2 Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 Gutiérrez et al. their shelf-life is more prolonged than that of liquid preparations. However, the process of freeze-drying increments the production cost and, therefore, the final price of antivenoms. Most antivenoms are dispensed in vials or ampoules in a volume of 10 mL, although presentations of larger volumes are also available. Antivenom manufacture and control involves four basic stages: (a) Immunization of animals with relevant snake venoms, (b) bleeding and fractionation of animal blood to obtain the active substance, (c) formulation and dispensing in the final containers, and (d) quality control of the final product. A detailed description of the various aspects involved in antivenom manufacture and quality control can be found in the recently published WHO Guidelines for the Production and Control and Regulation of Snake Antivenom Immunoglobulins [19]. Immunization Owing to the great diversity of snake species and the complexity and heterogeneity of venom composition, the selection of the venoms to be used for animal immunization in antivenom production is of paramount importance. Several criteria should be considered for this selection, such as: (a) Knowledge of the snake species that cause the highest toll of envenomings in a country or region. This, in turn, demands epidemiological information on snakebite envenomings, which is rather poor in many countries. (b) Knowledge of the immunological cross-neutralization between antivenoms and venoms of various snake species. This aspect requires research-based information on the ability of antivenoms to react and, more importantly, to neutralize the venoms of heterologous species, i.e. species whose venoms are not used in the immunization (e.g. [20]). Proteomic tools have been adapted for the detailed characterization of venoms and their immunoreactivity with antivenoms, a field called ‘antivenomics’ [21, 22]. (c) Knowledge of the clinical features of envenomings, in order to identify specific envenoming syndromes that could be clearly associated with particular species of snakes, thus allowing the selection of the appropriate antivenom for treatment. Consequently, the selection of venoms to be used in immunization should be based on a meticulous case by case analysis supported by epidemiological, clinical, immunological, and toxinological information. Unfortunately, in many cases, such selection has been decided on a rather arbitrary basis, a trend that should be reversed with the help of scientific information on snakes and their venoms. In general, polyspecific antivenoms are preferred, since their use does not require the precise identification of the snake species responsible for the accident; this is the case of viperid antivenoms used in Latin America where the majority of accidents are caused by Bothrops sp venoms [23, 24]. However, there are cases where monospecific antivenoms are preferred, either because the identification of the offending snake is easy, on the basis of clinical presentation (e.g. envenomings by South American Crotalus durissus [23]) or because the number of accidents by a given species is very low or its venom is difficult to obtain (e.g., envenomings by Atractaspis engaddensis, Micrurus sp, Lachesis sp, or Hypnale hypnale) [25-27]. The preparation of venom pools for immunization is a critical step to ensure the production of effective antivenoms. These pools should include venoms from many individuals of different geographical locations within the distribution range of the species. This is very important in the case of species with wide distribution ranges owing to the well- known phenomenon of intraspecies venom variability [28- 30]. Thus, knowledge on the biochemistry and proteomics of medically-relevant snake venoms is relevant for a more rigorous preparation of venom pools for immunization. Since many snake venoms, especially from viperid species, contain proteolytic enzymes, care should be taken as to ensure that venoms are rapidly frozen and freeze-dried after collection. Immunization with venoms should aim at obtaining a high neutralizing antibody titer without significantly harming the animals being immunized. These goals are usually achieved by the use of repeated injections of low doses of venom. Although some manufacturers inactivate venoms in order to reduce toxicity, this strategy may affect the structure of relevant epitopes, thus compromising antibody response; therefore, it is preferred to use native venoms for immunization. A strategy employing multisite injections of low volumes has been highly effective [31]. Immunization protocols are generally based on the use of Freund’s complete and incomplete adjuvants, during the first immunizations, followed by subsequent injections of venom with other adjuvants such as aluminum salts, bentonite, alginate or just saline solution [32-34]. The immune response of animals to venoms is complex and needs to be studied in further detail. For instance, a phenomenon of immune suppression has been described when venoms of some viperid species are injected [35, 36]. It is likely that the difficulty in obtaining high titers against some venoms is related to this largely unexplored phenomenon. The effects of venoms on the physiological status of immunized animals need to be also documented. In the case of a polyspecific viperid antivenom produced in Costa Rica, it was shown that immunization provoked mostly local tissue inflammation and a drop in hemoglobin concentration, without affecting other systemic parameters [37]. In the case of horses, the IgG isotype responsible for most of the neutralizing activity of antivenoms corresponds to IgG(T) [38], now classified as IgG3 and IgG5 [39]. Fractionation of Hyperimmune Plasma There are three basic formulations of antivenoms in terms of the active substance. The majority of manufacturers produce antivenoms based on divalent F(ab’)2 fragments, whereas other antivenoms contain whole IgG molecules, and few antivenoms are based on monovalent Fab fragments (Fig. 1). Whole IgG antivenoms: These are produced by fractionating hyperimmune horse plasma by either ammonium sulfate precipitation [40] or by caprylic acid (octanoic acid) precipitation of non-immunoglobulin plasma proteins at acidic pH [8, 9]. Caprylic acid fractionation yields antivenoms of higher yield and purity, as compared to ammonium sulfate-fractionated products [9]. Accordingly, clinical trials have demonstrated a better tolerability of Antivenoms for Snakebite Envenomings Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 3 caprylic acid-fractionated antivenom as compared to whole IgG antivenoms produced by ammonium sulfate fractionation [41]. Fig. (2) presents the basic fractionation scheme used in Costa Rica for the manufacture of antivenoms by using caprylic acid. F(ab’)2 antivenoms: Manufacture of F(ab’)2 antivenoms is based on the methodology developed by Pope [5, 6], using pepsin digestion, at acid pH, of plasma proteins, which cleaves the Fc fragment of immunoglobulins and also degrades many other non-immunoglobulin plasma proteins, such as albumin. Then, a heat treatment step, known as ‘thermocoagulation’, and a series of salting-out steps based on ammonium sulfate precipitation generate a highly purified F(ab’)2 preparation (e.g. [42, 43]) (Fig. 3). The yield of this procedure is affected by the effect that pepsin digestion and heating has on the IgGs. Some manufacturers include additional steps for F(ab’)2 purification, such as ion- exchange chromatography [10, 44], and the use of caprylic acid to eliminate lipoproteins that may cause turbidity in antivenom preparations. Fab antivenoms: Antivenoms made of monovalent Fab fragments are produced by the fractionation of sheep-derived plasma. After ammonium sulfate precipitation of IgGs, Fab fragments are generated by papain digestion at neutral pH. Preparations are further purified by ion-exchange chromatography or, in some cases, by affinity chromatography [11]. Quality Control of Antivenoms The manufacture of antivenoms involves a meticulous quality control, both in-process and in the final product. This includes a set of physicochemical and biological analyses such as: (a) neutralizing potency, expressed as Median Effective Dose (ED50); (b) pyrogenicity (either by