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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. [ABSTRACT FROM AUTHOR]

Abstract: The parenteral administration of antivenoms is the cornerstone of snakebite envenoming therapy. Efforts are made to ensure that antivenoms of adequate efficacy and safety are available world-wide. We address the main issues to be considered for the development and manufacture of improved antivenoms. Those include: (a) A knowledge-based composition design of venom mixtures used for immunization, based on biochemical, immunological, toxicological, taxonomic, clinical and epidemiological data; (b) a careful selection and adequate management of animals used for immunization; (c) well-designed immunization protocols; (d) sound innovations in plasma fractionation protocols to improve recovery, tolerability and stability of antivenoms; (e) the use of recombinant toxins as immunogens to generate antivenoms and the synthesis of engineered antibodies to substitute for animal-derived antivenoms; (f) scientific studies of the contribution of existing manufacturing steps to the inactivation or removal of viruses and other zoonotic pathogens; (g) the introduction of novel quality control tests; (h) the development of in vitro assays in substitution of in vivo tests to assess antivenom potency; and (i) scientifically-sound pre-clinical and clinical assessments of antivenoms. These tasks demand cooperative efforts at all main stages of antivenom development and production, and need concerted international partnerships between key stakeholders. [Copyright &y& Elsevier]

Abstract: We have evaluated for the first time the impact of a solvent/detergent (S/D) treatment on the quality and in vivo neutralization potency of horse-derived whole IgG antivenom used in the treatment of viperid snake bite envenoming in Central America. The S/D treatment by 1% tri (n-butyl) phosphate (TnBP) – 1% Triton X-45 at 22–25°C was applied either on starting plasma or on purified immunoglobulins. The S/D agents were removed from both fractions by extractions with oil. S/D-treated plasma was subjected to caprylic acid precipitation to purify the immunoglobulins. Products were formulated, sterile-filtered, and filled into 10-mL vials, stored at 5±3°C, and subjected to routine quality controls, SDS-PAGE, determination of anti-Bothrops asper venom antibody titre by ELISA, in vivo B. asper venom-neutralization potency tests, and safety test, comparatively with an antivenom manufactured by caprylic acid fractionation without S/D treatment. Results indicate that these conditions of S/D treatment on purified immunoglobulin yielded an antivenom of high turbidity that induced weight loss in animals. In contrast, antivenom fractionated from the S/D-treated plasma had physico-chemical and biological characteristics indistinguishable from those of the non-S/D-treated antivenom. S/D treatment of horse plasma may be considered to increase the viral safety of antivenoms. [Copyright &y& Elsevier]

Abstract: This work examined the presence of antibodies reacting with human erythrocytes in horse-derived antivenoms used in the treatment of snakebite envenomations, and assessed the efficacy of various fractionation protocols in the elimination of agglutinating antibodies. A number of antivenoms produced by various fractionation protocols were tested for direct agglutination of human erythrocytes. Reactions were observed visually and microscopically, and an indirect anti-equine globulin test was also used. In addition, rabbits and mice were injected intravenously with antivenoms to observe possible intravascular hemolysis and erythrocyte sequestration. All tested antivenoms agglutinated human erythrocytes, albeit to different extent, and also gave a positive anti-globulin test. Agglutination was due to IgG(T) subclass of antibodies. Pepsin digestion of horse IgG, to obtain F(ab′)2 fragments, reduced the direct agglutination, but not the indirect anti-globulin test. Ion-exchange chromatography of IgG in a strongly basic quaternary ammonium cellulose membrane abrogated direct agglutination and reduced the indirect anti-globulin test. Binding of antivenom antibodies to erythrocytes in vivo was demonstrated in rabbits, although there was no evidence of intravascular hemolysis or erythrocyte sequestration in rabbits and mice. It is concluded that anti-human erythrocyte antibodies are present in horse-derived antivenoms, and that fractionation of horse plasma by pepsin digestion, and especially by anion-exchange chromatography, reduces the titer of these antibodies. Our in vivo experimental results do not support a role for these antibodies in early adverse reactions occurring after antivenom administration. [Copyright &y& Elsevier]

Abstract: Caprylic acid purification of IgG, currently used in the manufacture of horse-derived antivenoms, was successfully adapted for the preparation of sheep and camel IgG. Sheep IgG had a molecular mass of ∼150kDa, whereas camel IgG presented two bands of molecular masses of ∼160 and 100kDa, the latter corresponding to heavy-chain IgG, which is devoid of light chains. Horse, sheep and camel IgGs were compared by several parameters aiming at predicting their potential for induction of early and late adverse reactions. Horse and sheep IgGs showed a higher anticomplementary activity than camel IgG, and also elicited a higher anti-IgG response than camel IgG, when injected in mice. Horse IgG agglutinated human type O+ erythrocytes, whereas no such reactivity was observed in sheep and camel IgG preparations. A novel procedure was used for the detection of antibodies in human serum against animal IgGs. It was found that a pool of human sera collected in Costa Rica had a higher titer of antibodies directed against horse and sheep IgGs than against camel IgG. Overall, camel IgG showed the lowest potential for the induction of adverse reactions among the three IgGs tested. [Copyright &y& Elsevier]

