ABSTRACT
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.
Subject(s)
Antivenins , Neutralization Tests , Animals , Horses/immunology , Antivenins/immunology , Antivenins/administration & dosage , Mice , Africa South of the Sahara , Antibodies, Neutralizing/immunology , Antibodies, Neutralizing/blood , Snake Venoms/immunology , Immune Sera/immunology , Elapid Venoms/immunology , Snake Bites/immunologyABSTRACT
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.
ABSTRACT
Venom-induced consumption coagulopathy and thrombocytopenia are common and potentially severe manifestations of viperid snakebite envenoming since they contribute to local and systemic hemorrhage. Therefore, the assessment of the efficacy of antivenoms to neutralize coagulopathic and thrombocytopenic toxins should be part of the preclinical evaluation of these drugs. To evaluate the efficacy of the polyvalent (Crotalinae) antivenom produced in Costa Rica, in this study we have used a mouse model of coagulopathy and thrombocytopenia induced by the venom of Bothrops asper, based on the bolus intravenous (i.v.) injection of venom. When venom and antivenom were incubated before injection, or when antivenom was administered i.v. immediately after venom injection, venom-induced hemostatic alterations were largely abrogated. We also studied the recovery rate of clotting parameters in conditions where antivenom was administered when mice were coagulopathic. Some parameters recovered more rapidly in antivenom-treated mice than in control envenomed animals, but others showed a spontaneous recovery without antivenom. This is due to a rapid clearance of plasma venom levels in these experimental conditions. This implies that models based on the bolus i.v. injection of venom have limitations for assessing the effect of antivenom in the recovery of clotting alterations once coagulopathy has developed. It is suggested that alternative models should be developed based on a slower systemic absorption of venom. Overall, our findings provide a protocol for the preclinical evaluation of antivenoms and demonstrate that the polyvalent antivenom is effective in neutralizing the toxins of B. asper venom responsible for coagulopathy and thrombocytopenia.
ABSTRACT
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.
Subject(s)
Lagomorpha , Naja , Animals , Rabbits , Elapidae , Antivenins/pharmacology , Immune Sera , Elapid VenomsABSTRACT
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.
ABSTRACT
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.
ABSTRACT
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.
Subject(s)
Viperidae , Africa South of the Sahara , Animals , Antivenins , Hemorrhage , Horses , Immune Sera , Rabbits , Sheep , Snake Venoms , SnakesABSTRACT
SARS-CoV-2 variants of concern show reduced neutralization by vaccine-induced and therapeutic monoclonal antibodies; therefore, treatment alternatives are needed. We tested therapeutic equine polyclonal antibodies (pAbs) that are being assessed in clinical trials in Costa Rica against five globally circulating variants of concern: alpha, beta, epsilon, gamma and delta, using plaque reduction neutralization assays. We show that equine pAbs efficiently neutralize the variants of concern, with inhibitory concentrations in the range of 0.146-1.078 µg/mL, which correspond to extremely low concentrations when compared to pAbs doses used in clinical trials. Equine pAbs are an effective, broad coverage, low-cost and a scalable COVID-19 treatment.
ABSTRACT
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.
Subject(s)
COVID-19/immunology , COVID-19/therapy , Coronavirus Nucleocapsid Proteins/immunology , Horses/immunology , SARS-CoV-2/immunology , Spike Glycoprotein, Coronavirus/immunology , Animals , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , COVID-19/virology , Coronavirus Nucleocapsid Proteins/genetics , Coronavirus Nucleocapsid Proteins/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Immunization/methods , Immunization, Passive/methods , Immunoglobulin G/immunology , Recombinant Proteins/immunology , Recombinant Proteins/metabolism , SARS-CoV-2/metabolism , SARS-CoV-2/physiology , Spike Glycoprotein, Coronavirus/genetics , Spike Glycoprotein, Coronavirus/metabolism , COVID-19 SerotherapyABSTRACT
Cobras are the most medically important elapid snakes in Africa. The African genera Naja and Hemachatus include snakes with neurotoxic and cytotoxic venoms, with shared biochemical, toxinological and antigenic characteristics. We have studied the antigenic cross-reactivity of four sub-Saharan Africa cobra venoms against an experimental monospecific Hemachatus haemachatus antivenom through comparative proteomics, preclinical assessment of neutralization, and third generation antivenomics. The venoms of H. haemachatus, N. annulifera, N. mossambica and N. nigricollis share an overall qualitative family toxin composition but depart in their proportions of three-finger toxin (3FTxs) classes, phospholipases A2 (PLA2s), snake venom metalloproteinases (SVMPs), and cysteine-rich secretory proteins (CRISPs). A monospecific anti-Hemachatus antivenom produced by Costa Rican Instituto Clodomiro Picado neutralized