Venom In Mice Research Articles

Sex-related differences in toxic manifestations induced by Bothrops atrox venom in mice

Lancehead snakes of the genus Bothrops are responsible for 90% of the snakebites in Latin America. The objective of this study was to assess the LD50, physical, and hematological manifestations induced by B. atrox venom in male and female mice inoculated by different routes. B. atrox venom was inoculated in male and female mice by intramuscular (IM), subcutaneous (SC), intravenous (IV), and intraperitoneal (IP) routes. B. atrox venom LD50 was lower in male than female groups, regardless of the injection route. However, it was the lowest when the venom was inoculated by the IP route. Moreover, comparisons between male and female responses according to the injected venom dose showed higher edema-forming, local hemorrhagic, dermonecrotic, and myotoxic activities in male than in female mice. While the minimal hemorrhagic, and necrotic doses were not statistically different between the two groups, the difference between males and females was more pronounced at high venom doses. Hematological parameter changes were also more significant in male than in female mice. The venom decreased the levels of total leukocytes after 24 h of injection in male and female mice, with a more profound decrease in the male group. The micronucleus test, a tool for genotoxicity assessment, documented the mutagenic effects of B. atrox on leucocytes. We demonstrate the higher susceptibility of male mice to B. atrox venom than females. Sex differences must be considered when conducting experimental studies on snake venoms.

Paraspecific neutralization capacity of polyvalent snake antivenom against Montivipera wagneri venom

PurposeThis study aims to measure the paraspecific neutralization capacity of nationally produced HSGM polyvalent snake antivenom (HSGM-PSAV), produced using Macrovipera lebetina obtusa, Montivipera xanthina, and Vipera ammodytes montandoni venom, against the lethal effect of the venom of Montivipera wagneri, which is endemic to the Eastern Black Sea and Eastern Anatolia regions of Turkey. MethodsThe neutralization capacity of HSGM-PSAV against the lethal effect of M. wagneri venom was studied using the potency determination testing method specified in the Turkish and European Pharmacopoeia. Lethal dose 50 (LD50) values of the venoms used in immunization, M. wagneri venom in mice, and effective dose 50 (ED50) values of HSGM-PSAV against four types of venoms were calculated using two-fold dilutions. ResultsHSGM-PSAV neutralized the lethal effect of M. wagneri venom in mice. The ED50 of the HSGM-PSAV against M. wagneri venom was calculated as 304.42 LD50/mL. ConclusionAs a result of this in-vivo study, it was determined that HSGM-PSAV neutralized M.wagneri venom above the antivenom neutralization capacity threshold values (≥50 LD50/mL) specified in the Turkish and European Pharmacopoeia. This result is important preclinical data regarding the usability of HSGM-PSAV in the treatment of poisoning due to M. wagneri bites.

Development and characterization of nanobody against envenomation by Naja naja oxiana

Snakebites are considered a significant health issue. Current antivenoms contain polyclonal antibodies, which vary in their specificity against different venom components. Development and characterization of next generation antivenoms including nanobodies against Naja naja oxiana was the main aim of this study. Crude venom was injected into the Sephadex G50 filtration gel chromatography column and then toxic fractions were obtained. Then the corresponding fraction was injected into the HPLC column and the toxic peaks were identified. N. naja oxiana venom was injected into a camel and specific nanobodies screening was performed against the toxic peak using phage display technique. The obtained results showed that among the 12 clones obtained, N24 nanobody was capable of neutralizing P1, the most toxic peak obtained from HPLC chromatography. The molecular weight of P1 was measured with a mass spectrometer and was found to be about seven kDa. The results of the neutralization test of crude N. naja oxiana venom with N24 nanobody showed that 250 μg of recombinant nanobody could neutralize the toxic effects of 20 μg equivalent to LD50 × 10 of crude venom in mice. The findings indicate the potential of the developed nanobody to serve as a novel antivenom therapy.

Protective Effects of the Aqueous Extract of Malva sylvestris Plant against the Lethality and Histopathological Damage in Experimental Envenoming of Hemiscorpius lepturus Venom in Balb/c Mice.