Abstract: Early adverse reactions occur in a number of patients treated with heterologous antivenoms and have been associated with anticomplementary activity (ACA). In order to reduce the ACA of equine whole IgG antivenoms produced by caprylic acid fractionation, three different fractionation protocols were compared: (a) routine caprylic acid fractionation; (b) caprylic acid fractionation followed by β-propiolactone treatment; and (c) caprylic acid fractionation followed by ion-exchange chromatography using a quaternary ammonium membrane. The three protocols yielded products with similar physicochemical characteristics and anti-Bothrops asper venom antibody titers, except that ion-exchange purified antivenom had a lower protein concentration. Antivenoms fractionated by using β-propiolactone or filtration through quaternary ammonium membrane had a significantly reduced in vitro ACA. A preparation of caprylic acid-fractionated antivenom was heated in order to induce the formation of protein aggregates; however, its ACA was similar to non-heated antivenom. None of the antivenoms affected the hemolytic activity of serum complement in rabbits after a bolus intravenous administration. It is concluded that (a) β-propiolactone and quaternary ammonium membranes significantly reduce in vitro ACA of caprylic acid-fractionated equine antivenom, and (b) the validity of in vitro ACA as a predictor of EAR needs to be reexamined in clinical and experimental studies, since it may not adequately predict in vivo complement activation by antivenoms. [Copyright &y& Elsevier]

Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods. [ABSTRACT FROM AUTHOR]

Abstract: Low pH treatment improves the tolerance to intravenous infusion, the stability, and the viral safety of various therapeutic immunoglobulins G preparations, but has never been evaluated for horse plasma-derived antivenoms. We have studied the impact of low pH formulation on the quality, safety, stability, potency and viral inactivation of a whole IgG antivenom used to treat viperid snake bite envenoming. Horse plasma-derived whole immunoglobulins purified by caprylic acid were incubated for 24 h at low pH in the presence of 4% sorbitol, then sterile-filtered and stored liquid at 2–8 °C. Appearance, aggregates, purity, safety tests in mice, venom antibody titre, and neutralization potency tests were controlled. Low pH treatment did not affect the physico-chemical characteristics, safety and potency of antivenom for at least 6 months of storage, but a major increase in aggregates was observed. In vitro antibody titre and in vivo neutralizing potency were maintained. There were ≥ 5.5 log inactivation of Herpes Simplex Virus-1, an enveloped virus, but no significant inactivation of the non-enveloped Poliovirus type 3. Low pH treatment appears feasible to improve the viral safety of antivenoms without affecting the neutralization potency. The possibility to formulate antivenoms at low pH requires further investigations to avoid formation of aggregates. [Copyright &y& Elsevier]

Snake venoms are mixtures of proteins whose physicochemical features confer them toxicity and immunogenicity. Animals (e.g., horses or sheep) immunized with snake venoms produce antibodies towards the venom proteins. Since these antibodies can neutralize the venom toxicity, they have been used to formulate snake antivenoms. The efficacy of the antivenoms is widely accepted, and standard venoms are expected to be representative of the snake's population that inhabit in the region where the antivenom is intended to be used. The representativeness of a single venom collected from a Crotalus simus snake, and its usefulness as standard venom to produce an antivenom is evaluated. The use of an "average venom" might be as representative of the population intended to be used, as the standard venom composed by many venom samples. Variations in the relative abundance concentration of crotoxin in the C. simus leads to different clinical manifestations, as well as differences in the neutralization efficacy of the antivenoms. A monovalent anti-Cs antivenom was produced from a single venom C. simus specimen, and its efficacy in neutralizing the lethal activity of 30 C. simus snakes was tested. Despite the variations in the relative abundance content of crotoxin found in the proteomes, the monovalent anti-Cs antivenom was successful in neutralize the toxicity caused by the variations on the venom composition of three different snake population used. Interestingly, it seems that the sex is not a key factor in the lethality of the venoms tested. The concept of representative venom mixtures for immunization should be revised for the case of C. simus on the populations found in Costa Rica, since it might use as less as one representative individual whose venom covers the mainly toxic enzymes. • The representativeness of a single Crotalus simus snake venom as an "average venom" should be considered. • The sex as a variable in the venom toxicity may not be a key factor in Crotalus simus venoms from Costa Rica. • The concept of representative venom mixture for antivenom production, in the case of Crotalus simus , should be revised. • The antivenom design should integrate a knowledge-base analysis of the snake venoms used. [ABSTRACT FROM AUTHOR]

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 PLA 2 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. • Preclinical support for Mexican Crotalus snakebite management was obtained. • Antigenic similarities between C. basiliscus and other Crotalus venoms were evidenced. • Crotalus basiliscus / Bothrops asper antivenom efficiently neutralized heterologous Crotalus venoms. [ABSTRACT FROM AUTHOR]

