Full Neutralization of Centruroidessculpturatus Scorpion Venom by Combining Two Human Antibody Fragments.

A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10-9 and 6.1 × 10-11 M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.

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.

Biochemical, electrophysiological and immunological characterization of the venom from Centruroides baergi, a new scorpion species of medical importance in Mexico.

In this communication the isolation, chemical and physiological characterization of three new toxins from the scorpion Centruroides baergi are reported. Their immunoreactive properties with scFvs generated in our group are described. The three new peptides, named Cb1, Cb2 and Cb3 affect voltage-dependent Na+ channels in a differential manner. These characteristics, explain the toxicity of this venom. Molecular interactions in real-time among these toxins and the best recombinant antibodies generated in our group, revealed that one of them was able to neutralize the main toxin of this venom (Cb1). These results represent an important advance for the neutralization of this venom and serve as the basis for generating new scFvs that will allow the neutralization of similar toxins from other venoms that have no yet been neutralized.

Novel Mutant Phospholipase D from Hemiscorpius lepturus Acts as A Highly Immunogen in BALB/c Mice Against the Lethality of Scorpion Venom.

Hemiscorpius lepturus (H. lepturus) which belongs to the Scorpionidae family, is the deadliest scorpion in Iran. It causes pathological manifestations like dermonecrosis, hemolysis, renal failure, necrotic ulcers, and in some cases, even death. The venom of this scorpion is well-known for its cytotoxic effects in comparison with the other venomous scorpions which show significant neurotoxic effects. Due to the painless nature of the sting of this scorpion, the clinical symptoms occur in victims 24 to 72 h post-sting. In our previous studies during the last decade, we demonstrated that the medical complications are attributable to the presence of phospholipase D (PLD) as a major toxin in the venom. With the purpose of designing and constructing a vaccine against H. lepturus for humans, animal model experiments were performed. To achieve this goal, non-toxic PLD was developed by mutation of two critical catalytic residues-His12 and His48-into alanines and the product was then denominated mut-rPLD1. The in-vivo tests showed that the mice immunized with interval doses of 10 µg of mut-rPLD1, were completely protected against 10× the LD100 of the venom. In conclusion, this mutant may be an effective vaccine candidate against scorpion envenomation by H. lepturus in future clinical studies.

Chitosan nanoparticles as a delivery platform for neurotoxin II from Androctonus australis hector scorpion venom: Assessment of toxicity and immunogenicity.

In recent years, biodegradable polymers based nanoparticles received high interest for the development of vaccine delivery vehicles. In this study, chitosan nanoparticles encapsulating Aah II toxin (AahII-CNPs) isolated from Androctonus australis hector venom, were investigated as vaccine delivery system. Particles obtained by ionotropic gelation were characterized for their size, surface charge, morphology and toxin release profile from Aah II-CNPs. Toxin-nanoparticles interactions were assessed by Fourier Transform Infrared Spectrometry and X-Ray Diffraction. An immunization protocol was designed in mice to investigate anti-toxin immunity and the protective status induced by different Aah II immune formulations. Unloaded chitosan nanoparticles presenting a spherical shape and smooth surface, were characterized by a size of 185 nm, a dispersion index (PDI) of 0.257 and a zeta potential of +34.6 mV. Aah II toxin was successfully entrapped into chitosan nanoparticles as revealed by FTIR and XRD data. Entrapment efficiency (EE) and Loading capacity (LC) were respectively of 96.66 and 33.5%. Aah II-CNPs had a diameter of 208 nm, a PDI of 0.23 and a zeta potential of +30 mV. Encapsulation of Aah II reduced its toxicity and protected mice until 10 LD50. Mice were immunized via a dual prime-boost scheme. Nanoentrapped Aah II immunogen elicited systemic innate and humoral immune responses as well as local spleen parenchyma hyperplasic alterations. Aah II-CNPs immunized mice withstood high lethal doses of native Aah II, one-month post-boost inoculation. This study provided encouraging and promising results for the development of preventive therapies against scorpion envenoming mainly for the populations at-risk.

