Towards better antivenoms: navigating the road to new types of snakebite envenoming therapies

Abstract Snakebite envenoming is a significant global health challenge, and for over a century, traditional plasma-derived antivenoms from hyperimmunized animals have been the primary treatment against this infliction. However, these antivenoms have several inherent limitations, including the risk of causing adverse reactions when administered to patients, batch-to-batch variation, and high production costs. To address these issues and improve treatment outcomes, the development of new types of antivenoms is crucial. During this development, key aspects such as improved clinical efficacy, enhanced safety profiles, and greater affordability should be in focus. To achieve these goals, modern biotechnological methods can be applied to the discovery and development of therapeutic agents that can neutralize medically important toxins from multiple snake species. This review highlights some of these agents, including monoclonal antibodies, nanobodies, and selected small molecules, that can achieve broad toxin neutralization, have favorable safety profiles, and can be produced on a large scale with standardized manufacturing processes. Considering the inherent strengths and limitations related to the pharmacokinetics of these different agents, a combination of them might be beneficial in the development of new types of antivenom products with improved therapeutic properties. While the implementation of new therapies requires time, it is foreseeable that the application of biotechnological advancements represents a promising trajectory toward the development of improved therapies for snakebite envenoming. As research and development continue to advance, these new products could emerge as the mainstay treatment in the future.

Generation of Multivalent Nanobody-Based Proteins with Improved Neutralization of Long α-Neurotoxins from Elapid Snakes.

Recombinantly produced biotherapeutics hold promise for improving the current standard of care for snakebite envenoming over conventional serotherapy. Nanobodies have performed well in the clinic, and in the context of antivenom, they have shown the ability to neutralize long α-neurotoxins in vivo. Here, we showcase a protein engineering approach to increase the valence and hydrodynamic size of neutralizing nanobodies raised against a long α-neurotoxin (α-cobratoxin) from the venom of the monocled cobraNaja kaouthia. Based on the p53 tetramerization domain, a panel of anti-α-cobratoxin nanobody-p53 fusion proteins, termed Quads, were produced with different valences, inclusion or exclusion of Fc regions for endosomal recycling purposes, hydrodynamic sizes, and spatial arrangements, comprising up to 16 binding sites. Measurements of binding affinity and stoichiometry showed that the nanobody binding affinity was retained when incorporated into the Quad scaffold, and all nanobody domains were accessible for toxin binding, subsequently displaying increased blocking potency in vitro compared to the monomeric format. Moreover, functional assessment using automated patch-clamp assays demonstrated that the nanobody and Quads displayed neutralizing effects against long α-neurotoxins from both N. kaouthia and the forest cobra N. melanoleuca. This engineering approach offers a means of altering the valence, endosomal recyclability, and hydrodynamic size of existing nanobody-based therapeutics in a simple plug-and-play fashion and can thus serve as a technology for researchers tailoring therapeutic properties for improved neutralization of soluble targets such as snake toxins.

An interactive database for the investigation of high-density peptide microarray guided interaction patterns and antivenom cross-reactivity.

Snakebite envenoming is a major neglected tropical disease that affects millions of people every year. The only effective treatment against snakebite envenoming consists of unspecified cocktails of polyclonal antibodies purified from the plasma of immunized production animals. Currently, little data exists on the molecular interactions between venom-toxin epitopes and antivenom-antibody paratopes. To address this issue, high-density peptide microarray (hdpm) technology has recently been adapted to the field of toxinology. However, analysis of such valuable datasets requires expert understanding and, thus, complicates its broad application within the field. In the present study, we developed a user-friendly, and high-throughput web application named "Snake Toxin and Antivenom Binding Profiles" (STAB Profiles), to allow straight-forward analysis of hdpm datasets. To test our tool and evaluate its performance with a large dataset, we conducted hdpm assays using all African snake toxin protein sequences available in the UniProt database at the time of study design, together with eight commercial antivenoms in clinical use in Africa, thus representing the largest venom-antivenom dataset to date. Furthermore, we introduced a novel method for evaluating raw signals from a peptide microarray experiment and a data normalization protocol enabling intra-microarray and even inter-microarray chip comparisons. Finally, these data, alongside all the data from previous similar studies by Engmark et al., were preprocessed according to our newly developed protocol and made publicly available for download through the STAB Profiles web application (http://tropicalpharmacology.com/tools/stab-profiles/). With these data and our tool, we were able to gain key insights into toxin-antivenom interactions and were able to differentiate the ability of different antivenoms to interact with certain toxins of interest. The data, as well as the web application, we present in this article should be of significant value to the venom-antivenom research community. Knowledge gained from our current and future analyses of this dataset carry the potential to guide the improvement and optimization of current antivenoms for maximum patient benefit, as well as aid the development of next-generation antivenoms.

