killer cells in hepatocellular carcinoma via the NKG2D-MICA pathway.

Previous studies have demonstrated that polypeptides extracted from scorpion venom (PESV) inhibited cell proliferation in several tumors, however, the effect on dysfunctional and exhausted natural killer cells which contribute to tumor escape from immune surveillance remain to be elucidated. In this study, we determined the effect of PESV on NK infiltration into H22 cells orthotopic transplantation tumors and on the expression of MHC class I chain-related proteins A (MICA) in HepG2 cells. We found that tumor growth in mice was significantly inhibited by PESV and the survival time of tumor-bearing mice treated with PESV was significantly prolonged. Moreover, levels of tumor-infiltrating NK cells, NKG2D protein, perforin and granzyme B mRNA were significantly increased in the group treated with PESV compared with the tumor-bearing control group. In addition, In addition, up-regulation of MICA by PESV enhances the susceptibility of HepG2 cells to NK lysis in vitro. These results indicate that the inhibitory effects of PESV on hepatic carcinoma are likely mediated by up-regulation of NK cell activity via the MICA-NKG2D pathway.

Optimal Neutralization of Centruroides noxius Venom Is Understood through a Structural Complex between Two Antibody Fragments and the Cn2 Toxin.

The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.

Effect of cytokine antibodies in the immunomodulation of inflammatory response and metabolic disorders induced by scorpion venom.

Androctonus australis hector (Aah) venom and its neurotoxins may affect the neuro-endocrine immunological axis due to their binding to ionic channels of axonal membranes. This binding leads to the release of neurotransmitters and immunological mediators accompanied by pathophysiological effects. Although the hyperglycemia induced by scorpion venom is clearly established, the involved mediators in these deregulations are unknown. The strong relationship between inflammation and the wide variety of physiological processes can suggest that the activation of the inflammatory response and the massive release of IL-6 and TNF-α release induced by the venom may induce hyperglycemia and various biological disorders. We therefore investigated in this study the contribution of IL-6 and TNF-α in the modulation of inflammatory response and metabolic disorder induced by Aah venom. Obtained results revealed that Aah venom induced inflammatory response characterized by significant increase of inflammatory cells in sera and tissues homogenates accompanied by hyperglycemia and hyperinsulinemia, suggesting that the venom induced insulin resistance. It also induced severe alterations in hepatic parenchyma associated to metabolic disorders and imbalanced redox status. Cytokine antagonists injected 30 min prior to Aah venom allowed a significant reduction of inflammatory biomarker and plasma glucose levels, they also prevented metabolic disorders, oxidative stress and hepatic tissue damage induced by Aah venom. In conclusion, IL-6 and TNF-α appear to play a crucial role in the inflammatory response, hyperglycemia and associated complications to glucose metabolism disorders (carbohydrate and fat metabolism disorders, oxidative stress and hepatic damage) observed following scorpion envenoming.

General characterization of Tityus fasciolatus scorpion venom. Molecular identification of toxins and localization of linear B-cell epitopes.

This communication describes the general characteristics of the venom from the Brazilian scorpion Tityus fasciolatus, which is an endemic species found in the central Brazil (States of Goiás and Minas Gerais), being responsible for sting accidents in this area. The soluble venom obtained from this scorpion is toxic to mice being the LD50 is 2.984 mg/kg (subcutaneally). SDS-PAGE of the soluble venom resulted in 10 fractions ranged in size from 6 to 10-80 kDa. Sheep were employed for anti-T. fasciolatus venom serum production. Western blotting analysis showed that most of these venom proteins are immunogenic. T. fasciolatus anti-venom revealed consistent cross-reactivity with venom antigens from Tityus serrulatus. Using known primers for T. serrulatus toxins, we have identified three toxins sequences from T. fasciolatus venom. Linear epitopes of these toxins were localized and fifty-five overlapping pentadecapeptides covering complete amino acid sequence of the three toxins were synthesized in cellulose membrane (spot-synthesis technique). The epitopes were located on the 3D structures and some important residues for structure/function were identified.

Androctonus australis hector venom contributes to the interaction between neuropeptides and mast cells in pulmonary hyperresponsiveness.

