We aim to use science for the benefit of human kind, and we aim for our science to have an impact. Our scientific discoveries build upon the work of previous great minds and the development of many molecular techniques, scientific methods, and technological breakthroughs, we have been able to contribute with novel insights within toxinology and antivenom development to the scientific community and society. With the support from generous donors, we have been able to make much of our work publicly available via publication in international peer-reviewed journals and popular science puplications. One can have an overview of our research output here or on our Google Scholar Account.
Peer-reviewed publications
2024
Three-finger toxins (3FTxs) have traditionally been obtained via venom fractionation of whole venoms from snakes. This method often yields functional toxins, but it can be difficult to obtain pure isoforms, as it is challenging to separate the many different toxins with similar physicochemical properties that generally exist in many venoms. This issue can be circumvented via the use of recombinant expression. However, achieving the correct disulfide bond formation in recombinant toxins is challenging and requires extensive optimization of expression and purification methods to enhance stability and functionality. In this study, we investigated the expression of α-cobratoxin, a well-characterized 3FTx from the monocled cobra (Naja kaouthia), in three different expression systems, namely Escherichia coli BL21 (DE3) cells with the csCyDisCo plasmid, Escherichia coli SHuffle cells, and Komagataella phaffii (formerly known as Pichia pastoris). While none of the tested systems yielded α-cobratoxin identical to the variant isolated from whole venom, the His6-tagged α-cobratoxin expressed in K. phaffii exhibited a comparable secondary structure according to circular dichroism spectra and similar binding properties to the α7 subunit of the nicotinic acetylcholine receptor. The findings presented here illustrate the advantages and limitations of the different expression systems and can help guide researchers who wish to express 3FTxs.
2023
Despite the considerable global impact of snakebite envenoming, available treatments remain suboptimal. Here, we report the discovery of a broadly-neutralizing human monoclonal antibody, using a phage display-based cross-panning strategy, capable of reducing the cytotoxic effects of venom phospholipase A2s from three different snake genera from different continents. This highlights the potential of utilizing monoclonal antibodies to develop more effective, safer, and globally accessible polyvalent antivenoms that can be widely used to treat snakebite envenoming.
Protein structure determination is a critical aspect of biological research, enabling us to understand protein function and potential applications. Recent advances in deep learning and artificial intelligence have led to the development of several protein structure prediction tools, such as AlphaFold2 and ColabFold. However, their performance has primarily been evaluated on well-characterised proteins, and comparisons using proteins with poor reference templates are lacking. In this study, we evaluated three modelling tools on their prediction of over 1000 snake venom toxin structures with no reference templates. Our findings show that AlphaFold2 (AF2) performed the best across all assessed parameters. We also observed that ColabFold (CF) only scored slightly worse than AF2, while being computationally less intensive. All tools struggled with regions of intrinsic disorder, such as loops and propeptide regions, and performed well in predicting the structure of functional domains. Overall, our study highlights the importance of exercising caution when working with proteins that have poor reference templates, are large, and contain flexible regions. Nonetheless, leveraging computational structure prediction tools can provide valuable insights into the modelling of protein interactions with different targets and reveal potential binding sites, active sites, and conformational changes, as well as into the design of potential molecular binders for reagent, diagnostic, or therapeutic purposes.
Recycling IgG antibodies bind to their target antigen at physiological pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the IgG antibodies to be recycled into circulation via FcRn-mediated cellular pathways, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve comparable therapeutic effect at lower doses than their non-recycling counterparts. The development of such antibodies is typically achieved by histidine doping of their variable regions or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. Both are strategies that may introduce sequence liabilities. Here, we present a methodology that employs a naïve antibody phage display library, consisting of natural variable domains, to discover antibodies that bind α-cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. As a result, an antibody was discovered that exhibits a 7-fold higher off-rate at pH 5.5 than pH 7.4 in bio-layer interferometry experiments. Interestingly, no histidine residues were found in its variable domains, and in addition, the antibody showed pH-dependent binding to a histidine-devoid antigen mutant. As such, the results demonstrate that pH-dependent antigen-antibody binding may not always be driven by histidine residues. By employing molecular dynamics simulations, different protonation states of titratable residues were found, which potentially could be responsible for the observed pH-dependent antigen binding properties of the antibody. Finally, given the typically high diversity of naïve antibody libraries, the methodology presented here can likely be applied to discover recycling antibodies against different targets ab initio without the need for histidine doping.
Ion channels play a crucial role in diverse physiological processes, including neurotransmission and muscle contraction. Venomous creatures exploit the vital function of ion channels by producing toxins in their venoms that specifically target these ion channels to facilitate prey capture upon a bite or a sting. Envenoming can therefore lead to ion channel dysregulation, which for humans can result in severe medical complications that often necessitate interventions such as antivenom administration. Conversely, the discovery of highly potent and selective venom toxins with the capability of distinguishing between different isoforms and subtypes of ion channels has led to the development of beneficial therapeutics that are now in the clinic. This review encompasses the historical evolution of electrophysiology methodologies, highlighting their contributions to venom and antivenom research, including venom-based drug discovery and evaluation of antivenom efficacy. By discussing the applications and advancements in patch-clamp techniques, this review underscores the profound impact of electrophysiology in unravelling the intricate interplay between ion channels and venom toxins, ultimately leading to the development of drugs for envenoming and ion channel-related pathologies.
Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.
Brazil is home to a multitude of venomous snakes; perhaps the most medically relevant of which belong to the Bothrops genus. Bothrops spp. are responsible for roughly 70% of all snakebites in Brazil, and envenomings caused by their bites can be treated with three types of antivenom: bothropic antivenom, bothro-lachetic antivenom, and bothro-crotalic antivenom. The choice to administer antivenom depends on the severity of the envenoming, while the choice of antivenom depends on availability and on how certain the treating physician is that the patient was bitten by a bothropic snake. The diagnosis of a bothropic envenoming can be made based on expert identification of the dead snake or a photo thereof or based on a syndromic approach wherein the clinician examines the patient for characteristic manifestations of envenoming. This approach can be very effective but requires staff that has been trained in clinical snakebite management, which, unfortunately, far from all relevant staff has.
Recycling antibodies can bind to their target antigen at neutral pH in the blood stream and release them upon endocytosis when pH levels drop, allowing the antibodies to be recycled into circulation via FcRn-mediated pathway, while the antigens undergo lysosomal degradation. This enables recycling antibodies to achieve the same therapeutic effect at lower doses than their non-recyclable counterparts. The development of such antibodies is typically achieved by histidine doping of the variable regions of specific antibodies or by performing in vitro antibody selection campaigns utilizing histidine doped libraries. While often successful, these strategies may introduce sequence liabilities, as they often involve mutations that may render the resultant antibodies to be non-natural. Here, we present a methodology that employs a naive antibody phage display library, consisting of natural variable domains, to discover antibodies that bind alpha-cobratoxin from the venom of Naja kaouthia in a pH-dependent manner. Upon screening of the discovered antibodies with immunoassays and bio-layer interferometry, a pH-dependent antibody was discovered that exhibits an 8-fold higher dissociation rate at pH 5.5 than 7.4. Interestingly, the variable domains of the pH-dependent antibody were found to be entirely devoid of histidines, demonstrating that pH-dependency may not always be driven by this amino acid. Further, given the high diversity available in a naive antibody library, the methodology presented here can likely be applied to discover pH-dependent antibodies against different targets ab initio without the need of histidine doping.
Snakebite envenoming is a global public health issue that causes significant morbidity and mortality, particularly in low-income regions of the world. The clinical manifestations of envenomings vary depending on the snake’s venom, with paralysis, haemorrhage, and necrosis being the most common and medically relevant effects. To assess the efficacy of antivenoms against dermonecrosis, a preclinical testing approach involves in vivo mouse models that mimic local tissue effects of cytotoxic snakebites in humans. However, current methods for assessing necrosis severity are time-consuming and susceptible to human error. To address this, we present the Venom Induced Dermonecrosis Analysis tool (VIDAL), a machine-learning-guided image-based solution that can automatically identify dermonecrotic lesions in mice, adjust for lighting biases, scale the image, extract lesion area and discolouration, and calculate the severity of dermonecrosis. We also introduce a new unit, the dermonecrotic unit (DnU), to better capture the complexity of dermonecrosis severity. Our tool is comparable to the performance of state-of-the-art histopathological analysis, making it an accessible, accurate, and reproducible method for assessing dermonecrosis. Given the urgent need to address the neglected tropical disease that is snakebite, high-throughput technologies such as VIDAL are crucial in developing and validating new and existing therapeutics for this debilitating disease.
Antibodies with cross-reactive binding and broad toxin-neutralizing capabilities are advantageous for treating indications such as infectious diseases and animal envenomings. Such antibodies have been successfully selected against closely related antigens using phage display technology. However, the mechanisms driving antibody cross-reactivity typically remain to be elucidated. Therefore, we sought to explore how a previously reported phage display-based cross-panning strategy drives the selection of cross-reactive antibodies using seven different snake toxins belonging to three protein (sub-)families: phospholipases A2, long-chain α-neurotoxins, and short-chain α-neurotoxins. We showcase how cross-panning can increase the chances of discovering cross-reactive single-chain variable fragments (scFvs) from phage display campaigns. Further, we find that the feasibility of discovering cross-reactive antibodies using cross-panning cannot easily be predicted by analyzing the sequence, structural, or surface similarity of the antigens alone. However, when antigens share the (exact) same functions, this seems to increase the chances of selecting cross-reactive antibodies, which may possibly be due to the existence of structurally similar motifs on the antigens.
Current snakebite antivenoms are based on polyclonal animal-derived antibodies, which can neutralize snake venom toxins in envenomed victims, but which are also associated with adverse reactions. Therefore, several efforts within antivenom research aim to explore the utility of recombinant monoclonal antibodies, such as human immunoglobulin G (IgG) antibodies, which are routinely used in the clinic for other indications. In this study, the feasibility of using tobacco plants as bioreactors for expressing full-length human monoclonal IgG antibodies against snake toxins was investigated. We show that the plant-produced antibodies perform similarly to their mammalian cell-expressed equivalents in terms of in vitro antigen binding. Complete neutralization was achieved by both the plant and mammalian cell-produced anti-α-cobratoxin antibody. The feasibility of using plant-based expression systems may potentially make it easier for laboratories in resource-poor settings to work with human monoclonal IgG antibodies.