Hemiscorpius lepturus envenomation is a serious health problem in the southern provinces of Iran. The antiserum produced in Iran to counteract this scorpion venom is not entirely effective due to the risk of anaphylactic shock and other adverse effects. Therefore, more efficient alternatives to treat patients deserve attention, and plants are extensively good candidates to be studied. This study aimed to assess the potential of the aqueous fraction of Malva sylvestris in inhibiting the toxic effects of H. lepturus venom. Injection of sub-lethal dose of H. lepturus venom leads to severe tissue damage in vital organs including the kidney, liver, heart and intestine, after 24 hours. By injecting 80 mg of the aqueous extract of M. sylvestris into the peritoneum helped treat the damaged tissues caused by H. lepturus venom in mice. Thus, Malva sylvestris could serve as an alternative treatment for scorpion sting envenomation and may be used as a drug to neutralize relevant toxic effects in patients stung by H. lepturus.

In vivo neutralization of coral snake venoms with an oligoclonal nanobody mixture in a murine challenge model

Oligoclonal mixtures of broadly-neutralizing antibodies can neutralize complex compositions of similar and dissimilar antigens, making them versatile tools for the treatment of e.g., infectious diseases and animal envenomations. However, these biotherapeutics are complicated to develop due to their complex nature. In this work, we describe the application of various strategies for the discovery of cross-neutralizing nanobodies against key toxins in coral snake venoms using phage display technology. We prepare two oligoclonal mixtures of nanobodies and demonstrate their ability to neutralize the lethality induced by two North American coral snake venoms in mice, while individual nanobodies fail to do so. We thus show that an oligoclonal mixture of nanobodies can neutralize the lethality of venoms where the clinical syndrome is caused by more than one toxin family in a murine challenge model. The approaches described may find utility for the development of advanced biotherapeutics against snakebite envenomation and other pathologies where multi-epitope targeting is beneficial.

Behavioral, biochemical and histopathological alterations induced in Swiss mice by Buthus boumalenii scorpion venom

The yellow scorpion, Buthus boumalenii, has long been recognized as a potentially dangerous species endemic to Morocco. However, there is currently no data on the in vivo toxicity or effects of Buthus boumalenii venom, as it has not been studied to the best of our knowledge. In order to achieve the objective of this study, the LD50 of B. boumalenii venom will be initially calculated, followed by determining its impact on vital organs using histological, biochemical, and behavioral analyses. The investigation also explores potential neurobehavioral impairments in Swiss mice at intervals of 3 hours, 6 hours, and 12 hours post-envenomation. The LD50 for Buthus boumalenii scorpion venom in mice was determined to be 353 µg/kg based on body weight. The findings of the study highlight the potential lethality of B. boumalenii. Observations related to the poisoning provide indications of potential tissue damage in specific vital organs. To sum up, the venom from this scorpion has the potential to result in considerable medical complications, including fatalities, particularly in individuals at risk. Consequently, healthcare practitioners should be knowledgeable about the diverse scorpion species in their regions and adhere to contemporary medical protocols when addressing scorpion envenomation.

Assessment of the Artemia salina toxicity assay as a substitute of the mouse lethality assay in the determination of venom-induced toxicity and preclinical efficacy of antivenom

Mice are routinely used in snake venom research but are costly and subject to pain and suffering. The crustacean Artemia salina could be an alternative to mice, but data to support its adoption in snake venom research is limited. The aim of the present study was to evaluate the suitability of A. salina as a surrogate of mice in assessing the toxicity of venoms and the preclinical efficacy of antivenoms. The toxicity of venoms from 22 snakes of medical importance in sub–Saharan Africa was evaluated in mice (intraperitoneally; i.p. and intravenously; i.v.) and in A. salina. Subsequently, the capacity of a commercial antivenom to neutralize the toxicity of these venoms in mice and A. salina was investigated. There was a positive correlation between the i.v. median lethal doses (LD50s) and the i.p. LD50s in mice (r = 0.804; p < 0.0001), a moderate correlation between the i.v. LD50s in mice and the median lethal concentrations (LC50s) in A. salina (r = 0.606; p = 0.003), and a moderate correlation between the i.p. LD50s in mice and the LC50s in A. salina (r = 0.426; p = 0.048). Moreover, there was a strong correlation between the i.p. median effective doses (ED50s) and the i.v. ED50s in mice (r = 0.941, p < 0.0001), between the i.p. ED50s in mice and the ED50s in A. salina (r = 0.818, p < 0.0001), and between the i.v. ED50s in mice and the ED50s in A. salina (r = 0.972, p < 0.0001). These findings present A. salina as a promising candidate for reducing reliance on mice in snake venom research. Future investigations should build upon these findings, addressing potential limitations and expanding the scope of A. salina in venom research and antivenom development.

Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (β-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide β-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

Apis mellifera syriaca Venom Modulates Splenic Cytokines Levels in BALB/c Mice.

Bee venoms are well-known for their important biological activities. More specifically, the venom of Apis mellifera syriaca was shown to exhibit various biological effects, including antimicrobial effects. It is suggested that the anti-microbial effect of venom could be accompanied by an immunomodulatory response in the host favoring anti-inflammatory responses. Thus, in this work, we investigated, for the first time, the immunomodulatory effects of A. mellifera syriaca venom in mice. Firstly, it was found that this venom exhibited mild toxicity in BALB/c mice after intraperitoneal injection with an LD50 of 3.8 mg/kg. We then investigated its immunomodulatory effects by evaluating the splenic levels of both pro- and anti-inflammatory cytokines in mice by ELISA. Interestingly, at 1 mg/kg, A. mellifera syriaca venom induced a decrease in IFN-γ, TNF-α, IL-4, and IL-10 at 24h postinjection. At a higher dose (3 mg/kg), an increase in IFN-γ and IL-4 levels was observed, while the levels of TNF-α and IL-10 remained low compared to the control. Altogether, these preliminary data suggest that A. mellifera syriaca venom exhibits anti-inflammatory effects at a sublethal dose (1 mg/kg), while at a higher dose (3 mg/kg), it induces inflammatory effects.

Aqueous Extract and Solvent Fractions of Hancornia speciosa Fruits, Rutin, and Chlorogenic Acid Attenuate the Edema, Inflammation, and Myonecrosis Caused by Bothrops jararaca Snake Venom in Mice

Aqueous Extract and Solvent Fractions of Hancornia speciosa Fruits, Rutin, and Chlorogenic Acid Attenuate the Edema, Inflammation, and Myonecrosis Caused by Bothrops jararaca Snake Venom in Mice

Agonists of melatonin receptors strongly promote the functional recovery from the neuroparalysis induced by neurotoxic snakes.

Snake envenoming is a major, but neglected, tropical disease. Among venomous snakes, those inducing neurotoxicity such as kraits (Bungarus genus) cause a potentially lethal peripheral neuroparalysis with respiratory deficit in a large number of people each year. In order to prevent the development of a deadly respiratory paralysis, hospitalization with pulmonary ventilation and use of antivenoms are the primary therapies currently employed. However, hospitals are frequently out of reach for envenomated patients and there is a general consensus that additional, non-expensive treatments, deliverable even long after the snake bite, are needed. Traumatic or toxic degenerations of peripheral motor neurons cause a neuroparalysis that activates a pro-regenerative intercellular signaling program taking place at the neuromuscular junction (NMJ). We recently reported that the intercellular signaling axis melatonin-melatonin receptor 1 (MT1) plays a major role in the recovery of function of the NMJs after degeneration of motor axon terminals caused by massive Ca2+ influx. Here we show that the small chemical MT1 agonists: Ramelteon and Agomelatine, already licensed for the treatment of insomnia and depression, respectively, are strong promoters of the neuroregeneration after paralysis induced by krait venoms in mice, which is also Ca2+ mediated. The venom from a Bungarus species representative of the large class of neurotoxic snakes (including taipans, coral snakes, some Alpine vipers in addition to other kraits) was chosen. The functional recovery of the NMJ was demonstrated using electrophysiological, imaging and lung ventilation detection methods. According to the present results, we propose that Ramelteon and Agomelatine should be tested in human patients bitten by neurotoxic snakes acting presynaptically to promote their recovery of health. Noticeably, these drugs are commercially available, safe, non-expensive, have a long bench life and can be administered long after a snakebite even in places far away from health facilities.

