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Neutralization of Centruroides tecomanus scorpion venom by the use of two human recombinant antibody fragments
Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)′2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms.
Key points
• Commercial antivenoms consist of predominantly purified equine F(ab)′2fragments.
• Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity.
• Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.
Antibody fragments are used in the clinic as important therapeutic proteins for treatment of indications where better tissue penetration and less immunogenic molecules are needed. Several expression platforms have been employed for the production of these recombinant proteins, from which E. coli and CHO cell-based systems have emerged as the most promising hosts for higher expression. Because antibody fragments such as Fabs and scFvs are smaller than traditional antibody structures and do not require specific patterns of glycosylation decoration for therapeutic efficacy, it is possible to express them in systems with reduced post-translational modification capacity and high expression yield, for example, in plant and insect cell-based systems. In this review, we describe different bioengineering technologies along with their opportunities and difficulties to manufacture antibody fragments with consideration of stability, efficacy and safety for humans. There is still potential for a new production technology with a view of being simple, fast and cost-effective while maintaining the stability and efficacy of biotherapeutic fragments.
Scorpionism in México is a public health problem caused by stings from the Centruroides scorpion family. The Ct1a and Ct17 toxins from the venom of the Centruroides tecomanus scorpion are the most abundant and toxic for mammals. This study describes the heterologous expression of recombinant proteins from genes encoding these toxins merged with thioredoxin in the vector pET-22b + and expressed in Escherichia coli BL21 (DE3). The yield of Ct1a and Ct17 recombinant toxins was 1.25 mg and 1.737 mg per liter of culture, respectively. These were purified by metal ion affinity chromatography and RP-HPLC and were used for immunization in rabbits, obtaining polyclonal antibodies that confer a positive immune response against the complete venom of Centruroides tecomanus. The serum was tested in vitro and in vivo, obtaining neutralization and protection against the venom. Both toxins were produced recombinantly and fused to the thioredoxin protein; remarkably, the recombinant toxins were excellent immunogens. 300 μl of each immunized rabbit serum was tested in mice, resulting in 50% of the mice protected with each serum, but when the sera were pooled, the protection increased to 83%. This communication reveals the possibility of producing a specific and regional antivenom with polyclonal antibodies that neutralize the complete venom of Centruroides tecomanus. The sequence similarity of Ct1a and Ct17 to Cn2, a toxin that recognizes sodium channels, allowed in silico modeling analysis and a proposal for the different toxicities of Ct1a and Ct17.
Centruroides huichol scorpion venom is lethal to mammals. Analysis of the venom allowed the characterization of four lethal toxins named Chui2, Chui3, Chui4, and Chui5. scFv 10FG2 recognized well all toxins except Chui5 toxin, therefore a partial neutralization of the venom was observed. Thus, scFv 10FG2 was subjected to three processes of directed evolution and phage display against Chui5 toxin until obtaining scFv HV. Interaction kinetic constants of these scFvs with the toxins were determined by surface plasmon resonance (SPR) as well as thermodynamic parameters of scFv variants bound to Chui5. In silico models allowed to analyze the molecular interactions that favor the increase in affinity. In a rescue trial, scFv HV protected 100% of the mice injected with three lethal doses 50 (LD50) of venom. Moreover, in mix-type neutralization assays, a combination of scFvs HV and 10FG2 protected 100% of mice injected with 5 LD50 of venom with moderate signs of intoxication. The ability of scFv HV to neutralize different toxins is a significant achievement, considering the diversity of the species of Mexican venomous scorpions, so this scFv is a candidate to be part of a recombinant anti-venom against scorpion stings in Mexico.
The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 μg/100 mg of cricket weight, but low amounts of peptides (0.8 μg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant β-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.
A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10⁻⁹ and 6.1 × 10⁻¹¹ M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.