Snakebite envenoming is a neglected tropical disease that causes over 100,000 deaths annually. Envenomings result in variable pathologies, but systemic neurotoxicity is among the most serious and is currently only treated with difficult to access and variably efficacious commercial antivenoms. Venom-induced neurotoxicity is often caused by α-neurotoxins antagonising the muscle-type nicotinic acetylcholine receptor (nAChR), a ligand-gated ion channel. Discovery of therapeutics targeting α-neurotoxins is hampered by relying on binding assays that do not reveal restoration of receptor activity or more costly and/or lower throughput electrophysiology-based approaches. Here, we report the validation of a screening assay for nAChR activation using immortalised TE671 cells expressing the γ-subunit containing muscle-type nAChR and a fluorescent dye that reports changes in cell membrane potential. Assay validation using traditional nAChR agonists and antagonists, which either activate or block ion fluxes, was consistent with previous studies. We then characterised antagonism of the nAChR by a variety of elapid snake venoms that cause muscle paralysis in snakebite victims, before defining the toxin-inhibiting activities of commercial antivenoms, and new types of snakebite therapeutic candidates, namely monoclonal antibodies, decoy receptors, and small molecules. Our findings show robust evidence of assay uniformity across 96-well plates and highlight the amenability of this approach for the future discovery of new snakebite therapeutics via screening campaigns. The described assay therefore represents a useful first-step approach for identifying α-neurotoxins and their inhibitors in the context of snakebite envenoming, and it should provide wider value for studying modulators of nAChR activity from other sources.
Beyond potency, a good developability profile is a key attribute of a biological drug. Selecting and screening for such attributes early in the drug development process can save resources and avoid costly late-stage failures. Here, we review some of the most important developability properties that can be assessed early on for biologics. These include the influence of the source of the biologic, its biophysical and pharmacokinetic properties, and how well it can be expressed recombinantly. We furthermore present in silico, in vitro, and in vivo methods and techniques that can be exploited at different stages of the discovery process to identify molecules with liabilities and thereby facilitate the selection of the most optimal drug leads. Finally, we reflect on the most relevant developability parameters for injectable versus orally delivered biologics and provide an outlook toward what general trends are expected to rise in the development of biologics.
Phage display technology is a powerful tool for selecting monoclonal antibodies against a diverse set of antigens. Within toxinology, however, it remains challenging to generate monoclonal antibodies against many animal toxins, as they are difficult to obtain from venom. Recombinant toxins have been proposed as a solution to overcome this challenge, but so far, few have been used as antigens to generate neutralizing antibodies. Here, we describe the recombinant expression of α-cobratoxin in E. coli and its successful application as an antigen in a phage display selection campaign. From this campaign, an scFv (single-chain variable fragment) was isolated with similar binding affinity to a control scFv generated against the native toxin. The selected scFv recognizes a structural epitope, enabling it to inhibit the interaction between the acetylcholine receptor and the native toxin in vitro. This approach represents the first entirely in vitro antibody selection strategy for generating neutralizing monoclonal antibodies against a snake toxin.
Single-domain antibodies (sdAbs) are exceptionally stable fragments derived from the antigen-binding domains of immunoglobulins. They can withstand extreme pH, high temperature, and proteolysis, making them suitable for controlling gastrointestinal (GI) infections in humans and animals. sdAbs may function in their native soluble form, although different derived protein formats and the use of delivery vehicles can be useful for improved oral delivery. We discuss selected examples of the use of orally delivered sdAbs for protecting humans and animals against GI infections caused by pathogenic bacteria, viruses, and parasites. We finally provide perspectives on how sdAbs may be applied industrially and what challenges should be overcome for orally delivered sdAbs to reach the market.
The neutralization capacity of different recombinant antivenoms are provieded and compared to the neutralization capacity of conventional antivenoms. The study highlights the potential of recombinant antibodies which may have higher neutralization capacities than conventional antivenoms.
Snakebite envenoming continues to claim many lives across the globe, necessitating the development of improved therapies. To this end, broadly-neutralizing human monoclonal antibodies may possess advantages over current plasma-derived antivenoms by offering superior safety and high neutralization capacity. Here, we report the establishment of a pipeline based on phage display technology for the discovery and optimization of high affinity broadly-neutralizing human monoclonal antibodies. This approach yielded a recombinant human antibody with superior broadly-neutralizing capacities in vitro and in vivo against different long-chain α-neurotoxins from elapid snakes. This antibody prevents lethality induced by Naja kaouthia whole venom at an unprecedented low molar ratio of one antibody per toxin and prolongs the survival of mice injected with Dendroaspis polylepis or Ophiophagus hannah whole venoms.
The identification of gastrointestinal helminth infections of humans and livestock almost exclusively relies on the detection of eggs or larvae in faeces, followed by manual counting and morphological characterisation to differentiate species using microscopy-based techniques. However, molecular approaches based on the detection and quantification of parasite DNA are becoming more prevalent, increasing the sensitivity, specificity and throughput of diagnostic assays. High-throughput sequencing, from single PCR targets through to the analysis of whole genomes, offers significant promise towards providing information-rich data that may add value beyond traditional and conventional molecular approaches; however, thus far, its utility has not been fully explored to detect helminths in faecal samples. In this study, low-depth whole genome sequencing, i.e. genome skimming, has been applied to detect and characterise helminth diversity in a set of helminth-infected human and livestock faecal material. The strengths and limitations of this approach are evaluated using three methods to characterise and differentiate metagenomic sequencing data based on (i) mapping to whole mitochondrial genomes, (ii) whole genome assemblies, and (iii) a comprehensive internal transcribed spacer 2 (ITS2) database, together with validation using quantitative PCR (qPCR). Our analyses suggest that genome skimming can successfully identify most single and multi-species infections reported by qPCR and can provide sufficient coverage within some samples to resolve consensus mitochondrial genomes, thus facilitating phylogenetic analyses of selected genera, e.g. Ascaris spp. Key to this approach is both the availability and integrity of helminth reference genomes, some of which are currently contaminated with bacterial and host sequences. The success of genome skimming of faecal DNA is dependent on the availability of vouchered sequences of helminths spanning both taxonomic and geographic diversity, together with methods to detect or amplify minute quantities of parasite nucleic acids in mixed samples.
2022
Venomous snakes are important parts of the ecosystem, and their behavior and evolution have been shaped by their surrounding environments over the eons. This is reflected in their venoms, which are typically highly adapted for their biological niche, including their diet and defense mechanisms for deterring predators. Sub-Saharan Africa is rich in venomous snake species, of which many are dangerous to humans due to the high toxicity of their venoms and their ability to effectively deliver large amounts of venom into their victims via their bite. In this study, the venoms of 26 of sub-Saharan Africa’s medically most relevant elapid and viper species were subjected to parallelized toxicovenomics analysis. The analysis included venom proteomics and in vitro functional characterization of whole venom toxicities, enabling a robust comparison of venom profiles between species. The data presented here corroborate previous studies and provide biochemical details for the clinical manifestations observed in envenomings by the 26 snake species. Moreover, two new venom proteomes (Naja anchietae and Echis leucogaster) are presented here for the first time. Combined, the presented data can help shine light on snake venom evolutionary trends and possibly be used to further improve or develop novel antivenoms.
Venom-induced haemorrhage constitutes a severe pathology in snakebite envenomings, especially those inflicted by viperid species. In order to both explore venom compositions accurately, and evaluate the efficacy of viperid antivenoms for the neutralisation of haemorrhagic activity it is essential to have available a precise, quantitative tool for empirically determining venom-induced haemorrhage. Thus, we have built on our prior approach and developed a new AI-guided tool (AHA) for the quantification of venom-induced haemorrhage in mice. Using a smartphone, it takes less than a minute to take a photo, upload the image, and receive accurate information on the magnitude of a venom-induced haemorrhagic lesion in mice. This substantially decreases analysis time, reduces human error, and does not require expert haemorrhage analysis skills. Furthermore, its open access web-based graphical user interface makes it easy to use and implement in laboratories across the globe. Together, this will reduce the resources required to preclinically assess and control the quality of antivenoms, whilst also expediting the profiling of hemorrhagic activity in venoms for the wider toxinology community.
Snakebite envenoming was reintroduced as a Category A Neglected Tropical Disease by the World Health Organization in 2017. Since then, increased attention has been directed towards this affliction and towards the development of a deeper understanding of how snake venoms exert their toxic effects and how antivenoms can counter them. However, most of our in vivo generated knowledge stems from the use of animal models which do not always accurately reflect how the pathogenic effects of snake venoms manifest in humans. Moreover, animal experiments are associated with pain, distress, and eventually animal sacrifice due to the toxic nature of snake venoms. Related to this, the implementation of the 3Rs principle (Replacement, Reduction, and Refinement) in the use of experimental animals in snakebite envenoming research is recommended by the World Health Organization. Therefore, more humane experimental designs and new in vitro/ex vivo alternatives for experimental animals are sought after. Here, we report the use of an organotypic model of human skin to further elucidate the pathophysiology of the dermonecrotic effects caused by the venom of the black-necked spitting cobra, Naja nigricollis, in humans. The goal of this study is to expand the repertoire of available models that can be used to study the local tissue damages induced by cytotoxic venoms.
Immunotargeting of tumor-specific antigens is a powerful therapeutic strategy. Immunotherapies directed at MHC-I complexes have expanded the scope of antigens and enabled the direct targeting of intracellular oncoproteins at the cell surface. We asked whether covalent drugs that alkylate mutated residues on oncoproteins could act as haptens to generate unique MHC-I-restricted neoantigens. Here, we report that KRAS G12C mutant cells treated with the covalent inhibitor ARS1620 present ARS1620-modified peptides in MHC-I complexes. Using ARS1620-specific antibodies identified by phage display, we show that these haptenated MHC-I complexes can serve as tumor-specific neoantigens and that a bispecific T cell engager construct based on a hapten-specific antibody elicits a cytotoxic T cell response against KRAS G12C cells, including those resistant to direct KRAS G12C inhibition. With multiple K-RAS G12C inhibitors in clinical use or undergoing clinical trials, our results present a strategy to enhance their efficacy and overcome the rapidly arising tumor resistance.