PoliVal-ICP antivenom is produced from plasma of horses immunized toward the venoms of Bothrops asper , Crotalus simus and Lachesis stenophrys . The antibody response induced by these venoms confers PoliVal-ICP the capacity to neutralize the venoms of the most important Central American viperids, including not only homologous venoms (i.e., venoms used as immunogen), but many heterologous venoms (i.e., venoms not used as immunogen). In this work, the individual contributions of homologous venoms to the paraspecificity of PoliVal-ICP were inferred from the capacity of experimental monospecific antivenoms toward venoms of B. asper (anti-Ba), C. simus (anti-Cs) and L. stenophrys (anti-Ls), and an experimental polyspecific antivenom (anti-Ba/Cs/Ls) to neutralize the lethality induced by different venoms in mice. It was found that all antivenoms neutralized their corresponding homologous venoms. Moreover, the anti-Ba antivenom cross-neutralized the venoms of Agkistrodon howardgloydi , Atropoides picadoi , Bothriechis lateralis , Bothriechis supraciliaris and Porthidium ophryomegas ; the anti-Cs antivenom cross-neutralized the venoms of B. lateralis , B. supraciliaris , Cerrophidion sasai and Porthidium nasutum ; and the anti-Ls antivenom cross-neutralized the venoms of B. lateralis , B. supraciliaris , C. sasai and Lachesis melanocephala . All venoms neutralized by any monospecific antivenom were also neutralized by the anti-Ba/Cs/Ls antivenom. Venoms of Atropoides mexicanus , Bothriechis nigroviridis and Bothriechis schlegelii were not neutralized by any experimental antivenom, thus explaining the limitations of PoliVal-ICP to neutralize these venoms. Consequently, an enlargement of the neutralization scope of PoliVal-ICP could be achieved by including these venoms in the group of those used as immunogens. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

EchiTAb + ICP is a pan-African antivenom used for the treatment of snakebite envenomation in rural sub-Saharan African communities, where the cold chain can be difficult to maintain. To develop a formulation of EchiTAb + ICP that can be distributed and stored without refrigeration, we submitted three different formulations of EchiTAb + ICP: control (i.e. liquid antivenom formulated without stabilizer), liquid antivenom stabilized with sorbitol, and freeze-dried antivenom formulated with sucrose, to an accelerated stability study (i.e. 38 ± 2 °C and 75% relative humidity for 6 months). We analyzed changes in color, residual humidity, reconstitution time (for freeze-dried preparation), pH, osmolality, total protein concentration, antibody monomers content, turbidity, bacterial endotoxins, and pre-clinical neutralizing efficacy of the lethal effect of Echis ocellatus venom at 0, 3 and 6 months. In the control formulation, instability was evidenced by the development of a yellow coloration and an increment in aggregation and turbidity, without change in its neutralizing activity. The sorbitol-stabilized formulation did not develop marked aggregation or turbidity, but instability was evidenced by the development of yellow coloration and a drop in the neutralizing potency. The freeze-dried formulation maintained its neutralizing potency and did not show marked signs of instability, thus indicating that freeze-drying could confer EchiTAb + ICP with improved thermal stability required for distribution and storage at room temperature in sub-Saharan Africa. [ABSTRACT FROM AUTHOR]

Enhancement of antivenom immune responses in horses through adjuvant technology improves antivenom production efficiency, but substantial local reactogenicity associated with some traditional veterinary adjuvants limits their usability. To explore modern adjuvant systems suitable for generating antivenom responses in horses, we first assessed their physicochemical compatibility with Bothrops asper snake venom. Liposome and nanoparticle aluminum adjuvants exhibited changes in particle size and phospholipid content after mixing with venom, whereas squalene emulsion-based adjuvants remained stable. Next, we evaluated serum antibody response magnitude and neutralization capacity in horses immunized with adjuvant-containing Echis ocellatus , Bitis arietans , Naja nigricollis , and Dendroaspis polylepis venom preparations. Whereas all tested adjuvants elicited significant neutralization capacity against the viperid venoms, the greatest antibody responses were generated by a squalene-in-water emulsion, thus representing a promising novel alternative for antivenom production. [Display omitted] • Lack of antivenoms with neutralizing activity against different snake venoms is a challenge to snake envenomation treatment. • Modern vaccine adjuvant technology has the potential to improve antivenom production. • Physicochemical compatibility of various adjuvant formulations with snake venom was characterized. • Immune responses elicited by selected adjuvant-containing venom preparations were evaluated in immunized horses. [ABSTRACT FROM AUTHOR]

The assessment of the preclinical neutralizing ability of antivenoms in Latin America is necessary to determine their scope of efficacy. This study was aimed at analyzing the neutralizing efficacy of a polyspecific bothropic-crotalic antivenom manufactured by BIRMEX in Mexico against lethal, hemorrhagic, defibrinogenating and in vitro coagulant activities of the venoms of Bothrops jararaca (Brazil), B. atrox (Perú and Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia), and B. asper (Costa Rica). Standard laboratory tests to determine these activities were used. In agreement with previous studies with bothropic antivenoms in Latin America, a pattern of cross-neutralization of heterologous venoms was observed. However, the antivenom had low neutralizing potency against defibrinogenating effect of the venoms of B. atrox (Colombia) and B. asper (Costa Rica), and failed to neutralize the in vitro coagulant activity of the venom of B. asper (Costa Rica) at the highest antivenom/venom ratio tested. It is concluded that, with the exception of coagulant and defibrinogenating activities of B. asper (Costa Rica) venom, this antivenom neutralizes toxic effects of various Bothrops sp venoms. Future studies are necessary to assess the efficacy of this antivenom against other viperid venoms. Rev. Biol. Trop. 65 (1): 345-350. Epub 2017 March 01. [ABSTRACT FROM AUTHOR]