the lethal activity of the homologous and heterologous neuro/cytotoxic (H. haemachatus) and cyto/cardiotoxic (N. mossambica and N. nigricollis) venoms of the three spitting cobras sampled, while it was ineffective against the lethal and toxic activities of the neurotoxic venom of the non-spitting snouted cobra N. annulifera. The ability of the anti-Hemachatus-ICP antivenom to neutralize toxic (dermonecrotic and anticoagulant) and enzymatic (PLA2) activities of spitting cobra venoms suggested a closer kinship of H. haemachatus and Naja subgenus Afrocobra spitting cobras than to Naja subgenus Uraeus neurotoxic taxa. These results were confirmed by third generation antivenomics. BIOLOGICAL SIGNIFICANCE: African Naja species represent the most widespread medically important elapid snakes across Africa. To gain deeper insight into the spectrum of medically relevant toxins, we compared the proteome of three spitting cobras (Hemachatus haemachatus, Naja mossambica and N. nigricollis) and one non-spitting cobra (N. annulifera). Three finger toxins and phospholipases A2 are the two major protein families among the venoms analyzed. The development of antivenoms of broad species coverage is an urgent need in sub-Saharan Africa. An equine antivenom raised against H. haemachatus venom showed cross-reactivity with the venoms of H. haemachatus, N. mossambica and N. nigricollis, while having poor recognition of the venom of N. annulifera. This immunological information provides clues for the design of optimum venom mixtures for the preparation of broad spectrum antivenoms.
Subject(s)
Antivenins , Hemachatus , Africa South of the Sahara , Animals , Elapid Venoms/toxicity , Elapidae , HorsesABSTRACT
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.
ABSTRACT
We have applied a combination of venomics, in vivo neutralization assays, and in vitro third-generation antivenomics analysis to assess the preclinical efficacy of the monospecific anti-Macrovipera lebetina turanica (anti-Mlt) antivenom manufactured by Uzbiopharm® (Uzbekistan) and the monospecific anti-Vipera berus berus antivenom from Microgen® (Russia) against the venom of Dagestan blunt-nosed viper, Macrovipera lebetina obtusa (Mlo). Despite their low content of homologous (anti-Mlt, 5-10%) or para-specific (anti-Vbb, 4-9%) F(ab')2 antibody fragments against M. l. obtusa venom toxins, both antivenoms efficiently recognized most components of the complex venom proteome's arsenal, which is made up of toxins derived from 11 different gene families and neutralized, albeit at different doses, key toxic effects of M. l. obtusa venom, i.e., in vivo lethal and hemorrhagic effects in a murine model, and in vitro phospholipase A2, proteolytic and coagulant activities. The calculated lethality neutralization potencies for Uzbiopharm® anti-Mlt and anti-Vbb Microgen® antivenoms were 1.46 and 1.77 mg/mL, indicating that 1 mL of Uzbiopharm® and Microgen® antivenoms may protect mice from 41 to 50 LD50s of Mlo venom, respectively. The remarkable degree of conservation of immunogenic determinants between species of the clades of European and Oriental viper, which evolved geographically segregated since the early Miocene, suggests an eventual window of opportunity for the treatment of envenomings by Eurasian snakes. Clearly, the rational use of heterologous antivenoms requires establishing their para-specificity landscapes. This paper illustrates the analytical power of combining in vitro and in vivo preclinical quantitative assays toward this goal.
ABSTRACT
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.
Subject(s)
Antivenins , Crotalid Venoms/toxicity , Crotalus , Animals , Bothrops , Crotalid Venoms/chemistry , Hemorrhage , Horses , Humans , Mexico , Neutralization Tests , Snake BitesABSTRACT
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.
Subject(s)
Antivenins/pharmacology , In Vitro Techniques/methods , Neutralization Tests/methods , Snake Venoms/antagonists & inhibitors , Animals , HumansABSTRACT
Snake antivenoms are formulations of animal immunoglobulins used in the treatment of snakebite envenomation. The general scheme for producing snake antivenoms has undergone few changes since its development more than a century ago; however, technological innovations have been introduced in the manufacturing process. These medicines must comply with identity, purity, safety and efficacy profiles, as requested by the current Good Manufacturing Practices (GMPs) applied to modern biopharmaceutical drugs. Industrial production of snake antivenoms comprises several stages, such as: 1) production of reference venom pools, 2) production of hyperimmune plasma, 3) purification of the antivenom immunoglobulins, 4) formulation of the antivenom, 5) stabilization of the formulation, and 6) quality control of in-process and final products. In this work, a general review of the existing technology used for the industrial manufacture of snake antivenoms is presented.
Subject(s)
Antivenins/pharmacology , Snake Venoms/antagonists & inhibitors , Technology, Pharmaceutical/methods , Technology, Pharmaceutical/standards , Animals , Quality ControlABSTRACT
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.
