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Recombinant Antivenoms
Abstract and Figures
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 combi-nation 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 the 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 ca-pable 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 iso-thermal titration calorimetry to bind to α-cobratoxin with a Kd of 20 μM. However, despite these positive results, much more research is needed before re-combinant 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.
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... 15,16 Very few peptides have been reported that neutralize the effect of snake venom toxins. However, peptides have a number of different therapeutic benefits, including low-cost synthesis, reproducibility, and engineerable pharmacokinetics, 15,17,18 which position peptides as a relevant pharmaceutical scaffold. Here, we report the discovery of an anti-myotoxin II peptide (JB006) using phage display technology. ...
... For phage display selection, two random linear peptide libraries, TriCo-16 Phage Display Peptide Library and TriCo-20 Phage Display Peptide Library from Creative Biolabs, were employed, following previously described protocols. 17,19 In short, five rounds of panning on directly coated myotoxin II were performed, followed by isolation of monoclonal phages and assessment of their ability to bind myotoxin II and two controls, human serum albumin and α-cobratoxin (≥99% purity, from Naja kaouthia, Latoxan), using ELISA. 17 For phages that displayed specific binding, their ssDNA was isolated and sequenced, and based on an assessment of their solubility and isoelectric point using http://pepcalc.com/peptide-solubility-calculator.php, ...
... 17,19 In short, five rounds of panning on directly coated myotoxin II were performed, followed by isolation of monoclonal phages and assessment of their ability to bind myotoxin II and two controls, human serum albumin and α-cobratoxin (≥99% purity, from Naja kaouthia, Latoxan), using ELISA. 17 For phages that displayed specific binding, their ssDNA was isolated and sequenced, and based on an assessment of their solubility and isoelectric point using http://pepcalc.com/peptide-solubility-calculator.php, peptides were selected for further analysis. ...
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.
... The incidence of envenoming is estimated to be in the order of 2-3 million per year, resulting in more than 100,000 deaths [2,3]. Although animal-derived antisera remain the cornerstone of snakebite therapy [4], biotechnological advances are driving the emergence of different antivenom formats based on human or camelid antibody scaffolds [5,6], which in the future may pave the way for recombinant oligoclonal mixtures of antivenom antibodies [7]. The potential benefits of recombinant antivenoms for treatment of snakebite envenoming include higher potency and fewer side effects (serum sickness and anaphylaxis is not uncommon from animal-derived antisera) due to the possibility of producing fully human antibody formats specifically targeting the medically relevant snake venom toxins [6,8]. ...
... Currently, animal-derived snakebite antivenoms are manufactured in three different structural formats: IgG-based, F(ab) 2 -based, and Fab-based [6,14] (see Fig 1). F(ab) 2 and Fab-based are generated with the intention of creating improved safety profiles through the removal of the Fc region of the animal-derived IgG antibodies by treatment with pepsin or papain during the manufacturing process [7]. Fab-based antivenoms have been shown to better reach and neutralize toxins in deep tissue than IgG-based antivenoms, but it comes at a cost of reduced serum half-life [14]. ...
... The IgG format may represent a more optimal solution for targeting systemically acting toxins, whereas the Fab format may potentially be more ideal against locally acting toxins. Other formats have been investigated, including single-chain variable fragments (scFv) and camelid single-domain antibodies (V H H fragments) [5][6][7], but none of these are have yet been tested in the clinic. ...
... The incidence of envenoming is estimated to be in the order of 2-3 million per year, resulting in more than 100,000 deaths [2,3]. Although animal-derived antisera remain the cornerstone of snakebite therapy [4], biotechnological advances are driving the emergence of different antivenom formats based on human or camelid antibody scaffolds [5,6], which in the future may pave the way for recombinant oligoclonal mixtures of antivenom antibodies [7]. The potential benefits of recombinant antivenoms for treatment of snakebite envenoming include higher potency and fewer side effects (serum sickness and anaphylaxis is not uncommon from animal-derived antisera) due to the possibility of producing fully human antibody formats specifically targeting the medically relevant snake venom toxins [6,8]. ...
... Currently, animal-derived snakebite antivenoms are manufactured in three different structural formats: IgG-based, F(ab) 2 -based, and Fab-based [6,14] (see Fig 1). F(ab) 2 and Fab-based are generated with the intention of creating improved safety profiles through the removal of the Fc region of the animal-derived IgG antibodies by treatment with pepsin or papain during the manufacturing process [7]. Fab-based antivenoms have been shown to better reach and neutralize toxins in deep tissue than IgG-based antivenoms, but it comes at a cost of reduced serum half-life [14]. ...
