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The performance of dynamic body-feed filtration (DBF) in the removal of bulky solids produced during the manufacturing of snake antivenoms using the caprylic acid method was evaluated. For this purpose, diatomites with different filterability properties were compared in a bench-scale study to assess their effectiveness in removing the precipitated material formed after the addition of caprylic acid to equine hyperimmune plasma. C1000 diatomite at a concentration of 90 g/L of precipitated plasma showed the best performance. Then, the process was scaled up to three batches of 50 L of hyperimmune horse plasma. At this pilot scale, 108 ± 4% of the immunoglobulins present following plasma precipitation were recovered after DBF. The antivenoms generated using this procedure met quality specifications. When compared to open filtration systems commonly used at an industrial scale by many antivenom manufacturers, DBF has a similar yield and produces filtrates with comparable physicochemical characteristics. However, DBF ensures the microbiological quality of the primary clarification in a way that open systems cannot. This is because: 1) DBF is performed in a single-use closed device of depth filters which prevents microbial contamination, and 2) DBF removes bulky material in few minutes instead of the more than 24 h needed by open filtration systems, thus reducing the risk of contamination. It was concluded that DBF is a cost-effective, easily validated, and GMP-compliant alternative for primary clarification following caprylic acid precipitation of plasma in snake antivenom production.
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Snakebite in the Middle East and North Africa (MENA) is a public health problem whose magnitude is not fully known. Several antivenoms are available in these regions, but these formulations are designed for restricted geographical settings. Many countries do not have local production of antivenoms and must access products whose clinical performance has not been demonstrated. We hypothesize that it is possible to unify the treatment for viperid snakebites of MENA in a single antivenom formulation. Hereby we describe the design, development and preclinical evaluation of an antivenom of broad geographical coverage for this region (MENAVip-ICP). We produced this antivenom from the plasma of horses immunized with eight medically important venoms of viperid snake species from MENA. For this, we used a strategy based on two stages: first, immunization of horses with North African (NA) venoms, followed by a second immunization stage, on the same horses, with MENA venoms. We purified antivenoms from both stages: the Anti-NA and the final product Anti-MENA (MENAVip-ICP). Anti-NA was considered as intermediate formulation and was purified with the intention to study the progression of the immunoglobulin immune response of the horses. Antivenoms from both stages neutralized lethal, hemorrhagic, and procoagulant activities of homologous venoms. Compared to Anti-NA, MENAVip-ICP improved the neutralization profile of intravenous lethality and in vitro procoagulant activities of venoms. A notable finding was the difference in the neutralization of lethality when MENAVip-ICP was assessed intraperitoneally versus intravenously in the murine model. Intraperitoneally, MENAVip-ICP appears more effective in neutralizing the lethality of all venoms. Furthermore, MENAVip-ICP neutralized the lethal activity of venoms of species from other regions of MENA, Central/East Asia, and Sub-Saharan Africa that were not included in the immunization protocol. Our results showed that MENAVip-ICP neutralizes the main toxic activities induced by viperid MENA venoms at the preclinical level. Consequently, it is a promising product that could be clinically assessed for the treatment of snakebite envenomings in this region.
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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.
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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
INTRODUCTION: Antivenom is a lifesaving medicine for treating snakebite envenoming, yet there has been a crisis in antivenom supply for many decades. Despite this, substantial quantities of antivenom stocks expire before use. This study has investigated whether expired antivenoms retain preclinical quality and efficacy, with the rationale that they could be used in emergency situations when in-date antivenom is unavailable. METHODS: Using WHO guidelines and industry test requirements, we examined the in vitro stability and murine in vivo efficacy of eight batches of the sub-Saharan African antivenom, South African Institute for Medical Research polyvalent, that had expired at various times over a period of 30 years. RESULTS: We demonstrate modest declines in immunochemical stability, with antivenoms older than 25 years having high levels of turbidity. In vitro preclinical analysis demonstrated all expired antivenoms retained immunological recognition of venom antigens and the ability to inhibit key toxin families. All expired antivenoms retained comparable in vivo preclinical efficacy in preventing the lethal effects of envenoming in mice versus three regionally and medically important venoms. CONCLUSIONS: This study provides strong rationale for stakeholders, including manufacturers, regulators and health authorities, to explore the use of expired antivenom more broadly, to aid in alleviating critical shortages in antivenom supply in the short term and the extension of antivenom shelf life in the longer term.
