RESUMEN
BACKGROUND: Snakebite envenomation inflicts a high burden of mortality and morbidity in sub-Saharan Africa. Antivenoms are the mainstay in the therapy of envenomation, and there is an urgent need to develop antivenoms of broad neutralizing efficacy for this region. The venoms used as immunogens to manufacture snake antivenoms are normally selected considering their medical importance and availability. Additionally, their ability to induce antibody responses with high neutralizing capability should be considered, an issue that involves the immunization scheme and the animal species being immunized. METHODOLOGY/PRINCIPAL FINDINGS: Using the lethality neutralization assay in mice, we compared the intrageneric neutralization scope of antisera generated by immunization of horses with monospecific, bispecific/monogeneric, and polyspecific/monogeneric immunogens formulated with venoms of Bitis spp., Echis spp., Dendroaspis spp., spitting Naja spp. or non-spitting Naja spp. It was found that the antisera raised by all the immunogens were able to neutralize the homologous venoms and, with a single exception, the heterologous congeneric venoms (considering spitting and non-spitting Naja separately). In general, the polyspecific antisera of Bitis spp, Echis spp, and Dendroaspis spp gave the best neutralization profile against venoms of these genera. For spitting Naja venoms, there were no significant differences in the neutralizing ability between monospecific, bispecific and polyspecific antisera. A similar result was obtained in the case of non-spitting Naja venoms, except that polyspecific antiserum was more effective against the venoms of N. melanoleuca and N. nivea as compared to the monospecific antiserum. CONCLUSIONS/SIGNIFICANCE: The use of polyspecific immunogens is the best alternative to produce monogeneric antivenoms with wide neutralizing coverage against venoms of sub-Saharan African snakes of the Bitis, Echis, Naja (non-spitting) and Dendroaspis genera. On the other hand, a monospecific immunogen composed of venom of Naja nigricollis is suitable to produce a monogeneric antivenom with wide neutralizing coverage against venoms of spitting Naja spp. These findings can be used in the design of antivenoms of wide neutralizing scope for sub-Saharan Africa.
Asunto(s)
Antivenenos , Pruebas de Neutralización , Animales , Caballos/inmunología , Antivenenos/inmunología , Antivenenos/administración & dosificación , Ratones , África del Sur del Sahara , Anticuerpos Neutralizantes/inmunología , Anticuerpos Neutralizantes/sangre , Venenos de Serpiente/inmunología , Sueros Inmunes/inmunología , Venenos Elapídicos/inmunología , Mordeduras de Serpientes/inmunologíaRESUMEN
Single-domain antibodies (sdAbs) hold promise for developing new biopharmaceuticals to treat neglected tropical diseases (NTDs), including snakebites, which are severe and occur frequently. In addition, limitations of conventional snakebite treatments, especially in terms of local action, and the global antivenom crisis incentivize the use of this biotechnological tool to design next-generation snakebite antivenoms. Conventional antivenoms for snakebite treatment are usually composed of immunoglobulin G or F(ab')2 fragments derived from the plasma of immunized animals. sdAbs, the smallest antigen-binding fragments, are derived from the variable domains of camelid heavy-chain antibodies. sdAbs may have some advantages over conventional antivenoms for local toxicity, such as better penetration into tissues due to their small size, and high solubility and affinity for venom antigens due to their unique antigen-binding loops and ability to access cryptic epitopes. We present an overview of current antivenom therapy in the context of sdAb development for toxin neutralization. Furthermore, strategies are presented for identifying snake venom's major toxins as well as for developing antisnake toxin sdAbs by employing proteomic tools for toxin neutralization.