the rabbit test or the Limulus amebocyte lysate (LAL) test); (c) identity test (by immunochemical methods); (d) protein concentration; (e) determination of pH; (f) purity (by SDS- PAGE or Fast Protein Liquid Chromatography, FPLC); (g) concentration of excipients (sodium chloride, etc.); (h) concentration of preservatives (phenol, cresols); (i) concentration of chemical agents used during fractionation (ammonium sulfate, caprylic acid, etc.); (j) residual moisture (in the case of freeze-dried products); and (k) detection of particulate matter. Manufacturers and regulatory agencies have specifications for these tests. A detailed account of these methods can be found in the WHO guidelines for antivenom production and control [19] and references therein. PHARMACOKINETIC CONSIDERATIONS Antivenom pharmacokinetics is affected by the molecular mass of the active substance, i.e. IgG, F(ab’)2 or Fab (see reviews [45, 46]). In general, Fab and F(ab’)2 fragments have larger volumes of distribution than IgG. Fab antivenoms distribute more rapidly to tissues than F(ab’)2 and IgG. On the other hand, Fab fragments have a shorter elimination half-life than F(ab’)2 and IgG, and Fab is cleared from the circulation 35 times faster than IgG due to renal elimination of the former [46, 47]. In contrast, F(ab’)2 and IgG are predominantly eliminated by extrarenal mechanisms [46, 47]. When compared in terms of net number of cycles per gram of tissue, IgG cycles 17 and 35 times more often than F(ab’)2 and Fab, respectively, in most organs [47]. In summary, Fab fragments have a larger volume of distribution and diffuse more rapidly than IgG and F(ab’)2 fragments, thus reaching higher interstitial fluid : plasma concentration ratios; on the other hand, Fab are cleared from the body much faster than F(ab’)2 and IgG [45, 46]. These pharmacokinetic differences have evident pharmacodynamic Fig. (1). Schematic representation of the three types of active substances constituting antivenoms currently in clinical use. Some manufacturers produce whole IgG antivenoms by purifying antibodies using either ammonium sulfate or caprylic acid fractionation. Many laboratories manufacture antivenoms made of divalent F(ab’)2 fragments, obtained by pepsin digestion of plasma at acid pH, thermocoagulation and ammonium sulfate purification of F(ab’)2. Few laboratories produce antivenoms made of monovalent Fab fragments by a process based on ammonium sulfate precipitation of IgG followed by papain digestion at neutral pH. Owing to their different molecular masses, these three types of active substance show different pharmacokinetic profiles. 4 Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 Gutiérrez et al. implications. In the case of viperid snake venoms, composed of high molecular mass toxins that may reach the central blood compartment at later time intervals after subcutaneous or intramuscular injection, as occurs in human envenoming cases, the rapid elimination of Fab fragments is a problem because, by the time toxins are reaching the bloodstream, Fab concentration in blood may be too low. This results in the phenomenon known as ‘recurrence of envenoming’, in which signs and symptoms of envenoming reappear several hours after antivenom infusion [48, 49]. Thus, a treatment protocol based on repeated administration of antivenom has been developed to compensate for this problem [49]. The selection of the most appropriate type of antivenom active substance has to be determined on the basis of a careful analysis of the toxicokinetics of venoms to be neutralized. In some cases, it is likely that a combination of IgG or F(ab’)2 fragment, which would remain in circulation for long time, and a low molecular mass fragment, such as Fab, which would diffuse rapidly to the extravascular compartment, may become a good combination to neutralize toxins that act on the circulation and those that rapidly diffuse to the tissue compartments [50]. NOVEL TECHNOLOGICAL POSSIBILITIES FOR THE MANUFACTURE AND CONTROL OF ANTIVENOMS Antivenoms of satisfactory safety and efficacy are produced with the methodologies described above. However, innovations in antivenom technology are being pursued. Some of these developments remain in the experimental realm, although their introduction in antivenom manufacture may be considered in the near future. Improvement of immunization schemes: Venoms are complex mixtures of hundreds of proteins, some of which are toxic whereas others are not. Thus, by immunizing animals with crude venoms, antibodies are raised against toxicologically-relevant and irrelevant venom proteins. If non-toxic proteins predominate, the immune response to relevant toxins may be reduced or affected. Therefore, it is necessary to develop novel immunization strategies aimed at directing the immune response against relevant toxic proteins. This could be achieved by the use of recombinant toxins, such as the sphingomyelinase D from the venoms of the medically-relevant spiders of the genus Loxosceles [51]. In the case of snake venoms, this strategy could be used for venoms whose toxicity depends on a single or few toxins, like the South American rattlesnake Crotalus durissus, in which case a single toxin, the dimeric phospholipase A2 complex crotoxin, is responsible for the predominant neurotoxic, myotoxic and renal alterations characteristic of these envenomings [52]. A promising novel avenue is DNA immunization, whereby animals are injected with cDNA coding for relevant toxins [53]. Moreover, by using bioinformatic tools, it is possible to design cDNA sequences encoding for immunogenic and structurally-relevant epitopes. The design of ‘epitope strings’, based on the preparation of single synthetic multiepitope DNA immunogens, has proved successful at the experimental level [54]. DNA immunization has the additional advantage of eliminating the need to collect and maintain snakes in captivity. On the other hand, it is necessary to develop novel adjuvants and immunization schemes on the basis of an improved knowledge on the immune responses of animals to venoms. The identification of possible immune-suppressor components of venoms and the design of more effective immunization schedules are important goals. Introduction of novel steps in plasma fractionation: Although the purity of many current antivenom preparations is satisfactory, it is necessary to explore novel ways to further increase the purity, and hence the safety, of the active substance in antivenoms. Examples of such innovations are the use of membrane-based methods involving precipitation, microfiltration and hydrophobic interaction-based membrane adsorption [55] and dedicated steps aimed at the removal or Fig. (2). Scheme of the basic methodology used for the manufacture of whole IgG antivenoms by caprylic acid precipitation of non- immunoglobulin plasma proteins. Antivenoms for Snakebite Envenomings Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 5 inactivation of viruses, such as pasteurization [10], nanofiltration [56] or solvent-detergent treatment [57]. Any innovation in the technology of antivenom production should be carefully analyzed from a cost-benefit perspective, in which the involved increments in production costs should be considered vis-à-vis the need to make antivenoms accessible at affordable prices to the countries and regions that require these immunobiologicals. Monoclonal antibodies: Monoclonal antibodies of high affinity and neutralizing capacity can be generated against snake venom toxins (see review [58]). However, the application of hybridoma technology for antivenom production has the disadvantage that most snake venoms are comprised of several hundred proteins, many of which are likely to play a role in toxicity of medically-relevant species [59, 60]. This would require a combination of several monoclonal antibodies, which increases the complexity of this approach. However, there are cases of venoms whose toxicity depends on a single or few toxins, thus opening a window of opportunity for monoclonal antibodies. Moreover, a monoclonal antibody preparation could be added to polyclonal antibody-based antivenoms to enhance the neutralizing titer against a particular toxin. The reactogenicity of monoclonal antibodies can be reduced by the generation of ‘chimeric’ or ‘humanized’ recombinant