In this communication the isolation, chemical and physiological characterization of three new toxins from the scorpion Centruroides baergi are reported. Their immunoreactive properties with scFvs generated in our group are described. The three new peptides, named Cb1, Cb2 and Cb3 affect voltage-dependent Na⁺ channels in a differential manner. These characteristics, explain the toxicity of this venom. Molecular interactions in real-time among these toxins and the best recombinant antibodies generated in our group, revealed that one of them was able to neutralize the main toxin of this venom (Cb1). These results represent an important advance for the neutralization of this venom and serve as the basis for generating new scFvs that will allow the neutralization of similar toxins from other venoms that have no yet been neutralized.
Because of their venom lethality towards mammals, scorpions of the Androctonus genus are considered a critical threat to human health in North Africa. Several decades of exploration have led to a comprehensive inventory of their venom components at chemical, pharmacological, and immunological levels. Typically, these venoms contain selective and high affinity ligands for the voltage-gated sodium (Nav) and potassium (Kv) channels that dictate cellular excitability. In the well-studied Androctonus australis and Androctonus mauretanicus venoms, almost all the lethality in mammals is due to the so-called α-toxins. These peptides commonly delay the fast inactivation process of Nav channels, which leads to increased sodium entry and a subsequent cell membrane depolarization. Markedly, their neutralization by specific antisera has been shown to completely inhibit the venom’s lethal activity, because they are not only the most abundant venom peptide but also the most fatal. However, the structural and antigenic polymorphisms in the α-toxin family pose challenges to the design of efficient serotherapies. In this review, we discuss past and present accomplishments to improve serotherapy against Androctonus scorpion stings.
The recombinant antibody fragments generated against the toxic components of scorpion venoms are considered a promising alternative for obtaining new antivenoms for therapy. Using directed evolution and site-directed mutagenesis, it was possible to generate a human single-chain antibody fragment with a broad cross-reactivity that retained recognition for its original antigen. This variant is the first antibody fragment that neutralizes the effect of an estimated 13 neurotoxins present in the venom of nine species of Mexican scorpions. This single antibody fragment showed the properties of a polyvalent antivenom. These results represent a significant advance in the development of new antivenoms against scorpion stings, since the number of components would be minimized due to their broad cross-neutralization capacity, while at the same time bypassing animal immunization.
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 aim of this study was to prepare specific F(ab')2 antivenom against Iraqi scorpion (Hottentotta saulcyi). Venom was obtained by electrical stimulation method, the scorpions venom storage capacity was 1.7 ± 0.4 mg and LD50 found to be 1.07 mg/kg by subcutaneous (s.c) route. Three local horses aging 3-4 years were selected for immunization. During the schedule each horse received 0.5-14 mg venom in 49 days by s.c route. The horses immune response was monitored by Ouchterlony double immunodiffusion method, however, older horse A showed the highest antibody titer (1:1056576). Hyperimmune plasma was purified by modified World Health Organization (WHO) protocol using pepsin and ammonium sulfate, whereas high purity and total removal of albumin was detected by serum protein electrophoresis (SPEP) method on cellulose acetate. ED50 of the antivenom was determined via s.c route and found to be 83 LD50/ml, neutralizing 1.78 mg (venom)/ml (antivenom). Protein concentration of the antivenom was 30 mg/ml, less than limited value (100 mg/ml) by WHO. Abnormal toxicity test showed no abnormal signs when 0.2 ml of the antivenom injected intraperitoneally in mice.
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.
Molecular recognition, which is the process of biological macromolecules interacting with each other or various small molecules with a high specificity and affinity to form a specific complex, constitutes the basis of all processes in living organisms. Proteins, an important class of biological macromolecules, realize their functions through binding to themselves or other molecules. A detailed understanding of the protein–ligand interactions is therefore central to understanding biology at the molecular level. Moreover, knowledge of the mechanisms responsible for the protein-ligand recognition and binding will also facilitate the discovery, design, and development of drugs. In the present review, first, the physicochemical mechanisms underlying protein–ligand binding, including the binding kinetics, thermodynamic concepts and relationships, and binding driving forces, are introduced and rationalized. Next, three currently existing protein-ligand binding models—the “lock-and-key”, “induced fit”, and “conformational selection”—are described and their underlying thermodynamic mechanisms are discussed. Finally, the methods available for investigating protein–ligand binding affinity, including experimental and theoretical/computational approaches, are introduced, and their advantages, disadvantages, and challenges are discussed.