... The IgG format may represent a more optimal solution for targeting systemically acting toxins, whereas the Fab format may potentially be more ideal against locally acting toxins. Other formats have been investigated, including single-chain variable fragments (scFv) and camelid single-domain antibodies (V H H fragments) [5][6][7], but none of these are have yet been tested in the clinic. ...
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.
... However, due to their heterologous nature, they have a propensity to cause immunological reactions in human recipients [90]. To circumvent this challenge of immunogenicity, and to improve other therapeutic properties, such as enhancing efficacy and reducing batch-tobatch variation, researchers worldwide have started to embrace different antibody technologies with the goal of developing recombinant antivenoms [89,91,92]. Recently, the discovery of the first fully human oligoclonal IgGs against animal toxins was reported [93]. ...
... About 5000 different species of mushrooms exist, with about 50 species being poisonous to humans [15]. In contrast to venom toxins that due to their size and proteinaceous nature are unable to be absorbed in the gastrointestinal tract, mushroom poisons comprise small oligopeptidic toxins that can be readily absorbed in the gastrointestinal tract and are thus toxic when ingested [91]. Most of the mushroom species involved in lethal poisonings are found in the genus Amanita, which produces amatoxins, phallotoxins, and virotoxins [99]. ...
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 opportunity 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 development 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.
... However, this number is unlikely to increase significantly, given the prospects of using transgenic (humanized) animals capable of producing human IgGs more suitable for human therapy and the advent of phage display technology. The latter is one of the most promising avenues for development of novel recombinant antivenoms [69,70]. Most commonly, phage display selection has been used to develop human single-chain variable fragments (scFvs) with important examples including the development of Serrumab against the toxins, Ts1 and Ts2, from the Brazilian yellow scorpion (T. ...
... Toxicovenomics builds upon venomics and includes in vivo toxicity data, which can be used to obtain an overview of venoms as pharmacological targets for antitoxin development and determine which toxins are essential to neutralize with antivenom in an envenoming case. The figure has been adapted from [70,115]. martensii) [92], and the honey bee (Apis mellifera) [93]. In addition, a multitude of transcriptomics studies have been performed, particularly on the venom glands from snakes, scorpions, and spiders [8,9]. ...
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.
... As an important part of this strategy, research and development on improved snakebite envenoming therapies is recommended. In this relation, a promising avenue that has gained interest in recent years, is the use of recombinant antivenoms based on carefully designed mixtures of human monoclonal antibodies targeting key toxins of medically important snake venoms (Laustsen, 2016). Some of the hypothesized benefits of using recombinant antivenoms include a reduced propensity to cause adverse reactions in patients and a higher content of therapeutically active antibodies (Kini et al., 2018). ...
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.
... For this reason, it is possible to use these peptides as scaffolds for the design of new inhibitors, even though they themselves are not useful due to their low affinity. [75,105] Laustsen, in his thesis work [106,107] screened linear peptides from two phage display peptide libraries against Naja kaouthia's α-cobratoxin. Initially two patterns were seen to be repeated in the selected sequences and six of those peptides were selected to assay. ...
Nowadays, treatment with specific antivenins is considered the only cure for snakebites accidents. However, access to antivenom obstructs the successful implementation of the World Health Organization international guidelines. In the last few years, natural organic compounds, peptides and proteins with the ability to inhibit snake toxins and obtained from different sources such as plant extracts and animal blood have been proposed as antivenoms. In this work, we will focus on the inhibitors of the main venom toxins, phospholipases A2 and metalloproteinases, and their application as novel antivenoms.
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.