The role and mechanism of NLRP3 in wasp venom-induced acute kidney injury

BackgroundInflammation and pyroptosis have crucial impacts on the development of acute kidney injury (AKI) and have been validated in a variety of existing AKI animal models. However, the mechanisms underlying wasp venom-induced AKI are still unclear. The involvement of nucleotide-binding oligomerization domain (NOD)-like receptor protein 3 (NLRP3) in some mouse models of AKI has been extensively documented, and its crucial function in controlling inflammation and pyroptosis has been highlighted. The objective of our study was to investigate the role and mechanism of NLRP3 in inflammation and pyroptosis associated with wasp venom-induced AKI. MethodsA mouse model of AKI induced by wasp venom pre-injected with an NLRP3 inhibitor was used to study the role and mechanism of NLRP3. To verify the importance of NLRP3, western blotting was performed to assess the expression of NLRP3, caspase-1 p20, and gasdermin D (GSDMD)-N. Additionally, quantitative real-time polymerase was used to determine the expression of NLRP3, caspase-1, and GSDMD. Furthermore, enzyme-linked immunosorbent assay was utilized to measure the levels of interleukin (IL)-1β and IL-18. ResultsNLRP3 was found to be the downstream signal of the stimulator of interferon genes in the wasp sting venom-induced AKI model. The administration of wasp venom in mice significantly upregulated the expression of NLRP3, leading to renal dysfunction, inflammation, and pyroptosis. Treatment with an NLRP3 inhibitor reversed the renal damage induced by wasp venom and attenuated pathological injury, inflammatory response, and pyroptosis. ConclusionNLRP3 activation is associated with renal failure, inflammatory response and pyroptosis in the hyper early phase of wasp venom-induced AKI. The inhibition of NLRP3 significantly weakened this phenomenon. These findings could potentially offer a viable therapeutic approach for AKI triggered by wasp venom.

Venom of the neotropical rattlesnake, Crotalus culminatus: Intraspecific variation, neutralization by antivenoms, and immunogenicity in rabbits

Crotalus culminatus is a medically significant species of rattlesnake in Mexico [1]. While the proteomic composition of its venom has been previously reported for both juvenile and adult specimens, there has been limited research into its functional properties, with only a few studies, including one focusing on coagulotoxicity mechanisms. In this study, we aimed to compare the biochemical and biological activities of the venom of juvenile and adult snakes. Additionally, we assessed antibody production using the venoms of juveniles and adults as immunogens in rabbits. Our findings reveal lethality and proteolytic activity differences between the venoms of juveniles and adults. Notably, juvenile venoms exhibited high proportions of crotamine, while adult venoms displayed a reduction of this component. A commercially available antivenom demonstrated effective neutralization of lethality of both juvenile and adult venoms in mice. However, it failed to neutralize the paralytic activity induced by crotamine, which, in contrast, was successfully inhibited by antibodies obtained from hyperimmunized rabbits. These results suggest the potential inclusion of C. culminatus venom from juveniles in commercial antivenom immunization schemes to generate antibodies targeting this small myotoxin.

Assessing the Efficacy of Monovalent and Commercialized Antivenoms for Neutralizing Moroccan Cobra Naja haje Venom: A Comparative Study.

In Morocco, eight species of venomous snakes belonging to the Viperidae and Elapidae families are responsible for severe envenomation cases. The species from the Elapidae family is only represented by the medically relevant cobra Naja haje, which is widely distributed in North Africa. However, there is little information on the systemic effects of Moroccan cobra venom on vital organs due to regional variations. It has been demonstrated that the venom of Naja haje from Egypt causes hemorrhage, while the venom of the Moroccan cobra is neurotoxic and devoid of systemic bleeding. This variability is known to significantly influence treatment efficacy against Naja haje cobra bites in the Middle East. In this study, we examined the pathophysiological mechanisms responsible for the lethality induced by Naja haje venom, as well as the evaluation of the neutralizing capacity of two antivenoms; the monospecific antivenom made for Naja haje only and the antivenom marketed in the Middle East and North Africa. We first determined the toxicity of Naja haje venom by LD50 test, then compared the neutralizing capacity of the two antivenoms studied by determining the ED50. We also performed histological analysis on Swiss mice envenomed and treated with these antivenoms to observe signs of cobra venom envenomation and the degree of reduction of induced systemic alterations. The results showed significant differences between both antivenoms in terms of neutralization. The monospecific antivenom was four times more effective than the marketed antivenom. These results were confirmed by a histological study, which showed that monospecific antivenoms neutralized severe signs of mortality, such as congestion of blood vessels in the heart and kidneys, pulmonary and renal edema, cytoplasmic vacuolization of hepatocytes in the liver, and infiltration of inflammatory cells in the brain and spleen. However, the polyvalent antivenom failed to protect all severe lesions induced by Naja haje venom in mice. These findings highlight the negative impact of geographic variation on the effectiveness of conventional antivenom therapy and confirm the need for a specific Naja haje antivenom for the effective treatment of cobra envenomation in Morocco.