antibodies [61, 62]. Recombinant antibody fragments: Recombinant scFv fragments have been produced against some toxins [63-67]. Another alternative is the use of recombinant ‘single domain antibodies’ comprised by the variable region of the heavy chain- only camelid IgG [68, 69]. These and other strategies in the field of antibody engineering offer interesting possibilities for the design of neutralizing antibody fragments of high affinity that could be applied to antivenom production. One problem here is that the pharmacokinetic profile of such small neutralizing molecules is characterized by a rapid elimination, thus precluding their efficacy in the case of envenomings by snakes in which toxins may reach the bloodstream at later time intervals. This problem could be circumvented by the engineering of molecules with varying half-lives. Improvement of the stability of antivenoms: Liquid antivenoms have to be stored at 2-8 ºC, thus precluding their distribution to places where the cold chain cannot be properly maintained, as in many low-income countries. Thus, the development of novel liquid antivenom formulations, stable at room temperature, is a highly relevant task. Possibilities include the use of stabilizers, such as sucrose or sorbitol [70, 71]. Reducing the use of animals in the assessment of antivenom potency: Despite the fact that the mouse lethality test remains as the gold standard for antivenom potency assessment [12, 19], there is a need to develop in vitro laboratory assays to assess the neutralizing ability of antivenoms, with the consequent reduction in the use of animals. Owing to the complexity and variability of snake venoms, this is a difficult goal. However, promising advances have been made using techniques such as enzyme immunoassays [72, 73], neutralization of enzymatic activities [74] and proteomic analysis of immunodepletion of venom components, a field known as ‘antivenomics’ [22]. Moreover, the use of fertile hens’ eggs to assess toxicity of venoms, at stages that precede the development of pain sensitivity, has been described [75]. As a deeper knowledge on the biochemical composition and mechanisms of action of venoms is gained, novel in vitro assays should be developed to evaluate the neutralizing ability of antivenoms. ANTIVENOM EFFICACY Antivenoms should be capable of neutralizing, in a timely fashion, the most relevant toxic components of venoms to which the antivenom is designed. Neutralization of venom toxins by antivenom antibodies can be Fig. (3). Scheme of the basic methodology used for the manufacture of F(ab’)2 fragment antivenoms by pepsin digestion at acid pH, thermocoagulation and ammonium sulfate precipitation. 6 Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 Gutiérrez et al. accomplished by, at least, four different mechanisms: (a) Antibody paratopes recognize epitopes located in the toxic site of a particular toxin. (b) The epitope may be located in the vicinity of the toxic site, neutralization being accomplished by steric hindrance. (c) Antibodies bind to an epitope at a molecular region distant from the toxic site, but antibody binding induces conformational changes in the toxin that reduce its capacity to interact with targets and to provoke toxicity. (d) Divalent IgG and F(ab’)2 molecules are able to form multivalent immunocomplexes with venom toxins, limiting the ability of toxins to interact with their targets and promoting the elimination of immunocomplexes by the mononuclear phagocyte system (Fig. 4). Preclinical Testing of Antivenom Efficacy The traditional way to assess the neutralizing potency of antivenoms is by determining their ability to neutralize the lethal activity of a venom in experimental animals, usually mice. For this, a ‘challenge dose’ of venom is selected (usually 3 to 5 Median Lethal Doses, LD50). This dose of venom is incubated with various dilutions of the antivenom and then aliquots of the mixtures, containing the challenge dose of venom, are injected in animals. Deaths are recorded and results are expressed as the Median Effective Dose (ED50), defined as the volume of antivenom, or the antivenom/venom ratio, in which 50% of the injected mice survive [12, 76]. Neutralizing activity is usually expressed in terms of mg venom, or number of LD50s, neutralized per mL antivenom. Most laboratories use the intravenous route in the assessment of antivenom ED50 [76], although other laboratories prefer the intraperitoneal route [77, 78]. Since this protocol, i.e. incubating venom and antivenom prior to injection, does not reproduce the actual circumstances of snakebite envenomings, some authors have used an alternative protocol based on the injection of venom in mice followed by the intravenous administration of antivenom [79]. However, the standardization of this procedure is not simple and, therefore, the protocol based on preincubation of venom and antivenom is used world wide in the assessment of antivenom neutralizing efficacy [19, 76]. Although the neutralization of lethal activity is the most important parameter for testing the capacity of antivenoms to neutralize snake venoms, the complex pathophysiology of snakebite envenoming involves various toxic effects whose neutralization should be tested for an adequate assessment of antivenom neutralizing efficacy. In the case of many viperid snake venoms, for instance, relevant effects include hemorrhagic, coagulant, defibrinogenating, myotoxic and necrotizing activities. A series of simple in vivo and in vitro laboratory tests have been developed to quantify these toxic activities and to analyze their neutralization by antivenoms [19, 20, 80, 81]. The basic protocol for these assays involves the selection of a challenge dose of venom and the incubation of this dose of venom with various dilutions of antivenom, followed by their testing in the corresponding assays systems. In this way, an overall picture of the antivenom neutralizing profile is achieved. It has been observed that some antivenoms are capable of neutralizing the lethal effect of a venom, but they are ineffective in the neutralization of a specific toxic effect. This principle is illustrated when anti-Bothrops sp antivenoms in Latin America are confronted with venoms of Lachesis sp. In this case, antivenoms are effective in the neutralization of lethality, but are ineffective in the neutralization of the clinically-relevant coagulant and defibrinogenating effects Fig. (4). Four mechanisms by which antivenom antibodies, or antibody fragments, may neutralize the toxic activity of snake venom toxins, here illustrated for the case of the phospholipase A2 from Agkistrodon piscivorus piscivorus (PDB code: 1VAP [123]), The mechanism based in the formation of multimolecular immune complexes between antibodies and toxins (upper left) occurs only in the case of divalent IgG and F(ab’)2 fragments, but not in the case of monovalent Fab fragments. The other three mechanisms of neutralization operate for the three types of active substance (IgG, F(ab’)2 and Fab). The sphere in the structure represents the calcium atom of the active site of the phospholipase A2 which is essential for catalytic activity. N corresponds to the N-terminus and C to the C-terminus of the protein. Adapted from [7]. Antivenoms for Snakebite Envenomings Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 7 induced by Lachesis sp venoms [82, 83]. In the case of venoms, such as those of many species of the family Elapidae, whose main toxic effect is neurotoxicity, the neutralization of lethality is a good parameter to assess the efficacy of antivenoms. Many studies have been published on the preclinical assessment of antivenom efficacy, evidencing a large extent of cross-reactivity in some cases, and the lack of neutralization of heterologous venoms in others [77, 84-88]. Such type of preclinical testing is required when a new antivenom is developed or when an existing antivenom is introduced in a new geographical setting. Clinical Assessment of Antivenom Efficacy The definitive proof of the efficacy and safety of antivenoms for the treatment of snakebite envenoming has to be shown in the clinical setting. Consequently, the design and development of clinical trials, including the appropriate training of medical and nursing staff in the performance of these trials, should be