Asunto(s)
Antivenenos , Proteómica , Anticuerpos de Dominio Único , Mordeduras de Serpientes , Venenos de Serpiente , Animales , Humanos , Antivenenos/inmunología , Proteómica/métodos , Anticuerpos de Dominio Único/inmunología , Anticuerpos de Dominio Único/farmacología , Mordeduras de Serpientes/tratamiento farmacológico , Mordeduras de Serpientes/inmunología , Venenos de Serpiente/inmunologíaRESUMEN
Systemic complement activation drives a plethora of pathological conditions, but its role in snake envenoming remains obscure. Here, we explored complement's contribution to the physiopathogenesis of Naja annulifera envenomation. We found that N. annulifera venom promoted the generation of C3a, C4a, C5a, and the soluble Terminal Complement Complex (sTCC) mediated by the action of snake venom metalloproteinases. N. annulifera venom also induced the release of lipid mediators and chemokines in a human whole-blood model. This release was complement-mediated, since C3/C3b and C5a Receptor 1 (C5aR1) inhibition mitigated the effects. In an experimental BALB/c mouse model of envenomation, N. annulifera venom promoted lipid mediator and chemokine production, neutrophil influx, and swelling at the injection site in a C5a-C5aR1 axis-dependent manner. N. annulifera venom induced systemic complementopathy and increased interleukin and chemokine production, leukocytosis, and acute lung injury (ALI). Inhibition of C5aR1 with the cyclic peptide antagonist PMX205 rescued mice from these systemic reactions and abrogated ALI development. These data reveal hitherto unrecognized roles for complement in envenomation physiopathogenesis, making complement an interesting therapeutic target in envenomation by N. annulifera and possibly by other snake venoms.
Asunto(s)
Activación de Complemento/inmunología , Complemento C5a/inmunología , Complemento C5a/metabolismo , Receptor de Anafilatoxina C5a/metabolismo , Venenos de Serpiente/inmunología , Animales , Biomarcadores , Quimiocinas/metabolismo , Modelos Animales de Enfermedad , Humanos , Hidrólisis , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Mediadores de Inflamación/metabolismo , Masculino , Ratones , Modelos Biológicos , Naja , Unión Proteica , Transducción de Señal , Mordeduras de SerpientesRESUMEN
Most antivenoms are produced by techniques developed over 50 years ago, with minor modifications. Herein we revise the core of traditional antivenom production processes aiming to optimize key determinants for both consistent antivenom production and the best balance between F(ab')2 quality and recovery. Factorial design analysis revealed that pepsin digestion of 1:3 saline diluted equine plasma for 60 min under pH: 3.20, 37 °C temperature and a 1:15 pepsin to protein ratio conditions, allowed to achieve maximal IgG to F(ab')2 conversion with minimal protein aggregate formation. Further downstream processing by salting out with ammonium sulfate was also studied by factorial analysis. The influence of ammonium sulfate (AS) concentration, temperature (T) and the albumin to total plasma protein ratio plasma (Alb:P) were assayed, revealing that both AS, T and their interaction have a significant impact in F(ab')2 quality and recovery. Taking into account the existing compromise between F(ab')2 monomer recovery and quality two alternative conditions were selected: 14 g/dl AS at 56 °C and, alternatively 16 g/dl AS at 30 °C. Reasonable yields (42%) and product quality (2.5% of aggregates) without significant changes in production cost of traditional methodologies was achieved under the optimized conditions found.
Asunto(s)
Antivenenos/inmunología , Caballos/inmunología , Fragmentos Fab de Inmunoglobulinas/inmunología , Pepsina A/metabolismo , Mordeduras de Serpientes/inmunología , Venenos de Serpiente/inmunología , Sulfato de Amonio/química , Sulfato de Amonio/metabolismo , Animales , Antivenenos/sangre , Antivenenos/metabolismo , Proteínas Sanguíneas/metabolismo , Caprilatos/química , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Electroforesis en Gel de Poliacrilamida , Caballos/sangre , Humanos , Fragmentos Fab de Inmunoglobulinas/sangre , Fragmentos Fab de Inmunoglobulinas/metabolismo , Papaína/metabolismo , Albúmina Sérica/metabolismo , Mordeduras de Serpientes/prevención & controlRESUMEN
Snakebites caused by Crotalus genus are the second most frequent in Brazil. Crotoxin is a beta-neurotoxin responsible for the main envenomation effects of Crotalus biting, while crotamine immobilizes the animal hind limbs, contributing to prey immobilization and to envenoming symptoms. As crotoxin and crotamine represent about 90% of Crotalus venom dry weight, these toxins are of great importance for antivenom therapy. In this sense, knowledge regarding the antigenicity/immunogenicity at the molecular level of these toxins can provide valuable information for the improvement of specific antivenoms. Therefore, the aims of this study are the identification of the B-cell epitopes from crotoxin and crotamine; and the characterization of the neutralizing potency of antibodies directed against the corresponding synthetic epitopes defined in the current study. Linear B-cell epitopes were identified using the Spot Synthesis technique probed with specific anti-C. d. terrificus venom horse IgG. One epitope of crotamine (F12PKEKICLPPSSDFGKMDCRW32) and three of crotoxin (L10LVGVEGHLLQFNKMIKFETR30; Y43CGWGGRGRPKDATDRCCFVH63 and T118YKYGYMFYPDSRCRGPSETC138) were identified. After synthesis in their soluble form, the peptides mixture correspondent to the mapped epitopes was entrapped in liposomes and used as immunogens for antibody production in rabbits. Anti-synthetic peptide antibodies were able to protect mice from the lethal activity of C. d. terrificus venom.