Freeze-drying is a process used to improve the stability of pharmaceutical proteins, including snake antivenoms. This additional step confers these with a higher stability in comparison to liquid formulations, especially in tropical regions where high temperatures could affect the activity of immunoglobulins. Currently, the knowledge about freeze-drying process conditions for snake antivenoms is very limited. Some of the scarce scientific works on this subject reported reconstitution times up to 90 min for these preparations, which could imply a delay in the beginning of the antivenom therapy at the clinical setting. Therefore there is a reasonable concern about whether freeze-dried antivenoms exhibit the desired attributes for solid pharmaceutical proteins. In this work, a physicochemical characterization of seven commercial freeze-dried snake antivenoms was performed based on tests recommended by the World Health Organization (WHO). No significant differences were observed between the products regarding macroscopic appearance of the solid cakes, reconstitution times, residual humidity and monomers content. On the other hand, total protein concentration, turbidity and electrophoretic profile were different among samples. Microscopic analysis by scanning electron microscopy showed no collapsed structure and, instead, most of the samples showed a characteristic protein morphology composed of smooth plates and channels. All the parameters tested in this study were according to literature recommendations and evidenced that, in spite of slight variations found for some products, formulation and freeze-drying conditions chosen by manufacturers are adequate to prevent aggregation and generate, in physicochemical terms, freeze-dried antivenoms of acceptable quality. [ABSTRACT FROM AUTHOR]

EchiTAb-plus-ICP is an antivenom prepared from plasma of horses hyperimmunized with the venoms of the carpet viper ( Echis ocellatus ), the puff adder ( Bitis arietans ) and the black-necked spitting cobra ( Naja nigricollis ). Therefore, the use of this antivenom has been limited to Western Africa. In order to expand the neutralization scope of EchiTAb-plus-ICP, we supplemented the immunogenic mixture with the venoms of B. arietans , the black mamba ( Dendroaspis polylepis ), the Mozambique spitting cobra ( Naja mossambica ), the snouted cobra ( N. annulifera ), and the rinkhals ( Hemachatus haemachatus ) from Swaziland. The ability of the expanded-scope antivenom, hereby named EchiTAb + ICP, to neutralize the venoms of B. arietans , D. polylepis , N. mossambica and H. haemachatus was similar to those of FAV Afrique and the SVA African antivenoms. In comparison to the SAIMR antivenom, the expanded-scope EchiTAb + ICP had lower ability to neutralize the venom of B. arietans , but similar ability to neutralize the venoms of D. polylepis , N. mossambica and H. haemachatus . Owing to its low protein concentration, the expanded-scope EchiTAb + ICP had lower ability to neutralize the venom of N. annulifera than FAV Afrique and the SAIMR antivenoms. However, when formulated at a protein concentration as high as FAV Afrique and SAIMR antivenoms, the expanded-scope EchiTAb + ICP showed similar capacity to neutralize this poorly immunogenic venom. Our results encourage the transition to the new EchiTAb + ICP antivenom, with an expanded neutralization scope that includes venoms of some of the most medically important elapids from Southern Africa. Clinical trials are required to determine the minimum effective-safe dose of the new EchiTAb + ICP for each type of envenomation. [ABSTRACT FROM AUTHOR]

A chicken-derived antivenom (ChDAv) towards taipan snake ( Oxyuranus scutellatus ) venom was produced by purifying anti-taipan IgY from egg yolks of hens immunized with taipan venom. The productivity, antivenomic profile, neutralization ability, pharmacokinetic properties and immunogenicity of the ChDAv were compared with those of an antivenom produced in horses (EDAv). We found that 382 eggs are required to produce the mass of anti-taipan antibodies contained in one liter of equine hyperimmune plasma, and that 63 chickens would be needed to generate the amount of anti-taipan antibodies annually produced by one horse. It was estimated that, in Costa Rica, the production of anti-taipan antibodies could be 40% cheaper if chickens were used as immunoglobulin source, instead of horses. During antivenomic assessment, ChDAv showed lower ability to immunocapture the α subunit of taipoxin, the most important neurotoxin in the venom. ChDAv showed a lower ability to neutralize the coagulant and lethal activities of taipan venom. ChDAv was more immunogenic in rabbits than EDAv, probably due to the fact that chickens are phylogenetically more distant to rabbits than horses. This finding may explain why clearance from rabbit bloodstream was faster for chicken-IgY than for equine-IgG in a pharmacokinetic study. In conclusion, the production of anti-taipan antivenom was less effective when chicken egg yolks were used as source of immunoglobulins instead of horses. [ABSTRACT FROM AUTHOR]

