Search Results (27)

Background: Accidents caused by the Loxosceles laeta spider constitute a health problem in South America. Envenomation can lead to severe systemic manifestations, eventually compromising the patient’s life. Most regional health authorities consider antivenom administration the basis of effective treatment in the most serious cases. The availability of spider venom is the primary bottleneck for antivenom production. Herein, we present a novel biotechnological approach, based on the expression of recombinant versions of the most relevant toxin in loxoscelism, sphingomyelinase D (SphD), in insect larvae (Spodoptera frugiperda). Methods: We produced two versions of SphD: one conserving its biological activities (wtSphD) and a second alternative that was designed to be genetically detoxified (dSphD). Two horses were subjected to three consecutive hyperimmunization cycles with dSphD. The horses’ plasma was extracted at the end of each cycle and used to produce Active Pharmaceutical Ingredients (APIs) of antivenoms at a pilot scale. Results: Dermonecrotic activity of wtSphD was completely neutralized with the sera obtained from one horse and partially with that of the other. In contrast, the APIs derived in both cases completely neutralized wtSphD dermonecrotic activity. Direct hemolysis of human red blood cells by wtSphD was also neutralized by sera and APIs. Conclusions: These results show venom replacement or complementation potential by recombinant dSphD produced in this novel platform. Full article

Yamakagashi (Rhabdophis tigrinus) is a widely distributed snake species in Japan. Yamakagashi causes venom-induced consumption coagulopathy (VICC) when the amount of infused venom is high, and bites can be fatal if antivenom treatment is delayed. However, yamakagashi antivenom is an unapproved treatment, and its storage capacity is limited, preventing its prompt administration. Therefore, we investigated the application of commercially available drugs, namely tranexamic acid and antithrombin III, in the treatment of VICC caused by yamakagashi venom in a rat model. Furthermore, we investigated the combination of each drug with recombinant thrombomodulin α. Administration of tranexamic acid or antithrombin III alone failed to extend rat survival or correct changes in blood coagulation markers, such as prothrombin time, fibrinogen concentrations, and D-dimer levels, in yamakagashi venom-treated rats. However, combined administration of recombinant thrombomodulin α and tranexamic acid extended rat survival and partially restored blood coagulation markers. Therefore, the combination of recombinant thrombomodulin α and tranexamic acid might represent a useful therapeutic regimen for yamakagashi venom exposure. Full article

Snakebite is a critical global public health issue, causing substantial mortality and morbidity, particularly in tropical and subtropical regions. The development of innovative antivenoms targeting snake venom toxins is therefore of paramount importance. In this study, we adopted an epitope-directed approach to design three degenerate 15-mer peptides based on amino acid sequence alignments of snake venom phospholipase A₂s (PLA₂s) and snake venom serine proteases (SVSPs) from snake (Crotalus atrox). By leveraging their immunogenic and inhibitory profiles, these peptides were specifically designed to target the Asp49 and Lys49 variants of PLA₂ and SVSP toxins. Groups of five mice were immunized with each peptide, and IgG mRNA was subsequently extracted from peripheral blood mononuclear cells (PBMCs) and spleen lymphocytes of the top three responders. The extracted mRNA was reverse-transcribed into complementary DNA (cDNA), and the variable regions of the IgG heavy and kappa chains were amplified using polymerase chain reaction (PCR). These amplified regions were then linked with a 66-nucleotide spacer to construct single-chain variable fragments (scFvs). Sequence analysis of 48 randomly selected plasmids from each PLA₂ and SVSP scFv library revealed that over 80% contained scFv sequences with notable diversity observed in the complementarity-determining regions (CDRs), particularly CDR3. Enzyme-linked immunosorbent assay (ELISA) results demonstrated that the SP peptide elicited a broader immune response in mice compared to the Asp49 peptide, implying the strong immunogenicity of the SP peptide. These scFvs represent a promising foundation for the development of recombinant human monoclonal antibodies targeting snake PLA₂ and SVSP toxins, providing a potential therapeutic strategy for the treatment of snakebites. Full article