Serum therapy remains the only specific treatment against envenoming, but anti-venoms are still prepared by fragmentation of polyclonal antibodies isolated from hyper-immunized horse serum. Most of these anti-venoms are considered to be efficient, but their production is tedious, and their use may be associated with adverse effects. Recombinant antibodies and smaller functional units are now emerging as credible alternatives and constitute a source of still unexploited biomolecules capable of neutralizing venoms. This review will be a walk through the technologies that have recently been applied leading to novel antibody formats with better properties in terms of homogeneity, specific activity and possible safety.
Centruroides tecomanus is a Mexican scorpion endemic of the State of Colima, that causes human fatalities. This communication describes a proteome analysis obtained from milked venom and a transcriptome analysis from a cDNA library constructed from two pairs of venom glands of this scorpion. High perfomance liquid chromatography separation of soluble venom produced 80 fractions, from which at least 104 individual components were identified by mass spectrometry analysis, showing to contain molecular masses from 259 to 44,392 Da. Most of these components are within the expected molecular masses for Na(+)- and K(+)-channel specific toxic peptides, supporting the clinical findings of intoxication, when humans are stung by this scorpion. From the cDNA library 162 clones were randomly chosen, from which 130 sequences of good quality were identified and were clustered in 28 contigs containing, each, two or more expressed sequence tags (EST) and 49 singlets with only one EST. Deduced amino acid sequence analysis from 53% of the total ESTs showed that 81% (24 sequences) are similar to known toxic peptides that affect Na(+)-channel activity, and 19% (7 unique sequences) are similar to K(+)-channel especific toxins. Out of the 31 sequences, at least 8 peptides were confirmed by direct Edman degradation, using components isolated directly from the venom. The remaining 19%, 4%, 4%, 15% and 5% of the ESTs correspond respectively to proteins involved in cellular processes, antimicrobial peptides, venom components, proteins without defined function and sequences without similarity in databases. Among the cloned genes are those similar to metalloproteinases.
We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.
Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are ‘discoverable’ through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.
Interactions within a protein structure and interactions between proteins in an assembly are essential considerations in understanding
molecular basis of stability and functions of proteins and their complexes. There are several weak and strong interactions
that render stability to a protein structure or an assembly. Protein Interactions Calculator (PIC) is a server which, given
the coordinate set of 3D structure of a protein or an assembly, computes various interactions such as disulphide bonds, interactions
between hydrophobic residues, ionic interactions, hydrogen bonds, aromatic–aromatic interactions, aromatic–sulphur interactions
and cation–π interactions within a protein or between proteins in a complex. Interactions are calculated on the basis of standard,
published criteria. The identified interactions between residues can be visualized using a RasMol and Jmol interface. The
advantage with PIC server is the easy availability of inter-residue interaction calculations in a single site. It also determines
the accessible surface area and residue-depth, which is the distance of a residue from the surface of the protein. User can
also recognize specific kind of interactions, such as apolar–apolar residue interactions or ionic interactions, that are formed
between buried or exposed residues or near the surface or deep inside.