Hackathons not only advance science itself but also help to educate young biotechnologists and artificial intelligence enthusiasts and to build capacity in digital biotechnology in low-income settings.
African trypanosomes, such as Trypanosoma brucei, are flagellated protozoa which proliferate in mammals and cause a variety of diseases in people and animals. In a mammalian host, the external face of the African trypanosome plasma membrane is covered by a densely packed coat formed of variant surface glycoprotein (VSG), which counteracts the host’s adaptive immune response by antigenic variation. The VSG is attached to the external face of the plasma membrane by covalent attachment of the C-terminus to glycosylphosphatidylinositol. As the trypanosome grows, newly synthesised VSG is added to the plasma membrane by vesicle fusion to the flagellar pocket, the sole location of exo- and endocytosis. Snake venoms contain dozens of components, including proteases and phospholipases A2. Here, we investigated the effect of Naja nigricollis venom on T. brucei with the aim of describing the response of the trypanosome to hydrolytic attack on the VSG. We found no evidence for VSG hydrolysis, however, N. nigricollis venom caused: (i) an enlargement of the flagellar pocket, (ii) the Rab11 positive endosomal compartments to adopt an abnormal dispersed localisation, and (iii) cell cycle arrest prior to cytokinesis. Our results indicate that a single protein family, the phospholipases A2 present in N. nigricollis venom, may be necessary and sufficient for the effects. This study provides new molecular insight into T. brucei biology and possibly describes mechanisms that could be exploited for T. brucei targeting.
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 cobra Naja 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.
Disintegrin-like/cysteine-rich (DC) proteins have long been regarded just as products of proteolysis of P-III snake venom metalloproteinases (SVMPs). However, here we demonstrate that a DC protein from the venom of Vipera ammodytes (Vaa; nose-horned viper), VaaMPIII-3, is encoded per se by a P-III SVMP-like gene that has a deletion in the region of the catalytic metalloproteinase domain and in part of the non-catalytic disintegrin-like domain. In this way, we justify the proposal of the introduction of a new subclass P-IIIe of SVMP-derived DC proteins. We purified VaaMPIII-3 from the venom of Vaa in a series of chromatographic steps. A covalent chromatography step based on thiol-disulphide exchange revealed that VaaMPIII-3 contains an unpaired Cys residue. This was demonstrated to be Cys6 in about 90% and Cys19 in about 10% of the VaaMPIII-3 molecules. We further constructed a three-dimensional homology model of VaaMPIII-3. From this model, it is evident that both Cys6 and Cys19 can pair with Cys26, which suggests that the intramolecular thiol-disulphide exchange has a regulatory function. VaaMPIII-3 is an acidic 21-kDa monomeric glycoprotein that exists in at least six N-glycoforms, with isoelectric points ranging from pH 4.5 to 5.1. Consistent with the presence of an integrin-binding motif in its sequence, SECD, VaaMPIII-3 inhibited collagen-induced platelet aggregation. It also inhibited ADP- and arachidonic-acid-induced platelet aggregation, but not ristocetin-induced platelet agglutination and the blood coagulation cascade.
The monocled cobra (Naja kaouthia) is among the most feared snakes in Southeast Asia due to its toxicity, which is predominantly derived from long-chain α-neurotoxins. The only specific treatment for snakebite envenoming is antivenom based on animal-derived polyclonal antibodies. Despite the lifesaving importance of these medicines, major limitations in safety, supply consistency, and efficacy create a need for improved treatments. Here, we describe the discovery and subsequent optimization of a recombinant human monoclonal immunoglobulin G antibody against α-cobratoxin using phage display technology. Affinity maturation by light chain-shuffling resulted in a significant increase in in vitro neutralization potency and in vivo efficacy. The optimized antibody prevented lethality when incubated with N. kaouthia whole venom prior to intravenous injection. This study is the first to demonstrate neutralization of whole snake venom by a single recombinant monoclonal antibody, thus providing a tantalizing prospect of bringing recombinant antivenoms based on human monoclonal or oligoclonal antibodies to the clinic.
Phage display technology can be used for the discovery of antibodies for research, diagnostic, and therapeutic purposes. In this review, we present and discuss key parameters that can be optimized when performing phage display selection campaigns, including the use of different antibody formats and advanced strategies for antigen presentation, such as immobilization, liposomes, nanodiscs, virus-like particles, and whole cells. Furthermore, we provide insights into selection strategies that can be used for the discovery of antibodies with complex binding requirements, such as targeting a specific epitope, cross-reactivity, or pH-dependent binding. Lastly, we provide a description of specialized phage display libraries for the discovery of bispecific antibodies and pH-sensitive antibodies. Together, these methods can be used to improve antibody discovery campaigns against all types of antigen.
Many snake venom toxins cause local tissue damage in prey and victims, which constitutes an important pathology that is challenging to treat with existing antivenoms. One of the notorious toxins that causes such effects is myotoxin II present in the venom of the Central and Northern South American viper, Bothrops asper. This Lys49 PLA2 homologue is devoid of enzymatic activity and causes myotoxicity by disrupting the cell membranes of muscle tissue. To improve envenoming therapy, novel approaches are needed, warranting the discovery and development of inhibitors that target key toxins that are currently difficult to neutralize. Here, we report the identification of a new peptide (JB006), discovered using phage display technology, that is capable of binding to and neutralizing the toxic effects of myotoxin II in vitro and in vivo. Through computational modeling, we further identify hypothetical binding interactions between the toxin and the peptide to enable further development of inhibitors that can neutralize myotoxin II.
Each year, thousands of people fall victim to envenomings caused by cobras. These incidents often result in death due to paralysis caused by α-neurotoxins from the three-finger toxin (3FTx) family, which are abundant in elapid venoms. Due to their small size, 3FTxs are among the snake toxins that are most poorly neutralized by current antivenoms, which are based on polyclonal antibodies of equine or ovine origin. While antivenoms have saved countless lives since their development in the late 18th century, an opportunity now exists to improve snakebite envenoming therapy via the application of new biotechnological methods, particularly by developing monoclonal antibodies against poorly neutralized α-neurotoxins. Here, we describe the use of phage-displayed synthetic antibody libraries and the development and characterization of six synthetic antibodies built on a human IgG framework and developed against α-cobratoxin – the most abundant long-chain α-neurotoxin from Naja kaouthia venom. The synthetic antibodies exhibited sub-nanomolar affinities to α-cobratoxin and neutralized the curare-mimetic effect of the toxin in vitro. These results demonstrate that phage display technology based on synthetic repertoires can be used to rapidly develop human antibodies with drug-grade potencies as inhibitors of venom toxins.
Snake venoms represent a danger to human health, but also a gold mine of bioactive proteins that can be harnessed for drug discovery purposes. The evolution of snakes and their venom has been studied for decades, particularly via traditional morphological and basic genetic methods alongside venom proteomics. However, while the field of genomics has matured rapidly over the past 2 decades, owing to the development of next-generation sequencing technologies, snake genomics remains in its infancy. Here, we provide an overview of the state of the art in snake genomics and discuss its potential implications for studying venom evolution and toxinology. On the basis of current knowledge, gene duplication and positive selection are key mechanisms in the neofunctionalization of snake venom proteins. This makes snake venoms important evolutionary drivers that explain the remarkable venom diversification and adaptive variation observed in these reptiles. Gene duplication and neofunctionalization have also generated a large number of repeat sequences in snake genomes that pose a significant challenge to DNA sequencing, resulting in the need for substantial computational resources and longer sequencing read length for high-quality genome assembly. Fortunately, owing to constantly improving sequencing technologies and computational tools, we are now able to explore the molecular mechanisms of snake venom evolution in unprecedented detail. Such novel insights have the potential to affect the design and development of antivenoms and possibly other drugs, as well as provide new fundamental knowledge on snake biology and evolution.
A major challenge in industrial pig production is the prevalence of post-weaning diarrhea (PWD) in piglets, often caused by enterotoxigenic Escherichia coli (ETEC). The increased use of antibiotics and zinc oxide to treat PWD has raised global concerns regarding antimicrobial resistance development and environmental pollution. Still, alternative treatments targeting ETEC and counteracting PWD are largely lacking. Here, we report the design of a pH, temperature, and protease-stable bivalent VHH-based protein BL1.2 that cross-links a F4+ ETEC model strain by selectively binding to its fimbriae. This protein inhibits F4+ ETEC adhesion to porcine epithelial cells ex vivo and decreases F4+ ETEC proliferation when administrated as a feed additive to weaned F4+ ETEC challenged piglets. These findings highlight the potential of a highly specific bivalent VHH-based feed additive in effectively delimiting pathogenic F4+ ETEC bacteria proliferation in piglets and may represent a sustainable solution for managing PWD while circumventing antimicrobial resistance development.
Animal venoms are complex mixtures containing peptides and proteins known as toxins, which are responsible for the deleterious effect of envenomations. Across the animal Kingdom, toxin diversity is enormous, and the ability to understand the biochemical mechanisms governing toxicity is not only relevant for the development of better envenomation therapies, but also for exploiting toxin bioactivities for therapeutic or biotechnological purposes. Most of toxinology research has relied on obtaining the toxins from crude venoms; however, some toxins are difficult to obtain because the venomous animal is endangered, does not thrive in captivity, produces only a small amount of venom, is difficult to milk, or only produces low amounts of the toxin of interest. Heterologous expression of toxins enables the production of sufficient amounts to unlock the biotechnological potential of these bioactive proteins. Moreover, heterologous expression ensures homogeneity, avoids cross-contamination with other venom components, and circumvents the use of crude venom. Heterologous expression is also not only restricted to natural toxins, but allows for the design of toxins with special properties or can take advantage of the increasing amount of transcriptomics and genomics data, enabling the expression of dormant toxin genes. The main challenge when producing toxins is obtaining properly folded proteins with a correct disulfide pattern that ensures the activity of the toxin of interest. This review presents the strategies that can be used to express toxins in bacteria, yeast, insect cells, or mammalian cells, as well as synthetic approaches that do not involve cells, such as cell-free biosynthesis and peptide synthesis. This is accompanied by an overview of the main advantages and drawbacks of these different systems for producing toxins, as well as a discussion of the biosafety considerations that need to be made when working with highly bioactive proteins.