Extract of Celosia leptostachya Leaves Inhibits Cobra Venom Toxins

Background: Celosia leptostachya belongs to Amaranthaceae plant family. Its leaves are used traditionally in the treatment of conditions, such as convulsion, eye infection and most notably to cure snakebites. This study investigated the inhibiting effect of the extract of C. leptostachya leaves against cobra snake venom in mice. Methods: We used 36 albino mice of mixed gender, weighing 20-25 g. They were divided into six groups of six rats each. Each rat was pre-treated with 0.4 mg/kg of cobra snake venom, and was subsequently given a graded dose of zero, 50, 100, 150, 200, or 250 mg/kg of the ethanol extract of C. leptostachya leaves. The animals were monitored for the survival rate. Various inhibition assays were performed to estimate the activities of acetyl cholinesterase, protease and adenosine triphosphatase of cobra venom, in the presence of 100-300 µg of the plant extract. Results: The extract inhibited the effects of cobra venom significantly after the intraperitoneal injection of the venom and extract at 20 minutes intervals. The results demonstrated that 100% of the mice survived if they received 100-250 mg/kg of the extract while only 83.3% survived with the extract at 50 mg/kg. The extract inhibited the venom’s acetyl cholinesterase, protease and adenosine triphosphatase. The inhibition occurred at higher percentages if the extract was given at 300 mg/kg. Conclusion: The plant extract significantly inhibited the snake venom, and its acetyl cholinesterase, protease and adenosine triphosphatase that mediated the venom’s toxicity.

Antibodies against a single fraction of Micrurus dumerilii venom neutralize the lethal effect of whole venom

The coralsnake Micrurus dumerilii (Elapidae) is reported to cause envenomings of medical importance. Previous studies characterized the protein composition of its venom, with phospholipase A2 (PLA2) proteins the most abundant. However, it is unknown which venom components are responsible for its lethal toxicity. Fractionation of M. dumerilii venom from Colombia was carried out using RP-HPLC and each fraction was screened for lethal effect in mice at a dose of 20 μg by intraperitoneal route. Results showed that only one fraction, F9, was lethal. This fraction displayed PLA2 activity, induced indirect hemolysis in vitro, as well as edema and myotoxicity in vivo. SDS-PAGE of unreduced F9 evidenced two bands of 8 and 15 kDa, respectively, consistent with the detection of proteins with masses of 13,217.77 Da, 7144.06 Da, and 7665.55 Da. Tryptic digestion of F9 followed by nESI-MS/MS revealed peptide sequences matching proteins of the three-finger toxin (3FTx) and PLA2 families. Immunization of a rabbit with F9 proteins elicited antibody titers up to 1:10,000 by ELISA. After serum fractionation with caprylic acid, the obtained IgG was able to neutralize the lethal effect of the complete venom of M. dumerilii using a challenge of 2 ×LD50 at the IgG/venom ratio of 50:1 (w/w). In conclusion, present results show that the lethal effect of M. dumerilii venom in mice is mainly driven by one fraction which contains 3FTx and PLA2 proteins. The antibodies produced against this fraction cross-recognized other PLA2s and neutralized the lethal effect of whole M. dumerilii venom, pointing out to the potential usefulness of F9 as a relevant antigen for improving current coral snake antivenoms.

Heterologous Expression and Immunogenic Potential of the Most Abundant Phospholipase A2 from Coral Snake Micrurus dumerilii to Develop Antivenoms.