actively promoted. Initially, phase II small-scale clinical trials are performed, involving a small number of envenomed patients [48, 89]. Placebo-controlled trials are generally unacceptable on ethical grounds for the clinical testing of antivenoms. The demonstration of antivenom efficacy should be carried out in phase III clinical trials which, ideally, should be controlled, randomized, blinded trials involving a large number of patients. Such types of trials have been performed with antivenoms since the 1970s [90], and a growing number of such studies have been published in various parts of the world [41, 91-93]. In some cases, however, open-label studies are performed, some of which compare results with historical controls [94]. Clinical trials should be based on robust clinical end-points to determine efficacy, such as halting of hemorrhagic manifestations and recovery of coagulation parameters in viperid envenomings. Clinical trials have demonstrated that, in general terms, antivenoms are highly effective in correcting local and systemic hemorrhage and clotting disturbances induced by viperid venoms and in preventing severe neurotoxic effects induced by elapid and some viperid venoms, provided antivenoms are administered in a timely fashion [95]. In contrast, the efficacy of antivenoms in the neutralization of toxins inducing local tissue damage and in reversing neurotoxic manifestations induced by presynaptically-acting neurotoxic phospholipases A2 is more limited [95]. In the case of local tissue damage, the rapid onset of these effects, and the often prolonged delay in antivenom administration, determine that, by the time antivenom is infused, significant local tissue pathology has already developed [95, 96]. ANTIVENOM SAFETY Adverse Reactions to Antivenom Administration The intravenous administration of animal-derived antivenoms may result in adverse reactions of variable severity [97, 98]. The incidence of such reactions varies depending on the product being administered, and may range from less than 6% to over 70% of the cases [99-101]. Adverse reactions to antivenoms may be pyrogenic reactions, resultant from the contamination of antivenoms with bacterial lipopolysaccharides [100]. However, since pyrogen testing is a routine procedure in quality control laboratories, these reactions are seldom reported. True anaphylactic reactions, i.e. reactions mediated by IgE, are also infrequent because most people receiving antivenom have not been previously sensitized to horse immunoglobulins. The most frequent adverse reaction corresponds to what has been called ‘early adverse reaction’ (EAR), which occurs within the first hours of antivenom infusion and is characterized by manifestations such as urticaria, itching, bronchospasm, angioedema, colic, nausea and hypotension [97]. Such EARs occur in people not previously sensitized with horse IgG and their incidence greatly varies among different products. These reactions are treated with antihistamines, steroids and adrenaline [41, 98, 99, 102]. The mechanisms involved in EARs have not been clearly identified. It has been proposed that antivenom-induced complement activation plays a key role in the onset of this reaction; the ability of antivenoms to activate human complement in vitro has been repeatedly demonstrated [41, 103-105]. However, the evidence of in vivo complement activation is not so clear [106]. It has been assumed that the cleavage of Fc fragment from horse IgG, by either pepsin or papain digestions, would reduce the anticomplementary effect of antivenoms and, consequently, would decrease the EARs to antivenom administration. Although in vitro evidence supports the concept that F(ab’)2 antivenoms have less anticomplementary activity than whole IgG antivenoms [104], F(ab’)2 antivenoms are still able to activate complement [104, 107]. More importantly, clinical trials have evidenced that some F(ab’)2 antivenoms and caprylic acid-fractionated IgG antivenoms induce a similar incidence of EARs [41, 100, 108]. It has been proposed that the critical issue in the generation of EARs by antivenoms lies in the physicochemical characteristics of these products, such as total protein concentration, presence of antibody aggregates, and presence of excipients, such as preservatives, that may contribute to these reactions [41, 104, 105, 109]. Thus, strategies aimed at further reducing the incidence of EARs to antivenom administration should be based on the development of products of good physicochemical profile and low protein concentration. In addition to complement activation, other mechanisms have been suggested as playing a role in the onset of EARs, such as: (a) The potential deleterious effects of preservatives included in the formulation of antivenoms [109]. (b) The presence of antibodies in antivenoms that react with blood cells and endothelium; this has been demonstrated in the case of anti-human erythrocyte antibodies in a number of antivenoms [110]. (c) The presence, in human plasma, of antibodies against horse IgGs, which develop probably as a consequence of sensitization to horse proteins through food ingestion or exposure to dander [111-113]. The presence of human antibodies against antivenom IgGs has been demonstrated [114]. In turn, antivenoms may also have antibodies against human plasma proteins. These immunological interactions may result, upon antivenom administration, in the formation of immune complexes, followed by complement activation and the generation of pharmacologically-active mediators that may participate in 8 Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 Gutiérrez et al. the pathogenesis of EARs. Despite the likeliness of this mechanism, a study with clinical samples did not find a correlation between the titer of anti-horse IgG antibodies and the development of EAR in patients bitten by snakes and receiving antivenom [114]. It is likely that several mechanisms are involved in EARs, and further research is necessary to identity them and to ascertain their role in this phenomenon. In addition to EARs, antivenom administration is also associated with late adverse reactions (LARs), which develop between 5 and 14 days after antivenom infusion and correspond to the typical type III hypersensitive phenomenon known as ‘serum sickness’ [115, 116]. It is based on the formation of immune complexes between antivenom IgG and human IgGs synthesized against horse IgG. Such complexes, after deposition in blood vessels and joints, induce complement activation, generation of anaphylatoxins and attraction of leukocytes, all of which contribute to the clinical manifestation of serum sickness, i.e. urticaria, itching, fever, arthralgia, myalgia and lymphadenopathy [102]. The incidence of LARs correlates with the dose of antivenom administered, i.e. the total load of foreign protein injected [116]. Microbial Safety of Antivenoms Antivenoms should be free of bacterial and fungal contamination, and some antivenoms contain preservatives in their formulation, such as phenol or cresols, to prevent microbial contamination [19]. Sterility is achieved through good manufacturing practices and by a membrane filtration step before dispensing in the final container. Animal-derived antivenoms have never been shown to transmit viruses to humans; however, recent reports of viral zoonotic diseases have raised concerns on the viral safety of antivenoms [19]. Horses and sheep harbor many types of viruses, some of which are pathogenic to humans [19, 117]. Consequently, the need to include steps in the manufacture of antivenoms aimed at removing or inactivating viruses in the final product is being considered. This includes a careful selection and monitoring of the animals used in antivenom production, including clinical examination, hematological and clinical chemistry laboratory testing, and screening for viruses using immunological and molecular tests [117]. In addition, the introduction of viral inactivation or removal steps in the fractionation protocol should be considered. Fortunately, some of the currently used steps in the purification of antivenoms, such as pepsin digestion at low pH and caprylic acid addition, are highly effective for viral inactivation, especially against enveloped viruses [117, 118]. In some cases, additional viral inactivation or removal procedures