Lung injury and respiratory distress syndrome are frequent symptoms observed in the most severe cases of scorpion envenomation. The uncontrolled transmigration of leukocyte cells into the lung interstitium and alveolar space and pulmonary edema may be the cause of death. Mast cells can release various inflammatory mediators known to be involved in the development of lung edema following scorpion venom injection. The present study was designed to determine the evidence of neurokinin 1 (NK1) receptor and the involvement of mast cell activation to induce pulmonary edema and to increase vascular permeability after Androctonus australis hector (Aah) venom administration. To this end, mast cells were depleted using compound 48/80 (C48/80). Furthermore, the involvement of tachykinin NK1 receptors expressed on mast cell membranes was elucidated by their blocking with an antagonist. On the other hand, the ability of Aah venom to increase vascular permeability and to induce edema was also assessed by measuring the amount of Evans blue dye (EBD) extravasation in bronchoalveolar lavage (BAL) fluid and in the lungs of mice. Pulmonary edema, as assessed by the levels of EBD extravasation, was completely inhibited in compound 48/80-treated animals. Depletion by stimuli non-immunological C48/80 component markedly reduced induced inflammatory response following the venom administration. The mast cells seem to play an important role in the development of lung injury and the increase of vascular permeability in mice following the subcutaneous administration of Aah scorpion venom through the NK1 receptor.

Assessment of immunogenic characteristics of Hemiscorpius lepturus venom and its cross-reactivity with venoms from Androctonus crassicauda and Mesobuthus eupeus.

Hemiscorpius lepturus (H. lepturus), one of the most venomous scorpions in tropical and sub-tropical areas, belongs to the Hemiscorpiidae family. Studies of antibodies in sera against the protein component of the venom from this organism can be of great use for the development of engineered variants of proteins for eventual use in the diagnosis/treatment of, and prevention of reactions to, stings. In the present in vitro study, the proteins of H. lepturus venom, which could specifically activate the production of immunoglobulin G (IgG) in victims accidently exposed to the venom from this scorpion, were evaluated and their cross-reactivity with venoms from two other important scorpion species including Androctonus crassicauda and Mesobuthus eupeus assessed. H. lepturus venom was analyzed with respect to its protein composition and its antigenic properties against antibodies found in sera collected from victims exposed to the venom of this scorpion within a previous 2-month period. The cross-reactivity of the H. lepturus venom with those from A. crassicauda and M. eupeus was assessed using ELISA and immunoblotting. Electrophoretic analysis of the venom of H. lepturus revealed several protein bands with weights of 8-116 KDa. The most frequent IgG-reactive bands in the test sera had weights of 34, 50, and 116 kDa. A weak cross-reactivity H. lepturus of venom with venoms from A. crassicauda and M. eupeus was detected. The results of immunoblotting and ELISA experiments revealed that H. lepturus venom activated the host immune response, leading to the production of a high titer of antibodies. Clearly, a determination of the major immunogenic components of H. lepturus venom could be valuable for future studies and ultimately of great importance for the potential production of recombinant or hypo-venom variants of these proteins.

Hp1404, a new antimicrobial peptide from the scorpion Heterometrus petersii.

Antimicrobial peptides have attracted much interest as a novel class of antibiotics against a variety of microbes including antibiotics resistant strains. In this study, a new cationic antimicrobial peptide Hp1404 was identified from the scorpion Heterometrus petersii, which is an amphipathic α-helical peptide and has a specific inhibitory activity against gram-positive bacteria including methicillin-resistant Staphylococcus aureus. Hp1404 can penetrate the membrane of S. aureus at low concentration, and disrupts the cellular membrane directly at super high concentration. S. aureus does not develop drug resistance after multiple treatments with Hp1404 at sub MIC concentration, which is possibly associated with the antibacterial mechanism of the peptide. In addition, Hp1404 has low toxicity to both mammalian cells (HC50  =  226.6 µg/mL and CC50 > 100 µg/mL) and balb-c mice (Non-toxicity at 80 mg/Kg by intraperitoneal injection and LD50  =  89.8 mg/Kg by intravenous injection). Interestingly, Hp1404 can improve the survival rate of the MRSA infected balb-c mice in the peritonitis model. Taken together, Hp1404 may have potential applications as an antibacterial agent.

[Epidemiological data on scorpion envenomation in Algeria]. / Données épidémiologiques sur l'envenimation scorpionique en Algérie.