Snakebite envenoming remains a major public health issue in Latin America and other rural tropical regions of the world, causing mortality and morbidity to hundreds of thousands. Particularly in Central America, the species Micrurus nigrocinctus is the most abundant and clinically relevant coral snake from the Elapidae family. The venom of M. nigrocinctus is predominantly composed of phospholipases A2 (PLA2s) and three-finger toxins (3FTxs), constituting 48% and 38% of the venom proteins, respectively. Snakebites from this species predominantly induce neurotoxic effects, although myotoxicity has been shown in animal models and may occur in humans. Myotoxicity is caused by PLA2s, while neurotoxicity is mainly caused by 3FTxs, with contribution of some PLA2s. Existing coral snake antivenoms are based on serum from immunized horses. These are complicated to manufacture due to difficulties in procuring scarce M. nigrocinctus venom and due to the poor immunogenicity of the venom toxins, making them less effective in the immunization process. Additionally, existing antivenoms may cause undesirable adverse reactions as they contain heterologous proteins that are not compatible with the human immune system. In this project, we employ phage display technology to discover human antibodies able to neutralize the medically most important toxins of M. nigrocinctus venom. After identification of scFv antibody fragments using the IONTAS phage display library, the most promising scFvs will be converted to the IgG format to gain improved half-life to provide prolonged protection against snake toxins in the circulatory system. We hope that with these initial steps we will pave the way for the development of a next-generation recombinant antivenom for M. nigrocinctus envenomings with improved efficacy and safety.
In Africa, Asia, Latin America, and parts of Oceania, envenoming after snakebite is a serious public health problem [1]. Conservative data suggest that between 1.2 and 5.5 million people suffer snakebites every year, resulting in 25,000 to 125,000 deaths and leaving approximately 400,000 victims with permanent sequelae [2,3]. Despite its significant impact on human health, this disease remains largely neglected by national and international health authorities, funding agencies, pharmaceutical companies, patients' organizations, and health advocacy groups [1,2]. Most initiatives aiming to study snakes, snake venoms, and snakebite envenoming and its treatment approach the problem from a biomedical and technological perspective. Notwithstanding the substantial scientific and clinical legacy generated through this view, significant gaps remain in our understanding of other highly relevant aspects of this problem and its solutions. The emerging field of global health has brought about a more holistic approach to health issues by incorporating a " biosocial approach " to the understanding of diseases and the circumstances behind their occurrence [4]. The centrepiece of this approach is the integration of biomedical aspects—including etiology, pathophysiology, diagnosis, and therapy—with the analysis of the social, economic, psychological, cultural, and political contexts in which diseases occur. Snakebite envenoming is predominantly a disease of the poor [5], with the highest incidence and severity seen in regions facing complex and interrelated social and economic problems. Understanding how this interplay of variables influences both the circumstances leading to snakebite injury and its consequences is crucial to developing successful strategies to mitigate the problem. A comprehensive multidisciplinary approach incorporating social research into the study of snakebite envenoming is needed. Hereby, we aim to increase awareness of the following areas where reinvigorated social research would be highly beneficial. A key issue undermining advocacy efforts to measure the impact of snakebite envenoming worldwide is the poor level of information on incidence, mortality, sequelae, and social suffering associated with this disease. Most studies are based on hospital statistics that greatly
''Excellent and very timely....It will undoubtedly become a standard reference for the application of circular dichroism (CD) to biomolecules.'' --- Quarterly Review of Biology, March 1997 ''[T]estament to the book's utility is the fact that during the course of my review I had to 'rescue' it from the desks of graduate students on an almost daily basis. In summary, this is a great book.'' --- American Scientist ''Well documented chapters provide a very good insight into the problems surrounding the conformation of biomacromolecules...An indispensible source of information.'' --- Nahrung, 42(2), 1998 Renowned experts present the first state-of-the-art description of circular dichroism spectroscopy (CD). Chapters present in-depth discussions of the history of the field, the theory of CD for application to globular proteins, membrane proteins, peptides, nucleic acids and their interactions, carbohydrates, and instrumentation. Discussions also feature new techniques using synchrotron radiation, vibrational Raman optical activity, and vibrational CD. More than 250 illustrations supplement the text.
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.
The production of snake antivenoms involves stages such as production of venom, immunization of animals to generate hyperimmune plasma, immunoglobulin purification, viral inactivation (or removal), and stabilization of the formulation. In order to manufacture products of satisfactory effectiveness and safety, antivenom design must be validated by preclinical and clinical studies. Moreover, during the industrial production, the quality of the products and of the entire manufacturing process (including management of clean rooms, production of water for injection, and sterilization or sanitization of the equipment) must be strictly evaluated. This chapter presents a practical description of the stages involved in the design, production, and quality control of snake antivenoms.