The scorpionism is an actual public health problem in several parts of the world because, either incidence, or severity of envenomations is high and managed with difficulty by health services, or for these two reasons at the same time. The treatment of scorpion envenomation is complex and controversial, in particular regarding the utility of the antivenoms and symptomatic treatments that must be associated. The authors reviewed the literature of last 30 years to discuss the epidemiologic importance of scorpionism and to point out the principal therapeutic or preventive measures. According to the most recent studies, seven areas were identified as at risk: north-Saharan Africa, Sahelian Africa, South Africa, Near and Middle-East, South India, Mexico and South Latin America, east of the Andes. These involve 2.3 billion at risk population. The annual number of scorpion stings exceeds 1.2 million leading to more than 3250 deaths (0.27%). Although adults are more often concerned, children experience more severe envenomations and among them, mortality is higher. Improvement of therapeutic management would reduce the lethality very significantly.
Previously, it was demonstrated that from the single chain fragment variable (scFv) 3F it is possible to generate variants capable of neutralizing the Cn2 and Css2 toxins, as well as their respective venoms (Centruroides noxius and Centruroides suffusus). Despite this success, it has not been easy to modify the recognition of this family of scFvs toward other dangerous scorpion toxins. The analysis of toxin-scFv interactions and in vitro maturation strategies allowed us to propose a new maturation pathway for scFv 3F to broaden recognition toward other Mexican scorpion toxins. From maturation processes against toxins CeII9 from C. elegans and Ct1a from C. tecomanus, the scFv RAS27 was developed. This scFv showed an increased affinity and cross-reactivity for at least 9 different toxins while maintaining recognition for its original target, the Cn2 toxin. In addition, it was confirmed that it can neutralize at least three different toxins. These results constitute an important advance since it was possible to improve the cross-reactivity and neutralizing capacity of the scFv 3F family of antibodies.
The methylotrophic yeast Pichia pastoris has been one of the most widely used organisms in recent years as an expression system for a wide variety of recombinant proteins with therapeutic potential. Its popularity as an alternative system to Escherichia coli is mainly due to the easy genetic manipulation and the ability to produce high levels of heterologous proteins, either intracellularly or extracellularly. Being a eukaryotic organism, P. pastoris carries out post-translational modifications that allow it to produce soluble and correctly folded recombinant proteins. This work, evaluated the expression capacity in P. pastoris of two single-chain variable fragments (scFvs) of human origin, 10FG2 and LR. These scFvs were previously obtained by directed evolution against scorpion venom toxins and are able to neutralize different toxins and venoms of Mexican species. The yield obtained in P. pastoris was higher than that obtained in bacterial periplasm (E. coli), and most importantly, biochemical and functional properties were not modified. These results confirm that P. pastoris yeast can be a good expression system for the production of antibody fragments of a new recombinant antivenom.
Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.
The peptide, denominated Ct1a, is a β-toxin of 66 amino acids, isolated from venom of the scorpion, Centruroides tecomanus, collected in Colima, Mexico. This toxin was purified using size exclusion, cationic exchange, and reverse phase chromatography. It is the most abundant toxin, representing 1.7% of the soluble venom. Its molecular mass of 7588.9 Da was determined by mass spectrometry. The amino acid sequence was determined by Edman degradation and confirmed by transcriptomic analysis. Since neurons of the suprachiasmatic nucleus (SCN) maintain a spontaneous firing rate (SFR), we evaluated the physiological effects of toxin Ct1a on these neurons. The SFR exhibited a bimodal concentration-dependent response: 100 nM of Ct1a increased the SFR by 223%, whereas 500 nM and 1000 nM reduced it to 42% and 7%, respectively. Control experiments, consisting of recordings of the SFR during a time similar to that used in Ct1a testing, showed stability throughout the trials. Experiments carried out with denatured Ct1a toxin (500 nM) caused no variation in SFR recordings. Action potentials of SCN neurons, before and after Ct1a (100 nM) showed changes in the time constants of depolarization and repolarization phases, amplitude, and half-time. Finally, recordings of hNav1.6 sodium currents indicated that Ct1a shifts the channel activation to a more negative potential and reduces the amplitude of the peak current. These results all demonstrate that toxin Ct1a affects the SFR of SCN neurons by acting upon sodium channels of sub-type 1.6, implicating them in regulation of the SFR of SCN neurons.