The pathogenesis of dermonecrosis induced by the venom of the African spitting cobra Naja nigricollis was investigated in a mouse model. Intradermal injection of venom induced a macroscopic necrotic lesion. Histological examination revealed early edema of the dermis, followed by blistering, loss of skin appendages and reduction in cellularity. By 24 h, necrosis of the dermis was evident, sections of epidermis were lost, and a fibrinoid hyaline material filled the damaged areas. Abundant inflammatory infiltrate was present in the hypodermis and basal dermis, and there was an increment in the expression of matrix metalloproteinases (MMPs). Thrombi were observed in blood vessels. Abundant cells were present in the dermis by 7 days. By 14 and 28 days, re-epithelization had occurred, collagen was widespread in the dermis, and few skin appendages were present. The RP-HPLC fractions that reproduced the necrotic activity were composed of low molecular mass cytotoxins of the three-finger toxin family and, to a lesser extent, of phospholipases A 2 (PLA 2 ). Inhibition of PLA 2 of venom by p -bromophenacyl bromide did not reduce the area of necrosis, but modified the appearance of necrotic regions. Depletion of neutrophils and inhibition of venom metalloproteinases and tissue MMPs did not affect dermonecrosis. IgG and F(ab′) 2 antivenoms were effective in the neutralization of dermonecrosis when incubated with venom prior to injection. However, when antivenoms were administered immediately after venom injection, dermonecrosis was reduced only to a partial extent, underscoring the difficulties in neutralizing this effect with antivenoms. [ABSTRACT FROM AUTHOR]

Two antivenoms prepared by using Echis ocellatus , Bitis arietans and Naja nigricollis venoms from different locations in sub-Saharan Africa were compared for their neutralizing ability. Both antivenoms were similarly effective in the neutralization of the venoms of the three species from different locations. However in the case of E. ocellatus venom, antivenom prepared using venom from Nigerian specimens was more effective than antivenom prepared with venom from Cameroon specimens in the neutralization of coagulant activity. [ABSTRACT FROM AUTHOR]

Freeze-drying is used to improve the long term stability of pharmaceutical proteins. Sugars and polyols have been successfully used in the stabilization of proteins. However, their use in the development of freeze-dried antivenoms has not been documented. In this work, whole IgG snake antivenom, purified from equine plasma, was formulated with different concentrations of sorbitol, sucrose or mannitol. The glass transition temperatures of frozen formulations, determined by Differential Scanning Calorimetry (DSC), ranged between −13.5 °C and −41 °C. In order to evaluate the effectiveness of the different stabilizers, the freeze-dried samples were subjected to an accelerated stability test at 40 ± 2 °C and 75 ± 5% relative humidity. After six months of storage at 40 °C, all the formulations presented the same residual humidity, but significant differences were observed in turbidity, reconstitution time and electrophoretic pattern. Moreover, all formulations, except antivenoms freeze-dried with mannitol, exhibited the same potency for the neutralization of lethal effect of Bothrops asper venom. The 5% (w:v) sucrose formulation exhibited the best stability among the samples tested, while mannitol and sorbitol formulations turned brown. These results suggest that sucrose is a better stabilizer than mannitol and sorbitol in the formulation of freeze-dried antivenoms under the studied conditions. [ABSTRACT FROM AUTHOR]

The toxicological profile of the venoms of the snakes Bothrops asper and Bothrops atrox from Ecuador was investigated, together with the venom of a population of B. asper formerly classified as ‘ Bothrops xanthogrammus’ . The three venoms exerted lethal, hemorrhagic, myotoxic, coagulant and defibrinogenating effects, in agreement with the characteristic toxicological profile of Bothrops sp venoms. A polyspecific antivenom (bothropic–crotalic–lachesic) manufactured in Costa Rica was assessed for its preclinical efficacy against the toxic activities of these Ecuadorian venoms. Antivenom was effective in the neutralization of the five activities tested in the three venoms. These observations are in agreement with previous reports on the extensive cross-reactivity and paraspecific neutralization of antivenoms manufactured in Latin America against the venoms of Bothrops sp snakes. [ABSTRACT FROM AUTHOR]

Abstract: Antivenom pharmacokinetics has been studied in heterologous models in which the animal species used as immunoglobulin source is different from that used as recipient. In these models, after intravenous administration of antivenom, the plasma concentration of immunoglobulins shows a rapid initial declining-phase followed by a slower terminal-phase, which has been associated with antivenom distribution and elimination, respectively. We have compared pharmacokinetic parameters for equine-derived antivenom in homologous (horse) and heterologous (cow) models. It was found that the maximum concentration is lower in cows than in horses. Additionally, the steady-state distribution volume is higher in cows as compared to horses. On the other hand, models were not different in the time required to reach the maximum concentration, the area under the concentration/time curve, the half-life of decay during the slowest phase, the systemic clearance and the mean residence time. Similar results were obtained in a rabbit model, in which the pharmacokinetics was also affected by passive immunization of rabbits with anti-equine IgG. We conclude that, in addition to other physiological differences (e.g. cardiac frequency, plasmatic volume, glomerular filtration rate, etc.) between animal models, the ability to remove foreign immunoglobulins might influence the way in which the plasma concentration of antivenom decreases over time, thereby distorting the pharmacokinetic predictions based on non-compartmental models. [Copyright &y& Elsevier]