Venom diversity in the Neotropical scorpion genus Tityus: Implications for antivenom design emerging from molecular and immunochemical analyses across endemic areas of scorpionism.

Scorpions of the Neotropical genus Tityus are responsible for most severe envenomations in the Caribbean, South America, and Lower Central America (LCA). Although Tityus is taxonomically complex, contains high toxin polymorphism, and produces variable clinical manifestations, treatment is limited to antivenoms produced against species with restricted distributions. In this study, we explored the compositional and antigenic diversity of Tityus venoms to provide improved guidelines for the use of available antivenoms at a broader geographic scale. We used immunoblotting, competitive ELISA, and in vivo studies to compare reactivity against commercial antivenoms from Brazil, Venezuela, and Mexico, as well as MALDI-TOF mass spectrometry, cDNA sequencing, and phylogenetic analyses to assess venom sodium channel-active toxin (NaTx) content from medically important Tityus populations inhabiting Brazil, Colombia, Costa Rica, Ecuador, Panama, Trinidad and Tobago, and Venezuela. Additionally, we raised rabbit antibodies against Tityus venoms from LCA to test for cross-reactivity with congeneric species. The results suggest that Tityus spp. possess high venom antigenic diversity, underlying the existence of four toxinological regions in Tropical America, based on venom composition and immunochemical criteria: LCA/Colombia/Amazonia (Region I), Venezuela (Region II), southeast South America (Region III), and a fourth region encompassing species related to toxinologically divergent Tityus cerroazul. Importantly, our molecular and cross-reactivity results highlight the need for new antivenoms against species inhabiting Region I, where scorpions may produce venoms that are not significantly reactive against available antivenoms.

Scorpion envenomation and inflammation: Beyond neurotoxic effects.

Scorpion envenomation results in a wide range of clinical manifestations that are mostly attributed to the activation of the autonomic nervous system by venom toxins. In fact, sympathetic and parasympathetic disturbances play important roles during poisoning. However, scorpion venom also induces a complex hyperinflammatory state that occurs parallel to systemic inflammatory response syndrome and acute sepsis. After a scorpion sting, innate immune cells are exposed to the venom molecules, which bind to pattern recognition receptors and activate pro-inflammatory pathways that contribute toward the promotion of severe symptoms, such as pulmonary edema, and eventually lead to death. In this review, we highlight studies that pointed out inflammation as a major pathological facet of scorpion envenomation, so as to provide novel targets to improve therapeutics for scorpionism.

Proposed reduction of the in vivo pyrogen test by the in vitro LAL assay for the quality control of anticrotallic, antiscorpion, antirabies and antitetanus sera.

The rationale for a formal study of the LAL (kinetic chromogenic method) assay for anticrotallic (SAC), antirabies (SAR), antitetanus (SAT) and antiscorpion (SAE) sera involved the determination of parameters required by the Brazilian National Health Surveillance Agency (ANVISA), the USA Food and Drug Administration (FDA), USP (United States Pharmacopeia, 39) and ICH (International Conference on Harmonization). The curve correlation coefficients obtained with the standard endotoxin control ranged from -0.980 to -1.000 in all experiments. Endotoxin recovery added to the SAC, SAR, SAT and SAE samples, at the working dilutions (1:10, 1:10, 1:10 and 1, 100 respectively), met the criteria required by the FDA, USP and Brazilian ANVISA for the Inhibition-Potentiation test. The applied methodology for the four analyzed sera fulfilled the required criteria for all performance parameters. Thus, the present study demonstrated that the in vivo pyrogen test can be potentially replaced by the LAL assay for all assessed sera samples displaying higher sensitivity and following the 3 Rs principle, in addition to maintaining quality control in Sanitary Surveillance.

Induction of two independent immunological cell death signaling following hemoglobinuria -induced acute kidney injury: In vivo study.