Viperid snake venoms are notably abundant in metalloproteinases (proteins) (SVMPs), which are primarily responsible for inducing hemorrhage and disrupting the hemostatic process and tissue integrity in envenomed victims. In this study, barnettlysin-III (Bar-III), a hemorrhagic P-III SVMP, was purified from the venom of the Peruvian snake Bothrops barnetti. Bar-III has a molecular mass of approximately 50 kDa and is a glycosylation-dependent functional metalloproteinase. Some biochemical properties of Bar-III, including the full amino acid sequence deduced from its cDNA, are reported. Its enzymatic activity is increased by Ca²⁺ ions and inhibited by an excess of Zn²⁺. Synthetic metalloproteinase inhibitors and EDTA also inhibit its proteolytic action. Bar-III degrades several plasma and ECM proteins, including fibrin(ogen), fibronectin, laminin, and nidogen. Platelets play a key role in hemostasis and thrombosis and in other biological process, such as inflammation and immunity, and platelet activation is driven by the platelet signaling receptors, glycoprotein (GP)Ib-IX-V, which binds vWF, and GPVI, which binds collagen. Moreover, Bar-III inhibits vWF- and convulxin-induced platelet aggregation in human washed platelets by cleaving the recombinant A1 domain of vWF and GPVI into a soluble ectodomain fraction of ~55 kDa (sGPVI). Bar-III does not reduce the viability of cultured endothelial cells; however, it interferes with the adhesion of these cells to fibronectin, vitronectin, and RGD peptides, as well as their migration profile. Bar-III binds specifically to the surface of these cells, and part of this interaction involves α5β1 integrin receptors. These results contribute to a better comprehension of the pathophysiology of snakebite accidents/incidents and could be used as a tool to explore novel and safer anti-venom therapeutics. Full article

Snake venoms are cocktails of biologically active molecules that have evolved to immobilize prey, but can also induce a severe pathology in humans that are bitten. While animal-derived polyclonal antivenoms are the primary treatment for snakebites, they often have limitations in efficacy and can cause severe adverse side effects. Building on recent efforts to develop improved antivenoms, notably through monoclonal antibodies, requires a comprehensive understanding of venom toxins. Among these toxins, snake venom metalloproteinases (SVMPs) play a pivotal role, particularly in viper envenomation, causing tissue damage, hemorrhage and coagulation disruption. One of the current challenges in the development of neutralizing monoclonal antibodies against SVMPs is the large size of the protein and the lack of existing knowledge of neutralizing epitopes. Here, we screened a synthetic human antibody library to isolate monoclonal antibodies against an SVMP from saw-scaled viper (genus Echis) venom. Upon characterization, several antibodies were identified that effectively blocked SVMP-mediated prothrombin activation. Cryo-electron microscopy revealed the structural basis of antibody-mediated neutralization, pinpointing the non-catalytic cysteine-rich domain of SVMPs as a crucial target. These findings emphasize the importance of understanding the molecular mechanisms of SVMPs to counter their toxic effects, thus advancing the development of more effective antivenoms. Full article

Alternative recombinant sources of antivenoms have been successfully generated. The application of such strategies requires the characterization of the venoms for the development of specific neutralizing molecules against the toxic components. Five toxic peptides to mammals from the Mexican scorpion Centruroides villegasi were isolated by chromatographic procedures by means of gel filtration on Sephadex G-50, followed by ion-exchange columns on carboxy-methyl-cellulose (CMC) resins and finally purified by high-performance chromatography (HPLC) columns. Their primary structures were determined by Edman degradation. They contain 66 amino acids and are maintained well packed by four disulfide bridges, with molecular mass from 7511.3 to 7750.1 Da. They are all relatively toxic and deadly to mice and show high sequence identity with known peptides that are specific modifiers of the gating mechanisms of Na⁺ ion channels of type beta-toxin (β-ScTx). They were named Cv1 to Cv5 and used to test their recognition by single-chain variable fragments (scFv) of antibodies, using surface plasmon resonance. Three different scFvs generated in our laboratory (10FG2, HV, LR) were tested for recognizing the various new peptides described here, paving the way for the development of a novel type of scorpion antivenom. Full article