Abstract: Purity is a characteristic that, together with effectiveness and safety, must be tested to determine the quality of biopharmaceutical products. In therapeutic immunoglobulins, such as human intravenous immunoglobulin (IVIG), purity is evaluated on the basis of physicochemical properties, and is usually assessed by chromatography and electrophoresis. However, in the case of antivenoms these methods fail to discriminate between antibodies towards venom antigens, which constitute the active substance, and antibodies towards non-venom antigens, which are the major impurities in most of the current formulations. The assessment of this aspect of purity requires the use of the immunochemical methods. In this study, it was demonstrated that antivenoms showing physicochemical purity higher than 90% might present immunochemical purity lower than 40%. It is proposed that a comprehensive analysis of antivenom purity should combine physicochemical and immunochemical parameters. In addition, these results are crucial to decide the more appropriate strategies to improve antivenom purity. Taking into account that the current methods of antivenom purification remove most non-antibodies proteins, we propose that efforts must be primarily directed to the improvement of immunization protocols to enhance the antibody response towards venom components in hyperimmunized animals, and secondarily, in the realm of immunoglobulin purification technology. [Copyright &y& Elsevier]

Abstract: The venom proteomes of populations of the highly venomous taipan snake, Oxyuranus scutellatus, from Australia and Papua New Guinea (PNG), were characterized by reverse-phase HPLC fractionation, followed by analysis of chromatographic fractions by SDS-PAGE, N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of tryptic peptides. Proteins belonging to the following seven protein families were identified in the two venoms: phospholipase A2 (PLA2), Kunitz-type inhibitor, metalloproteinase (SVMP), three-finger toxin (3FTx), serine proteinase, cysteine-rich secretory proteins (CRISP), and coagulation factor V-like protein. In addition, C-type lectin/lectin-like protein and venom natriuretic peptide were identified in the venom of specimens from PNG. PLA2s comprised more than 65% of the venoms of these two populations. Antivenoms generated against the venoms of these populations showed a pattern of cross-neutralization, corroborating the immunological kinship of these venoms. Toxicity experiments performed in mice suggest that, at low venom doses, neurotoxicity leading to respiratory paralysis represents the predominant mechanism of prey immobilization and death. However, at high doses, such as those injected in natural bites, intravascular thrombosis due to the action of the prothrombin activator may constitute a potent and very rapid mechanism for killing prey. [Copyright &y& Elsevier]

Abstract: The efficacy and safety of two polyvalent horse-derived antivenoms in Bothrops asper envenomings were tested in a randomized, double-blind, clinical trial performed in Colombia. Both antivenoms were manufactured from the same pool of hyperimmune plasma. Antivenom A was made of F(ab′)2 fragments, generated by pepsin digestion and caprylic acid precipitation, whereas antivenom B consisted of whole IgG molecules produced by caprylic acid precipitation followed by ion-exchange chromatography. Besides the different nature of the active substance, antivenom B had higher protein concentration, slightly higher turbidity and aggregate content. No significant differences were observed in the efficacy of antivenoms. Both halted local and systemic bleeding (P = 0.40) within 6–12 h of treatment in 100% of the cases, and restored blood coagulation (P = 0.87) within 6–24 h in 84.7% of patients, and within 48 h in all of them, in agreement with restoration of plasma fibrinogen concentration. Venom concentrations in serum dropped significantly (P < 0.001), to very low levels, 1 h after antivenom infusion. Nevertheless, eight patients (11.1%), four for each antivenom, presented recurrence of venom antigenaemia at different times, from 6 to 96 h, with clinical significance (recurrent coagulopathy) only in one group B patient (2.9%). Serum creatine kinase (CK) activity was increased, as a consequence of local myonecrosis. There was no significant difference (P = 0.51) in the incidence of early adverse reactions to antivenom administration (28.9% for patients of group A and 20.6% for patients of group B), most of the reactions being mild, mainly cutaneous. The most frequent complications were cellulitis (16.7%), abscess formation (5.6%), acute renal failure (8.3%), and compartmental syndrome (5.6%). In conclusion, IgG and F(ab′)2 antivenoms, prepared by caprylic acid fractionation, presented similar efficacy and safety profiles for the treatment of B. asper envenomings in Colombia. [Copyright &y& Elsevier]