Micrurus dumerilii is a coral snake of clinic interest in Colombia. Its venom is mainly composed of phospholipases A2 being MdumPLA2 the most abundant protein. Nevertheless, Micrurus species produce a low quantity of venom, which makes it difficult to produce anticoral antivenoms. Therefore, in this work, we present the recombinant expression of MdumPLA2 to evaluate its biological activities and its immunogenic potential to produce antivenoms. For this, a genetic construct rMdumPLA2 was cloned into the pET28a vector and expressed heterologously in bacteria. His-rMdumPLA2 was extracted from inclusion bodies, refolded in vitro, and isolated using affinity and RP-HPLC chromatography. His-rMdumPLA2 was shown to have phospholipase A2 activity, a weak anticoagulant effect, and induced myonecrosis and edema. The anti-His-rMdumPLA2 antibodies produced in rabbits recognized native PLA2, the complete venom of M. dumerilii, and a phospholipase from another species of the Micrurus genus. Antibodies neutralized 100% of the in vitro phospholipase activity of the recombinant toxin and a moderate percentage of the myotoxic activity of M. dumerilii venom in mice. These results indicate that His-rMdumPLA2 could be used as an immunogen to improve anticoral antivenoms development. This work is the first report of an M. dumerilii functional recombinant PLA2.

Evaluation of Anti-Venom Potential of Areca catechu Seed Extract on Bungarus caeruleus Venom

Areca catechu seeds and their extract/s are currently used to treat various ailments and infections including snakebites. The purpose of this investigation was to assess the inhibiting/neutralizing effect of ethyl acetate and aqueous ethanolic seed extracts of A. catechu on Bungarus caeruleus (krait) venom. The enzyme activities and their inhibition were evaluated using standard procedures (in vitro). In vivo studies were conducted using chick embryos and murine models. The extracts inhibited hyaluronidase and phospholipase A2 activities. Protease activity was neutralized by the aqueous ethanolic extract only. The IC50 value of aqueous ethanolic extract for hyaluronidase was 0.001 g/mL, while that for the ethyl acetate extract for phospholipase A2 was 0.006 g/mL. In addition, both the extracts neutralized the indirect hemolysis and fibrinogenolytic activity induced by B. caeruleus venom. The LD50 for the chick embryos was 4.9 µg/egg. The 50 and 100 µg aqueous ethanolic extracts neutralized the LD50 and the challenging dose (3LD50) of venom effectively in the chick embryo model. The LD50 of B. caeruleus venom in mice was 0.1927 µg/kg; the extract extended the survival time of the mice from 25 min to 30 and 35 min in 1:10 and 1:20 ((w/w) venom:extract) ratios, respectively. The extract also neutralized myotoxic activity. The A. catechu seed extract showed promising inhibitory properties against B. caeruleus venom. In this regard, academia and industries should work collaboratively to develop and formulate a cost-effective first-aid drug.

Blood plasma proteome alteration after local tissue damage induced by Bothrops erythromelas snake venom in mice

Snakes of the genus Bothrops are responsible the most snakebites in the Brazil, causing a diverse and complex pathophysiological condition. Bothrops erythromelas is the main specie of medical relevance found in the Caatinga from the Brazilian Northeast region. The pathophysiological effects involving B. erythromelas snakebite as well as the organism reaction in response to this envenomation are not so explored. Thus, edema was induced in mice paws using 2.5 μg or 5.0 μg of B. erythromelas venom, and the percentage of edema was measured. Plasma was collected 30 minutes after the envenomation-induced in mice and analyzed by mass spectrometry. It was identified a total of 112 common plasma proteins differentially abundant among experimental groups, which are involved with the complement system and coagulation cascades, oxidative stress, neutrophil degranulation, platelets degranulation and inflammatory response. Apolipoprotein A1 (Apoa), serum amyloid protein A-4 (Saa4), adiponectin (Adipoq) showed up-regulated in mice plasma after injection of venom, while fibulin (Fbln1), factor XII (F12) and vitamin K-dependent protein Z (Proz) showed down-regulated. The results indicate a protein pattern of thrombo-inflammation to the B. erythromelas snakebite, evidencing potential biomarkers for monitoring this snakebite, new therapeutic targets and its correlations with the degree of envenomation once showed modulations in the abundance among the different groups according to the amount of venom injected into the mice.

Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity.

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.