Asunto(s)
Crotalus/inmunología , Epítopos/inmunología , Liposomas , Venenos de Serpiente/inmunología , Secuencia de Aminoácidos , Anafilaxia/inmunología , Anafilaxia/prevención & control , Animales , Antivenenos/administración & dosificación , Antivenenos/inmunología , Crotoxina/química , Crotoxina/inmunología , Modelos Animales de Enfermedad , Mapeo Epitopo , Epítopos/administración & dosificación , Epítopos/química , Femenino , Inmunoglobulina G/inmunología , Ratones , Modelos Moleculares , Pruebas de Neutralización , Péptidos/química , Péptidos/inmunología , Conformación Proteica , Conejos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Envenomation by viperid snakes is characterized by systemic thrombotic syndrome and prominent local inflammation. To date, the mechanisms underlying inflammation and blood coagulation induced by Viperidae venoms have been viewed as distinct processes. However, studies on the mechanisms involved in these processes have revealed several factors and signaling molecules that simultaneously act in both the innate immune and hemostatic systems, suggesting an overlap between both systems during viper envenomation. Moreover, distinct classes of venom toxins involved in these effects have also been identified. However, the interplay between inflammation and hemostatic alterations, referred as to thromboinflammation, has never been addressed in the investigation of viper envenomation. Considering that platelets are important targets of viper snake venoms and are critical for the process of thromboinflammation, in this review, we summarize the inflammatory effects and mechanisms induced by viper snake venoms, particularly from the Bothrops genus, which strongly activate platelet functions and highlight selected venom components (metalloproteases and C-type lectins) that both stimulate platelet functions and exhibit pro-inflammatory activities, thus providing insights into the possible role(s) of thromboinflammation in viper envenomation.
Asunto(s)
Inflamación/inmunología , Mordeduras de Serpientes/inmunología , Venenos de Serpiente/inmunología , Trombosis/inmunología , Animales , Coagulación Sanguínea , Hemostasis , Humanos , Lectinas Tipo C/metabolismo , Metaloproteasas/metabolismo , Activación Plaquetaria , ViperidaeRESUMEN
Most colubrid snake venoms have been poorly studied, despite the fact that they represent a great resource for biological, ecological, toxinological and pharmacological research. Herein, we explore the venom delivery system of the Aesculapian False Coral Snake Erythrolamprus aesculapii as well as some biochemical and toxicological properties of its venom. Its Duvernoy's venom gland is composed of serous secretory cells arranged in densely packed secretory tubules, and the most striking feature of its fang is their double-curved shape, exhibiting a beveled bladelike appearance near the tips. Although E. aesculapii resembles elapid snakes of the genus Micrurus in color pattern, this species produces a venom reminiscent of viperid venoms, containing mainly tissue-damaging toxins such as proteinases. Prominent hemorrhage developed both locally and systemically in mice injected with the venom, and the minimum hemorrhagic dose was found to be 18.8 µg/mouse; the lethal dose, determined in mice, was 9.5⯱â¯3.7⯵g/g body weight. This work has toxicological implications that bites to humans by E. aesculapii could result in moderately severe local (and perhaps systemic) hemorrhage and gives insight into future directions for research on the venom of this species.