Chronic kidney failure following lancehead bite envenoming: a clinical report from the Amazon region

Snakebite envenoming can be a life-threatening condition, for which emergency care is essential. The Bothrops (lancehead) genus is responsible for most snakebite-related deaths and permanent loss of function in human victims in Latin America. Bothrops spp. venom is a complex mixture of different proteins that are known to cause local necrosis, coagulopathy, and acute kidney injury. However, the long-term effects of these viper envenomings have remained largely understudied. Case presentation: Here, we present a case report of a 46-years old female patient from Las Claritas, Venezuela, who was envenomed by a snake from the Bothrops genus. The patient was followed for a 10-year period, during which she presented oliguric renal failure, culminating in kidney failure 60 months after the envenoming. Conclusion: In Latin America, especially in Brazil, where there is a high prevalence of Bothrops envenoming, it may be relevant to establish long-term outpatient programs. This would reduce late adverse events, such as chronic kidney disease, and optimize public financial resources by avoiding hemodialysis and consequently kidney transplantation.(AU)

High-density peptide microarray exploration of the antibody response in a rabbit immunized with a neurotoxic venom fraction.

Polyvalent snakebite antivenoms derive their therapeutic success from the ability of their antibodies to neutralize venom toxins across multiple snake species. This ability results from a production process involving immunization of large mammals with a broad suite of toxins present in venoms. As a result of immunization with this wide range of toxins, many polyvalent antivenoms have a high degree of cross-reactivity to similar toxins in other snake venoms - a cross-reactivity which cannot easily be deconvoluted. As a proof of concept, we aimed at exploring the opposite scenario by performing a high-throughput evaluation of the extent of cross-reactivity of a polyclonal mixture of antibodies that was raised against only a single snake venom fraction. For this purpose, a venom fraction containing short neurotoxin 1 (SN-1; Uniprot accession number P01416, three-finger toxin (3FTx) family), which is the medically most important toxin from the notorious black mamba (Dendroaspis polylepis), was employed. Following immunization of a rabbit, a specific polyclonal antibody response was confirmed by ELISA and immunodiffusion. Subsequently, these antibodies were investigated by high-density peptide microarray to reveal linear elements of recognized epitopes across 742 3FTxs and 10 dendrotoxins. This exploratory study demonstrates in a single immunized animal that cross-reactivity between toxins of high similarity may be difficult to obtain when immunizing with a single 3FTx containing venom fraction. Additionally, this study explored the influence of employing different lengths of peptides in high-density peptide microarray experiments for identification of toxin epitopes. Using 8-mer, 12-mer, and 15-mer peptides, a single linear epitope element was identified in SN-1 with high precision.

Exploring the venom of the forest cobra snake: Toxicovenomics and antivenom profiling of Naja melanoleuca.

A toxicovenomic analysis of the venom of the forest cobra, N. melanoleuca, was performed, revealing the presence of a total of 52 proteins by proteomics analysis. The most abundant proteins belong to the three-finger toxins (3FTx) (57.1wt%), which includes post-synaptically acting α-neurotoxins. Phospholipases A2 (PLA2) were the second most abundant group of proteins (12.9wt%), followed by metalloproteinases (SVMPs) (9.7wt%), cysteine-rich secretory proteins (CRISPs) (7.6wt%), and Kunitz-type serine proteinase inhibitors (3.8wt%). A number of additional protein families comprised each <3wt% of venom proteins. A toxicity screening of the fractions, using the mouse lethality test, identified toxicity in RP-HPLC peaks 3, 4, 5 and 8, all of them containing α-neurotoxins of the 3FTx family, whereas the rest of the fractions did not show toxicity at a dose of 0.53mg/kg. Three polyspecific antivenoms manufactured in South Africa and India were tested for their immunoreactivity against crude venom and fractions of N. melanoleuca. Overall, antivenoms immunorecognized all fractions in the venom, the South African antivenom showing a higher titer against the neurotoxin-containing fractions. This toxicovenomic study identified the 3FTx group of α-neurotoxins in the venom of N. melanoleuca as the relevant targets to be neutralized. BIOLOGICAL SIGNIFICANCE: A toxicovenomic analysis of the venom of the forest cobra, also known as black cobra, Naja melanoleuca, was performed. Envenomings by this elapid species are characterized by a progressive descending paralysis which starts with palpebral ptosis and, in severe cases, ends up with respiratory arrest and death. A total of 52 different proteins were identified in this venom. The most abundant protein family was the three-finger toxin (3FTx) family, which comprises almost 57.1wt% of the venom, followed by phospholipases A2 (PLA2) (12.9wt%). In addition, several other protein families were identified in a much lower percentage in the venom. A toxicity screening of the fractions, using the mouse lethality assay, identified four peaks as those having toxicity higher than that of the crude venom. These fractions predominantly contain α-neurotoxins of the 3FTx family. This toxicovenomic characterization agrees with the clinical and experimental manifestations of envenomings by this species, in which a strong neurotoxic effect predominates. Therefore, our findings suggest that immunotherapy against envenomings by N. melanoleuca should be directed towards the neutralization of 3FTxs; this has implications for the improvement of current antivenoms and for the development of novel antivenoms based on biotechnological approaches. A screening of the immunoreactivity of three antivenoms being distributed in sub-Saharan Africa revealed that they immunoreact with the fractions containing α-neurotoxins, although with different antibody titers.