Abstract: Bothrops asper is the species that induces the highest incidence of snakebite envenomation in southern Mexico, Central America and parts of northern South America. The intraspecies variability in HPLC profile and toxicological activities between the venoms from specimens collected in Mexico (Veracruz) and Costa Rica (Caribbean and Pacific populations) was investigated, as well as the cross-neutralization by antivenoms manufactured in these countries. Venoms differ in their HPLC profiles and in their toxicity, since venom from Mexican population showed higher lethal and defibrinogenating activities, whereas those from Costa Rica showed higher hemorrhagic and in vitro coagulant activities. In general, antivenoms were more effective in the neutralization of homologous venoms. Overall, both antivenoms effectively neutralized the various toxic effects of venoms from the two populations of B. asper. However, antivenom raised against venom from Costa Rican specimens showed a higher efficacy in the neutralization of defibrinogenating and coagulant activities, thus highlighting immunochemical differences in the toxins responsible for these effects associated with hemostatic disturbances in snakebite envenoming. These observations illustrate how intraspecies venom variation may influence antivenom neutralizing profile. [Copyright &y& Elsevier]

Abstract: Administration of antivenoms to treat snakebite envenomings has the potential risk of inducing early adverse reactions. The mechanisms involved in these reactions are unclear. In this study, polyspecific antivenom consisting of whole IgG purified from equine plasma by caprylic acid precipitation was administered intravenously to non-envenomed horses (n = 47) and cows (n = 20) at a dose of 0.4 mL/kg. It has been reported that, in humans, this formulation (administered at a dose of 0.4 mL/kg) induces mild noticeable early adverse reactions, such as fever, vomiting, diarrhea, urticaria, generalized rash, tachypnea or tachycardia, in about 15–20% of the patients. Unexpectedly, none of the animals receiving antivenom in our study showed any evidence of early adverse reaction. Moreover, no late adverse reactions, i.e. serum sickness, were observed during 40 days after antivenom administration. Unlike studies performed in envenomed humans, our present results were obtained in a group of non-envenomed individuals. It is concluded that, in addition to the physicochemical characteristics of the formulation, other unknown factors must determine the occurrence of adverse reactions in snakebite envenomed humans treated with equine-derived antivenoms. [Copyright &y& Elsevier]

Abstract: Polyspecific bothropic/crotalic and bothropic/lachesic antivenoms were produced in Bolivia by immunizing two donkeys with the venoms of Bothrops mattogrossensis and Crotalus durissus terrificus and one llama with the venoms of B. mattogrossensis and Lachesis muta. These antivenoms are currently being used for snakebite envenomation in Bolivia. The rationale for using these animals is that donkeys and llamas are better adapted than horses to the high altitudes in South America and constitute good alternatives for antivenom production in these regions. Plasma was fractionated by caprylic acid precipitation of non-immunoglobulin plasma proteins, to obtain whole IgG preparations. Donkey-derived antivenom showed one band of 150kDa when analyzed by SDS–PAGE, whereas llama antivenom presented two immunoglobulin bands, of 170kDa and 120kDa, the latter corresponding to the heavy-chain antibodies present in camelid sera. The effectiveness of these antivenoms to neutralize lethal, hemorrhagic, myotoxic, edema-forming, and defibrinogenating activities of the venom of B. mattogrossensis from Bolivia, a species formerly known as Bothrops neuwiedii, was assessed at the experimental level. Although llama antivenom has a total protein concentration four times lower than donkey antivenom, both preparations have similar neutralizing capacity against all toxic activities assessed. Llama and donkey IgG-based antivenoms are effective in the neutralization of B. mattogrossensis venom and represent valuable alternatives for antivenom manufacture in highland regions of South America. [Copyright &y& Elsevier]

Abstract: A preclinical assessment was performed on the neutralizing efficacy of a whole IgG polyspecific antivenom (EchiTAb-Plus-ICP®), designed for the treatment of snakebite envenomings in Nigeria. It was generated by immunizing horses with the venoms of Echis ocellatus, Bitis arietans and Naja nigricollis, the most medically important species in Nigeria. Antivenom was tested against the venoms of E. ocellatus, Echis leucogaster, Echis pyramidum leakeyi, B. arietans, Bitis gabonica, Bitis rhinoceros and Bitis nasicornis. The neutralization of the venom toxins responsible for the lethal, hemorrhagic, coagulant and local necrotizing activities was assessed, since these are the most significant effects that characterize envenoming by these species. Echis sp venoms exerted lethal, hemorrhagic, coagulant and necrotizing effects, whereas the Bitis sp venoms tested induced lethality, hemorrhage and necrosis, but were devoid of coagulant activity. The antivenom was effective in the neutralization of all effects tested in all venoms. Highest neutralization was achieved against the venoms of E. ocellatus and B. arietans, and the lowest neutralizing potency was against the venom of B. nasicornis, a species that has a low clinical relevance. It is concluded that EchiTAb-Plus-ICP®, whilst specifically designed for Nigeria, has a good preclinical neutralizing profile against homologous and heterologous viperid venoms from other sub-Saharan African locations. It therefore constitutes a promising therapeutic option for the treatment of snakebite envenoming in this region. [Copyright &y& Elsevier]