could be introduced in antivenom manufacture, such as pasteurization [10], storage and formulation at acid pH [117] and nanofiltration [56]. HOW TO ENSURE THAT ANTIVENOMS REACH THE PEOPLE THAT NEED THEM Despite significant advances in our understanding of venom composition and variability and in the technological platforms for antivenom manufacture, there is a growing concern for the inaccessibility of antivenoms in many regions of the world, particularly in sub-Saharan Africa, some regions of Asia and Latin America, and Papua-New Guinea [13, 14, 119, 120]. Therefore, the issue of antivenom manufacture and distribution clearly goes beyond the scientific and technological realms, as it involves political, economic and public health considerations as well. It is necessary to ensure that antivenoms are accessible, at affordable prices, to low-income countries where they are needed. This includes international cooperative efforts to enhance the technological capacity of local antivenom manufacturing laboratories, together with innovative purchasing schemes, involving public and private sectors [120]. Moreover, the issue of antivenom distribution within countries is a critical one, since it should be based on a rigorous epidemiological account of snakebite envenomings and the identification of the most vulnerable regions requiring antivenoms. The use of geographical information system (GIS) technologies [121] and the introduction of compulsory notification of snakebite cases represent necessary tools to have an accurate picture of the areas where antivenoms have to be deployed. Issues associated with antivenom distribution are closely related to antivenom stability and with a meticulous analysis of the cold chain system in different regions, especially in remote rural areas where these accidents are frequent, in order to decide which type of antivenom preparation is most suitable for each region. Furthermore, the strengthening of public health systems, particularly in rural areas where snakebites are frequent, is also mandatory, since many regions of the world are characterized by the absence of health posts in areas of high incidence of snakebites. Finally, the adequate training of health staff, including physicians, nurses and other personnel, on the correct diagnosis of snakebite envenoming and on the adequate use of antivenom is especially important, because even if antivenom is accessible, its incorrect use may result in clinical consequences or in the misuse of this precious drug [120, 122]. Thus, the issue of antivenom manufacture and control has to be closely linked and integrated with public health areas dealing with antivenom accessibility and its correct distribution and use in regions of the world characterized by a high incidence of snakebite envenoming. On a broader perspective, the issue of public education on the prevention and management of snakebite envenomings, especially in rural areas of high snakebite incidence, should complement the tasks discussed above, since a timely transportation of patients to health centers is a crucial aspect in the management of these envenomings. A concerted global effort, being currently promoted by the World Health Organization [14] and by the Global Snake Bite Initiative of the International Society on Toxinology [119], is necessary to fulfill these goals. ACKNOWLEDGEMENTS The authors thank their colleagues at Instituto Clodomiro Picado and other groups in various countries of Latin America and abroad for productive discussions on the subject of antivenom production and control. Part of the results presented in this review derived from studies supported by Vicerrectoría de Investigación, Universidad de Costa Rica, and by the program CYTED (project 206AC0281). Antivenoms for Snakebite Envenomings Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 9 REFERENCES [1] Bon, C. Serum Therapy was Discovered 100 Years Ago. In: Envenomings and Their Treatments; Bon, C., Goyffon, M., Eds.; Fondation Marcel Mérieux: Lyon, 1996; pp. 3-9. [2] Vital-Brazil, O. History of the primordia of snake-bite accident serotherapy. Mem. Inst. Butantan., 1987, 49, 7-20. [3] Winkel, K.D.; Mirtschin, P.; Pearn, J. Twentieth century toxinology and antivenom development in Australia. Toxicon, 2006, 48, 738-754. [4] Zhang, Y.; Cremer, P.S. Interactions between macromolecules and ions: the Hofmeister series. Curr. Op. Chem. Biol., 2006, 10, 158- 163. [5] Pope, C.G. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. I. True digestion of the proteins. Br. J. Exp. Pathol., 1939, 20, 132-149. [6] Pope, C.G. The action of proteolytic enzymes on the antitoxins and proteins in immune sera. II. Heat denaturation after partial enzyme action. Br. J. Exp. Pathol., 1939, 20, 201-212. [7] Gutiérrez, J.M.; León, G. Snake Antivenoms. Technological, Clinical and Public Health Issues. In: Animal Toxins: State of the Art. Perspectives in Health and Biotechnology; de Lima, M.E., Pimenta, A.M.C., Martin-Euclaire, M.F., Zingali, R.B., Rochat, H., Eds,; Editora UFMG: Belo Horizonte, 2009; pp. 393-421. [8] Dos Santos, M.C.; D’Imperio Lima, M.R.; Furtado, G.C.; Colletto, G.M.; Kipnis, T.L.; Dias da Silva, W. Purification of F(ab’)2 anti- snake venom by caprylic acid: a fast method for obtaining IgG fragments with high neutralization activity, purity and yield. Toxicon, 1989, 27, 297-303. [9] Rojas, G.; Jiménez, J.M.; Gutiérrez, J.M. Caprylic acid fractionation of hyperimmune horse plasma: description of a simple procedure for antivenom production. Toxicon, 1994, 32, 351-63. [10] Grandgeorge, M.; Véron, J.L.; Lutsch, C.; Makula, M.F.; Riffard, P.; Pépin, S.; Scherrmann, J.M. Preparation of Improved F(ab’)2 Antivenoms. An Example: New Polyvalent European Viper Antivenom (Equine). In: Envenomings and Their Treatments; Bon, C., Goyffon, M., Eds,; Fondation Marcel Mérieux: Lyon, 1996; pp. 161-172. [11] Al-Abdulla, I.; Garnvwa, J.M.; Rawat, S.; Smith, D.S.; Landon, J.; Nasidi, A. Formulation of a liquid ovine Fab-based antivenom for the treatment of envenomation by the Nigerian carpet viper (Echis ocellatus). Toxicon 2003, 42, 399-404. [12] Theakston, R.D.G.; Warrell, D.A.; Griffiths, E. Report of a WHO workshop on the standardization and control of antivenoms. Toxicon, 2003, 41, 541-557. [13] Gutiérrez, J.M.; Theakston, R.D.G.; Warrell, D.A. Confronting the neglected problem of snake bite envenoming: the need for a global partnership. PLoS Med., 2006, 3, e412. [14] World Health Organization. Rabies and Envenomigs. A Neglected Public Health Issue; World Health Organization: Geneva, 2007, pp. 1-32. [15] Meier, J. Commercially Available Antivenoms (“Hyperimune Sera”, “Antivenins”, “Antisera”) for Antivenom Treatment. In: Handbook of Clinical Toxicology of Animal Venoms and Poisons; Meier, J.; White, J., Eds,; CRC Press: Boca Raton, 1995; pp. 689- 721. [16] Lalloo, D.G.; Theakston, R.D.G. Snake antivenoms. J. Toxicol. Clin. Toxicol., 2003, 41, 317-327. [17] Gutiérrez, J.M.; Higashi, H.G.; Wen, F.H.; Burnouf, T. Strengthening antivenom production in Central and South American public laboratories: report of a workshop. Toxicon, 2007, 49, 30-35. [18] Fernández, G.P.; Segura, A.; Herrera, M.; Velasco, W.; Solano, G.; Gutiérrez, J.M.; León, G. Neutralization of Bothrops mattogrossensis snake venom from Bolivia: Experimental evaluation of llama and donkey antivenoms produced by caprylic acid precipitation. Toxicon, 2010, 55, 642-645. [19] World Health Organization. WHO Guidelines for the Production, Control and Regulation of Snake Antivenom Immunoglobulins; World Health Organization: Geneva, 2010. [20] Gutiérrez, J.M.; Rojas, G.; Bogarín, G.; Lomonte, B. Evaluation of the Neutralizing Ability of Antivenoms for the Treatment of Snake Bite Envenoming in Central America. In: Envenomings and Their Treatments; Bon, C.; Goyffon, M., Eds,; Fondation Marcel Mérieux: Lyon, 1996; pp. 223-231. [21] Gutiérrez, J.M. ; Lomonte, B. ; León, G. ; Alape-Girón, A. ; Flores- Díaz, M. ; Sanz, L. ; Angulo, Y. ; Calvete, J.J. Snake venomics and antivenomics: Proteomic tools in the design and control of antivenoms for the treatment of snakebite envenoming. J. Proteomics, 2009, 165-182. [22] Calvete, J.J.; Sanz, L.; Angulo, Y.; Lomonte, B.; Gutiérrez, J.M. Venoms, venomics, antivenomics. FEBS Lett. 2009, 583, 1736- 1743. [23] Fan, H.W.; Cardoso, J.L. Clinical