The scorpion envenomation is a major public health problem in Algeria. Given this fact, the Ministry of Health has developed a national strategy for prevention and control based on the training of health personnel, information, education and communication, and standardization of care on the basis of a therapeutic consensus. The monitoring and evaluation activities are carried out by epidemiological indicators through the implementation of an information system based in the services of Epidemiology, INSP (National Institute of Public Health) and Prevention Department of the Health Ministry. The information carriers are report cards implemented in different health facilities that collect data on bites and deaths from scorpion envenomation. Summaries of notifications from the wilayas are collected monthly, and processed by the Epi info software using monitoring indicators. From 1991 to 2010, there has been a stagnation in the number of stings with an average of 50,000 cases per year, but mortality decreased from more than 100 deaths in the last fifty years to 50 nowadays. The higher proportion of stings was recorded during the summer period. The most affected group is from 15 to 49 years which constitute the workforce, but children from 5 to 14 years rank first in terms of mortality. But these rates vary across years and regions. Despite all these efforts, the scorpion envenomation in Algeria remains of concern and our main challenges are to strengthen cross-sectional actions at the local level and improving the quality of care.

[Report of the 4th International Conference on Envenomations by Snakebites and Scorpion Stings in Africa, Dakar, April 25-29, 2011]. / 4(e) Conférence internationale sur les envenimations par morsures de serpent et piqûres de scorpion en Afrique : Dakar, 25-29 avril 2011.

The authors present a summary of the proceedings and the recommendations of the Fourth International Conference on Envenomations by Snakebites and Scorpion Stings in Africa, held from 25 to 29 April 2011 in Dakar. After a two-day workshop for Senegalese health personnel on the most relevant aspects of the management of envenomations, about 270 participants met to share their experiences in the field. Nearly a hundred oral and poster presentations were made on the epidemiology of snakebites and scorpion stings in Africa, the composition and action of venoms and the manufacture and use of antivenoms. The last day was devoted to an institutional debate involving experts, representatives of national health authorities and concerned professionals (physicians, pharmacists, nurses and traditional healers) as well as members of the pharmaceutical industry to discuss and elaborate a set of recommendations. It was agreed that it is necessary to improve knowledge of the epidemiological situation by case reporting. Quality control of antivenoms and procedures for their registration at the level of national health authorities should aim at improving the distribution of safe and effective antivenoms in peripheral health centers for the better assessment of victims. It was also recommended that adequate training should be provided for health personnel in all aspects of medical management of envenomations. Equitable distribution of funding and the establishment of a network of African experts were also discussed in the conference.

Isolation and characterization of a human antibody fragment specific for Ts1 toxin from Tityus serrulatus scorpion.

Scorpion stings are a common event that occurs in tropical and subtropical areas of the world, being a public health problem in certain countries. In most places, medical treatment relays on antivenoms obtained from the sera of hyper-immunized horses, however some efforts are being made to prepare specific antibodies of human origin, using phage display methodology. This communication describes the strategy followed for obtaining a protective human single chain antibody (scFv) capable of partially neutralizing the effect of Ts1, the major toxin isolated from the venom of the Brazilian scorpion Tityus serrulatus. Phage display technique allowed the isolation of scFv 15e from a human library of antibodies, after four rounds of selection against Ts1. This clone codes for 124 amino acids belonging to the family VH6 and 114 amino acids of family VK4. This scFv also recognizes toxins from the scorpions Tityus packyurus and Tityus cambridgei from the Amazonian region. Mice challenged with a LD(50) of Ts1 in the presence of this scFv were substantially resistant to intoxication. ScFv 15e is a leading compound for the development of better anti-scorpion antidotes.

[Emergency immunotherapy: snake and scorpion antivenoms]. / Immunothérapie d'urgence: antivenins de serpent et de scorpion.

Passive immunotherapy was discovered in 1894. It is the only etiological treatment of envenomations by snakes or scorpions. Immunotherapy is based on administration of antibodies produced by an animal hyperimmunised against venom. Improvement of whole antivenomous sera was obtained, first by separating the antibodies from other components of blood plasma, then by using enzyme digestion of immunoglobulins G and, finally, by purifying the final product. Efficacy and also tolerance were significantly increased. Antivenom administration should be performed through the intravascular route to ensure better diffusion and to facilitate the complexion with the venom. The sale of antivenoms, in particular in Africa, is considerably reduced since about thirty years and is not in adequacy with the epidemiologic needs. In addition to the high cost of the current products, the difficulties of supplying, storage and use seem at the origin of this rejection by both the health staff and the victims who resort massively to traditional medicine. Whereas WHO reminds the rules of production and use of the antivenoms to producers and authorities in charge of drug regulations, it appears that the accessibility of the antivenoms depends on a concerted effort from all the actors, coordinated by health authorities: producers, distributers, manufacturers and public. Beyond technological improvements necessary in antivenom production, the strategy to be implemented must include the strict evaluation of the needs based on epidemiologic studies, the share of cost recovery between all the actors, and the optimization of the therapeutic protocol to make it applicable in remote health centres. Once confidence towards immunotherapy will be restored, mortality will be significantly reduced.