Toxicovenomics and antivenom profiling of the Eastern green mamba snake (Dendroaspis angusticeps).

A toxicovenomic study was performed on the venom of the green mamba, Dendroaspis angusticeps. Forty-two different proteins were identified in the venom of D. angusticeps, in addition to the nucleoside adenosine. The most abundant proteins belong to the three-finger toxin (3FTx) (69.2%) and the Kunitz-type proteinase inhibitor (16.3%) families. Several sub-subfamilies of the 3FTxs were identified, such as Orphan Group XI (Toxin F-VIII), acetylcholinesterase inhibitors (fasciculins), and aminergic toxins (muscarinic toxins, synergistic-like toxins, and adrenergic toxins). Remarkably, no α-neurotoxins were identified. Proteins of the Kunitz-type proteinase inhibitor family include dendrotoxins. Toxicological screening revealed a lack of lethal activity in all RP-HPLC fractions, except one, at the doses tested. Thus, the overall toxicity depends on the synergistic action of various types of proteins, such as dendrotoxins, fasciculins, and probably other synergistically-acting toxins. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality. These antivenoms also showed a pattern of broad immunorecognition of the different HPLC fractions by ELISA and immunoprecipitated the crude venom by gel immunodiffusion. The synergistic mechanism of toxicity constitutes a challenge for the development of effective recombinant antibodies, as it requires the identification of the most relevant synergistic toxins. BIOLOGICAL SIGNIFICANCE: Envenomings by elapid snakes of the genus Dendroaspis, collectively known as mambas, represent a serious medical problem in sub-Saharan Africa. The development of novel antivenoms and of recombinant neutralizing antibodies demands the identification of the most relevant toxins in these venoms. In this study, a bottom-up approach was followed for the study of the proteome of the venom of the Eastern green mamba, D. angusticeps. Forty-two different proteins were identified, among which the three-finger toxin (3FTx) family, characteristic of elapid venoms, was the most abundant, followed by the Kunitz-type proteinase inhibitor family. In addition, several other protein families were present in the venom, together with the nucleoside adenosine. No α-neurotoxins were identified within the family of 3FTxs in the venom of D. angusticeps, in contrast to the venom of Dendroaspis polylepis, in which α-neurotoxins are largely responsible for the toxicity. With one exception, HPLC fractions from D. angusticeps venom did not kill mice at the doses tested. This underscores that the toxicity of the whole venom is due to the synergistic action of various components, such as fasciculins and dendrotoxins, and probably other synergistically-acting toxins. Thus, the venoms of these closely related species (D. angusticeps and D. polylepis) seem to have different mechanisms to subdue their prey, which may be related to different prey preferences, as D. angusticeps is predominantly arboreal, whereas D. polylepis lives mostly in open bush country and feeds mainly on mammals. It is therefore likely that the predominant clinical manifestations of human envenomings by these species also differ, although in both cases neurotoxic manifestations predominate. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality in mice and showed a pattern of broad immunorecognition of the various venom fractions. It is necessary to identify the toxins responsible for the synergistic mode of toxicity in this venom, since they are the targets for the development of recombinant antibodies for the treatment of envenomings.

Danger in the reef: Proteome, toxicity, and neutralization of the venom of the olive sea snake, Aipysurus laevis.

Four specimens of the olive sea snake, Aipysurus laevis, were collected off the coast of Western Australia, and the venom proteome was characterized and quantitatively estimated by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses. A. laevis venom is remarkably simple and consists of phospholipases A2 (71.2%), three-finger toxins (3FTx; 25.3%), cysteine-rich secretory proteins (CRISP; 2.5%), and traces of a complement control module protein (CCM; 0.2%). Using a Toxicity Score, the most lethal components were determined to be short neurotoxins. Whole venom had an intravenous LD50 of 0.07 mg/kg in mice and showed a high phospholipase A2 activity, but no proteinase activity in vitro. Preclinical assessment of neutralization and ELISA immunoprofiling showed that BioCSL Sea Snake Antivenom was effective in cross-neutralizing A. laevis venom with an ED50 of 821 µg venom per mL antivenom, with a binding preference towards short neurotoxins, due to the high degree of conservation between short neurotoxins from A. laevis and Enhydrina schistosa venom. Our results point towards the possibility of developing recombinant antibodies or synthetic inhibitors against A. laevis venom due to its simplicity.