The main important clinical signs in acute kidney injury (AKI) after sever Hemiscorpius lepturus envenomation in patients is associated with proteinuria, hemolysis and hemoglobinuria. Unfortunately, our limited knowledge of molecular cell death mechanism in H. lepturus induced AKI restricts the development of desirable therapeutics. So, in the present study, the potential role of necroptosis and ferroptosis in H. lepturus induced AKI were investigated in male albino mice. The animals were administrated by SC injection of venom (1, 2.5, 5 and 10 mg/kg) based on LD50 determination. After 1 and 7 days, urinalysis, stereological assessments and gene expression of Ngal, Tnf-α, Tlr-4, Ripk3, Mlkl and Acsl4 were evaluated by real time PCR. Our data revealed that upregulation of renal Ngal expression is associated with the gene over expression of Tnf-α, Tlr-4, Ripk3 and Mlkl in venom treated kidneys. We observed that the Malondialdehyde (MDA) level was increased in dose-dependent manner similar to Acsl4 gene over expression suggesting a main role of ferroptosis in hemoglobinuria mediated AKI following envenomation. Moreover, transcriptional enhancement of Tlr-4and Tnf-α receptor can cause phosphorylation of Ripk3-Mlkl complex, collapse of membrane potential and DAMPs release which intensified the inflammation cytokines in kidney. Taken together, it supposes co-existence of two separate pathways of regulated necrosis and inflammatory environment provides a promising outlook in prevention and management of hemoglobinuria induced AKI following envenomation in clinical practice.

Serotherapy against Voltage-Gated Sodium Channel-Targeting αToxins from Androctonus Scorpion Venom.

Because of their venom lethality towards mammals, scorpions of the Androctonus genus are considered a critical threat to human health in North Africa. Several decades of exploration have led to a comprehensive inventory of their venom components at chemical, pharmacological, and immunological levels. Typically, these venoms contain selective and high affinity ligands for the voltage-gated sodium (Nav) and potassium (Kv) channels that dictate cellular excitability. In the well-studied Androctonusaustralis and Androctonusmauretanicus venoms, almost all the lethality in mammals is due to the so-called α-toxins. These peptides commonly delay the fast inactivation process of Nav channels, which leads to increased sodium entry and a subsequent cell membrane depolarization. Markedly, their neutralization by specific antisera has been shown to completely inhibit the venom's lethal activity, because they are not only the most abundant venom peptide but also the most fatal. However, the structural and antigenic polymorphisms in the α-toxin family pose challenges to the design of efficient serotherapies. In this review, we discuss past and present accomplishments to improve serotherapy against Androctonus scorpion stings.

Generation of a Broadly Cross-Neutralizing Antibody Fragment against Several Mexican Scorpion Venoms.

The recombinant antibody fragments generated against the toxic components of scorpion venoms are considered a promising alternative for obtaining new antivenoms for therapy. Using directed evolution and site-directed mutagenesis, it was possible to generate a human single-chain antibody fragment with a broad cross-reactivity that retained recognition for its original antigen. This variant is the first antibody fragment that neutralizes the effect of an estimated 13 neurotoxins present in the venom of nine species of Mexican scorpions. This single antibody fragment showed the properties of a polyvalent antivenom. These results represent a significant advance in the development of new antivenoms against scorpion stings, since the number of components would be minimized due to their broad cross-neutralization capacity, while at the same time bypassing animal immunization.

Preparation of F(ab')2 antivenom in Iraq against scorpion (Hottentotta saulcyi) venom.