In Mexico, scorpion sting envenomation (SSE) is a significant public health issue that has engaged the attention of health authorities for more than a century. Rigorously characterized today, scorpion sting incidence is stable around 230 stings per 100,000 population, i.e. 300,000 annual stings treated in Mexican health centers and hospitals. Higher incidence is observed mainly in central and Pacific Mexico. Scorpion populations thrive in populated places, particularly in impoverished areas. Scorpion stings occur in houses. This could explain similar incidence according to gender and age. The number of scorpion stings has remained stable since the mid-2000s. In contrast, mortality, which was over 1500 deaths per year before the 1960s, underwent a dramatic drop after the 1970s, from 500 deaths per year to fewer than 50 annual deaths today. Case fatality rates have shown similar trend. We noted a significantly higher specific mortality in males than in females (0.199 and 0.168 per 100,000 respectively; P < 1.9·10⁻⁶). Three causes explained the drop in mortality and case fatality rate, a) ongoing improvement in hospital care, particularly in terms of supportive standardized treatments, b) the use of highly purified immunoglobulin F(ab')2 fragments after 1995 and, c) increasing access to health services for most of the Mexican population. The authors retrace the history of the management of SSE, including the development of antivenoms, in Mexico between 1905 and today.
Scorpion envenomation is common in the tropical and subtropical regions. It poses a major public health problem with some patients having serious clinical manifestations and severe complications including death. Old World and New World scorpions are usually contrasted because of differences in venom composition, clinical presentation and severity, and, accordingly, different therapeutic approaches. The majority of scorpion stings are either dry or result in low amounts of injected venom, thus explaining why up to 95% of scorpion stings ensue only in local signs. For a clinical envenomation to occur, it has been suggested that the interaction between the quantity of venom introduced in the body of the prey and the distribution volume should ensue in a critical threshold of scorpion toxin plasma concentration. In this case, there is a massive release of neurohormonal mediators (mainly catecholamine), with systemic vasoconstrictor effects eliciting a sharp increase in systemic arterial pressure and LV-filling pressure and decreased cardiac output. This early phase of cardiac dysfunction, also called “vascular phase”, is followed by a severe cardiomyopathy, a form of Takotsubo cardiomyopathy, involving both ventricles and reversible in days to weeks. The more comprehensive understanding of the disease pathophysiology has allowed for a well-codified symptomatic treatment, thus contributing to a substantial reduction in the death toll of scorpion envenomation over the past few decades. The standard intensive-care treatment (when available) overcomes envenomation’s consequences such as acute pulmonary edema and cardiogenic shock. Even though it continues to inspire many evaluative studies, immunotherapy seems less attractive because of the major role held by mediators in the pathogenesis of envenomation, and unfavorable pharmacokinetic properties to existing sera compared to venom. Meta-analyses of controlled trials of immunotherapy in severe scorpion envenomation reached similar conclusions: there is an acceptable level of evidence in favor of the use of scorpion antivenom (Fab’2) against Centruroides sp. in USA/Mexico, while there is still a need for a higher level of evidence for immunotherapy in the Old World envenomation.
Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na+-channels revealed a typical β-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other β-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions.
Background: An effective therapy against envenoming should be a priority in view of the high number scorpion stings and snakebites. Serum therapy is still widely applied to treat the envenomation victims; however this approach suffers from several shortcomings. The employment of monoclonal antibodies might be an outcome as these molecules are at the core of a variety of applications from protein structure determination to cancer treatment. The progress of activities in the twilight zone between genetic and antibody engineering have led to the development of a unique class of antibody fragments. These molecules possess several benefits and lack many possible disadvantages over classical antibodies. Within recombinant antibody formats, nanobodies or single domain antigen binding fragments derived from heavy chain only antibodies in camelids occupy a privileged position. Scope of review: In this paper we will briefly review the common methods of envenomation treatment and focus on details of various in vivo research activities that investigate the performance of recombinant, monoclonal nanobodies in venom neutralization. Major conclusions: Nanobodies bind to their cognate target with high specificity and affinity, they can be produced in large quantities from microbial expression systems and are very robust even when challenged with harsh environmental conditions. Upon administering, they rapidly distribute throughout the body and seem to be well tolerated in humans posing low immunogenicity. General significance: Scorpion and snake envenomation is a major issue in developing countries and nanobodies as a venom-neutralizing agent can be considered as a valuable and promising candidate in envenomation therapy.