Over 32,000 individuals succumb to snake envenoming in sub-Saharan Africa (sSA) annually. This results from several factors, including a lack of antivenom products capable of neutralising the venoms of diverse snake species in this region. Most manufacturers produce polyvalent antivenoms targeting 3 to 16 clinically important snake species in sSA. However, specific products are unavailable for many others, especially those with a restricted geographic distribution. While next-generation antivenoms, comprising a cocktail of broadly neutralising antibodies, may offer an effective solution to this problem, given the need for their clinical validation, recombinant antivenoms are far from being available to snakebite victims. One of the strategies that could immediately address this issue involves harnessing the cross-neutralisation potential of existing products. Therefore, we assessed the neutralisation potency of PANAF-Premium antivenom towards the venoms of 14 medically important snakes from 13 countries across sSA for which specific antivenom products are unavailable. Preclinical assays in a murine model of snake envenoming revealed that the venoms of most snake species under investigation were effectively neutralised by this antivenom. Thus, this finding highlights the potential use of PANAF-Premium antivenom in treating bites from diverse snakes across sSA and the utility of harnessing the cross-neutralisation potential of antivenoms. Full article

Today, the production and use of various samples of recombinant protein/polypeptide toxins is known and is actively developing. This review presents state-of-the-art in research and development of such toxins and their mechanisms of action and useful properties that have allowed them to be implemented into practice to treat various medical conditions (including oncology and chronic inflammation applications) and diseases, as well as to identify novel compounds and to detoxify them by diverse approaches (including enzyme antidotes). Special attention is given to the problems and possibilities of the toxicity control of the obtained recombinant proteins. The recombinant prions are discussed in the frame of their possible detoxification by enzymes. The review discusses the feasibility of obtaining recombinant variants of toxins in the form of protein molecules modified with fluorescent proteins, affine sequences and genetic mutations, allowing us to investigate the mechanisms of toxins’ bindings to their natural receptors. Full article

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. Full article

Snakebite is a neglected tropical disease that causes considerable death and disability in the tropical world. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Antivenoms are the mainstay therapy for treating the toxic effects of snakebite, but despite saving thousands of lives annually, these therapies are associated with limited cross-snake species efficacy due to venom variation, which ultimately restricts their therapeutic utility to particular geographical regions. In this study, we sought to explore the potential of ssDNA aptamers as toxin-specific inhibitory alternatives to antibodies. As a proof of principle model, we selected snake venom serine protease toxins, which are responsible for contributing to venom-induced coagulopathy following snakebite envenoming, as our target. Using SELEX technology, we selected ssDNA aptamers against recombinantly expressed versions of the fibrinogenolytic SVSPs ancrod from the venom of C. rhodostoma and batroxobin from B. atrox. From the resulting pool of specific ssDNA aptamers directed against each target, we identified candidates that exhibited low nanomolar binding affinities to their targets. Downstream aptamer-linked immobilised sorbent assay, fibrinogenolysis, and coagulation profiling experiments demonstrated that the candidate aptamers were able to recognise native and recombinant SVSP toxins and inhibit the toxin- and venom-induced prolongation of plasma clotting times and the consumption of fibrinogen, with inhibitory potencies highly comparable to commercial polyvalent antivenoms. Our findings demonstrate that rationally selected toxin-specific aptamers can exhibit broad in vitro cross-reactivity against toxin isoforms found in different snake venoms and are capable of inhibiting toxins in pathologically relevant in vitro and ex vivo models of venom activity. These data highlight the potential utility of ssDNA aptamers as novel toxin-inhibiting therapeutics of value for tackling snakebite envenoming. Full article

Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies. Full article

Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD₅₀) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD₅₀ of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico. Full article