Asunto(s)
Colubridae/anatomía & histología , Venenos de Serpiente/química , Venenos de Serpiente/toxicidad , Animales , Antivenenos/inmunología , Glándulas Exocrinas/anatomía & histología , Femenino , Hemorragia/inducido químicamente , Humanos , Masculino , Maxilar/ultraestructura , Ratones , Microscopía Electrónica de Rastreo , Proteolisis , Mordeduras de Serpientes , Venenos de Serpiente/inmunología , Diente/ultraestructuraRESUMEN
Emulsions are crucial in the treatment of snake bites to bust the antibody response of the inmunogen. The widely used Freund's emulsion typically combines 50/50 water-oil (W/O) phase. However, its use is limited because it is associated with tissue damage. We formulated and characterized a Pickering Emulsion 70/30 (W/O) that uses a chemically modified hydrophobic hydroxyapatite as surfactant. This Pickering emulsion has similar rheologic behavior to Freund's emulsion 50/50, but with lower oil and surfactant concentration. Evaluation of cell recruitment, antibody response and adhering tissue in mice immunized with B. asper of Pacific venom and treated with Freund's and Pickering 70/30 emulsions resulted in similar adjuvant activity (only 18% lower in Pickering 70/30 emulsion). However, Pickering 70/30 emulsions minimized negative side effects in the host animals and showed better ease of flow that favors injection of the host. Our results open up room for optimization and improvement of Pickering emulsion based on modified nanoparticles for medical applications.
Asunto(s)
Adyuvantes Inmunológicos/química , Anticuerpos/metabolismo , Durapatita/química , Emulsiones/química , Nanopartículas/química , Venenos de Serpiente/inmunología , Animales , Ratones , Venenos de Serpiente/química , Serpientes/metabolismo , Tensoactivos/químicaRESUMEN
Envenoming and deaths resulting from snakebites are a particularly important public health problem in rural tropical areas of Africa, Asia, Latin America, and New Guinea. In 2015, The Lancet highlighted snake-bite envenoming as a neglected tropical disease and urged the world to increase antivenom production. In Brazil, around 20,000 snakebites occur per year affecting mostly agricultural workers and children, of which 1% is caused by coral snakes (Micrurus sp.). Although human envenoming by coral snakes is relatively rare due to their semifossorial habits and nonaggressive behavior, they are always considered severe due to the neurotoxic, myotoxic, hemorrhagic, and cardiovascular actions of their venom, which is highly toxic when compared to the venom of other Brazilian venomous snakes as Bothrops sp. (pit vipers), Crotalus sp. (rattlesnakes), and Lachesis sp. (bushmasters). The production of antivenom serum is an important public health issue worldwide and the maintenance of venomous snakes in captivity essential to obtain high-quality venom. Though more than 30 species of Brazilian coral snakes exist, the specific antivenom serum produced with the venom of two species, Micrurus corallinus and M. frontalis, is able to neutralize the accidents caused by the genus in general. M. corallinus is considered a difficult species to maintain in captivity and concerned about this difficulty the Laboratory of Herpetology (LH) at Instituto Butantan, over the last 10 yr, has given special attention to its maintenance in captivity. In more than 20 yr of maintenance, LH has made some changes to improve Micrurus captive husbandry and welfare. The objective of this study was to verify the factors influencing the survival rates of coral snakes in captivity through data generated from 289 M. corallinus from the LH snake facility in the last 10 yr. We observed that survival rates increased significantly with the improvement of nutritional adequacy that included freezing food items before offering them to coral snakes, as well as the development of a new pasty diet to force-feed anorexic animals. Another important factor responsible for increasing life expectancy was the shift of the cage's substrate from Sphagnum to bark in 2010, aiding in the eradication of Blister Disease, which used to be responsible for the death of several coral snakes in previous years.
Asunto(s)
Crianza de Animales Domésticos , Bienestar del Animal , Antivenenos/metabolismo , Serpientes de Coral/fisiología , Venenos de Serpiente/inmunología , Animales , Brasil , Humanos , Esperanza de Vida , Mordeduras de Serpientes , Tasa de SupervivenciaRESUMEN
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.