The increment in the number of scorpion envenoming cases in Mexico is mainly associated to the rapid growth of the urban areas, and consequently, to the invasion of natural habitats of these arachnids. On the other hand, there is a great diversity of scorpion species, so it is indispensable to identify those of medical importance, which we now know are many more than the 7-8 previously reported as dangerous to humans. Because different LD50 values have been reported for the venom of the same species, probably due to variations in the experimental conditions used, in this work we determined the LD50 values for the venoms of 13 different species of scorpions using simple but systematic procedures. This information constitutes a referent on the level of toxicity of medically important scorpion species from Mexico and establishes the bases for a more comprehensive assessment of the neutralizing capacity of current and developing antivenoms.
A previously undescribed toxic peptide named Cl13 was purified from the venom of the Mexican scorpion Centruroides limpidus. It contains 66 amino acid residues, including four disulfide bonds. The physiological effects assayed in 7 different subtypes of voltage gated Na⁺-channels, showed that it belongs to the β-scorpion toxin type. The most notorious effects were observed in subtypes Nav1.4, Nav1.5 and Nav1.6. Although having important sequence similarities with two other lethal toxins from this scorpion species (Cll1 m and Cll2), the recently developed single chain antibody fragments (scFv) of human origin were not capable of protecting against Cl13. At the amino acid sequence level, in 3 stretches of peptide Cl13 (positions 7-9, 30-38 and 62-66) some differences with respect to other similar toxins are observed. Some of these differences coincide with contact points with the human antibody fragments.
Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 μg/ml concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 μg/ml concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 μg/ml.
Abstract In Brazil, scorpion envenomation is an important public health problem. The yellow scorpion, Tityus serrulatus (Ts), is considered the most dangerous species in the country, being responsible for the most severe clinical cases of envenomation. Currently, the administration of serum produced in horses is recognized and used as a treatment for accidents with scorpions. However, horse herds' maintenance is costly and the antibodies are heterologous, which can cause anaphylaxis and Serum Sickness. In the present work, a human monoclonal fragment antibody, Serrumab, has been analysed. Toxin neutralizing effects of Serrumab were evaluated using a two-electrode voltage-clamp technique. The results show that Serrumab presented a high neutralizing effect against Ts β-toxins (Ts1, 43.2% and Ts2, 68.8%) and none or low neutralizing effect against α-toxins (Ts3, 0% and Ts5, 10%). Additional experiments demonstrated that Serrumab was also able to neutralize the action of toxins from other scorpion genus (Css II, 45.96% and Lqh III, 100%/β- and α-toxins, respectively). This work indicated that Serrumab is able to neutralize many toxins in Ts venom, and could being considered as a neutralizing antibody for formulating a human anti-scorpion serum in Brazil. Additionally, this work demonstrated that Serrumab could neutralize different toxins from distinct scorpion genus. All these results reinforce the idea that Serrumab is a scFv antibody with multiple neutralizing capacities and a promising candidate for inclusion in scorpion anti-venoms against different genera.