2021
The Middle East and Northern Africa, collectively known as the MENA region, are inhabited by a plethora of venomous animals that cause up to 420,000 bites and stings each year. To understand the resultant health burden and the key variables affecting it, this review describes the epidemiology of snake, scorpion, and spider envenomings primarily based on heterogenous hospital data in the MENA region and the pathologies associated with their venoms. In addition, we discuss the venom composition and the key medically relevant toxins of these venomous animals, and, finally, the antivenoms that are currently in use to counteract them. Unlike Asia and sub-Saharan Africa, scorpion stings are significantly more common (approximately 350,000 cases/year) than snakebites (approximately 70,000 cases/year) and present the most significant contributor to the overall health burden of envenomings, with spider bites being negligible. However, this review also indicates that there is a substantial lack of high-quality envenoming data available for the MENA region, rendering many of these estimates speculative. Our understanding of the venoms and the toxins they contain is also incomplete, but already presents clear trends. For instance, the majority of snake venoms contain snake venom metalloproteinases, while sodium channel–binding toxins and potassium channel–binding toxins are the scorpion toxins that cause most health-related challenges. There also currently exist a plethora of antivenoms, yet only few are clinically validated, and their high cost and limited availability present a substantial health challenge. Yet, some of the insights presented in this review might help direct future research and policy efforts toward the appropriate prioritization of efforts and aid the development of future therapeutic solutions, such as next-generation antivenoms.
Throughout history, pandemics like the Black Death or the Spanish Flue have periodically wiped out large parts of populations. Today, history repeats itself with the on-going SARS-CoV-2 pandemic, which manifests that pandemics still hold the potential to cause devastating impact to global health and economy. To prevent severe illness and death, epidemics have been fought with polyclonal antibodies derived from immunized animals, since this technology was invented by Behring and Kitasato in 1890 [1]. Since then, major advances have occurred in the field of immunotherapy, enabled particularly by the advent of recombinant DNA technology and a range of discovery methodologies that allow for the generation and manufacture of (human) monoclonal antibodies and antibody fragments [2]. These advances strengthen our ability to react to infectious diseases with pandemic potential and have already been put to utility in a number of cases [3]. As an example, during the 2014–2016 Ebola outbreak in West Africa, a mixture of three monoclonal antibodies (ZMappTM) originating from two prior antibody mixtures (MB003 and Zmab) were combined and rapidly used for treatment of Ebola infected patients despite lacking prior clinical trials to prove safety and efficacy [4]. The antibody mixture was shown to be beneficial to the patients, although it did not reach statistical significance compared to standard of care [5]. In addition, therapeutic monoclonal antibodies have been, or are currently being developed for H1N1 [6], SARS, MERS [7], and SARS-CoV-2 [8]. While vaccine development is supreme to reduce mortality in a pandemic situation, and convalescent plasma or small molecules sometimes can be used for treatment, an urgent need also exists for fast development of therapeutic monoclonal antibodies as a complement. These antibodies can be administered to groups responding weakly to vaccination, such as immunocompromised patients, or prophylactically as a first line of defense for people at high risk of being infected and/or becoming severely ill. In addition, if prompt testing to detect infected people is in place, an opportunity may exist to administer antibodies as a precaution and treatment before clinical manifestations occur, thereby possibly reducing the severity of an infection.
Snakebite envenoming is a major cause of morbidity and mortality in rural communities throughout the tropics. Generally, the main clinical features of snakebites are local swelling, tissue necrosis, shock, spontaneous systemic hemorrhage, incoagulable blood, paralysis, rhabdomyolysis, and acute kidney injury. These clinical manifestations result from complex biochemical venom constituents comprising of cytotoxins, hemotoxins, neurotoxins, myotoxins, and other substances. Timely diagnosis of envenoming and identification of the responsible snake species is clinically challenging in many parts of the world and necessitates prompt and thorough clinical assessment, which could be supported by the development of reliable, affordable, widely-accessible, point-of-care tests. Conventional antivenoms based on polyclonal antibodies derived from animals remain the mainstay of therapy along with supportive medical and surgical care. However, while antivenoms save countless lives, they are associated with adverse reactions, limited potency, and are relatively inefficacious against presynaptic neurotoxicity and in preventing necrosis. Nevertheless, major scientific and technological advances are facilitating the development of new molecular and immunologic diagnostic tests, as well as a new generation of antivenoms comprising human monoclonal antibodies with broader and more potent neutralization capacity and less immunogenicity. Repurposed pharmaceuticals based on small molecule inhibitors (e.g., marimastat and varespladib) used alone and in combination against enzymatic toxins, such as metalloproteases and phospholipase A2s, have shown promise in animal studies. These orally bioavailable molecules could serve as early interventions in the out-of-hospital setting if confirmed to be safe and efficacious in clinical studies. Antivenom access can be improved by the usage of drones and ensuring constant antivenom supply in remote endemic rural areas. Overall, the improvement of clinical management of snakebite envenoming requires sustained, coordinated, and multifaceted efforts involving basic and applied sciences, new technology, product development, effective clinical training, implementation of existing guidelines and therapeutic approaches, supported by improved supply of existing antivenoms.
Actinoporins constitute a family of α pore-forming toxins produced by sea anemones. The soluble fold of these proteins consists of a β-sandwich flanked by two α-helices. Actinoporins exert their activity by specifically recognizing sphingomyelin at their target membranes. Once there, they penetrate the membrane with their N-terminal α-helices, a process that leads to the formation of cation-selective pores. These pores kill the target cells by provoking an osmotic shock on them. In this review, we examine the role and relevance of the structural features of actinoporins, down to the residue level. We look at the specific amino acids that play significant roles in the function of actinoporins and their fold. Particular emphasis is given to those residues that display a high degree of conservation across the actinoporin sequences known to date. In light of the latest findings in the field, the membrane requirements for pore formation, the effect of lipid composition, and the process of pore formation are also discussed.
Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.
Protein-lipid interactions are crucial events from a biochemical point of view, like the interaction of proteins with the cell plasma membrane, and their study is of great importance. Actinoporins are a very powerful tool to study this kind of interactions, since they are soluble proteins in an aqueous environment, capable of inserting into membranes when they have the adequate composition. In fact, actinoporins have been used to study protein-lipid interactions for many years now. Sometimes it is not possible to use real biological membranes in the experiments, so model membranes need to be used. This article aims to give a thorough description of many of the techniques used to study actinoporin-lipid interactions, using both biological and model membranes: Hemolysis, release of vesicles content, surface plasmon resonance, isothermal titration calorimetry, fluorescence-based measurements, etc. Some of these techniques measure the actinoporins activity and some measure their binding properties. The combination of all the techniques described can offer valuable information about the thermodynamics and the kinetics of the actinoporin-lipid interaction.
Sticholysins are pore-forming toxins produced by sea anemones that are members of the actinoporin family. They exert their activity by forming pores on membranes, provided they have sphingomyelin. To assemble into pores, specific recognition, binding, and oligomerization are required. While recognition and binding have been extensively studied, delving into the oligomerization process and the stoichiometry of the pores has been more difficult. Here, we present evidence that these toxins are capable of oligomerizing in solution and suggesting that the interaction of sticholysin II (StnII) with its isoform sticholysin I (StnI) is stronger than that of StnI with itself. We also show that the stoichiometry of the final, thermodynamically stable StnI pores is, at least, heptameric. Furthermore, our results indicate that this association maintains its oligomerization number when StnII is included, indicating that the stoichiometry of StnII is also of that order, and not tetrameric, as previously thought. These results are compatible with the stoichiometry observed for the crystallized pore of FraC, another very similar actinoporin produced by a different sea anemone species. Our results also indicate that the stoichiometry of actinoporin pores in equilibrium is conserved regardless of the particular composition of a given pore ensemble, which we have shown for mixed sticholysin pores.
Snakebite envenoming is predominantly an occupational disease of the rural tropics, causing death or permanent disability to hundreds of thousands of victims annually. The diagnosis of snakebite envenoming is commonly based on a combination of patient history and a syndromic approach. However, the availability of auxiliary diagnostic tests at the disposal of the clinicians vary from country to country, and the level of experience within snakebite diagnosis and intervention may be quite different for clinicians from different hospitals. As such, achieving timely diagnosis, and thus treatment, is a challenge faced by treating personnel around the globe. For years, much effort has gone into developing novel diagnostics to support diagnosis of snakebite victims, especially in rural areas of the tropics. Gaining access to affordable and rapid diagnostics could potentially facilitate more favorable patient outcomes due to early and appropriate treatment. This review aims to highlight regional differences in epidemiology and clinical snakebite management on a global scale, including an overview of the past and ongoing research efforts within snakebite diagnostics. Finally, the review is rounded off with a discussion on design considerations and potential benefits of novel snakebite diagnostics.
For years, a discussion has persevered on the benefits and drawbacks of antibody discovery using animal immunization versus in vitro selection from non-animal-derived recombinant repertoires using display technologies. While it has been argued that using recombinant display libraries can reduce animal consumption, we hold that the number of animals used in immunization campaigns is dwarfed by the number sacrificed during preclinical studies. Thus, improving quality control of antibodies before entering in vivo studies will have a larger impact on animal consumption. Both animal immunization and recombinant repertoires present unique advantages for discovering antibodies that are fit for purpose. Furthermore, we anticipate that machine learning will play a significant role within discovery workflows, refining current antibody discovery practices.
This study reports the isolation, structural, biochemical, and functional characterization of a novel phosphodiesterase from Crotalus durissus collilineatus venom (CdcPDE). CdcPDE was successfully isolated from whole venom using three chromatographic steps and represented 0.7% of total protein content. CdcPDE was inhibited by EDTA and reducing agents, demonstrating that metal ions and disulfide bonds are necessary for its enzymatic activity. The highest enzymatic activity was observed at pH 8–8.5 and 37 °C. Kinetic parameters indicated a higher affinity for the substrate bis(p-nitrophenyl) phosphate compared to others snake venom PDEs. Its structural characterization was done by the determination of the protein primary sequence by Edman degradation and mass spectrometry, and completed by the building of molecular and docking-based models. Functional in vitro assays showed that CdcPDE is capable of inhibiting platelet aggregation induced by adenosine diphosphate in a dose-dependent manner and demonstrated that CdcPDE is cytotoxic to human keratinocytes. CdcPDE was recognized by the crotalid antivenom produced by the Instituto Butantan. These findings demonstrate that the study of snake venom toxins can reveal new molecules that may be relevant in cases of snakebite envenoming, and that can be used as molecular tools to study pathophysiological processes due to their specific biological activities.