Abstract: A novel method of high performance liquid chromatography with UV detection for the quantification of caprylic acid in formulations of therapeutic immunoglobulins was developed and validated. Samples have interfering proteins that were removed by ultrafiltration in a centrifugal filter unit of 10kDa nominal molecular weight limit. Then, compounds present in ultrafiltrates were separated on an Eclipse XDB-C8 5μm column (150mm×4.6mm i.d.), using a mixture of acetonitrile–water (60:40, v/v) as the mobile phase at a flow rate of 1mL/min. The UV detection was performed at 210nm. The method was found to be precise and accurate, with a linearity range from 400μg/mL to 600μg/mL (r =0.9948). The limit of detection (LOD) and the limit of quantification (LOQ) were found to be 13.46μg/mL and 44.85μg/mL, respectively. To illustrate the usefulness of the method in the in-process and final quality control for production of therapeutic immunoglobulin formulations, permeates obtained from the industrial diafiltration step in the manufacture of equine-derived snake antivenoms and ten batches of finished product were analyzed. [Copyright &y& Elsevier]

Abstract: Liquid formulations of antivenom require a cold chain for their distribution and storage, especially in tropical countries characterized by high temperature and humidity (climatic zone IV). Since cold chain is often deficient in many regions, there is a need to develop novel formulations of liquid antivenoms of higher stability at room temperatures. The effect of addition of the polyols mannitol and sorbitol on the thermal stability of caprylic acid-fractionated equine whole IgG antivenoms was assessed in preparations having different concentrations of protein and phenol. Results evidenced that: (1) turbidity increases proportionally to phenol and protein concentration. (2) After one year of storage at 25 °C, caprylic acid-purified antivenoms, formulated with or without polyols, did not show evidences of instability. (3) Formulation of antivenoms with 2.0 M sorbitol prevents the appearance of turbidity after one year storage at 37 °C; however, there was a partial loss in neutralizing potency in these conditions. Results suggest that formulation based on sorbitol is an option to obtain liquid whole IgG antivenoms of higher stability at tropical room temperatures. [Copyright &y& Elsevier]

The proteomic, enzymatic, toxicological, and immunogenic profiles of the venom of C. d. pifanorum were studied. It was found that venom of C. d. pifanorum is composed of 63% phospholipases A 2 (PLA 2 s), 13% serine proteinases (SVSPs), 8% bradykinin-potentiating peptides (BPPs), 4% L-amino acid oxidases (LAAOs), 3% metalloproteinases (SVMPs), and other minor components. This composition allows the venom to exert lethal, PLA 2 , myotoxic, coagulant and defibrinogenating activities, but no azocaseinolytic or hemorrhagic activities. The addition of C. d. pifanorum venom to the group of venoms used as immunogens to produce the Central American antivenom PoliVal-ICP (i.e., venoms of Bothrops asper , Crotalus simus and Lachesis stenophrys) resulted in 1) the expansion of the neutralization scope of the antivenom to cover the venom of C. d. pifanorum and other antigenically related venom (i.e., C. s. scutulatus venom), 2) improvement of the neutralizing potency towards the venom of C. simus , and 3) no significant reduction of the neutralization of venoms of B. asper and L. stenophrys. It was concluded that supplementation of the immunogens used to produce PoliVal-ICP with the venom of C. d. pifanorum is a viable alternative to expand the neutralization scope of the antivenom. The venom of Crotalus durissus pifanorum from Venezuela has a proteomic profile like those of other subspecies of the South American rattlesnake C. durissus , with predominance of phospholipases A 2 (especially crotoxin) and serine proteinases. This explains a toxicological profile characterized by neurotoxicity, myotoxicity, and coagulopathies, but being devoid of hemorrhagic activity. The antivenom used in Central America (PoliVal-ICP) includes the venom of C. simus , which has a different composition, in the immunizing strategy. Accordingly, this antivenom does not neutralize C. d. pifanorum venom. The addition of C. d. pifanorum venom to the immunizing mixture of PoliVal-ICP expands the neutralizing scope of this antivenom, to cover additional rattlesnake venoms, while not affecting the response to C. simus , Bothrops asper and Lachesis stenophrys venoms. This represents an improvement of the current PoliVal-ICP. [Display omitted] • The venom of Crotalus durissus. pifanorum is composed predominantly of phospholipases A 2 (PLA 2) and serine proteinases. • C. d. pifanorum venom exerts lethal, PLA 2 , myotoxic, coagulant and defibrinogenating activities. • Antivenom raised against venoms of B. asper , C. simus and L. stenophrys does not neutralize the venom of C. d. pifanorum. • Antibodies induced by C. d. pifanorum venom neutralize lethality of C. s. scutulatus venom. • C. d. pifanorum venom does not reduce the antibody response towards venoms of B. asper , C. simus or L. stenophrys. [ABSTRACT FROM AUTHOR]

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 A 2 (PLA 2 s), 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 (PLA 2) 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. 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 A 2 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. [Display omitted] • H. haemachatus , N. annulifera , N. mossambica and N. nigricollis venoms share an overall qualitative family toxin composition. • Cytotoxic 3FTxs are the most toxins in the venoms of these snakes. • A closer immunological kinship exists between H. haemachatus, N. mossambica and N. nigricollis than to N. annulifera venom. [ABSTRACT FROM AUTHOR]