Yamakagashi (Rhabdophis tigrinus) inhabits Japan widely, and incidents involving its bites occur every year. Its bite causes disseminated intravascular coagulation when the amount of infused venom is high, and it can be fatal if treatment with Yamakagashi antivenom is delayed. Although Yamakagashi antivenom is used for treating Yamakagashi bites, it is an unapproved drug and its capacity for storage is limited. Hence, it is difficult to administer to patients promptly. As a therapeutic agent for this bite, we investigated the application of recombinant thrombomodulin alpha, a commercially available disseminated intravascular coagulation therapeutic agent. Its therapeutic effect on Yamakagashi venom was confirmed in a coagulation system of human plasma using in vitro Yamakagashi venom as well as a rat experimental model of disseminated intravascular coagulation using in vivo Yamakagashi venom. The administration of recombinant thrombomodulin alpha induced an effect that prolonged the blood coagulation time of Yamakagashi venom in vitro, and the drug was administered in vivo within 0.5 h after the administration of Yamakagashi venom to save rats. Blood coagulation markers such as platelet count, prothrombin time, fibrinogen concentration, and D-dimer levels recovered to normal values in rats. Therefore, recombinant thrombomodulin alpha may be used as a therapeutic agent for Yamakagashi bites. Full article

In Colombia, the genus Micrurus includes 30 species, of which M. mipartitus and M. dumerilii are the most widely distributed. Micrurus causes less than 3% of the approximately 5000 cases of snakebite per year. The elapid envenomation caused by the snakes from the Micrurus genus, are characterized by the severity of their clinical manifestations, due to the venom neurotoxic components such as three-finger toxins (3FTx) and phospholipases (PLA₂). The treatment for snakebites is the administration of specific antivenoms, however, some of them have limitations in their neutralizing ability. A strategy proposed to improve antivenoms is to produce antibodies against the main components of the venom. The aim of this work was to produce an antivenom, using an immunization protocol including the main 3FTx and PLA₂ responsible for M. mipartitus lethality. The antibody titers were determined by ELISA in rabbits’ serum. The immunized animals elicited a response against toxins and whole venom. The Immunoglobulin G (IgGs) obtained were able to neutralize the lethal effect of their homologous toxins. A combination of antivenom from M. mipartitus with antitoxins improved their neutralizing ability. In the same way, a mixture of anti 3FTx and PLA₂ protected the mice from a 1.5 median lethal dose (LD₅₀) of M. mipartitus venom. The results showed that this might be a way to improve antibody titers specificity against the relevant toxins in M. mipartitus venom and indicated that there is a possibility to develop and use recombinant 3FTx and PLA₂ toxins as immunogens to produce antivenoms. Additionally, this represents an alternative to reduce the amount of venom used in anti-coral antivenom production. Full article

Three-finger toxins (3FTXs) are the most clinically relevant components in cobra (genus Naja) venoms. Administration of the antivenom is the recommended treatment for the snakebite envenomings, while the efficacy to cross-neutralize the different cobra species is typically limited, which is presumably due to intra-specific variation of the 3FTXs composition in cobra venoms. Targeting the clinically relevant venom components has been considered as an important factor for novel antivenom design. Here, we used the recombinant type of long-chain α-neurotoxins (P01391), short-chain α-neurotoxins (P60770), and cardiotoxin A3 (P60301) to generate a new immunogen formulation and investigated the potency of the resulting antiserum against the venom lethality of three medially important cobras in Asia, including the Thai monocled cobra (Naja kaouthia), the Taiwan cobra (Naja atra), and the Thai spitting cobra (Naja Siamensis) snake species. With the fusion of protein disulfide isomerase and the low-temperature settings, the correct disulfide bonds were built on these recombinant 3FTXs (r3FTXs), which were confirmed by the circular dichroism spectra and tandem mass spectrometry. Immunization with r3FTX was able to induce the specific antibody response to the native 3FTXs in cobra venoms. Furthermore, the horse and rabbit antiserum raised by the r3FTX mixture is able to neutralize the venom lethality of the selected three medically important cobras. Thus, the study demonstrated that the r3FTXs are potential immunogens in the development of novel antivenom with broad neutralization activity for the therapeutic treatment of victims involving cobra snakes in countries. Full article