2020
Venoms constitute complex mixtures of many different molecules arising from evolution in processes driven by continuous prey–predator interactions. One of the most common compounds in these venomous cocktails are pore-forming proteins, a family of toxins whose activity relies on the disruption of the plasmatic membranes by forming pores. The venom of sea anemones, belonging to the oldest lineage of venomous animals, contains a large amount of a characteristic group of pore-forming proteins known as actinoporins. They bind specifically to sphingomyelin-containing membranes and suffer a conformational metamorphosis that drives them to make pores. This event usually leads cells to death by osmotic shock. Sticholysins are the actinoporins produced by Stichodactyla helianthus. Three different isotoxins are known: Sticholysins I, II, and III. They share very similar amino acid sequence and three-dimensional structure but display different behavior in terms of lytic activity and ability to interact with cholesterol, an important lipid component of vertebrate membranes. In addition, sticholysins can act in synergy when exerting their toxin action. The subtle, but important, molecular nuances that explain their different behavior are described and discussed throughout the text. Improving our knowledge about sticholysins behavior is important for eventually developing them into biotechnological tools.
Background: 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.
Widow spiders are among the few spider species worldwide that can cause serious envenoming in humans. The clinical syndrome resulting from Latrodectus spp. envenoming is called latrodectism and characterized by pain (local or regional) associated with diaphoresis and nonspecific systemic effects. The syndrome is caused by α-latrotoxin, a ~130 kDa neurotoxin that induces massive neurotransmitter release. Due to this function, α-latrotoxin has played a fundamental role as a tool in the study of neuroexocytosis. Nevertheless, some questions concerning its mode of action remain unresolved today. The diagnosis of latrodectism is purely clinical, combined with the patient’s history of spider bite, as no analytical assays exist to detect widow spider venom. By utilizing antibody phage display technology, we here report the discovery of the first recombinant human monoclonal immunoglobulin G antibody (TPL0020_02_G9) that binds α-latrotoxin from the Mediterranean black widow spider (Latrodectus tredecimguttatus) and show neutralization efficacy ex vivo. Such antibody can be used as an affinity reagent for research and diagnostic purposes, providing researchers with a novel tool for more sophisticated experimentation and analysis. Moreover, it may also find therapeutic application in future.
Venomous snakebites cause >100 000 deaths every year, in many cases via potent depression of human neuromuscular signaling by snake α-neurotoxins. Emergency therapy still relies on antibody-based antivenom, hampered by poor access, frequent adverse reactions, and cumbersome production/purification. Combining high-throughput discovery and subsequent structure–function characterization, we present simple peptides that bind α-cobratoxin (α-Cbtx) and prevent its inhibition of nicotinic acetylcholine receptors (nAChRs) as a lead for the development of alternative antivenoms. Candidate peptides were identified by phage display and deep sequencing, and hits were characterized by electrophysiological recordings, leading to an 8-mer peptide that prevented α-Cbtx inhibition of nAChRs. We also solved the peptide:α-Cbtx cocrystal structure, revealing that the peptide, although of unique primary sequence, binds to α-Cbtx by mimicking structural features of the nAChR binding pocket. This demonstrates the potential of small peptides to neutralize lethal snake toxins in vitro, establishing a potential route to simple, synthetic, low-cost antivenoms.
Snake ‘dry bites’ are characterized by the absence of venom being injected into the victim during a snakebite incident. The dry bite mechanism and diagnosis are quite complex, and the lack of envenoming symptoms in these cases may be misinterpreted as a miraculous treatment or as proof that the bite from the perpetrating snake species is rather harmless. The circumstances of dry bites and their clinical diagnosis are not well-explored in the literature, which may lead to ambiguity amongst treating personnel about whether antivenom is indicated or not. Here, the epidemiology and recorded history of dry bites are reviewed, and the clinical knowledge on the dry bite phenomenon is presented and discussed. Finally, this review proposes a diagnostic and therapeutic protocol to assist medical care after snake dry bites, aiming to improve patient outcomes.
In the field of antivenom research, development, and manufacture, it is often advised to follow the World Health Organization’s (WHO) guidelines for the production, control, and regulation of snake antivenom immunoglobulins, which recommend the use of preincubation assays to assess the efficacy of snakebite therapeutics. In these assays, venom and antivenom are mixed and incubated prior to in vivo administration to rodents, which allows for a standardizable comparison of antivenoms with similar characteristics. However, these assays are not necessarily sufficient for therapeutics with significantly different pharmacological properties than antibody-based antivenoms, such as small molecule inhibitors, nanoparticles, and other modalities. To ensure that the in vivo therapeutic utility of completely novel toxin-neutralizing molecules with no history of use in envenoming therapy and variable pharmacokinetics is properly evaluated, such molecules must also be tested in preclinical rescue assays, where rodents are first challenged with appropriate doses of venoms or toxins, followed by the administration of neutralizing modalities after an appropriate time delay to better mimic the real-life scenarios faced by human snakebite victims. Such an approach takes the venom (or toxin) toxicokinetics, the drug pharmacokinetics, and the drug pharmacodynamics into consideration. If new modalities are only assessed in preincubation assays and not subjected to evaluation in rescue assays, the publication of neutralization data may unintentionally misrepresent the actual therapeutic efficacy and suitability of the modality being tested, and thus potentially misguide strategic decision making in the research and development of novel therapies for snakebite envenoming.
Snake venoms are mixtures of toxins that vary extensively between and within snake species. This variability has serious consequences for the management of the world’s 1.8 million annual snakebite victims. Advances in ‘omic’ technologies have empowered toxinologists to comprehensively characterize snake venom compositions, unravel the molecular mechanisms that underpin venom variation, and elucidate the ensuing functional consequences. In this review, we describe how such mechanistic processes have resulted in suites of toxin isoforms that cause diverse pathologies in human snakebite victims and we detail how variation in venom composition can result in treatment failure. Finally, we outline current therapeutic approaches designed to circumvent venom variation and deliver next-generation treatments for the world’s most lethal neglected tropical disease.
Snakebite envenoming is a neglected tropical disease that affects millions of people across the globe. It has been suggested that recombinant antivenoms based on mixtures of human monoclonal antibodies, which target key toxins of medically important snake venom, could present a promising avenue toward the reduction of morbidity and mortality of envenomated patients. However, since snakebite envenoming is a disease of poverty, it is pivotal that next-generation therapies are affordable to those most in need; this warrants analysis of the cost dynamics of recombinant antivenom manufacture. Therefore, we present, for the first time, a bottom-up analysis of the cost dynamics surrounding the production of future recombinant antivenoms based on available industry data. We unravel the potential impact that venom volume, abundance of medically relevant toxins in a venom, and the molecular weight of these toxins may have on the final product cost. Furthermore, we assess the roles that antibody molar mass, manufacturing and purification strategies, formulation, antibody efficacy, and potential cross-reactivity play in the complex cost dynamics of recombinant antivenom manufacture. Notably, according to our calculations, it appears that such next-generation antivenoms based on cocktails of monoclonal immunoglobulin Gs (IgGs) could be manufacturable at a comparable or lower cost to current plasma-derived antivenoms, which are priced at USD 13-1120 per treatment. We found that monovalent recombinant antivenoms based on IgGs could be manufactured for USD 20-225 per treatment, while more complex polyvalent recombinant antivenoms based on IgGs could be manufactured for USD 48-1354 per treatment. Finally, we investigated the prospective cost of manufacturing for recombinant antivenoms based on alternative protein scaffolds, such as DARPins and nanobodies, and highlight the potential utility of such scaffolds in the context of low-cost manufacturing. In conclusion, the development of recombinant antivenoms not only holds a promise for improving therapeutic parameters, such as safety and efficacy, but could possibly also lead to a more competetive cost of manufacture of antivenom products for patients worldwide.
Broadly-neutralizing monoclonal antibodies are of high therapeutic utility against infectious diseases caused by bacteria and viruses, as well as different types of intoxications. Snakebite envenoming is one such debilitating pathology, which is currently treated with polyclonal antibodies derived from immunized animals. For the development of novel envenoming therapies based on monoclonal antibodies with improved therapeutic benefits, new discovery approaches for broadly-neutralizing antibodies are needed. Here, we present a methodology based on phage display technology and a cross-panning strategy that enables the selection of cross-reactive monoclonal antibodies that can broadly neutralize toxins from different snake species. This simple in vitro methodology is immediately useful for the development of broadly-neutralizing (polyvalent) recombinant antivenoms with broad species coverage, but may also find application in the development of broadly-neutralizing antibodies against bacterial, viral, and parasitic agents that are known for evading therapy via resistance mechanisms and antigen variation.
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 (https://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.
Bushmasters (Lachesis spp) and lancehead vipers (Bothrops spp) are two of the most dangerous snakes found in Latin America. Victims of envenoming by these snakes require urgent administration of antivenom. Here, we report the identification of a small set of broadly neutralizing human monoclonal single-chain variable fragment (scFv) antibodies targeting key phospholipases A2 from Lachesis and Bothrops spp using phage display technology and demonstrate their in vitro efficacy using a hemolysis assay.
Scorpion venom may cause severe medical complications and untimely death if injected into the human body. Neurotoxins are the main components of scorpion venom that are known to be responsible for the pathological manifestations of envenoming. Besides neurotoxins, a wide range of other bioactive molecules can be found in scorpion venoms. Advances in separation, characterization, and biotechnological approaches have enabled not only the development of more effective treatments against scorpion envenomings, but have also led to the discovery of several scorpion venom peptides with interesting therapeutic properties. Thus, scorpion venom may not only be a medical threat to human health, but could prove to be a valuable source of bioactive molecules that may serve as leads for the development of new therapies against current and emerging diseases. This review presents both the detrimental and beneficial properties of scorpion venom toxins and discusses the newest advances within the development of novel therapies against scorpion envenoming and the therapeutic perspectives for scorpion toxins in drug discovery.