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Antivenenos , Mordeduras de Serpientes , Ratones , Humanos , Animales , Antivenenos/uso terapéutico , Mordeduras de Serpientes/tratamiento farmacológico , Ponzoñas/uso terapéuticoRESUMEN
As injectable therapeutics, snake antivenoms must meet specifications for endotoxin content. The Limulus amebocyte lysate (LAL) test was used to evaluate the endotoxin content in several commercially available antivenoms released for clinical use. It was found that some products have endotoxin concentrations higher than the accepted limit for these contaminants. These results emphasize the need to include endotoxin determination as part of the routine evaluation of antivenoms by manufacturers and regulatory agencies.
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Snakebite envenomation is a neglected tropical disease posing a high toll of mortality and morbidity in sub-Saharan Africa. Polyspecific antivenoms of broad effectiveness and specially designed for this region require a detailed understanding of the immunological features of the mamba snake (Dendroaspis spp.) venoms for the selection of the most appropriate antigen combination to produce antivenoms of wide neutralizing scope. Monospecific antisera were generated in rabbits against the venoms of the four species of mambas. The toxic effects of the immunization scheme in the animals were evaluated, antibody titers were estimated using immunochemical assays, and neutralization of lethal activity was assessed. By the end of the immunization schedule, rabbits showed normal values of the majority of hematological parameters tested. No muscle tissue damage was noticed, and no alterations in most serum chemical parameters were observed. Immunological analyses revealed a variable extent of cross-reactivity of the monospecific antisera against the heterologous venoms. The venoms of D. jamesoni and D. viridis generated the antisera with broader cross-reactivity by immunochemical parameters. The venoms of D. polylepis and D. viridis generated the antisera with better cross-neutralization of lethality, although the neutralizing ability of all antisera was lower than 0.16 mg venom/mL antiserum against either homologous or heterologous venoms. These experimental results must be scaled to large animal models used in antivenom manufacture at industrial level to assess whether these predictions are reproducible.
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BACKGROUND: Envenomations by African snakes represent a high burden in the sub-Sahara region. The design and fabrication of polyspecific antivenoms with a broader effectiveness, specially tailored for its use in sub-Saharan Africa, require a better understanding of the immunological features of different Naja spp. venoms of highest medical impact in Africa; and to select the most appropriate antigen combinations to generate antivenoms of wider neutralizing scope. METHODOLOGY/PRINCIPAL FINDINGS: Rabbit-derived monospecific antisera were raised against the venoms of five spitting cobras and six non-spitting cobras. The effects of immunization in the animal model were assessed, as well as the development of antibody titers, as proved by immunochemical assays and neutralization of lethal, phospholipase A2 and dermonecrotic activities. By the end of the immunization schedule, the immunized rabbits showed normal values of all hematological parameters, and no muscle tissue damage was evidenced, although alterations in aspartate aminotransferase (AST) and alkaline phosphatase (ALP) suggested a degree of hepatic damage caused mainly by spitting cobra venoms. Immunologic analyses revealed a considerable extent of cross-reactivity of monospecific antisera against heterologous venoms within the spitting and no-spitting cobras, yet some antisera showed more extensive cross-reactivity than others. The antisera with the widest coverage were those of anti-Naja ashei and anti-N. nigricollis for the spitting cobras, and anti-N. haje and anti-N. senegalensis for the non-spitting cobras. CONCLUSIONS/SIGNIFICANCE: The methods and study design followed provide a rationale for the selection of the best combination of venoms for generating antivenoms of high cross-reactivity against cobra venoms in sub-Saharan Africa. Results suggest that venoms from N. ashei, N. nigricollis within the spitting cobras, and N. haje and N. senegalensis within the non-spitting cobras, generate antisera with a broader cross-reactivity. These experimental results should be translated to larger animal models used in antivenom elaboration to assess whether these predictions are reproduced.