Snake venomics of monocled cobra (Naja kaouthia) and investigation of human IgG response against venom toxins.

The venom proteome of the monocled cobra, Naja kaouthia, from Thailand, was characterized by RP-HPLC, SDS-PAGE, and MALDI-TOF-TOF analyses, yielding 38 different proteins that were either identified or assigned to families. Estimation of relative protein abundances revealed that venom is dominated by three-finger toxins (77.5%; including 24.3% cytotoxins and 53.2% neurotoxins) and phospholipases A2 (13.5%). It also contains lower proportions of components belonging to nerve growth factor, ohanin/vespryn, cysteine-rich secretory protein, C-type lectin/lectin-like, nucleotidase, phosphodiesterase, metalloproteinase, l-amino acid oxidase, cobra venom factor, and cytidyltransferase protein families. Small amounts of three nucleosides were also evidenced: adenosine, guanosine, and inosine. The most relevant lethal components, categorized by means of a 'toxicity score', were α-neurotoxins, followed by cytotoxins/cardiotoxins. IgGs isolated from a person who had repeatedly self-immunized with a variety of snake venoms were immunoprofiled by ELISA against all venom fractions. Stronger responses against larger toxins, but lower against the most critical α-neurotoxins were obtained. As expected, no neutralization potential against N. kaouthia venom was therefore detected. Combined, our results display a high level of venom complexity, unveil the most relevant toxins to be neutralized, and provide prospects of discovering human IgGs with toxin neutralizing abilities through use of phage display screening.

Unveiling the nature of black mamba (Dendroaspis polylepis) venom through venomics and antivenom immunoprofiling: Identification of key toxin targets for antivenom development.

The venom proteome of the black mamba, Dendroaspis polylepis, from Eastern Africa, was, for the first time, characterized. Forty- different proteins and one nucleoside were identified or assigned to protein families. The most abundant proteins were Kunitz-type proteinase inhibitors, which include the unique mamba venom components 'dendrotoxins', and α-neurotoxins and other representatives of the three-finger toxin family. In addition, the venom contains lower percentages of proteins from other families, including metalloproteinase, hyaluronidase, prokineticin, nerve growth factor, vascular endothelial growth factor, phospholipase A2, 5'-nucleotidase, and phosphodiesterase. Assessment of acute toxicity revealed that the most lethal components were α-neurotoxins and, to a lower extent, dendrotoxins. This venom also contains a relatively high concentration of adenosine, which might contribute to toxicity by influencing the toxin biodistribution. ELISA immunoprofiling and preclinical assessment of neutralization showed that polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of D. polylepis venom, albeit showing different potencies. Antivenoms had higher antibody titers against α-neurotoxins than against dendrotoxins, and displayed high titers against less toxic proteins of high molecular mass. Our results reveal the complexity of D. polylepis venom, and provide information for the identification of its most relevant toxins to be neutralized by antivenoms. BIOLOGICAL SIGNIFICANCE: The black mamba, D. polylepis, is one of the most feared snakes in the world, owing to the potency of its venom, the severity and rapid onset of clinical manifestations of envenomings, and its ability to strike fast and repeatedly. The present study reports the first proteomic analysis of this venom. Results revealed a complex venom constituted predominantly by proteins belonging to the Kunitz-type proteinase inhibitor family, which comprises the dendrotoxins, and to α-neurotoxins of the three-finger toxin family. The proteins showing highest acute toxicity were α-neurotoxins, which induce post-synaptic blockade of the neuromuscular junctions, followed by dendrotoxins, which inhibit the voltage-dependent potassium channels. The combination of these two types of toxins in the venom underscores the presence of a dual strategy that results in a highly effective mechanism for prey subduction. This complex toxic arsenal is likely to provide D. polylepis with high trophic versatility. The rapid onset and severity of neurotoxic clinical manifestations in envenomings by D. polylepis demand the rapid administration of effective and safe antivenoms. Preclinical tests showed that an antivenom from South Africa and two antivenoms from India were effective in the neutralization of this venom, albeit differing in their potency. Moreover, ELISA immunoprofiling of these antivenoms against all venom fractions revealed that antivenoms have higher titers against α-neurotoxins than against dendrotoxins, thus underscoring the need to develop improved immunization strategies. The results of this investigation identified the most relevant toxins present in D. polylepis venom, which need to be targeted by antivenoms or other type of inhibitors.