A correct alignment is an essential requirement in homology modeling. Yet in order to bridge the structural gap between template and target, which may not only involve loop rearrangements, but also shifts of secondary structure elements and repacking of core residues, high-resolution refinement methods with full atomic details are needed. Here, we describe four approaches that address this “last mile of the protein folding problem” and have performed well during CASP8, yielding physically realistic models: YASARA, which runs molecular dynamics simulations of models in explicit solvent, using a new partly knowledge-based all atom force field derived from Amber, whose parameters have been optimized to minimize the damage done to protein crystal structures. The LEE-SERVER, which makes extensive use of conformational space annealing to create alignments, to help Modeller build physically realistic models while satisfying input restraints from templates and CHARMM stereochemistry, and to remodel the side-chains. ROSETTA, whose high resolution refinement protocol combines a physically realistic all atom force field with Monte Carlo minimization to allow the large conformational space to be sampled quickly. And finally UNDERTAKER, which creates a pool of candidate models from various templates and then optimizes them with an adaptive genetic algorithm, using a primarily empirical cost function that does not include bond angle, bond length, or other physics-like terms. Proteins 2009. © 2009 Wiley-Liss, Inc.
9C2 is a murine monoclonal IgG that participates in the neutralization of Androctonus australis hector scorpion venom. It recognizes AahI and AahIII, two of the three main neurotoxins responsible for almost all the toxicity of the venom when injected into mammals. Using PCR we cloned the antibody variable region coding genes from 9C2 hybridoma cells and constructed a gene encoding a single-chain antibody variable fragment molecule (scFv). This scFv was produced in the periplasm of Escherichia coli in a soluble and functional form and purified in a single step using protein L−agarose beads yielding 1–2 mg·L−1 of bacterial culture. scFv9C2 was predominantly monomeric but also tended to form dimeric and oligomeric structures, all capable of binding toxin AahI. The affinity of scFv and the parental mAb for toxin AahI and homologous toxin AahIII was of the same magnitude, in the nanomolar range. Similarly, purified forms of scFv9C2 completely inhibited the binding of toxin AahI to rat brain synaptosomes. Finally, scFv9C2 was efficient in protecting mice against the toxic effects of AahI after injection of the toxin and scFv to mice by the intracerebroventricular route in a molar ratio as low as 0.36 : 1. Thus, we produced a recombinant scFv that reproduces the recognition properties of the parent antibody and neutralizes the scorpion neurotoxin AahI, thereby opening new prospects for the treatment of envenomation.
Biologically relevant macromolecules, such as proteins, do not operate as static, isolated entities. On the contrary, they are involved in numerous interactions with other species, such as proteins, nucleic acid, membranes, small molecule ligands, and also, critically, solvent molecules. These interactions often display a remarkable degree of specificity and high affinity. Fundamentally, the biological processes rely on molecular organisation and recognition events. Binding between two interacting partners has both enthalpic (ΔH) and entropic (-TΔS) components, which means the recognition event is associated with changes of both the structure and dynamics of each counterpart. Like any other spontaneous process, binding occurs only when it is associated with a negative Gibbs' free energy of binding ( Δ G ), which may have differing thermodynamic signatures, varying from enthalpy- to entropy-driven. Thus, the understanding of the forces driving the recognition and interaction require a detailed description of the binding thermodynamics, and a correlation of the thermodynamic parameters with the structures of interacting partners. Such an understanding of the nature of the recognition phenomena is of a great importance for medicinal chemistry and material research, since it enables truly rational structure-based molecular design.
The standard method of dealing with sensitivity of dosage-mortality data is the probit technique developed by Bliss and Fisher. This paper provides an alternative technique based on a special system for obtaining such data. It has some advantages when observations must be taken on individuals rather than groups of individuals, and it may be preferred in certain other situations.
A methodology using biosensor technology for combined kinetic and thermodynamic analysis of biomolecular interactions is described. Rate and affinity constants are determined with BIAcore. Thermodynamics parameters, changes in free energy, enthalpy and entropy, are evaluated from equilibrium data and by using rate constants and transition state theory. The methodology using van't Hoff theory gives complementary information to microcalorimetry, since only the direct binding is measured with BIAcore whereas microcalorimetry measures all components, including e.g. hydration effects. Furthermore, BIAcore gives possibilities to gain new information by thermodynamic analysis of the rate constants.