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Lagomorpha , Naja , Animales , Conejos , Elapidae , Antivenenos/farmacología , Sueros Inmunes , Venenos ElapídicosRESUMEN
During the production of snake antivenoms, the animals used as immunoglobulin source are subjected to processes that could deteriorate their physical condition. Therefore, these conditions must be carefully designed and validated. In this work, the immunization and bleeding protocols applied to horses used to produce the African polyspecific antivenom EchiTAb-plus-ICP were evaluated regarding their effects on the horses' health. The study focused on horses that had been previously immunized with venoms and then received periodic booster venom injections for antivenom production. It was found that the periodic immunization with 5 mg of a mixture of venoms of Bitis arietans, Echis ocellatus, Dendroaspis polylepis, and Naja nigricollis did not induce systemic signs of envenomation, and only caused mild swelling at the injection site, which did not evolve to abscesses, fistulas, or fibrosis. Three consecutive days of bleeding, collecting 6-8 L of blood per day, and self-transfusing the red blood cells (RBC) in the second and third days, did not induce evident cardiorespiratory alterations. However, this procedure caused significant reductions in RBC, hematocrit, hemoglobin, and total plasma protein values. Seven weeks after bleeding, these parameters were recovered, and horses were ready for the next immunization/bleeding cycle. The intravenous administration of equine albumin, at a dose of 2 g/kg body weight, increased the apparent plasma volume and the albumin concentration. However, this procedure induced early adverse reactions and transient alterations of the serum levels of the enzyme gamma-glutamyl transferase (GGT), thus suggesting some degree of hepatic injury. It was concluded that immunization and bleeding as described in this work do not cause significant clinical alterations in the horse's health, except for a transient drop in some hematological parameters. The albumin-based fluid therapy used does not hasten the recovery after bleeding but instead induces adverse events in the animals.
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Adjuvant emulsions are widely used to enhance the antibody response in animals used as immunoglobulin source to produce snake antivenoms. We tested the performance of four commercial emulsion adjuvants (Montanide, Freund, Carbigen, and Emulsigen-D) and an experimental adjuvant (QH-769) in the antibody response of horses towards venoms of the African snakes Bitis arietans, Echis ocellatus, Dendroaspis polylepis and Naja nigricollis. Montanide, Freund and Carbigen adjuvants generated the highest immune response but induced moderate/severe local lesions at the site of injection. In contrast, Emulsigen-D and QH-769 adjuvants generated the lowest immune response and low incidence of local lesions. No evidence of systemic alterations was observed in the horses immunized with any of the adjuvants. It is suggested that the use of Montanide or Freund-based emulsions in the first immunization steps, followed by the use of Emulsigen-D, QH-769 or similar adjuvants in the following injections, could result in a satisfactory immune response against snake venoms, while not inducing serious local deleterious effects.
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BACKGROUND: Snakebite envenomation exerts a heavy toll in sub-Saharan Africa. The design and production of effective polyspecific antivenoms for this region demand a better understanding of the immunological characteristics of the different venoms from the most medically important snakes, to select the most appropriate venom combinations for generating antivenoms of wide neutralizing scope. Bitis spp. and Echis spp. represent the most important viperid snake genera in Africa. METHODOLOGY/PRINCIPAL FINDINGS: Eight rabbit-derived monospecific antisera were raised against the venoms of four species of Bitis spp. and four species of Echis spp. The effects of immunization in the rabbits were assessed, as well as the development of antibody titers, as judged by immunochemical assays and neutralization of lethal, hemorrhagic, and in vitro coagulant effects. At the end of immunizations, local and pulmonary hemorrhage, together with slight increments in the plasma activity of creatine kinase (CK), were observed owing to the action of hemorrhagic and myotoxic venom components. Immunologic analyses revealed a considerable extent of cross-reactivity of monospecific antisera against heterologous venoms within each genus, although some antisera provided a more extensive cross-reactivity than others. The venoms that generated antisera with the broadest coverage were those of Bitis gabonica and B. rhinoceros within Bitis spp. and Echis leucogaster within Echis spp. CONCLUSIONS/SIGNIFICANCE: The methodology followed in this study provides a rational basis for the selection of the best combination of venoms for generating antivenoms of high cross-reactivity against viperid venoms in sub-Saharan Africa. Results suggest that the venoms of B. gabonica, B. rhinoceros, and E. leucogaster generate antisera with the broadest cross-reactivity within their genera. These experimental results in rabbits need to be translated to large animals used in antivenom production to assess whether these predictions are reproduced in horses or sheep.