Toxicology of Snake Bites in South America. In: Handbook of Clinical Toxicology of Animal Venoms and Poisons; Meier, J.; White, J., Eds.; CRC Press: Boca Raton, 1995; pp. 667-688. [24] Warrell, D.A. Epidemiology, Clinical Features and Management of Snakebites in Central and South America In: The Venomous Reptiles of the Western Hemisphere; Campbell, J.A.; Lamar, W.W., Eds.; Cornell University Press: Ithaca and London, 2004; Vol. I, pp. 709-761. [25] Bolaños, R.; Cerdas, L.; Abalos, J.W. Venoms of coral snakes (Micrurus spp): report of a multivalent antivenin for the Americas. Bull. Pan Am. Health Org., 1978, 12, 23-27. [26] Ismail, M.; Al-Ahaidib, M.S.; Abdoon, N.; Abd-Elsalam, M.A. Preparation of a novel antivenom against Atractaspis and Walterinnesia venoms. Toxicon, 2007, 49, 8-18. [27] Ariaratnam, C.A.; Thuraisingam, V.; Kularatne, S.A.; Sheriff, M.H.; Theakstomn, R.D.G.; de Silva, A.; Warrell, D.A. Frequent and potentially fatal envenoming by hump-nosed pit vipers (Hypnale hypnale and H. nepa) in Sri Lanka: lack of effective antivenom. Trans. R. Soc. Trop. Med. Hyg., 2008, 102, 1120-1126. [28] Chippaux, J.P.; Williams, V.; White, J. Snake venom variability: methods of study, results and interpretation. Toxicon, 1991, 29, 1279-1303. [29] Alape-Girón, A.; Sanz, L.; Escolano, J.; Flores-Díaz, M.; Madrigal, M.; Sasa, M.; Calvete, J.J. Snake venomics of the lancehead pitviper Bothrops asper: geographic, individual and ontogenetic variations. J. Proteome Res., 2008, 7, 3556-3571. [30] Barlow, A.; Pook, C.E.; Harrison, R.A.; Wüster, W. Coevolution of diet and prey-specific venom activity supports the role of selection in snake venom evolution. Proc. Biol. Sci., 2009, 276, 1443-1449. [31] Chotwiwatthanakun, C.; Pratapaphon, R.; Akesowan, S.; Sriprapat, S.; Ratanabanangkoon, K. Production of potent polyvalent antivenom against three elapid venoms using a low dose, low volume, multi-site immunization protocol. Toxicon, 2001, 39, 1487-1494. [32] Christensen, P.A. 1955. South African Snake Venoms and Antivenoms. The South African Institute for Medical Research: Johannesburg, 1955, 129 p. [33] Sriprapat, S.; Aeksowan, S.; Sapsutthipas, S.; Chotwiwatthanakun, C.; Suttijitpaisal, P.; Pratanaphon, R.; Khow, O.; Sitprija, V.; Ratanabanangkoon, K. The impact of a low dose, low volume, multi-site immunization on the production of therapeutic antivenoms in Thailand. Toxicon, 2003, 41, 57-64. [34] Gutiérrez, J.M.; Rojas, E.; Quesada, L.; León, G.; Núñez, J.; Laing, G.D.; Sasa, M.; Renjifo, J.M.; Nasidi, A.; Warrell, D.A.; Theakston, R.D.G.; Rojas, G. Pan-African polyspecific antivenom produced by caprylic acid purification of horse IgG: an alternative to the antivenom crisis in Africa. Trans. R. Soc. Trop. Med. Hyg., 2005, 99, 468-475. [35] Cardoso, D.F.; Mota, I. Effect of Crotalus venom on the humoral and cellular immune response. Toxicon, 1997, 35, 607-612. [36] Stephano, M.A.; Guidolin, R.; Higashi, H.G.; Tambourgi, D.V.; Sant'Anna, O.A. The improvement of the therapeutic anti-Lachesis muta serum production in horses. Toxicon, 2005, 45, 467-473. [37] Angulo, Y.; Estrada, R.; Gutiérrez, J.M. Clinical and laboratory alterations in horses during immunization with snake venoms for the production of polyvalent (Crotalinae) antivenom. Toxicon, 1997, 35, 81-90. [38] Fernandes, I.; Lima, E.X.; Takehara, H.A.; Moura-da-Silva, A.M.; Tanjoni, I.; Gutiérrez, J.M. Horse IgG isotypes and cross- neutralization of two snake antivenoms produced in Brazil and Costa Rica. Toxicon 2000, 38, 633-644. [39] Steinbach, F.; Deeg, C.; Mauel, S.; Wagner, B. Equine immunology: offspring of the serum horse. Trends Immunol., 2002, 23, 223-225. [40] Bolaños, R. Antivenenos. In: Manual de Procedimientos. Producción y Pruebas de Control en la Preparación de Antisueros Diftérico, Tetánico, Botulínico, Antivenenos y de la Gangrena 10 Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 Gutiérrez et al. Gaseosa; Organización Panamericana de la Salud: Washington, D.C., 1977; pp. 104-141. [41] Otero, R.; Gutiérrez, J.M.; Rojas, G.; Núñez, V.; Díaz, A.; Miranda, E.; Uribe, A.F.; Silva, J.F.; Ospina, J.G.; Medina, Y.; Toro, M.F.; García, M.E.; León, G.; García, M.; Lizano, S.; De La Torre, J.; Márquez, J.; Mena, Y.; González, N.; Arenas, L.C.; Puzón, A.; Blanco, N.; Sierra, A.; Espinal, M.E.; Arboleda, M.; Jiménez, J.C.; Ramírez, P.; Díaz, M.; Guzmán, M.C.; Barros, J.; Henao, S.; Ramírez, A.; Macea, U.; Lozano, R.; A randomized blinded clinical trial of two antivenoms, prepared by caprylic acid or ammonium sulphate fractionation of IgG in Bothrops and Porthidium snake bites in Colombia: correlation between safety and biochemical characteristics of antivenoms. Toxicon, 1999, 37, 895-908. [42] Raw, I.; Guidolin, R.; Higashi, H.G.; Kelen, E.M.A. Antivenins in Brazil: Preparation. In: Handbook of Natural Toxins, Reptile Venoms and Toxins; Tu, A.T., Ed.; Marcel Dekker: New York, 1991, Vol 5, pp. 557-581. [43] Jadhav, S.S.; Kapre, S.V. Antivenom production in India. In: Handbook of Natural Toxins. Reptile Venoms and Toxins; Tu, A.T., Ed.; Marcel Dekker: New York, 1991, Vol 5, 583-610. [44] Jones, R.G.A.; Landon, J. A protocol for ‘enhanced pepsin digestion’: a step by step method for obtaining pure antibody fragments in high yield from serum. J. Immunol. Meth., 2003, 275, 239-250. [45] Scherrmann, J.M. Antibody treatment of toxin poisoning-recent advances. J. Toxicol. Clin. Toxicol., 1994, 32, 363-375. [46] Gutiérrez, J.M.; León, G.; Lomonte, B. Pharmacokinetic- pharmacodynamic relationships of immunoglobulin therapy for envenomation. Clin. Pharmacokinet., 2003, 42, 721-741. [47] Covell, D.G.; Barbet, J.; Holton, O.D.; Black, C.D.; Parker, R.J.; Weinstein, J.N. Pharmacokinetics of monoclonal immunoglobulin G1, F(ab’)2, and Fab’ in mice. Cancer Res., 1986. 46, 3969-3978. [48] Ariaratnam, C.A.; Meyer, W.P.; Perera, G.; Eddleston, M.; Kuleratne, S.A.; Attapattu, W.; Sheriff, R.; Richards, A.M.; Theakston, R.D.G.; Warrell, D.A. A new monospecific ovine Fab fragment antivenom for treatment of envenoming by the Sri Lankan Russell’s viper (Daboia russelii russelli): a preliminary dose-finding and pharmacokinetic study. Am. J. Trop. Med. Hyg., 1999, 61, 259-65. [49] Boyer, L.V. ; Seifert, S.A. ; Clark, R.F.; Williams, S.R.; Nordt, S.P.; Walter, F.G.; Dart, R.C. Recurrent and persistent coagulopathy following pit viper envenomation. Arch. Intern. Med., 1999, 159, 706-710. [50] Gutiérrez, J.M. ; Lomonte, B. ; León, G. ; Rucavado, A. ; Chaves, F. ; Angulo, Y. Trends in snakebite envenomation therapy: scientific, technological and public health considerations. Curr. Pharm. Des., 2007, 13, 2935-2950. [51] Olvera, A.; Ramos-Cerrillo, B.; Estévez, J.; Clement, H.; de Roodt, A.; Paniagua-Solís, J.; Vázquez, H.; Zavaleta, A.; Arruz, M.S.; Stock, R.P.; Alagón, A. North and South American Loxosceles spiders: development of a polyvalent antivenom with recombinant sphingomyelinases D as antigens. Toxicon, 2006, 48, 64-74. [52] Azevedo-Marques, M.M.; Hering, S.E.; Cupo, P. Acidente Crotálico. In: Animais Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes; Cardoso, J.L.C.; França, F.O.S.; Wen, F.H.; Málaque, C.M.S.; Haddad, V., Eds.; Sarvier: Sao Paulo, 2003; pp. 91-98. [53] Harrison, R.A. Development of venom toxin-specific antibodies by DNA immunization: rationale and strategies to improve therapy of viper envenoming. Vaccine 2004, 22, 1648-1655. [54] Wagstaff, S.C.; Laing, G.D.; Theakston, R.D.G.; Papaspyridis, C.; Harrison, R.A. Bioinformatics and multiepitope DNA immunization to design rational snake antivenom. PLoS Med., 2006, 3, e184. [55] Wang, L.; Sun, X.; Ghosh, R. Purification of equine IgG using membrane based enhanced hybrid bioseparation technique: a potential method for manufacturing hyperimmune antibody. Biotechnol. Bioeng., 2008, 99, 625-633. [56] Burnouf, T.; Radosevich, M. Nanofiltration of plasma-derived biopharmaceutical products. Haemophilia, 2003, 9, 24-37. [57] Segura. A.; León, G.; Su, C.Y.; Gutiérrez, J.M.; Burnouf, T. Assessment of the impact of solvent/detergent treatment on the quality and potency of a whole IgG equine antivenom. Biologicals, 2009, 37, 306-312. [58] Ménez, A. Molecular immunology of snake toxins. Pharmac. Ther., 1985, 30, 91-113. [59] Serrano, S.M.T.; Shannon, J.D.; Wang, D.; Camargo, A.C.; Fox, J.W.. A multifaceted analysis of viperid snake venoms by two- dimensional gel electrophoresis: an approach to understanding venom proteomics. Proteomics, 2005, 5, 501-510. [60] Calvete, J.J.; Juárez, P.; Sanz, L. Snake venomics. Strategy and applications. J. Mass Spectrom., 2007, 42, 1405-1414. [61] Cardoso, D.F.; Yamaguchi, I.K.; Moura da Silva, A.M. Produção de Soros Antitoxinas e Perspectivas de Modernização por Técnicas de Biologia Molecular. In: Animais Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes; Cardoso, J.L.C.; França, F.O.S.; Wen, F.H.; Málaque, C.M.S.; Haddad, V., Eds.; Sarvier: Sao Paulo, 2003, pp. 367-379. [62] Selisko, B.; Cosio, G.; García, C.; Becerril, B.; Possani, L.D.; Horjales, E. Bacterial expression, purification and functional characterization of a recombinant chimeric Fab derived from murine mAb BCF2 that neutralizes the venom of the scorpion Centruroides noxius Hoffmann. Toxicon, 2004, 43, 43-51. [63] Vaughan, T.J.; Williams, A.J.; Pritchard, K.; Osbourn, J.K.; Pope, A.R.; Earnshaw, J.C.; McCafferty, J.; Hodits, R.A.; Wilton, J.; Johnson, K.S. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nature Biotechnol., 1996, 14, 309-14, [64] Lafaye, P.; Choumet, V.; Demangel, C.; Bon, C.; Mazié, J.C. Biologically active human anti-crotoxin scFv isolated from a semi- synthetic phage library. Immunotechnology, 1997, 3, 117-125. [65] Stewart, C.S.; MacKenzie, C.R.; Hall, J.C. Isolation, characterization and pentamerization of -cobrotoxin specific single-domain antibodies from a naïve phage display library: preliminary findings for antivenom development. Toxicon, 2007, 49, 699-709. [66] Juárez-González, V.R.; Riaño-Umbarila, L.; Quintero-Hernández, V.; Olamendi-Portugal, T.; Ortiz-León, M.; Possani, L.D.; Becerril, B. Directed evolution, phage display and combination of evolved mutants: a strategy to recover the neutralization properties of the scFv version of BCF2 a neutralizing monoclonal antibody specific to scorpion toxin Cn2. J. Mol. Biol., 2005, 346, 1287-1297. [67] Kulkeaw, K.; Sakolvaree, Y.; Srimanote, P.; Tongtawe, P.; Maneewatch, S.; Sookrung, N.; Tungtrongchitr, A.; Tapchaisri, P.; Kurazono, H.; Chaicumpa, W. Human monoclonal scFv neutralize lethal Thai cobra, Naja kaouthia, neurotoxin. J. Proteomics, 2009, 72, 270-282. [68] Lauwereys, M.; Gharoudi, M.A.; Desmyter, A.; Kinne, J.; Hölzer, W.; De Genst, E.; Wyns, L.; Muyldermans, S. Potent enzyme inhibitors derived from dromedary heavy-chain antibodies. EMBO J, 1998, 17, 3512-20. [69] Omidfar, K.; Rasaee, M.J.; Moditahedi, H.; Forouzandeh, M.; Taghikhani, M.; Golmakani, N. Production of a novel camel single- domain antibody specific for the type III mutant EGFR. Tumor Biol., 2004, 25, 296-305. [70] Rodrigues-Silva, R.; Antunes, G.F.; Velarde, D.T.; Santoro, M.M. Thermal stability studies of hyperimmune horse antivenoms. Toxicon, 1999, 37, 33-45. [71] Segura, A.; Herrera, M.; González, E.; Vargas, M.; Solano, G.; Gutiérrez, J.M.; León, G. Stability of equine IgG antivenoms obtained by caprylic acid precipitation: towards a liquid formulation stable at tropical room temperature. Toxicon, 2009, 53, 609-615. [72] Maria, W.S.; Pacheco, B.G.; Barbosa, C.F.; Velarde, D.T.; Chavez- Olórtegui, C. Determination of the neutralizing potency of horse antibothropic and anticrotalic antivenoms in blood samples collected on filter paper. Toxicon, 2001, 39, 1607-1609. [73] Rial, A.; Morais, V.; Rossi, S.; Massaldi, H. A new ELISA for determination of potency in snake antivenoms. Toxicon, 2006, 48, 462-466. [74] Gutiérrez, J.M.; Avila, C.; Rojas, G.; Cerdas L. An alternative in vitro method for testing the potency of the polyvalent antivenom produced in Costa Rica. Toxicon, 1988, 26, 411-413. [75] Sells, P.G.; Laing, G.D.; Theakston, R.D.G. An in vivo but insensate model for the evaluation of antivenoms (ED50) using fertile hens’ eggs. Toxicon, 2001, 39, 665-668. [76] World Health Organization. Progress in the Characterization of Venoms and Standardization of Antivenoms. World Health Organization: Geneva, 1981; pp. 1-43. Antivenoms for Snakebite Envenomings Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 11 [77] Bogarín, G.; Morais, J.F.; Yamaguchi, I.K.; Stephano, M.A.; Marcelino, J.R.; Nishikawa, A.K.; Guidolin, R.; Rojas, G.; Higashi, H.G.; Gutiérrez, J.M. Neutralization of crotaline snake venoms from Central and South America by antivenoms produced in Brazil and Costa Rica. Toxicon, 2000, 38, 1429-1441. [78] Araújo, H.P.; Bourguignon, S.C.; Boller, M.A.; Dias, A.A.; Lucas, E.P.; Santos, I.C.; Delgado, I.F. Potency evaluation of antivenoms in Brazil: the national control laboratory experience between 2000 and 2006. Toxicon, 2008, 51, 502-514. [79] Gutiérrez, J.M.; Rojas, G.; Cerdas, L. Ability of a polyvalent antivenom to neutralize the venom of Lachesis muta melanocephala, a new Costa Rican subspecies of the bushmaster. Toxicon, 1987, 25, 713-20. [80] Theakston, R.D.G.; Reid, H.A. Development of simple standard assay procedures for the characterization of snake venom. Bull. World Health Organ., 1983, 61, 949-956. [81] Instituto Clodomiro Picado. Determinación de Actividades Tóxicas de Venenos de Serpientes y su Neutralización por Antivenenos. Manual de Métodos de Laboratorio. Instituto Clodomiro Picado: San José, 2008, pp. 1-31. [82] Gutiérrez, J.M.; Chaves, F.; Rojas, E.; Elizondo, J.; Avila, C.; Cerdas, L. Production of monovalent anti-Bothrops asper antivenom: development of immune response in horses and neutralizing ability. Rev. Biol. Trop., 1988, 36, 511-517. [83] Colombini, M.; Fernandes, I.; Cardoso, D.F.; Moura-da-Silva, A.M. Lachesis muta muta venom: immunological differences compared with Bothrops atrox venom and importance of specific antivenom therapy. Toxicon, 2001, 39, 711-719. [84] Theakston, R.D.G.; Laing, G.D.; Fielding, C.M.; Lascano, A.F.; Touzet, J.M.; Vallejo, F.; Guderian, R.H.; Nelson, S.J.; Wüster, W., Richards, A.M. Treatment of snake bites by Bothrops species and Lachesis muta in Ecuador: laboratory screening of candidate antivenoms. Trans. R. Soc. Trop. Med. Hyg., 1995, 89, 550-554. [85] Gutiérrez, J.M.; Gené, J.A.; Rojas, G.; Cerdas, L. Neutralization of proteolytic and hemorrhagic activities of Costa Rican snake venoms by a polyvalent antivenom. Toxicon, 1985, 23, 887-893. [86] De Roodt, A. ; Dolab, J.A. ; Fernández, T. ; Segre, L. ; Hajos, S.E. Cross-reactivity and heterologous neutralization of crotaline antivenoms used in Argentina. Toxicon, 1998, 36, 1025-38. [87] Segura, A.; Villalta, M.; Herrera, M.; León, G.; Harrison, R.; Durfa, N.; Nasidi, A.; Calvete, J.J.; Theakston, R.D.G.; Warrell, D.A.; Gutiérrez, J.M. Preclinical assessment of the efficacy of a new antivenom (EchiTAb-Plus-ICP®) for the treatment of viper envenoming in sub-Saharan Africa. Toxicon, 2010, 55, 369-374 [88] Lomonte, B.; León, G.; Angulo, Y.; Rucavado, A.; Núñez, V. Neutralization of Bothrops asper venom by antibodies, natural products, and synthetic drugs: contributions to understanding snakebite envenomings and their treatment. Toxicon 2009, 54, 1012-1028. [89] Meyer, W.P.; Habib, A.G.; Onayade, A.A.; Yakubu, A.; Smith, D.C.; Nasidi, A.; Daudu, I.J.; Warrell, D.A.; Theakston, R.D.G. First clinical experiences with a new ovine Fab Echis ocellatus snake bite antivenom in Nigeria: randomized comparative trial with Institute Pasteur Serum (IPSER) Africa antivenom. Am. J. Trop. Med. Hyg., 1997, 56, 291-300. [90] Warrell, D.A.; Davidson, N.M.; Omerod, L.D.; Pope, H.M.; Watkins, B.J.; Greenwood, B.M.; Reid, H.A. Bites by the saw- scaled or carpet viper (Echis carinatus): trial of two specific antivenoms. Br. Med. J., 1974, 4, 437-440. [91] Cardoso, J.L.C.; Fan, H.W.; França, F.O.S.; Jorge, M.T.; Leite, R.P.; Nishioka, S.A.; Ávila, A.; Sano-Martins, I.S.; Tomy, S.C.; Santoro, M.L.; Chudzinski, A.M.; Castro, S.C.B.; Kamiguti, A.S.; Kelen, E.M.A.; Hirata, M.H.; Mirnadola, R.M.S.; Theakston, R.D.G.; Warrell, D.A. Randomized comparative trial of three antivenoms in the treatment of envenoming by lance-headed vipers (Bothrops jararaca) in São Paulo, Brazil. Q. J. Med., 1993, 86, 315-325. [92] Smalligan, R.; Cole, J.; Brito, N.; Laing, G.D.; Mertz, B.L.; Manock, S.; Maudlin, J.; Quist, B.; Holland, G.; Nelson, S.; Lalloo, D.G.; Rivadeneira, G.; Barragan, M.E.; Dolley, D.; Eddleston, M.; Warrell, D.A.; Theakston, R.D.G. Crotaline snake bite in the Ecuadorian Amazon: randomized double blind comparative