Molecular systematics of the neotropical scorpion genus Tityus (Buthidae): the historical biogeography and venom antigenic diversity of toxic Venezuelan species.

We provide a mitochondrial DNA-based phylogenetic hypothesis for 21 Tityus species collected in Venezuela, Trinidad, Brazil and Panama, including 12 taxa known to be toxic to humans. Our phylogenetic reconstruction is based on 850 nucleotides of the combined cytochrome oxidase subunit I and 16S rRNA genes for most species, and centered on Venezuelan scorpions owing to the detailed taxonomic and biogeographic information available for Tityus in this region. The principal phylogenetic result was the strong support for mtDNA clades representing geographical groupings associated with the Perijá mountain range, the Mérida Andes, or the central and eastern coastal ranges in Venezuela, suggesting that vicariance has been a potent force in the diversification of local scorpions. Venezuelan Tityus species have been organized by González-Sponga into three artificial morphological groups, "androcottoides", "discrepans", and "nematochirus", based on the array of ventral carinae in metasomal segments II-IV. We also incorporated a fourth morphological group ("Tityus clathratus"), recently documented in Venezuela. Our results do not support the clustering of the species in the "androcottoides" and "discrepans" morphological groups, which include the majority of taxa of medical importance, but provided support for the "nematochirus" species group. T. clathratus was found to cluster with the Brazilian T. serrulatus and T. bahiensis. Divergence times of most clades are consistent with major events in the geological history of northern Venezuela and suggest that many Venezuelan Tityus species formed in the late Miocene and the Pliocene. In turn, we used the Tityus mtDNA phylogeny to determine the potential utility of phylogenetic systematics to predict Tityus venom antigenic reactivity by testing the recognition of T. nororientalis, T. discrepans, T. zulianus, T. perijanensis, and T. clathratus venoms by anti-T. discrepans horse antibodies. Cross-reactivity was significantly higher for the closely related eastern (T. nororientalis) and central coastal (T. discrepans) species in comparison to the distantly related Andean (T. zulianus) and Perijá (T. perijanensis) species. Reactivity of T. clathratus low mol. mass toxic components towards anti-T. serrulatus and anti-T. discrepans antivenoms was low, suggesting that venom components produced by the subgenus Archaeotityus (which encompass "clathratus" species) diverge antigenically from other Tityus scorpions.

Antepartum fetal death following a yellow scorpion sting.

Scorpion envenomation in pregnant victims has been scarcely studied. We would like to suggest an association between yellow scorpion sting during the third trimester of pregnancy and adverse fetal outcome. The particular deleterious mechanism of scorpion venom has not been elucidated yet.

Design of antibody-reactive peptides from discontinuous parts of scorpion toxins.

The Amm VIII protein was previously isolated from the venom of the scorpion Androctonus mauretanicus mauretanicus. Despite 87% identity with AaH II, the most toxic alpha-type scorpion toxin, Amm VIII is not toxic to mice. However, antisera against Amm VIII protect mice from AaH II lethal action. Here, we report that the Amm VIII protein elicits antibodies that only recognize discontinuous-type epitopes since we could not observe any antibody binding to overlapping 12-mer peptides covering the whole Amm VIII sequence. By using a new bioinformatic tool, 24 peptides mimicking discontinuous regions of Amm VIII were designed in silico, then prepared by Spot synthesis. Seven of these discontinuous-continuous peptides were recognized by anti-Amm VIII antibodies. Analysis of the 3D location of the segments that compose the antigenically reactive discontinuous-continuous peptides, allowed us to group those antigenic segments into three regions of Amm VIII, putatively corresponding to discontinuous antigenic regions of alpha-type scorpion toxins. Anti-Amm VIII antibodies were also found to cross-react towards several of the discontinuous-continuous peptides designed from the AaH II structure, pointing to a possible involvement of the corresponding discontinuous epitopes in the capacity displayed by anti-Amm VIII antibodies to neutralize AaH II. Altogether, our results show that it is possible to design antibody-reactive peptides from discontinuous parts of scorpion toxins. The position of the reactive segments in the structural context of scorpion toxins highlights the antigenic properties of the Amm VIII anatoxin and concurs to explain the capacity of anti-Amm VIII antibodies to neutralize the potent AaH II toxin.