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Viperidae , África del Sur del Sahara , Animales , Antivenenos , Hemorragia , Caballos , Sueros Inmunes , Conejos , Ovinos , Venenos de Serpiente , SerpientesRESUMEN
The lethality neutralization assay in mice is the gold standard for the evaluation of the preclinical efficacy and specification fulfillment of snake antivenoms. However, owing to the animal suffering involved, this assay is a candidate to be replaced by in vitro alternatives or, at least, improved by the reduction of the number of animals used per experiment, the introduction of analgesia, and the refinement of the test. Since these tests are usually run for 24 or 48 h, one possibility to refine it is to shorten the endpoint observation time of the assay and so limiting the duration of suffering. To assess the effect of this modification of the standard procedure on the analytical properties of the assay, we compared the median lethal dose (LD50) and median effective dose (ED50) values, estimated through observation times of 6, 24 and 48 h. We used African and Latin American snake venoms and several batches of two polyspecific antivenoms. A significant correlation was found between LD50 and ED50 values estimated at the three observation times. Although some LD50 and ED50 values were significantly different at these time points, results of 6 h were robust enough to be used in the characterization of new antivenoms, the verification of specification compliance, and the parallel comparison of formulations. Our observations support the modification of the standard procedures used for assessing neutralizing ability of antivenoms by carrying out the observations at 6 h instead of 24 or 48 h, with the consequent reduction in the suffering inflicted upon mice during these assays. However, the shortening of the observation time in the lethality tests must be validated for each venom and antivenom before its introduction in the routine procedures.
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In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.
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COVID-19/inmunología , COVID-19/terapia , Proteínas de la Nucleocápside de Coronavirus/inmunología , Caballos/inmunología , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/inmunología , Animales , Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , COVID-19/virología , Proteínas de la Nucleocápside de Coronavirus/genética , Proteínas de la Nucleocápside de Coronavirus/metabolismo , Ensayo de Inmunoadsorción Enzimática , Humanos , Inmunización/métodos , Inmunización Pasiva/métodos , Inmunoglobulina G/inmunología , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Sueroterapia para COVID-19RESUMEN
Despite vaccines are the main strategy to control the ongoing global COVID-19 pandemic, their effectiveness could not be enough for individuals with immunosuppression. In these cases, as well as in patients with moderate/severe COVID-19, passive immunization with anti-SARS-CoV-2 immunoglobulins could be a therapeutic alternative. We used caprylic acid precipitation to prepare a pilot-scale batch of anti-SARS-CoV-2 intravenous immunoglobulins (IVIg) from plasma of donors immunized with the BNT162b2 (Pfizer-BioNTech) anti-COVID-19 vaccine (VP-IVIg) and compared their in vitro efficacy and safety with those of a similar formulation produced from plasma of COVID-19 convalescent donors (CP-IVIg). Both formulations showed immunological, physicochemical, biochemical, and microbiological characteristics that meet the specifications of IVIg formulations. Moreover, the concentration of anti-RBD and ACE2-RBD neutralizing antibodies was higher in VP-IVIg than in CP-IVIg. In concordance, plaque reduction neutralization tests showed inhibitory concentrations of 0.03-0.09 g/L in VP-IVIg and of 0.06-0.13 in CP-IVIg. Thus, VP-IVIg has in vitro efficacy and safety profiles that justify their evaluation as therapeutic alternative for clinical cases of COVID-19. Precipitation with caprylic acid could be a simple, feasible, and affordable alternative to produce formulations of anti-SARS-CoV-2 IVIg to be used therapeutically or prophylactically to confront the COVID-19 pandemic in middle and low-income countries.