Toxin synergism is a complex biochemical phenomenon, where different animal venom proteins interact either directly or indirectly to potentiate toxicity to a level that is above the sum of the toxicities of the individual toxins. This provides the animals possessing venoms with synergistically enhanced toxicity with a metabolic advantage, since less venom is needed to inflict potent toxic effects in prey and predators. Among the toxins that are known for interacting synergistically are cytotoxins from snake venoms, phospholipases A2 from snake and bee venoms, and melittin from bee venom. These toxins may derive a synergistically enhanced toxicity via formation of toxin complexes by hetero-oligomerization. Using a human keratinocyte assay mimicking human epidermis in vitro, we demonstrate and quantify the level of synergistically enhanced toxicity for 12 cytotoxin/melittin-PLA2 combinations using toxins from elapids, vipers, and bees. Moreover, by utilizing an interaction-based assay and by including a wealth of information obtained via a thorough literature review, we speculate and propose a mechanistic model for how toxin synergism in relation to cytotoxicity may be mediated by cytotoxin/melittin and PLA2 complex formation.
Antibiotics are often administered with antivenom following snakebite envenomings in order to avoid secondary bacterial infections. However, to this date, no studies have evaluated whether antibiotics may have undesirable potentiating effects on snake venom. Herein, we demonstrate that four commonly used antibiotics affect the enzymatic activities of proteolytic snake venom toxins in two different in vitro assays. Similar findings in vivo could have clinical implications for snakebite management and require further examination.
2019
Honey bees can be found all around the world and fulfill key pollination roles within their natural ecosystems, as well as in agriculture. Most species are typically docile, and most interactions between humans and bees are unproblematic, despite their ability to inject a complex venom into their victims as a defensive mechanism. Nevertheless, incidences of bee stings have been on the rise since the accidental release of Africanized bees to Brazil in 1956 and their subsequent spread across the Americas. These bee hybrids are more aggressive and are prone to attack, presenting a significant healthcare burden to the countries they have colonized. To date, treatment of such stings typically focuses on controlling potential allergic reactions, as no specific antivenoms against bee venom currently exist. Researchers have investigated the possibility of developing bee antivenoms, but this has been complicated by the very low immunogenicity of the key bee toxins, which fail to induce a strong antibody response in the immunized animals. However, with current cutting-edge technologies, such as phage display, alongside the rise of monoclonal antibody therapeutics, the development of a recombinant bee antivenom is achievable, and promising results towards this goal have been reported in recent years. Here, current knowledge on the venom biology of Africanized bees and current treatment options against bee envenoming are reviewed. Additionally, recent developments within next-generation bee antivenoms are presented and discussed.
Each year, millions of humans fall victim to animal envenomings, which may either be deadly or cause permanent disability to the effected individuals. The Nobel Prize-winning discovery of serum therapy for the treatment of bacterial infections (tetanus and diphtheria) paved the way for the introduction of antivenom therapies for envenomings caused by venomous animals. These antivenoms are based on polyclonal antibodies derived from the plasma of hyperimmunized animals and remain the only specific treatment against animal envenomings. Following the initial development of serum therapy for snakebite envenoming by French scientists in 1894, other countries with high incidences of animal envenomings, including Brazil, Australia, South Africa, Costa Rica, and Mexico, started taking up antivenom production against local venomous animals over the course of the twentieth century. These undertakings revolutionized envenoming therapy and have saved innumerous patients worldwide during the last 100 years. This review describes in detail the above-mentioned historical events surrounding the discovery and the application of serum therapy for envenomings, as well as it provides an overview of important developments and scientific breakthroughs that were of importance for antibody-based therapies in general. This begins with discoveries concerning the characterization of antibodies, including the events leading up to the elucidation of the antibody structure. These discoveries further paved the way for other milestones in antibody-based therapies, such as the introduction of hybridoma technology in 1975. Hybridoma technology enabled the expression and isolation of monoclonal antibodies, which in turn formed the basis for the development of phage display technology and transgenic mice, which can be harnessed to directly obtain fully human monoclonal antibodies. These developments were driven by the ultimate goal of producing potent neutralizing monoclonal antibodies with optimal pharmacokinetic properties and low immunogenicity. This review then provides an outline of the most recent achievements in antivenom research, which include the application of new biotechnologies, the development of the first human monoclonal antibodies that can neutralize animal toxins, and efforts toward creating fully recombinant antivenoms. Lastly, future perspectives in the field of envenoming therapies are discussed, including rational engineering of antibody cross-reactivity and the use of oligoclonal antibody mixtures.
Introduction: Monoclonal antibody-based therapies now represent the single-largest class of molecules undergoing clinical investigation. Although a handful of different monoclonal antibodies have been clinically approved for bacterial and viral indications, including rabies, therapies based on monoclonal antibodies are yet to fully enter the fields of neglected tropical diseases and other infectious diseases. Areas covered: This review presents the current state-of-the-art in the development and use of monoclonal antibodies against neglected tropical diseases and other infectious diseases, including viral, bacterial, and parasitic infections, as well as envenomings by animal bites and stings. Additionally, a short section on mushroom poisonings is included. Key challenges for developing antibody-based therapeutics are discussed for each of these fields. Expert opinion: Neglected tropical diseases and other infectious diseases represent a golden oppor- tunity for academics and technology developers for advancing our scientific capabilities within the understanding and design of antibody cross-reactivity, use of oligoclonal antibody mixtures for multi- target neutralization, novel immunization methodologies, targeting of evasive pathogens, and devel- opment of fundamentally novel therapeutic mechanisms of action. Furthermore, a huge humanitarian and societal impact is to gain by exploiting antibody technologies for the development of biotherapies against diseases, for which current treatment options are suboptimal or non-existent.
Snakebite envenoming is a devastating Neglected Tropical Disease, the treatment of which has seen relatively little innovation since the invention of antivenom serotherapy in 1894. Current antivenoms have been and continue to be invaluable in saving thousands of lives. However, these medicines are associated with a number of drawbacks pertaining to availability, safety, and efficacy. Fortunately, with the advent of novel methodologies, such as antibody discovery technologies, high-throughput drug discovery approaches, and improved methods for protein engineering, we are starting to see scientific advances in the field. This review presents relevant engineering and design considerations for exploiting these methodologies to develop next-generation antivenoms with improved safety, efficacy, and affordability. The pros and cons of different treatment modalities will be discussed with regards to immunogenicity, the suitability of preclinical efficacy assays, availability of discovery methods, economic viability of production schemes, and possible regulatory approval paths.
Snake venom metalloproteinases (SVMPs) and snake venom serine proteinases (SVSPs) are among the most abundant enzymes in many snake venoms, particularly among viperids. These proteinases are responsible for some of the clinical manifestations classically seen in viperid envenomings, including hemorrhage, necrosis, and coagulopathies. The objective of this study was to investigate the enzymatic activities of these proteins using a high-throughput peptide library to screen for the proteinase targets of the venoms of five viperid (Echis carinatus, Bothrops asper, Daboia russelii, Bitis arietans, Bitis gabonica) and one elapid (Naja nigricollis) species of high medical importance. The proteinase activities of these venoms were each tested against 360 peptide substrates, yielding 2160 activity profiles. A nonlinear regression model that accurately described the observed enzymatic activities was fitted to the experimental data, allowing for the comparison of cleavage rates across species. In this study, previously unknown protein targets of snake venom proteinases were identified, potentially implicating novel human and animal proteins that may be involved in the pathophysiology of viper envenomings. The functional relevance of these targets was further evaluated and discussed. These new findings may contribute to our understanding of the clinical manifestations and underlying biochemical mechanisms of snakebite envenoming by viperid species.
Animal toxins present a major threat to human health worldwide, predominantly through snakebite envenomings, which are responsible for over 100,000 deaths each year. To date, the only available treatment against snakebite envenoming is plasma-derived antivenom. However, despite being key to limiting morbidity and mortality among snakebite victims, current antivenoms suffer from several drawbacks, such as immunogenicity and high cost of production. Consequently, avenues for improving envenoming therapy, such as the discovery of toxin-sequestering monoclonal antibodies against medically important target toxins through phage display selection, are being explored. However, alternative binding protein scaffolds that exhibit certain advantages compared to the well-known immunoglobulin G scaffold, including high stability under harsh conditions and low cost of production, may pose as possible low-cost alternatives to antibody-based therapeutics. There is now a plethora of alternative binding protein scaffolds, ranging from antibody derivatives (e.g., nanobodies), through rationally designed derivatives of other human proteins (e.g., DARPins), to derivatives of non-human proteins (e.g., affibodies), all exhibiting different biochemical and pharmacokinetic profiles. Undeniably, the high level of engineerability and potentially low cost of production, associated with many alternative protein scaffolds, present an exciting possibility for the future of snakebite therapeutics and merit thorough investigation. In this review, a comprehensive overview of the different types of binding protein scaffolds is provided together with a discussion on their relevance as potential modalities for use as next-generation antivenoms.
2018
In this review, the different approaches that have been employed with the aim of developing novel antivenoms against animal envenomings are presented and discussed. Reported efforts have focused on the use of innovative immunization strategies, small molecule inhibitors against enzymatic toxins, endogenous animal proteins with toxin-neutralizing capabilities, and recombinant monoclonal antibodies. Harnessing either of these approaches, antivenom development may benefit from an in-depth understanding of venom compositions and the medical importance of individual venom toxins. Focus is thus also directed towards the different omics technologies (particularly venomics, antivenomics, and toxicovenomics) that are being used to uncover novel animal toxins, shed light on venom complexity, and provide directions for how to determine the medical relevance of individual toxins within whole venoms. Finally, techniques for assessing antivenom specificity and cross-reactivity are reviewed, with special focus on antivenomics and high-density peptide microarray technology.
Snakebite envenoming is a neglected tropical disease that each year claims the lives of 80,000–140,000 victims worldwide. The only effective treatment against envenoming involves intravenous administration of antivenoms that comprise antibodies that have been isolated from the plasma of immunized animals, typically horses. The drawbacks of such conventional horse-derived antivenoms include their propensity for causing allergenic adverse reactions due to their heterologous and foreign nature, an inability to effectively neutralize toxins in distal tissue, a low content of toxin-neutralizing antibodies, and a complex manufacturing process that is dependent on husbandry and procurement of snake venoms. In recent years, an opportunity to develop a fundamentally novel type of antivenom has presented itself. By using modern antibody discovery strategies, such as phage display selection, and repurposing small molecule enzyme inhibitors, next-generation antivenoms that obviate the drawbacks of existing plasma-derived antivenoms could be developed. This article describes the conceptualization of a novel therapeutic development strategy for biosynthetic oligoclonal antivenom (BOA) for snakebites based on recombinantly expressed oligoclonal mixtures of human monoclonal antibodies, possibly combined with repurposed small molecule enzyme inhibitors.