Asunto(s)
Antivenenos/administración & dosificación , Antivenenos/biosíntesis , Mordeduras de Serpientes/tratamiento farmacológico , Picaduras de Arañas/tratamiento farmacológico , Animales , Antivenenos/inmunología , Humanos , Venenos de Serpiente/inmunología , Venenos de Araña/inmunologíaRESUMEN
Epitope identification is essential for developing effective antibodies that can detect and neutralize bioactive proteins. Computational prediction is a valuable and time-saving alternative for experimental identification. Current computational methods for epitope prediction are underused and undervalued due to their high false positive rate. In this work, we targeted common properties of linear B-cell epitopes identified in an individual protein class (metalloendopeptidases) and introduced an alternative method to reduce the false positive rate and increase accuracy, proposing to restrict predictive models to a single specific protein class. For this purpose, curated epitope sequences from metalloendopeptidases were transformed into frame-shifted Kmers (3 to 15 amino acid residues long). These Kmers were decomposed into a matrix of biochemical attributes and used to train a decision tree classifier. The resulting prediction model showed a lower false positive rate and greater area under the curve when compared to state-of-the-art methods. Our predictions were used for synthesizing peptides mimicking the predicted epitopes for immunization of mice. A predicted linear epitope that was previously undetected by an experimental immunoassay was able to induce neutralizing-antibody production in mice. Therefore, we present an improved prediction alternative and show that computationally identified epitopes can go undetected during experimental mapping.
Asunto(s)
Anticuerpos Neutralizantes/biosíntesis , Biología Computacional/métodos , Epítopos de Linfocito B/inmunología , Venenos de Serpiente/inmunología , Algoritmos , Secuencia de Aminoácidos , Aminoácidos/química , Animales , Árboles de Decisión , Mapeo Epitopo , Epítopos de Linfocito B/química , Femenino , Inmunización , Metaloproteasas/metabolismo , Ratones Endogámicos BALB C , Modelos Moleculares , Péptidos/química , Curva ROC , Reproducibilidad de los ResultadosRESUMEN
Snake venoms are complex mixtures of organic and inorganic compounds, including proteins belonging to the protease (serine and metalloproteinases), oxidase (L-amino acid oxidases), and phospholipase (especially phospholipases A2) enzyme classes. These toxins account for the serious deleterious effects of snake envenomations, such as tissue necrosis, neurotoxicity, and hemorrhage. In addition to their toxic effects, snake venom toxins have served as important tools for investigating the mechanisms underlying envenomation and discovering new pharmacologically active compounds with immunotherapeutic potential. In this sense, the present review discusses the new findings and therapeutic perspectives in the immune modulating potential of enzymatic toxins from snake venoms belonging to the classes metalloproteinase, serine protease, L-amino acid oxidase, and phospholipase A2.