This chapter reviews that the popularity of using biosensors to characterize biomolecular interactions continues to grow. These instruments are capable of providing information about the energetics of macromolecular interactions without labeling. While commercial instruments such as BIACORE are relatively easy to use, extracting accurate rate constants for a reaction requires careful experimental technique and robust methods of data analysis. It discusses how biosensors can be used as a biophysical tool to determine equilibrium constants and kinetic rate constants for binding events. By measuring reactions at different temperatures, it is possible to extract thermodynamic information about the system. The model system used in this study involves the interaction of interleukin 2 (IL-2) ligand with the α subunit of its receptor. The chapter concludes that BIACORE is a powerful tool used to measure the reaction kinetics and equilibrium constants for biological molecules. Improved methods for data collection and processing make it possible to dramatically improve the quality of biosensor data. The ability to describe binding data with simple interaction models validates the technology as a tool for kinetic analysis. Recording the temperature dependence of reaction parameters makes it possible to extract some thermodynamic information about the binding reaction. Together, kinetic and thermodynamic data will increase the understanding of biomolecular recognition events.
This study describes the construction of a library of single-chain antibody fragments (scFvs) from a single human donor by individual amplification of all heavy and light variable domains (1.1 x 10(8) recombinants). The library was panned using the phage display technique, which allowed selection of specific scFvs (3F and C1) capable of recognizing Cn2, the major toxic component of Centruroides noxius scorpion venom. The scFv 3F was matured in vitro by three cycles of directed evolution. The use of stringent conditions in the third cycle allowed the selection of several improved clones. The best scFv obtained (6009F) was improved in terms of its affinity by 446-fold, from 183 nm (3F) to 410 pm. This scFv 6009F was able to neutralize 2 LD(50) of Cn2 toxin when a 1 : 10 molar ratio of toxin-to-antibody fragment was used. It was also able to neutralize 2 LD(50) of the whole venom. These results pave the way for the future generation of recombinant human antivenoms.
We discuss basic physical-chemical principles underlying the formation of stable macromolecular complexes, which in many cases are likely to be the biological units performing a certain physiological function. We also consider available theoretical approaches to the calculation of macromolecular affinity and entropy of complexation. The latter is shown to play an important role and make a major effect on complex size and symmetry. We develop a new method, based on chemical thermodynamics, for automatic detection of macromolecular assemblies in the Protein Data Bank (PDB) entries that are the results of X-ray diffraction experiments. As found, biological units may be recovered at 80-90% success rate, which makes X-ray crystallography an important source of experimental data on macromolecular complexes and protein-protein interactions. The method is implemented as a public WWW service.
Voltage-gated potassium channels of the ether-a-go-go related gene (ERG) family are implicated in many important cellular processes. Three such genes have been cloned (erg1, erg2 and erg3) and shown to be expressed in the central nervous system (CNS) of mammalians. This communication describes the isolation and characterization of two isoforms of scorpion toxin (CeErg4 and CeErg5, systematic nomenclature gamma-KTx1.7 and gamma-KTx1.8, respectively) that can discriminate the various subtypes of ERG channels of human and rat. These peptides were purified from the venom of the Mexican scorpion Centruroides elegans elegans. They contain 42 amino acid residues, tightly folded by four disulfide bridges. Both peptides block in a reversible manner human and rat ERG1 channels, but have no effect on human ERG2. They also block completely and irreversibly the rat ERG2 and the human ERG3 channels hence are excellent tools for the discrimination of the various sub-types of ion-channels studied.
Immunoreactivity and pharmacokinetics of horse anti-scorpion venom F(ab')2-scorpion venom interactions
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Casos nuevos de Intoxicación por picadura de alacrán (T63.2, X22) por fuente de notificación. Dirección General de Epidemiología
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