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Specimens of the Crotalus genus represent a potential snakebite problem in Mexico, and despite the great number of species of Crotalus present in this country, only a few of them are relevant from a medical point of view. Crotalus envenomed patients can present a range of signs and symptoms, depending on the species involved, and their treatment is indistinctly with either of the anti-viperid antivenoms available in the Mexican Public Health System. One of these antivenoms is produced by immunization of horses with a mixture of only two venoms: Crotalus basiliscus and Bothrops asper venoms. In light of the high variability found in Crotalus species venom composition, it is important to demonstrate the cross-neutralization of this antivenom against other Crotalus species. Therefore, in this work the toxic variability of eight medically important Crotalus venoms from Mexico and its neutralization by the Crotalus basiliscus/Bothrops asper antivenom were assessed. The present study evidenced the variability of toxic and enzymatic activities among the following Crotalus venoms: (1) Crotalus atrox, (2) Crotalus basiliscus, (3) Crotalus culminatus, (4) Crotalus simus, (5) Crotalus tzabcan, (6) Crotalus scutulatus salvini, (7) Crotalus scutulatus scutulatus-A, and (8) Crotalus scutulatus scutulatus-B. All venoms studied possess lethal and hemorrhagic activity on a murine model, although there are important variations among the species; in contrast, the PLA2 activity was similar for all venoms. Interestingly, only C. simus venom exhibited coagulant activity on human plasma under 100 µg. The antivenom neutralized the lethality and all the other assessed activities for all venoms tested. However, the dose required varied depending on the venom and the evaluated activity. Our preclinical data support the recommendation of using this antivenom to clinically manage Crotalus snakebites produced by the species assessed in this study. Nonetheless, only clinical trials could categorically validate these results.
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Antivenenos , Venenos de Crotálidos/toxicidad , Crotalus , Animales , Bothrops , Venenos de Crotálidos/química , Hemorragia , Caballos , Humanos , México , Pruebas de Neutralización , Mordeduras de SerpientesRESUMEN
Snakebite envenoming is a relevant public health problem in French Guiana, and Bothrops atrox is responsible for the vast majority of envenomings in this overseas French territory. The preclinical efficacy of freeze-dried antivenoms manufactured in Costa Rica (Polival-ICP®) and Mexico (Antivipmyn Tri®) was assessed against the lethal, hemorrhagic, in vitro coagulant, and myotoxic effects of Bothrops atrox venom from French Guiana. Antivenoms differ in protein concentration and in the type of active principle (IgG and F (ab')2, respectively). Polival-ICP® showed significantly higher neutralizing activity against lethal, hemorrhagic and in vitro coagulant activities of the venom. Antivenoms neutralized myotoxic effect to a similar extent. In the case of lethal activity, Antivipmyn Tri® did not neutralize the effect at the highest antivenom level tested (1 mg venom/mL antivenom).
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Antivenenos/farmacología , Bothrops , Mordeduras de Serpientes , Animales , Antivenenos/uso terapéutico , Costa Rica , Guyana Francesa , Hemorragia , MéxicoRESUMEN
There is an urgent need to strengthen the implementation of the 3Rs principle (Replacement, Reduction and Refinement) in the use of experimental animals in toxinological research and in the assessment of the neutralizing efficacy of snake antivenoms. This is a challenging task owing to the inherent complexity of snake venoms. The state of the art on this topic is hereby reviewed, with emphasis on the studies in which a correlation has been observed between in vivo toxicity tests and in vitro surrogate assays, particularly in the study of lethal activity of venoms and its neutralization. Correlations have been described with some venoms-antivenoms when using: (a) enzyme immunoassays, (b) hemagglutination, (c) enzyme assays (proteinase, phospholipase A2), (d) in vitro coagulant effect on plasma, (e) cell culture assays for cytotoxicity, (f) functional assays for assessing neurotoxicity in vitro, (g) use of hens' eggs, and (h) antivenomics. Additionally, the routine introduction of analgesia in these assays and the design of more 'humane' protocols for the lethality test are being pursued. It is expected that the next years will witness a growing awareness of the relevance of the 3Rs principles in antivenom testing, and that new in vitro alternatives and more 'humane' experimental designs will emerge in this field.
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Antivenenos/farmacología , Técnicas In Vitro/métodos , Pruebas de Neutralización/métodos , Venenos de Serpiente/antagonistas & inhibidores , Animales , HumanosRESUMEN
The hump-nosed pit viper Hypnale hypnale is responsible for a high number of snakebite cases in southwestern India and Sri Lanka. Although most patients only develop local signs and symptoms of envenoming, there is a growing body of evidence indicating that these envenomings may be associated with systemic alterations, including acute kidney injury. In this study we evaluated the renal toxicity of H. hypnale venom by using a perfused isolated rat kidney system and by assessing cytotoxicity in two different renal tubular cell lines in culture. The venom caused alterations in several renal functional parameters, such as reduction on perfusion pressure, renal vascular resistance, and sodium and chloride tubular transport, whereas glomerular filtration rate and urinary flow initially decreased and then increased after venom perfusion. In addition, this venom was cytotoxic to proximal and distal renal tubular cells in culture, with predominance of necrosis over apoptosis. Moreover, the venom affected the mitochondrial membrane potential and induced an increment in reactive oxygen species in these cells. Taken together, our results demonstrate a nephrotoxic activity of H. hypnale venom in these experimental models, in agreement with clinical observations.