Asunto(s)
Enzimas/química , Enzimas/metabolismo , Venenos de Serpiente/química , Venenos de Serpiente/enzimología , Toxinas Biológicas/química , Toxinas Biológicas/metabolismo , Animales , Enzimas/inmunología , Humanos , Inmunomodulación , Mordeduras de Serpientes/inmunología , Mordeduras de Serpientes/metabolismo , Mordeduras de Serpientes/patología , Mordeduras de Serpientes/terapia , Venenos de Serpiente/inmunología , Venenos de Serpiente/uso terapéutico , Toxinas Biológicas/inmunologíaRESUMEN
Polyclonal antibodies raised in Balb-c mice against BnSP-7, a Lys-49 phospholipase A2, were used to measure cross reactivity against other snake venoms. Using ELISA, these antibodies were able to recognize PLA2s isoforms present in venoms of botropic snakes at 1:6400, 1:3200 and 1:100 ratios (w/w). These antibodies highly recognized proteins of low molecular weight (â¼14,000) from crude snake venom Bp and Bm by Western Blotting. PLA2 these venoms, by alignment of primary structures demonstrated high identity with BnSP-7 PLA2, especially in the C-terminal region. However, the crude snake venom Bd and Bj, showed low recognition. The PLA2 activity of Bothrops pauloensis, Bothrops moojeni venoms or BpPLA2-TXI was inhibited significantly when anti-BnSP-7 antibodies were incubated at 1:10 and 1:20 ratios (venoms or toxin:anti-BnSP-7, w/w), respectively. The myotoxic effect induced by the same venoms was also reduced significantly at 1:1, 1:10 and 1:20 ratios, by decreased creatine kinase levels. The anti-PLA2 polyclonal antibodies effectively recognized PLA2s from Bothrops pauloensis and Bothrops moojeni venoms, and neutralized specific catalytic and myotoxic activity.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Bothrops/inmunología , Reacciones Cruzadas/inmunología , Venenos de Crotálidos/inmunología , Fosfolipasas A2/inmunología , Venenos de Serpiente/inmunología , Secuencia de Aminoácidos , Animales , Western Blotting , Bothrops/clasificación , Bothrops/metabolismo , Venenos de Crotálidos/metabolismo , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratones Endogámicos BALB C , Pruebas de Neutralización , Fosfolipasas A2/genética , Fosfolipasas A2/metabolismo , Homología de Secuencia de Aminoácido , Venenos de Serpiente/metabolismo , Especificidad de la EspecieRESUMEN
L-amino acid oxidases from snake venoms have been described to possess various biological functions. In this study, we investigated the inflammatory responses induced in vivo and in vitro by CR-LAAO, an L-amino acid oxidase isolated from Calloselasma rhodostoma venom, and its antitumor potential. CR-LAAO induced acute inflammatory responses in vivo, with recruitment of neutrophils and release of IL-6, IL-1ß, LTB4 and PGE2. In vitro, IL-6 and IL-1ß production by peritoneal macrophages stimulated with CR-LAAO was dependent of the activation of the Toll-like receptors TLR2 and TLR4. In addition, CR-LAAO promoted apoptosis of HL-60 and HepG2 tumor cells mediated by the release of hydrogen peroxide and activation of immune cells, resulting in oxidative stress and production of IL-6 and IL-1ß that triggered a series of events, such as activation of caspase 8, 9 and 3, and the expression of the pro-apoptotic gene BAX. We also observed that CR-LAAO modulated the cell cycle of these tumor cells, promoting delay in the G0/G1 and S phases. Taken together, our results suggest that CR-LAAO could serve as a potential tool for the development of novel immunotherapeutic strategies against cancer, since this toxin promoted apoptosis of tumor cells and also activated immune cells against them.
Asunto(s)
L-Aminoácido Oxidasa/metabolismo , Venenos de Serpiente/enzimología , Viperidae/metabolismo , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasas/metabolismo , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Línea Celular Tumoral , Citocinas/metabolismo , Humanos , Inmunoterapia , Mediadores de Inflamación/metabolismo , L-Aminoácido Oxidasa/inmunología , L-Aminoácido Oxidasa/farmacología , L-Aminoácido Oxidasa/uso terapéutico , Neoplasias/inmunología , Neoplasias/metabolismo , Neoplasias/terapia , Infiltración Neutrófila , Neutrófilos/inmunología , Neutrófilos/metabolismo , Neutrófilos/patología , Estrés Oxidativo/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Venenos de Serpiente/inmunología , Venenos de Serpiente/farmacología , Venenos de Serpiente/uso terapéutico , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 4/metabolismoRESUMEN
This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year.(AU)
Asunto(s)
Animales , Venenos de Serpiente/inmunología , Proteómica , AntivenenosRESUMEN
This work offers a general overview on the evolving strategies for the proteomic analysis of snake venoms, and discusses how these may be combined through diverse experimental approaches with the goal of achieving a more comprehensive knowledge on the compositional, toxic, and immunological characteristics of venoms. Some recent developments in this field are summarized, highlighting how strategies have evolved from the mere cataloguing of venom components (proteomics/venomics), to a broader exploration of their immunological (antivenomics) and functional (toxicovenomics) characteristics. Altogether, the combination of these complementary strategies is helping to build a wider, more integrative view of the life-threatening protein cocktails produced by venomous snakes, responsible for thousands of deaths every year.(AU)