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A toxicovenomic study was performed on the venom of the green mamba, Dendroaspis angusticeps. Forty-two different proteins were identified in the venom of D. angusticeps, in addition to the nucleoside adenosine. The most abundant proteins belong to the three-finger toxin (3FTx) (69.2%) and the Kunitz-type proteinase inhibitor (16.3%) families. Several sub-subfamilies of the 3FTxs were identified, such as Orphan Group XI (Toxin F-VIII), acetylcholinesterase inhibitors (fasciculins), and aminergic toxins (muscarinic toxins, synergistic-like toxins, and adrenergic toxins). Remarkably, no α-neurotoxins were identified. Proteins of the Kunitz-type proteinase inhibitor family include dendrotoxins. Toxicological screening revealed a lack of lethal activity in all RP-HPLC fractions, except one, at the doses tested. Thus, the overall toxicity depends on the synergistic action of various types of proteins, such as dendrotoxins, fasciculins, and probably other synergistically-acting toxins. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality. These antivenoms also showed a pattern of broad immunorecognition of the different HPLC fractions by ELISA and immunoprecipitated the crude venom by gel immunodiffusion. The synergistic mechanism of toxicity constitutes a challenge for the development of effective recombinant antibodies, as it requires the identification of the most relevant synergistic toxins.
Biological significance:
Envenomings by elapid snakes of the genus Dendroaspis, collectively known as mambas, represent a serious medical problem in sub-Saharan Africa. The development of novel antivenoms and of recombinant neutralizing antibodies demands the identification of the most relevant toxins in these venoms. In this study, a bottom-up approach was followed for the study of the proteome of the venom of the Eastern green mamba, D. angusticeps. Forty-two different proteins were identified, among which the three-finger toxin (3FTx) family, characteristic of elapid venoms, was the most abundant, followed by the Kunitz-type proteinase inhibitor family. In addition, several other protein families were present in the venom, together with the nucleoside adenosine. No α-neurotoxins were identified within the family of 3FTxs in the venom of D. angusticeps, in contrast to the venom of Dendroaspis polylepis, in which α-neurotoxins are largely responsible for the toxicity. With one exception, HPLC fractions from D. angusticeps venom did not kill mice at the doses tested. This underscores that the toxicity of the whole venom is due to the synergistic action of various components, such as fasciculins and dendrotoxins, and probably other synergistically-acting toxins. Thus, the venoms of these closely related species (D. angusticeps and D. polylepis) seem to have different mechanisms to subdue their prey, which may be related to different prey preferences, as D. angusticeps is predominantly arboreal, whereas D. polylepis lives mostly in open bush country and feeds mainly on mammals. It is therefore likely that the predominant clinical manifestations of human envenomings by these species also differ, although in both cases neurotoxic manifestations predominate. Polyspecific antivenoms manufactured in South Africa and India were effective in the neutralization of venom-induced lethality in mice and showed a pattern of broad immunorecognition of the various venom fractions. It is necessary to identify the toxins responsible for the synergistic mode of toxicity in this venom, since they are the targets for the development of recombinant antibodies for the treatment of envenomings.
Inducible expression systems in which T7 RNA polymerase transcribes coding sequences cloned under control of a T7lac promoter efficiently produce a wide variety of proteins in Escherichia coli. Investigation of factors that affect stability, growth, and induction of T7 expression strains in shaking vessels led to the recognition that sporadic, unintended induction of expression in complex media, previously reported by others, is almost certainly caused by small amounts of lactose. Glucose prevents induction by lactose by well-studied mechanisms. Amino acids also inhibit induction by lactose during log-phase growth, and high rates of aeration inhibit induction at low lactose concentrations. These observations, and metabolic balancing of pH, allowed development of reliable non-inducing and auto-inducing media in which batch cultures grow to high densities. Expression strains grown to saturation in non-inducing media retain plasmid and remain fully viable for weeks in the refrigerator, making it easy to prepare many freezer stocks in parallel and use working stocks for an extended period. Auto-induction allows efficient screening of many clones in parallel for expression and solubility, as cultures have only to be inoculated and grown to saturation, and yields of target protein are typically several-fold higher than obtained by conventional IPTG induction. Auto-inducing media have been developed for labeling proteins with selenomethionine, 15N or 13C, and for production of target proteins by arabinose induction of T7 RNA polymerase from the pBAD promoter in BL21-AI. Selenomethionine labeling was equally efficient in the commonly used methionine auxotroph B834(DE3) (found to be metE) or the prototroph BL21(DE3).