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Venenos de Crotálidos/toxicidad , Riñón/efectos de los fármacos , Animales , Línea Celular , Técnicas In Vitro , Túbulos Renales/efectos de los fármacos , Masculino , Ratas , Ratas Wistar , Sri LankaRESUMEN
The protein composition and toxinological profile of the venom of the African spitting elapid Hemachatus haemachatus (Ringhals) were characterized by bottom-up proteomics and functional in vitro and in vivo assays. Venom is composed of abundant three-finger toxins (3FTxs; 63.3%), followed by phospholipases A2 (PLA2s; 22.8%), snake venom metalloproteinases (SVMPs; 7.1%), cysteine-rich secretory proteins (CRISPs; 4.1%) and Kunitz type protease inhibitors (KTPIs; 1.5%). 3FTxs are the main responsible for lethality and myotoxicity in mice and in vitro anticoagulant activity. In contrast to closely related spitting species, whose venom 3FTxs induces dermonecrosis, the 3FTxs of H. haemachatus did not induce dermonecrotic activity. The venom showed in vitro PLA2 activity, and most likely PLA2s contribute to some extent in venom lethality, as judged by partial reduction in toxicity after inhibition of their catalytic activity. Despite its relatively high content of SVMPs, compared to most elapids, the venom of H. haemachatus did not exert hemorrhagic effect, proteolytic activity on azocasein or defibrinogenating activity. Toxicovenomic characterization of H. haemachatus venom revealed that RP-HPLC fractions with higher abundance of 3FTxs presented lethal activity, while fractions with high content of PLA2s did not, underscoring the role of 3FTxs in the pathophysiology caused by this venom. BIOLOGICAL SIGNIFICANCE: The proteomic composition and toxinological profile of the venom of Ringhals snake, Hemachatus haemachatus, a cobra-like spitting snake endemic to southern Africa, were investigated. In vitro, Ringhals venom showed anticoagulant and phospholipase A2 activities, but was devoid of proteolytic activity on azocasein. In mice, venom induced lethality and myotoxicity, but no local hemorrhage or dermonecrosis. The lack of dermonecrotic activity is in sharp contrast to venoms of closely related spitting cobras which present a similar relative abundance of 3FTxs but are potently dermonecrotic. 3FTxs, the most abundant protein family in the venom, are predominantly responsible for toxic effects. PLA2 enzyme inactivation experiments suggest that H. haemachatus venom lethality is not dependent on PLA2s, but instead is more related to neurotoxic or cardiotoxic 3FTxs. The characterization of this venom, based on proteomic and toxicovenomic approaches, is useful for more in depth studies associated with biogeography, phylogeny, toxinology and antivenom efficacy towards the venom of this species, and its association with related elapids.
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Venenos Elapídicos , Hemachatus/metabolismo , Fosfolipasas A2/metabolismo , Fosfolipasas A2/toxicidad , Proteoma/metabolismo , Animales , Venenos Elapídicos/metabolismo , Venenos Elapídicos/toxicidad , Ratones , SudáfricaRESUMEN
Bothrops lanceolatus is an endemic viperid species in the Lesser Caribbean island of Martinique. Envenomings by this species are characterized by local and systemic effects, among which the development of thrombosis in various organs is the most severe complication. An experimental toxicological characterization of this venom was performed using in vivo mouse tests and various in vitro assays. The venom induced lethal, local and systemic hemorrhagic, edema-forming, myotoxic, thrombocytopenic, proteinase and phospholipase A2 activities. The preclinical efficacy of a batch of monospecific Bothrofav® antivenom currently in use in Martinique was assessed. The antivenom was highly effective in the neutralization of all activities tested, in agreement with its described clinical efficacy. This batch of antivenom showed a higher preclinical efficacy as compared to a previous batch used in the past.