Asunto(s)
Animales , Venenos de Serpiente/inmunología , Proteómica , AntivenenosRESUMEN
BACKGROUND: Envenoming by coral snakes (Elapidae: Micrurus), although not abundant, represent a serious health threat in the Americas, especially because antivenoms are scarce. The development of adequate amounts of antielapidic serum for the treatment of accidents caused by snakes like Micrurus corallinus is a challenging task due to characteristics such as low venom yield, fossorial habit, relatively small sizes and ophiophagous diet. These features make it difficult to capture and keep these snakes in captivity for venom collection. Furthermore, there are reports of antivenom scarcity in USA, leading to an increase in morbidity and mortality, with patients needing to be intubated and ventilated while the toxin wears off. The development of an alternative method for the production of an antielapidic serum, with no need for snake collection and maintenance in captivity, would be a plausible solution for the antielapidic serum shortage. METHODS AND FINDINGS: In this work we describe the mapping, by the SPOT-synthesis technique, of potential B-cell epitopes from five putative toxins from M. corallinus, which were used to design two multiepitope DNA strings for the genetic immunisation of female BALB/c mice. Results demonstrate that sera obtained from animals that were genetically immunised with these multiepitope constructs, followed by booster doses of recombinant proteins lead to a 60% survival in a lethal dose neutralisation assay. CONCLUSION: Here we describe that the genetic immunisation with a synthetic multiepitope gene followed by booster doses with recombinant protein is a promising approach to develop an alternative antielapidic serum against M. corallinus venom without the need of collection and the very challenging maintenance of these snakes in captivity.
Asunto(s)
Antivenenos/inmunología , Antivenenos/farmacología , Elapidae/inmunología , Venenos de Serpiente/inmunología , Tecnología Farmacéutica/métodos , Vacunas de ADN/inmunología , Animales , Modelos Animales de Enfermedad , Elapidae/genética , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Femenino , Ratones Endogámicos BALB C , Pruebas de Neutralización , Mordeduras de Serpientes/terapia , Venenos de Serpiente/genética , Vacunación/métodos , Vacunas de ADN/administración & dosificación , Vacunas de Subunidad/administración & dosificación , Vacunas de Subunidad/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunologíaRESUMEN
BACKGROUND: Snake envenoming is a significant public health problem in underdeveloped and developing countries. In sub-Saharan Africa, it is estimated that 90,000-400,000 envenomations occur each year, resulting in 3,500-32,000 deaths. Envenomings are caused by snakes from the Viperidae (Bitis spp. and Echis spp.) and Elapidae (Naja spp. and Dendroaspis spp.) families. The African continent has been suffering from a severe antivenom crisis and current antivenom production is only sufficient to treat 25% of snakebite cases. Our aim is to develop high-quality antivenoms against the main snake species found in Mozambique. METHODS: Adult horses primed with the indicated venoms were divided into 5 groups (B. arietans; B. nasicornis + B. rhinoceros; N. melanoleuca; N. mossambica; N. annulifera + D. polylepis + D. angusticeps) and reimmunized two times for antivenom production. Blood was collected, and plasma was separated and subjected to antibody purification using caprylic acid. Plasmas and antivenoms were subject to titration, affinity determination, cross-recognition assays and in vivo venom lethality neutralization. A commercial anti-Crotalic antivenom was used for comparison. RESULTS: The purified antivenoms exhibited high titers against B. arietans, B. nasicornis and B. rhinoceros (5.18 x 106, 3.60 x 106 and 3.50 x 106 U-E/mL, respectively) and N. melanoleuca, N. mossambica and N. annulifera (7.41 x 106, 3.07 x 106 and 2.60 x 106 U-E/mL, respectively), but lower titers against the D. angusticeps and D. polylepis (1.87 x 106 and 1.67 x 106 U-E/mL). All the groups, except anti-N. melanoleuca, showed significant differences from the anti-Crotalic antivenom (7.55 x 106 U-E/mL). The affinity index of all the groups was high, ranging from 31% to 45%. Cross-recognition assays showed the recognition of proteins with similar molecular weight in the venoms and may indicate the possibility of paraspecific neutralization. The three monospecific antivenoms were able to provide in vivo protection. CONCLUSION: Our results indicate that the anti-Bitis and anti-Naja antivenoms developed would be useful for treating snakebite envenomations in Mozambique, although their effectiveness should to be increased. We propose instead the development of monospecific antivenoms, which would serve as the basis for two polyvalent antivenoms, the anti-Bitis and anti-Elapidae. Polyvalent antivenoms represent an increase in treatment quality, as they have a wider range of application and are easier to distribute and administer to snake envenoming victims.