Snakes, scorpions, and spiders are venomous animals that pose a threat to human health, and severe envenomings from the bites or stings of these animals must be treated with antivenom. Current antivenoms are based on plasma-derived immunoglobulins or immunoglobulin fragments from hyper-immunized animals. Although these medicines have been life-saving for more than 120 years, opportunities to improve envenoming therapy exist. In the later decades, new biotechnological tools have been applied with the aim of improving the efficacy, safety, and affordability of antivenoms. Within the avenues explored, novel immunization strategies using synthetic peptide epitopes, recombinant toxins (or toxoids), or DNA strings as immunogens have demonstrated potential for generating antivenoms with high therapeutic antibody titers and broad neutralizing capacity. Furthermore, these approaches circumvent the need for venom in the production process of antivenoms, thereby limiting some of the complications associated with animal captivity and venom collection. Finally, an important benefit of innovative immunization approaches is that they are often compatible with existing antivenom manufacturing setups. In this review, we compile all reported studies examining venom-independent innovative immunization strategies for antivenom development. In addition, a brief description of toxin families of medical relevance found in snake, scorpion, and spider venoms is presented, as well as how biochemical, bioinformatic, and omics tools could aid the development of next-generation antivenoms.
The black mamba (Dendroaspis polylepis) is one of the most feared snake species of the African savanna. It has a potent, fast-acting neurotoxic venom comprised of dendrotoxins and α-neurotoxins associated with high fatality in untreated victims. Current antivenoms are both scarce on the African continent and present a number of drawbacks as they are derived from the plasma of hyper-immunized large mammals. Here, we describe the development of an experimental recombinant antivenom by a combined toxicovenomics and phage display approach. The recombinant antivenom is based on a cocktail of fully human immunoglobulin G (IgG) monoclonal antibodies capable of neutralizing dendrotoxin-mediated neurotoxicity of black mamba whole venom in a rodent model. Our results show the potential use of fully human monoclonal IgGs against animal toxins and the first use of oligoclonal human IgG mixtures against experimental snakebite envenoming.
Antivenom cross-reactivity has been investigated for decades to determine which antivenoms can be used to treat snakebite envenomings from different snake species. Traditionally, the methods used for analyzing cross-reactivity have been immunodiffusion, immunoblotting, enzyme-linked immunosorbent assay (ELISA), enzymatic assays, and in vivo neutralization studies. In recent years, new methods for determination of cross-reactivity have emerged, including surface plasmon resonance, antivenomics, and high-density peptide microarray technology. Antivenomics involves a top-down assessment of the toxin-binding capacities of antivenoms, whereas high-density peptide microarray technology may be harnessed to provide in-depth knowledge on which toxin epitopes are recognized by antivenoms. This review provides an overview of both the classical and new methods used to investigate antivenom cross-reactivity, the advantages and disadvantages of each method, and examples of studies using the methods. A special focus is given to antivenomics and high-density peptide microarray technology as these high-throughput methods have recently been introduced in this field and may enable more detailed assessments of antivenom cross-reactivity.
Snakebite envenoming is a neglected tropical disease that requires immediate attention. Conventional plasma-derived snakebite antivenoms have existed for more than 120 years and have been instrumental in saving thousands of lives. However, both a need and an opportunity exist for harnessing biotechnology and modern drug development approaches to develop novel snakebite antivenoms with better efficacy, safety, and affordability. For this to be realized, though, development approaches, clinical testing, and manufacturing must be feasible for any novel treatment modality to be brought to the clinic. Here, we present engineering, manufacturing, and regulatory considerations that need to be taken into account for any development process for a novel antivenom product, with a particular emphasis on novel antivenoms based on mixtures of monoclonal antibodies. We highlight key drug development challenges that must be addressed, and we attempt to outline some of the important shifts that may have to occur in the ways snakebite antivenoms are designed and evaluated.
Trypanosoma brucei is a parasitic protozoan species capable of infecting insects, whose bite transmits African sleeping sickness (trypanosomiasis) in humans. Current treatments are becoming ineffective due to the parasite’s ability to avoid the lytic immunogenic response of the host. The parasite achieves this avoidance by modifying the composition of its outer coat, which is mainly composed of Variable Surface Glycoprotein (VSG). Snake venoms are composed of toxic proteins and peptides, with or without enzymatic activity, and a range of other molecules that may influence physiological processes. Previously, it has been demonstrated that viper venoms are able to kill certain parasitic species, but elapid snake venoms have never been investigated. The venom of the elapid Naja nigricollis (black-necked spitting cobra) is mainly composed of cytotoxic three-finger toxins (cytotoxins) that interfere with and disrupt cellular membranes with high target specificity. Here, we investigated how T. brucei is affected when this parasite is subjected to whole venom of N. nigricollis.
With the introduction of powerful mass spectrometry equipment into the field of snake venom proteomics, a large body of venomics data is accumulating. To allow for better comparison between venom compositions from different snake species and to provide an online database containing this data, we devised the Snake Venomics Display toolbox for visualization of snake venomics data on linear scales. This toolbox is freely available to be used online at https://tropicalpharmacology.com/tools/snake-venomics-display/ and allows researchers to visualize venomics data in a Relative Abundance (%) visualization mode and in an Absolute Abundance (mg) visualization mode, the latter taking venom yields into account. The curated venomics data for all snake species included in this database is also made available in a downloadable Excel file format. The Snake Venomics Display toolbox represents a simple way of handling snake venomics data, which is better suited for large data sets of venom compositions from multiple snake species.
Antibody discovery has become increasingly important in almost all areas of modern medicine. Different antibody discovery approaches exist, but one that has gained increasing interest in the field of toxinology and antivenom research is phage display technology. In this review, the lifecycle of the M13 phage and the basics of phage display technology are presented together with important factors influencing the success rates of phage display experiments. Moreover, the pros and cons of different antigen display methods and the use of naïve versus immunized phage display antibody libraries is discussed, and selected examples from the field of antivenom research are highlighted. This review thus provides in-depth knowledge on the principles and use of phage display technology with a special focus on discovery of antibodies that target animal toxins.
Antibody technologies are being increasingly applied in the field of toxinology. Fuelled by the many advances in immunology, synthetic biology, and antibody research, different approaches and antibody formats are being investigated for the ability to neutralize animal toxins. These different molecular formats each have their own therapeutic characteristics. In this review, we provide an overview of the advances made in the development of toxin-targeting antibodies, and discuss the benefits and drawbacks of different antibody formats in relation to their ability to neutralize toxins, pharmacokinetic features, propensity to cause adverse reactions, formulation, and expression for research and development (R&D) purposes and large-scale manufacturing. A research trend seems to be emerging towards the use of human antibody formats as well as camelid heavy-domain antibody fragments due to their compatibility with the human immune system, beneficial therapeutic properties, and the ability to manufacture these molecules cost-effectively.
2017
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.
Mambas (genus Dendroaspis) are among the most feared of the medically important elapid snakes found in sub-Saharan Africa, but many facets of their biology, including the diversity of venom composition, remain relatively understudied. Here, we present a reconstruction of mamba phylogeny, alongside genus-wide venom gland transcriptomic and high-resolution top-down venomic analyses. Whereas the green mambas, D. viridis, D. angusticeps, D. j. jamesoni and D. j. kaimosae, express 3FTx-predominant venoms, black mamba (D. polylepis) venom is dominated by dendrotoxins I and K. The divergent terrestrial ecology of D. polylepis compared to the arboreal niche occupied by all other mambas makes it plausible that this major difference in venom composition is due to dietary variation. The pattern of intrageneric venom variability across Dendroaspis represented a valuable opportunity to investigate, in a genus-wide context, the variant toxicity of the venom, and the degree of paraspecific cross-reactivity between antivenoms and mamba venoms. To this end, the immunological profiles of the five mamba venoms were assessed against a panel of commercial antivenoms generated for the sub-Saharan Africa market. This study provides a genus-wide overview of which available antivenoms may be more efficacious in neutralising human envenomings caused by mambas, irrespective of the species responsible. The information gathered in this study lays the foundations for rationalising the notably different potency and pharmacological profiles of Dendroaspis venoms at locus resolution. This understanding will allow selection and design of toxin immunogens with a view to generating a safer and more efficacious pan-specific antivenom against any mamba envenomation.
Snakebite antivenom is a 120 years old invention based on polyclonal mixtures of antibodies purified from the blood of hyper-immunized animals. Knowledge on antibody recognition sites (epitopes) on snake venom proteins is limited, but may be used to provide molecular level explanations for antivenom cross-reactivity. In turn, this may help guide antivenom development by elucidating immunological biases in existing antivenoms. In this study, we have identified and characterized linear elements of B-cell epitopes from 870 pit viper venom protein sequences by employing a high-throughput methodology based on custom designed high-density peptide microarrays. By combining data on antibody-peptide interactions with multiple sequence alignments of homologous toxin sequences and protein modelling, we have determined linear elements of antibody binding sites for snake venom metalloproteases (SVMPs), phospholipases A2s (PLA2s), and snake venom serine proteases (SVSPs). The studied antivenom antibodies were found to recognize linear elements in each of the three enzymatic toxin families. In contrast to a similar study of elapid (non-enzymatic) neurotoxins, these enzymatic toxins were generally not recognized at the catalytic active site responsible for toxicity, but instead at other sites, of which some are known for allosteric inhibition or for interaction with the tissue target. Antibody recognition was found to be preserved for several minor variations in the protein sequences, although the antibody-toxin interactions could often be eliminated completely by substitution of a single residue. This finding is likely to have large implications for the cross-reactivity of the antivenom and indicate that multiple different antibodies are likely to be needed for targeting an entire group of toxins in these recognized sites.