The aim of this study was to prepare specific F(ab')2 antivenom against Iraqi scorpion (Hottentotta saulcyi). Venom was obtained by electrical stimulation method, the scorpions venom storage capacity was 1.7 ±â€¯0.4 mg and LD50 found to be 1.07 mg/kg by subcutaneous (s.c) route. Three local horses aging 3-4 years were selected for immunization. During the schedule each horse received 0.5-14 mg venom in 49 days by s.c route. The horses immune response was monitored by Ouchterlony double immunodiffusion method, however, older horse A showed the highest antibody titer (1:1056576). Hyperimmune plasma was purified by modified World Health Organization (WHO) protocol using pepsin and ammonium sulfate, whereas high purity and total removal of albumin was detected by serum protein electrophoresis (SPEP) method on cellulose acetate. ED50 of the antivenom was determined via s.c route and found to be 83 LD50/ml, neutralizing 1.78 mg (venom)/ml (antivenom). Protein concentration of the antivenom was 30 mg/ml, less than limited value (100 mg/ml) by WHO. Abnormal toxicity test showed no abnormal signs when 0.2 ml of the antivenom injected intraperitoneally in mice.

Ts1 from the Brazilian scorpion Tityus serrulatus: A half-century of studies on a multifunctional beta like-toxin.

The Tityus serrulatus scorpion species represents a serious human health threat to in Brazil because it is among the animals that produces the most dangerous venoms for mammals in South America. Its venom has provided several highly selective ligands that specifically interact with sodium and potassium channels. During the past decades, several international groups published an increasing amount of data on the isolation and the chemical, pharmacological and immunological characterisation of its main ß-toxin, Ts1. In this review, we compiled the best available past and recent knowledge on Ts1. Aside from its intricate purification, the state-of-the-art understanding concerning its pharmacological activities is presented. Its solved three-dimensional structure is shown, as well as the possible surface areas of contact between Ts1 and its diverse voltage-gated Na+ channel targets. Organisations of the gene and the precursor encoding Ts1 are also tackled based on available cDNA clones or on information obtained from polymerase chain reactions of stretches of scorpion DNA. At last, the immunological studies complete with Ts1 to set up an efficient immunotherapy against the Tityus serrulatus venom are summarized.

Developing recombinant phospholipase D1 (rPLD1) toxoid from Iranian Hemiscorpius lepturus scorpion and its protective effects in BALB/c mice.

Hemiscorpius lepturus (H. lepturus) is one of the most dangerous scorpions and the most medically important scorpion in Iran. The clinical signs of H. lepturus envenomation, including dermonecrosis, hematuria, renal failure and early death, are attributed to phospholipase D activity. This study was conducted to develop a novel recombinant phospholipase D1 (rPLD1) toxoid and investigate its immunogenicity and protective effects against the lethality of H. lepturus venom. The lethal protein recombinant phospholipase D1 was expressed from PLD H. lepturus venom gland. The rPLD1 toxin was converted into toxoid (the first toxoid of H. lepturus PLD) with a 0.25% concentration of formalin and stored for ten days at room temperature. In the toxicity test, the lethal activity of recombinant phospholipase D1 was fully inhibited. When it reached up to 3 times higher than the maximal effective concentration of the purified toxin (11.1 µg), rPLD1 toxoid was used. The sphingomyelinase activity was inhibited when up to 5.4 times of the LD100 of the purified toxin (20 µg), toxoid was used. It was then used to produce an antibody in BALB/c as an antigen and the mice were then challenged with rPLD1 toxin and the whole venom. The immunogenicity of rPLD1 toxoid was evaluated and the maximum titer of the raised antibodies was determined by ELISA assay. The optimum titer for anti-rPLD1 toxoid sera was obtained at the third intraperitoneal injection of rPLD1 toxoid, and a high titer was reached at the fourth injection in the mice. This toxoid increased the amount of antibodies and produced a protective antiserum against the whole venom of H. lepturus and rPLD1 toxin. The in-vivo test results showed that the mice were completely resistant against 200 times the LD100 of recombinant phospholipase D1 and the whole venom of H. lepturus. To conclude, rPLD1 can be used in toxoid form as an immunogen in the production of a new generation of neutralizing antibodies against the lethality and toxicity of H. lepturus whole venom.

Development of a new approach of immunotherapy against scorpion envenoming: Avian IgYs an alternative to equine IgGs.