trial of three South American polyspecific antivenoms. Br. Med. J., 2004, 329, 1129-1135. [93] Otero, R.; León, G.; Gutiérrez, J.M.; Rojas, G.; Toro, M.F.; Barona, J.; Rodríguez, V.; Díaz, A.; Núñez, V.; Quintana, J.C.; Ayala, S.; Mosquera, D.; Conrado, L.L.; Fernández, D.; Arroyo, Y.; Paniagua, C.A.; López, M.; Ospina, C.E.; Alzate, C.; Fernández, J.; Meza, J.J.; Silva, J.F.; Ramírez, P.; Fabra, P.E.; Ramírez, E.; Córdoba, E.: Arrieta, A.B.; Warrell, D.A.; Theakston, R.D.G. Efficacy and safety of two whole IgG polyvalent antivenoms, refined by caprylic acid fractionation with or without -propiolactone, in the treatment of Bothrops asper bites in Colombia. Trans. R. Soc. Trop. Med. Hyg., 2006, 100, 1173-1182. [94] Chippaux, J.P.; Massougbodji, A.; Stock, R.; Alagón, A. Clinical trial of an F(ab’)2 polyvalent antivenom for African snake bites in Benin. Am. J. Trop. Med. Hyg., 2007, 77, 538-546. [95] Warrell, D.A. Clinical Features of Envenoming from Snake Bites. In: Envenomings and Their Treatments; Bon, C.; Goyffon, M., Eds.; Fondation Marcel Mérieux : Lyon, 1996, pp. 63-76. [96] Gutiérrez, J.M.; León, G. ; Rojas, G.; Lomonte, B.; Rucavado, A.; Chaves, F. Neutralization of local tissue damage induced by Bothrops asper (terciopelo) snake venom. Toxicon, 1998, 36, 1529- 1538. [97] Warrell, D.A. Clinical Toxicology of Snakebite in Africa and the Middle East/Arabian Peninsula. In: Handbook of Clinical Toxicology of Animal Venoms and Poisons; Meier, J.; White, J., Eds.; CRC Press: Boca Raton, 1995; pp. 433-492. [98] Fan, H.W. Soroterapia. In: Animais Peçonhentos no Brasil. Biologia, Clínica e Terapêutica dos Acidentes; Cardoso, J.L.C.; França, F.O.S.; Wen, F.H.; Málaque, C.M.S.; Haddad, V., Eds.; Sarvier: Sao Paulo, 2003; pp. 380-393. [99] Chippaux, J.P.; Lang, J.; Eddine, S.A.; Fagot, P.; Rage, V.; Peyrieux, J.C.; Le Mener, V. Clinical safety of a polyvalent F(ab’)2 equine antivenom in 223 African snake envenomations: a field trial in Cameroon. Trans. R. Soc. Trop. Med. Hyg., 1998, 92, 657-62. [100] Otero-Patiño, R.; Cardoso, J.L.C.; Higashi, H.G.; Núñez, V.; Díaz, A.; Toro, M.F.; García, M.E.; Sierra, A.; García, L.F.; Moreno, A.M.; Medina, M.C.; Castañeda, N.; Silva-Díaz, J.F.; Murcia, M.; Cárdenas, S.Y.; Dias da Silva, W.D. A randomized, blinded, comparative trial of one pepsin-digested and two whole IgG antivenoms for Bothrops snake bites in Urabá, Colombia. Am. J. Trop. Med. Hyg., 1998, 58, 183-189. [101] Moran, N.F.; Newman, W.J.; Theakston, R.D.G.; Warrell, D.A.; Wilkinson, D. High incidence of early anaphylactoid reaction to SAIMR polyvalent snake antivenom. Trans. R. Soc. Trop. Med. Hyg., 1998, 92, 69-70. [102] Warrell, D.A. WHO/SEARO Guidelines for the clinical management of snake bites in the Southeast Asian region. Southeast Asian J. Trop. Med. Public Health, 1999, 30 (Suppl 1), 1-85. [103] Sutherland, S.K. Serum reactions. An analysis of commercial antivenoms and the possible role of anticomplementary activity in de-novo reactions to antivenoms and antitoxins. Med. J. Aust., 1977, 1, 613-615. [104] León, G.; Monge, M.; Rojas, E.; Lomonte, B.; Gutiérrez, J.M. Comparison between IgG and F(ab’)2 polyvalent antivenoms: neutralization of systemic effects produced by Bothrops asper venom in mice, extravasation to muscle tissue, and potential for induction of adverse reactions. Toxicon, 2001, 39, 793-801. [105] Herrera, M.; León, G.; Segura, A.; Meneses, F.; Lomonte, B.; Chippaux, J.P.; Gutiérrez, J.M. Factors associated with adverse reactions induced by caprylic acid-fractionated whole IgG preparations: comparison between horse, sheep and camel IgGs. Toxicon, 2005, 46, 775-81. [106] Malasit, P.; Warrell, D.A.; Chanthavanich. P.; Viravan, C.; Mongkolsapaya, J.; Singhthong, B.; Supich, C. Prediction, prevention, and mechanism of early (anaphylactic) antivenom reactions in victims of snake bites. Br. Med. J., 1986, 292, 17-20. [107] Morais, J.F.; de Freitas, M.C.; Yamaguchi, I.K.; Dos Santos, M.C.; Dias da Silva, W. Snake antivenoms from hyperimmunized horses: comparison of the antivenom activity and biological properties of their whole IgG and F(ab’)2 fragments. Toxicon, 1994, 32, 725-734. [108] Otero-Patiño, R.; Silva-Haad, J.J.; Barona-Acevedo, M.J.; Toro- Castaño, M.F.; Quintana-Castillo, J.C.; Díaz Cadavid, A.; Vásquez- Vélez, I.C.; Rodríguez-Rivera, V.; Delgado-Figueroa, C.I.; Fernández-Ceballos, M.; Ayala-Audiber, S.; Conrado-Chaverra, N.L.; Marín-Restrepo, C.A.; Ramírez-Giraldo, C.E.; Arrieta-Vides, A.B.; Córdoba-Julio, E.A.; Ruiz-Zabaleta, T.I.; García-Castañeda, M.V.; Aguirre-Agudelo, A.S.; Londoño-Guerra, J.J.; Ospina- Bedoya, N.; Macías-Prada, D.M.; Jaramillo-Delgado, O.F.; Peláez- Agudelo, H.D.; Espinal-Saldarriaga, M.E.; Camargo-Ballestas, 12 Inflammation & Allergy - Drug Targets, 2011, Vol. 10, No. 5 Gutiérrez et al. J.M. Accidente botrópico en Colombia: estudio multicéntrico de la eficacia y seguridad de Antivipmyn-Tri, un antiveneno polivalente producido en México. Iatreia, 2007, 20, 244-262. [109] García, M.; Monge, M.; León, G.; Lizano, S.; Segura, E.; Solano, G.; Rojas, G.; Gutiérrez, J.M. Effect of preservatives on IgG aggregation, complement-activating effect and hypotensive activity of horse polyvalent antivenom used in snakebite envenomation. Biologicals, 2002, 30, 143-151. [110] León, G.; Rodríguez, M.A.; Rucavado, A.; Lomonte, B.; Gutiérrez, J.M. Anti-human erythrocyte antibodies in horse-derived antivenoms used in the treatment of snakebite envenomations. Biologicals, 2007, 35, 5-11. [111] Ayuso, R.; Lehrer, S.B.; López, M.; Reese, G.; Ibáñez, M.D.; Esteban, M.M.; Ownby, D.R.; Schwartz, H. Identification of bovine IgG as a major cross-reactive vertebrate meat allergen. Allergy, 2000, 55, 348-354. [112] Hunter, W.M.; Budd, P.S. Circulating antibodies to ovine and bovine immunoglobulin in healthy subjects: a hazard for immunoassays. Lancet, 1980, 1136. [113] Bernhisel-Broadbent, J.; Yolken, R.H.; Sampson, H.A. Allergenicity of orally administered immunoglobulin preparations in food-allergic children. Pediatrics, 1991, 87, 208-214. [114] León, G.; Segura, A.; Herrera, M.; Otero, R.; França, F.O.; Barbaro, K.C.; Cardoso, J.L.; Wen, F.H.; de Medeiros, C.R.; Prado, J.C.; Málaque, C.M.; Lomonte, B.; Gutiérrez, J.M. Human heterophylic antibodies against equine immunoglobulins: assessment of their role in the early adverse reactions to antivenom administration. Trans. R. Soc. Trop. Med. Hyg., 2008, 102, 1115- 1119. [115] Corrigan, P.; Russell, F.E.; Wainschel, J. Clinical reactions to antivenin. In: Toxins: Animal, Plant and Microbial; Rosenberg, P., Ed.; Pergamon Press: Oxford, 1978; pp. 457-65. [116] LoVecchio, F.; Klemens, J.; Roundy, E.B.; Klemens, A. Serum sickness following administration of Antivenin (Crotalidae) Polyvalent in 181 cases of presumed rattlesnake envenomation. Wilderness Environ. Med., 2003, 14, 220-221. [117] Burnouf, T.; Griffiths, E.; Padilla, A.; Seddik, S.; Stephano, M.A.; Gutiérrez, J.M. Assessment of the viral safety of antivenoms fractionated from equine plasma. Biologicals, 2004, 32, 115-128. [118] Burnouf, T.; Terpstra, F.; Habib, G.; Seddik, S. Assessment of viral inactivation during pH 3.3 pepsin digestion and caprylic acid treatment of antivenoms. Biologicals, 2007, 35, 329-334. [119] Williams, D.; Gutiérrez, J.M.; Harrison, R.; Warrell, D.A.; White, J.; Winkel, K.D.; Gopalakrishnakone, P. An antidote for snake bite: the global snake bite initiative. Lancet, 2010, 375, 89-91. [120] Gutiérrez, J.M.; Williams, D.; Fan, H.W.; Warrell, D.A. Snakebite envenoming from a global perspective: towards an integrated approach. Toxicon, 2010, 56, 1223-1235. [121] Leynaud, G.C.; Reati, G.J. Identificación de las zonas de riesgo ofídico en Córdoba, Argentina, mediante el programa SIGEpi. Rev. Panam. Salud Pública, 2009, 26, 64-69. [122] Gutiérrez, J.M.; Fan, H.W.; Silvera, C.L.; Angulo, Y. Stability, distribution and use of antivenoms for snakebite envenomation in Latin America: report of a workshop. Toxicon, 2009, 53, 625-630. [123] Han, S.K.; Yoon, E.T.; Scott, D.L.; Sigler, P.B.; Cho, W. Structural aspects of interfacial adsorption. A crystallographic and site- directed mutagenesis study of the phospholipase A2 from the venom of Agkistrodon piscivorus piscivorus. J. Biol. Chem., 1997, 272, 3573-3582. Received: August 2, 2010 Revised: February 15, 2011 Accepted: February 17, 2011