Snakebite envenoming is a major public health burden in tropical parts of the developing world. In sub-Saharan Africa, neglect has led to a scarcity of antivenoms threatening the lives and limbs of snakebite victims. Technological advances within antivenom are warranted, but should be evaluated not only on their possible therapeutic impact, but also on their cost-competitiveness. Recombinant antivenoms based on oligoclonal mixtures of human IgG antibodies produced by CHO cell cultivation may be the key to obtaining better snakebite envenoming therapies. Based on industry data, the cost of treatment for a snakebite envenoming with a recombinant antivenom is estimated to be in the range USD 60–250 for the Final Drug Product. One of the effective antivenoms (SAIMR Snake Polyvalent Antivenom from the South African Vaccine Producers) currently on the market has been reported to have a wholesale price of USD 640 per treatment for an average snakebite. Recombinant antivenoms may therefore in the future be a cost-competitive alternative to existing serum-based antivenoms.
Snakebite envenomings represent a neglected public health issue in many parts of the rural tropical world. Animal-derived antivenoms have existed for more than a hundred years and are effective in neutralizing snake venom toxins when timely administered. However, the low immunogenicity of many small but potent snake venom toxins represents a challenge for obtaining a balanced immune response against the medically relevant components of the venom. Here, we employ high-throughput sequencing of the immunoglobulin (Ig) transcriptome of mice immunized with a three-finger toxin and a phospholipase A2 from the venom of the Central American coral snake, Micrurus nigrocinctus. Although exploratory in nature, our indicate results showed that only low frequencies of mRNA encoding IgG isotypes, the most relevant isotype for therapeutic purposes, were present in splenocytes of five mice immunized with 6 doses of the two types of toxins over 90 days. Furthermore, analysis of Ig heavy chain transcripts showed that no particular combination of variable (V) and joining (J) gene segments had been selected in the immunization process, as would be expected after a strong humoral immune response to a single antigen. Combined with the titration of toxin-specific antibodies in the sera of immunized mice, these data support the low immunogenicity of three-finger toxins and phospholipases A2found in M. nigrocinctusvenoms, and highlight the need for future studies analyzing the complexity of antibody responses to toxins at the molecular level.
Antivenoms against bites and stings from snakes, spiders, and scorpions are associated with immunological side effects and high cost of production, since these therapies are still derived from the serum of hyper-immunized production animals. Biotechnological innovations within envenoming therapies are thus warranted, and phage display technology may be a promising avenue for bringing antivenoms into the modern era of biologics. Although phage display technology represents a robust and high-throughput approach for the discovery of antibody-based antitoxins, several pitfalls may present themselves when animal toxins are used as targets for phage display selection. Here, we report selected critical challenges from our own phage display experiments associated with biotinylation of antigens, clone picking, and the presence of amber codons within antibody fragment structures in some phage display libraries. These challenges may be detrimental to the outcome of phage display experiments, and we aim to help other researchers avoiding these pitfalls by presenting their solutions.
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 0.53 mg/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.
2016
Snakebite envenoming is a serious condition requiring medical attention and administration of antivenom. Current antivenoms are antibody preparations obtained from the plasma of animals immunised with whole venom(s) and contain antibodies against snake venom toxins, but also against other antigens. In order to better understand the molecular interactions between antivenom antibodies and epitopes on snake venom toxins, a high-throughput immuno-profiling study on all manually curated toxins from Dendroaspis species and selected African Naja species was performed based on custom-made high-density peptide microarrays displaying linear toxin fragments. By detection of binding for three different antivenoms and performing an alanine scan, linear elements of epitopes and the positions important for binding were identified. A strong tendency of antivenom antibodies recognizing and binding to epitopes at the functional sites of toxins was observed. With these results, high-density peptide microarray technology is for the first time introduced in the field of toxinology and molecular details of the evolution of antibody-toxin interactions based on molecular recognition of distinctive toxic motifs are elucidated.
The cost of producing antivenoms from recombinant human antibodies to counter the shortage of animal-derived antisera against snakebites is not as prohibitive as you imply (Nature 537, 26–28; 2016).
We estimate that 500–2,000 kilograms of therapeutically active antibodies would be needed to produce enough antivenom to treat the 1 million or so people bitten annually by snakes in sub-Saharan Africa. On the basis of production data for monoclonal antibodies (N. Hammerschmidt et al. Biotechnol. J. 9, 766–775; 2014) and for oligoclonal antibody mixtures (S. K. Rasmussen et al. Arch. Biochem. Biophys. 526, 139–145; 2012), we calculate that antivenoms created from a mixture of recombinant antibodies could be produced on this scale for US$55–65 per gram.
A typical African snakebite could therefore be treated with a pan-African recombinant-antibody antivenom for $30–150. This compares favourably with the wholesale cost of a typical dose of conventional antiserum ($60–600, which includes packaging and transport, as well as production, costs).
Synergism between venom toxins exists for a range of snake species. Synergism can be derived from both intermolecular interactions and supramolecular interactions between venom components, and can be the result of toxins targeting the same protein, biochemical pathway or physiological process. Few simple systematic tools and methods for determining the presence of synergism exist, but include co-administration of venom components and assessment of Accumulated Toxicity Scores. A better understanding of how to investigate synergism in snake venoms may help unravel strategies for developing novel therapies against snakebite envenoming by elucidating mechanisms for toxicity and interactions between venom components.
Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest in using bioactive toxins from spiders and scorpions for drug discovery purposes and for solving crystal structures of membrane-embedded receptors. Additionally, the identification and isolation of a myriad of spider and scorpion toxins has allowed research within next generation antivenoms to progress at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation immunization strategies. The increasing number of discovery and development efforts within this field may point towards an upcoming transition from serum-based antivenoms towards therapeutic solutions based on modern biotechnology.
With an annual 5 million cases, 150,000 deaths, and about 400,000 amputations, snakebite envenoming is an ever-present threat in many parts of the rural tropical world. Parental administration of animal-derived, serum-based antivenoms remains the mainstay of snakebite envenoming therapy. However, the high level of immunogenicity of such heterologous medicines leads to severe side effects in human recipients. In order to bring antivenoms into the modern era of biopharmaceuticals, it is important to have a thorough understanding of snake venom toxins and to have an optimal antitoxin discovery strategy. In this thesis, a novel approach is presented on how to develop synthetic and recombinant antivenoms based on a range of different molecules, including peptides, nanobodies, antibodies, and antibody fragments. This approach is based on toxicovenomics and phage display selection.
In the work behind this thesis, a systematic method for selecting key toxins for antitoxin discovery was developed (the Toxicity Score). The combination of this approach with the venomics strategy was a central element in the establishment of the new field of toxicovenomics – the study of snake venom proteomes in relation to the pathophysiological effects of their toxins. Four toxicovenomics studies were performed on the venoms of Dendroaspis polylepis (Black mamba), Dendroaspis angusticeps (Eastern green mamba), Naja kaouthia (Monocled cobra), and Aipysurus laevis (Olive sea snake). These studies not only estimated the quantitative venom proteomes of these snakes and identified the medically most relevant toxins responsible for the pathophysiological effects of the venoms, but also revealed mechanistic differences between the venoms. As an example, the venoms from the black mamba, the green mamba, and the olive sea snake showed synergistic behaviors, while the venom from the monocled cobra displayed a dominance of non-synergistically-acting α-neurotoxins. α-neurotoxins played a major role in venom toxicity for all venoms, which cause flaccid paralysis in rodent models.
Several drug discovery programs based on phage display selection were carried out, aiming at finding antitoxins against the medically relevant toxins identified in the toxicovenomic studies. A few hundreds of peptide-displaying phages, dozens of nanobody-displaying phages, and over a thousand human scFv-displaying phages were selected and screened. Among these, dozens of promising peptidic antitoxins with inhibitory effects against elapid neurotoxins were identified. In two-electrode voltage clamp assays using Xenopus laevis (African clawed frog) oocytes, Peptide 33535 was capable of abrogating α-cobratoxin induced inhibition (at a concentration of 40 μM peptide and 100 μM α-cobratoxin) of the nicotinic acetylcholine receptor, responsible for neuromuscular transmission. This peptide was shown by isothermal titration calorimetry to bind to α-cobratoxin with a Kd of 20 μM. However, despite these positive results, much more research is needed before recombinant or synthetic antivenoms may reach the clinic and benefit victims of snakebite envenoming.
It is the hope that the work presented here will help enable that snakebite victims around the world will gain access to inexpensive and safe recombinant antivenom with high efficacy.
The snake is the symbol of medicine due to its association with Asclepius, the Greek God of medicine, and so with good reasons. More than 725 species of venomous snakes have toxins specifically evolved to exert potent bioactivity in prey or victims, and snakebites constitute a public health hazard of high impact in Asia, Africa, Latin America, and parts of Oceania. Parenteral administration of antivenoms is the mainstay in snakebite envenoming therapy. However, despite well-demonstrated efficacy and safety of many antivenoms worldwide, they are still being produced by traditional animal immunization procedures, and therefore present a number of drawbacks. Technological advances within biopharmaceutical development and medicinal chemistry could pave the way for rational drug design approaches against snake toxins. This could minimize the use of animals and bring forward more effective therapies for snakebite envenomings. In this review, current state-of-the-art in biopharmaceutical antitoxin development is presented together with an overview of available bioinformatics and structural data on snake venom toxins. This growing body of scientific and technological tools could define the basis for introducing a rational drug design approach into the field of snakebite envenoming therapy.
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.
2015
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.
For more than 100 years, antivenoms have been produced by traditional methods of immunization of large mammals with mixtures of snake venoms (World Health Organization, 2010 and Gutiérrez et al., 2011). With the introduction of proteomic and transcriptomic tools in the molecular analysis of both venoms (venomics) (Calvete, 2014) and antivenoms (antivenomics) (Calvete, 2011 and Calvete et al., 2014), in combination with the toxicological assessment of venoms, a more in-depth understanding of venom composition and antivenom efficacy is being built. As retrieved from current public databases on Elapidae, values for Median Lethal Dose (LD50) are known for 203 toxins, belonging to seven protein sub-families, originating from 40 species (Fig. 1). Furthermore, the number of elapids for which venom-wide proteomics or transcriptomics studies have been reported has now reached 49 out of 355 described species (our unpublished data; http://www.reptile-database.org). Information is now available for a considerable number of species of high medical relevance.
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.
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.
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