Asunto(s)
Antivenenos/inmunología , Caballos/inmunología , Inmunoglobulina G/inmunología , Venenos de Serpiente/inmunología , Serpientes/clasificación , Animales , Antivenenos/clasificación , Mozambique , Venenos de Serpiente/clasificaciónRESUMEN
Snakebite envenoming constitutes an important public health problem in Latin America and some countries of the Caribbean. The advances and pending tasks in the study and control of this neglected tropical disease in this region are reviewed in the light of a roadmap proposed in 2006. Significant progress has been achieved in the study of snake venoms, particularly regarding venom proteomics, i.e.'venomics', and the analysis of the mechanism of action of toxins. Likewise, a deeper understanding has been gained in the preclinical efficacy of antivenoms produced in the region. In contrast, despite advances made in the study of clinical manifestations of envenomings and safety and efficacy of antivenoms at the clinical level, much remains to be done in this subject. Improvements have occurred in antivenom manufacturing technologies and availability, although there are still countries where there is insufficient supply of antivenoms, or where manufacture has to be improved. In spite of considerable efforts in some countries in prevention, accessibility to treatment, and training of health staff in the management of envenomings, important challenges remain for the region as a whole, with the long term goal of reducing the impact of this disease in terms of personal and social suffering.
Asunto(s)
Antivenenos/uso terapéutico , Educación Médica Continua/normas , Adhesión a Directriz , Salud Pública , Mordeduras de Serpientes/tratamiento farmacológico , Venenos de Serpiente/análisis , Animales , Antivenenos/farmacología , Región del Caribe/epidemiología , Guías como Asunto , Conocimientos, Actitudes y Práctica en Salud , Humanos , América Latina/epidemiología , Proteómica/tendencias , Control de Calidad , Mordeduras de Serpientes/epidemiología , Mordeduras de Serpientes/inmunología , Venenos de Serpiente/inmunología , Venenos de Serpiente/toxicidad , Serpientes , Especificidad de la EspecieRESUMEN
BACKGROUND: The snake Bothrops atrox is responsible for the majority of envenomings in the northern region of South America. Severe local effects, including hemorrhage, which are mainly caused by snake venom metalloproteinases (SVMPs), are not fully neutralized by conventional serum therapy. Little is known about the immunochemistry of the P-I SVMPs since few monoclonal antibodies (mAbs) against these molecules have been obtained. In addition, producing toxin-neutralizing mAbs remains very challenging. METHODOLOGY/PRINCIPAL FINDINGS: Here, we report on the set-up of a functional screening based on a synthetic peptide used as a biosensor to select neutralizing mAbs against SVMPs and the successful production of neutralizing mAbs against Atroxlysin-I (Atr-I), a P-I SVMP from B. atrox. Hybridomas producing supernatants with inhibitory effect against the proteolytic activity of Atr-I towards the FRET peptide Abz-LVEALYQ-EDDnp were selected. Six IgG1 Mabs were obtained (named mAbatr1 to mAbatr6) and also two IgM. mAbatrs1, 2, 3 and 6 were purified. All showed a high specific reactivity, recognizing only Atr-I and B. atrox venom in ELISA and a high affinity, showing equilibrium constants in the nM range for Atr-I. These mAbatrs were not able to bind to Atr-I overlapping peptides, suggesting that they recognize conformational epitopes. CONCLUSIONS/SIGNIFICANCE: For the first time a functional screening based on a synthetic biosensor was successfully used for the selection of neutralizing mAbs against SVMPs.