Subject(s)
Elapid Venoms , Hemachatus/metabolism , Phospholipases A2/metabolism , Phospholipases A2/toxicity , Proteome/metabolism , Animals , Elapid Venoms/metabolism , Elapid Venoms/toxicity , Mice , South AfricaABSTRACT
Haemorrhage is a common clinical manifestation in envenomings caused by bites from snakes of the family Viperidae. Therefore, knowing the haemorrhagic potential of venoms and the capacity of antivenoms to neutralise this effect are of paramount relevance in toxinology. The most widely used method for quantifying haemorrhage involves the intradermal injection of venom (or a mixture of venom/antivenom) in mice, and the assessment of the resulting haemorrhagic area in the inner side of the skin. Although this method allows a straightforward assessment of the haemorrhagic activity of a venom, it does not account for haemorrhagic lesions having a similar area but differing in the depth and intensity of haemorrhage. We have developed an approach that allows the assessment of both area and intensity of a venom-induced haemorrhagic lesion using computational tools and propose a unit to represent the combination of these two factors as a measure of haemorrhage intensity, namely haemorrhagic unit (HaU). A strong correlation was observed between haemoglobin extracted from a haemorrhagic lesion and the associated HaUs. The method was used to determine the haemorrhagic activity of the venoms of Bothrops asper, Echis ocellatus and Crotalus basiliscus and the haemorrhage neutralising capabilities of the three associated antivenoms. Overall, the ease of use, as well as the time involved in this new method, makes its implementation very feasible in the determination of haemorrhagic activity of venoms and its neutralisation by antivenoms in the murine model.
Subject(s)
Hemorrhage/chemically induced , Image Processing, Computer-Assisted/methods , Viper Venoms/toxicity , Animals , Dose-Response Relationship, Drug , Injections, Intradermal , Mice , Software , Viper Venoms/administration & dosageABSTRACT
A mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys is used as immunogen to produce the polyspecific Central American antivenom (PoliVal-ICP). In this work, we studied the ability of each of these venoms to modulate the antibody response induced by the other two venoms included in the immunization mixture. For that, equine monospecific, bispecific and polyspecific antivenoms were prepared and compared regarding their ability to neutralize the phospholipase A2, coagulant and lethal activities of each venom, and their anti-venom antibodies concentration. Results indicate that there is low cross-reactivity and cross-neutralization between venoms of B. asper, C. simus and L. stenophrys, hence justifying the use of all of them as immunogens for the production of the Central American antivenom. It was also found that the venom of B. asper reduces the anti-crotalic response while the venom of C. simus does not affect the anti-bothropic response. On the other hand, the venoms of B. asper and C. simus increase the anti-lachesic response, and L. stenoprhys venom reduced both the anti-bothropic and anti-crotalic responses. On the basis of these results, the immunization strategy can be adjusted by preventing or taking advantage of cross-immunomodulation between venoms, in order to maximize the antibody response towards all venoms. Immune responses can be improved by injecting horses with several immunogen mixtures, composed by one or two of the three venoms, and administering them at different times during the immunization, eventually generating a high titer against the three venoms. Our results suggest that addressing the issue of immunomodulation by venoms might improve antivenom manufacture worldwide.
Subject(s)
Antivenins/immunology , Antivenins/pharmacology , Cross Reactions/immunology , Viper Venoms/immunology , Viperidae , Animals , Antibody Formation , Blood Coagulation/drug effects , Central America , Female , Horses/immunology , Humans , Immunomodulation , Male , Mice , Phospholipases A2/metabolismABSTRACT
Animal-derived antivenoms constitute the mainstay in the therapy of snakebite envenoming. The efficacy of antivenoms to neutralize toxicity of medically-relevant snake venoms has to be demonstrated through meticulous preclinical testing before their introduction into the clinical setting. The gold standard in the preclinical assessment and quality control of antivenoms is the neutralization of venom-induced lethality. In addition, depending on the pathophysiological profile of snake venoms, the neutralization of other toxic activities has to be evaluated, such as hemorrhagic, myotoxic, edema-forming, dermonecrotic, in vitro coagulant, and defibrinogenating effects. There is a need to develop laboratory assays to evaluate neutralization of other relevant venom activities. The concept of the 3Rs (Replacement, Reduction, and Refinement) in Toxinology is of utmost importance, and some advances have been performed in their implementation. A significant leap forward in the study of the immunological reactivity of antivenoms against venoms has been the development of "antivenomics", which brings the analytical power of mass spectrometry to the evaluation of antivenoms. International partnerships are required to assess the preclinical efficacy of antivenoms against snake venoms in different regions of the world in order to have a detailed knowledge on the neutralizing profile of these immunotherapeutics.