Antivenom treatment has been largely used against scorpion stings. Despite their efficacy, the use of mammalian antivenoms may cause adverse effects due to the immune system activation. IgYs from hyperimmunized laying hens against venoms could be a promising alternative to equine IgGs due to the various benefits that these antibodies can provide. Here we report the preparation of specific IgYs by immunizing laying hens with Aah (Androctonus australis hector) scorpion venom. IgYs were isolated from egg yolks by water dilution and salt precipitation methods; they were characterized by sodium dodecyl sulfate polyacrylamide gel electrophoresis, western blot and ELISA. The efficiency of these immunoglobulins on the pathophysiological effects induced by Aah venom was assessed by histological and metabolical analysis of the aorta and the heart. The inflammatory response was assessed by evaluating the granulocyte tissue infiltration and oxidative/nitrosative status. Results revealed high IgYs titers against Aah venom by ELISA. Overall, these IgYs seem to protect efficiently mice against envenomation and neutralized the lethal effects of scorpion venom with a high efficacy; the median effective dose (ED50) was 221 µl/2 LD50; i.e. an amount of 79.23 mg of IgY scan neutralize 1 mg of Aah venom. IgY antibodies neutralize effectively the Aah venom lethality and could prevent severe pathological effects induced by scorpion venom and could be used as an effective alternative to equine IgGs against scorpion envenoming.

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.

Genotoxicity evaluation induced by Tityus serrulatus scorpion venom in mice.

Tityus serrulatus is the scorpion associated with the most severe cases of scorpion envenoming in Brazil. However, there are no studies reporting the genotoxic effects of this venom in natural or experimental envenomations. It is well known that DNA-damage responses are providing opportunities for improving disease detection and management. In this study was evaluating the genotoxicity of the T. serrulatus venom in different organs (hippocampus, cortex, striatum, blood, heart, lung, liver and kidney) and periods in mice experimentally envenomed. ELISA and the Comet assays were used to quantification of venoms antigens and DNA damage, respectively. Forty-eight Swiss mice were divided into five groups and 0.5 DL50 of T. serrulatus venom (0.90 mg/kg) was administered intraperitoneally in each animal. Euthanasia was performed by cervical dislocation in the period of 0h (control group) 1h, 2h, 6h and 12h, where it the tissues were removed. The results showed high DNA damage in all structures analyzed, suggesting that T. serrulatus venom presented genotoxic activity or some secondary effect generated by venom injection. In the ELISA test, toxic circulant antigens were verified in practically all organs at the time intervals analyzed. Therefore, the distribution of the venom changes from organ to organ. We conclude that scorpion envenoming affects DNA in all organs analyzed even when the venom concentration is lower or no detectable, DNA damage persists.

Safety and efficiency of active immunization with detoxified antigen against scorpion venom: side effect evaluation.

OBJECTIVE: The efficiency and safety of vaccine are the most important properties, however, as any medication, it can induce side effects. This prophylactic therapy could be used to prevent the lethal and pathophysiological effects induced after scorpion envenomation. METHODS: In this study, detoxified venom associated to alum adjuvant (V*alum) is used as a vaccine against scorpion venom for immunization of mice. We evaluate the safety and the inflammatory response of this vaccine. We also investigated the protective effect of this formulation against the toxicity of native Androctonus australis hector venom. RESULTS: Results showed no adverse events occurred after immunization of animals. This active immunization of animals did not cause change in vascular permeability, no edema formation in the studied organs. Furthermore, there are no IgE production in sera, nor change in the morphology of the mast cells in skin tissues. However, low inflammatory response triggered by activating the recruitment of eosinophils associated to IL-4 and IL-5 release was observed. All immunized animals are protected from the toxic effects of native venom until 6 LD50 and to 7 LD50 after the second challenge. CONCLUSION: This safe vaccine preparation seems to induce a long-term protection without any risk of deleterious inflammatory response.