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Each year, millions of humans fall victim to animal envenomings, which may either be deadly or cause permanent disability to the effected individuals. The Nobel Prize-winning discovery of serum therapy for the treatment of bacterial infections (tetanus and diphtheria) paved the way for the introduction of antivenom therapies for envenomings caused by venomous animals. These antivenoms are based on polyclonal antibodies derived from the plasma of hyperimmunized animals and remain the only specific treatment against animal envenomings. Following the initial development of serum therapy for snakebite envenoming by French scientists in 1894, other countries with high incidences of animal envenomings, including Brazil, Australia, South Africa, Costa Rica, and Mexico, started taking up antivenom production against local venomous animals over the course of the twentieth century. These undertakings revolutionized envenoming therapy and have saved innumerous patients worldwide during the last 100 years. This review describes in detail the above-mentioned historical events surrounding the discovery and the application of serum therapy for envenomings, as well as it provides an overview of important developments and scientific breakthroughs that were of importance for antibody-based therapies in general. This begins with discoveries concerning the characterization of antibodies, including the events leading up to the elucidation of the antibody structure. These discoveries further paved the way for other milestones in antibody-based therapies, such as the introduction of hybridoma technology in 1975. Hybridoma technology enabled the expression and isolation of monoclonal antibodies, which in turn formed the basis for the development of phage display technology and transgenic mice, which can be harnessed to directly obtain fully human monoclonal antibodies. These developments were driven by the ultimate goal of producing potent neutralizing monoclonal antibodies with optimal pharmacokinetic properties and low immunogenicity. This review then provides an outline of the most recent achievements in antivenom research, which include the application of new biotechnologies, the development of the first human monoclonal antibodies that can neutralize animal toxins, and efforts toward creating fully recombinant antivenoms. Lastly, future perspectives in the field of envenoming therapies are discussed, including rational engineering of antibody cross-reactivity and the use of oligoclonal antibody mixtures.

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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.

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The venom proteomes of Bothrops atrox from Colombia, Brazil, Ecuador, and Perú were characterized using venomic and antivenomic strategies. Our results evidence the existence of two geographically differentiated venom phenotypes. The venom from Colombia comprises at least 26 different proteins belonging to 9 different groups of toxins. PI-metalloproteinases and K49-PLA(2) molecules represent the most abundant toxins. On the other hand, the venoms from Brazilian, Ecuadorian, and Peruvian B. atrox contain predominantly PIII-metalloproteinases. These toxin profiles correlate with the venom phenotypes of adult and juvenile B. asper from Costa Rica, respectively, suggesting that paedomorphism represented a selective trend during the trans-Amazonian southward expansion of B. atrox through the Andean Corridor. The high degree of crossreactivity of a Costa Rican polyvalent (Bothrops asper, Lachesis stenophrys, Crotalus simus) antivenom against B. atrox venoms further evidenced the close evolutionary kinship between B. asper and B. atrox. This antivenom was more efficient immunodepleting proteins from the venoms of B. atrox from Brazil, Ecuador, and Perú than from Colombia. Such behaviour may be rationalized taking into account the lower content of poorly immunogenic toxins, such as PLA(2) molecules and PI-SVMPs in the paedomorphic venoms. The immunological profile of the Costa Rican antivenom strongly suggests the possibility of using this antivenom for the management of snakebites by B. atrox in Colombia and the Amazon regions of Ecuador, Perú and Brazil.

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Cardiovascular function is affected by many mechanisms, including the autonomic system, the kallikrein-kinin system (KKS), the renin-angiotensin system (RAS) and the endothelin system. The function of these systems seems to be fairly well preserved throughout the vertebrate scale, but evolution required several adaptations. Snakes are particularly interesting for studies related to the cardiovascular function because of their elongated shape, their wide variation in size and length, and because they had to adapt to extremely different habitats and gravitational influences. To keep the normal cardiovascular control the snakes developed anatomical and functional adaptations and interesting structural peculiarities are found in their autonomic, KKS, RAS and endothelin systems. Our laboratory has characterized some biochemical, pharmacological and physiological properties of these systems in South American snakes. This review compares the components and function of these systems in snakes and other vertebrates, and focuses on differences found in snakes, related with receptor or ligand structure and/or function in autonomic system, RAS and KKS, absence of components in KKS and the intriguing identity between a venom and a plasma component in the endothelin system.

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Intraspecific variation in Crotalus durissus terrificus venom composition was studied in relation to crotamine activity. Crotamine induces paralysis in extension of hind legs of mice and myonecrosis in skeletal muscle cells. To determine whether the venom of crotamine-negative rattlesnake contains a quantity of myotoxin incapable of inducing paralysis, we have developed a very sensitivity immunological assay method, an enzyme-linked immunoabsorbent assay (ELISA), capable of detecting 0.6 ng of purified crotamine. The parallel-lines analysis of ELISA data showed to be useful because it shows the reliability of the experimental conditions. A variation in the amount of myotoxin in the crotamine-positive venom was observed, but not less than 0.1 mg of crotamine per mg of venom. It was not possible to detect it in crotamine-negative venom even at high venom concentrations.

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Se estudió la actividad fibrinolítica del veneno Lachesis muta stenophyrs y de su enzima fibrinogenolítica. Ratas Wistar cateterizadas e la arteria carótida y vena yugular fueron inoculadas con el veneno crudo o la enzima. Se monitoreó los cambios en la presión arterial, frecuencia cardíaca y electrocardiograma. La enzima indujo una mayor reducción del fibrinógeno en el veneno crudo sin causar alteraciones cardiovasculares o histológicas. In vitro el veneno crudo coaguló la sangre mientras que la enzima redujo el fibrinógeno en un 23 por ciento. Los resultados sugieren el uso potencial de la enzima fibrinogenolítica como agente antitrombótico

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A phospholipase myotoxin (MOO-1) and a non-phospholipase myotoxin (JSU-5) were studied for their antigenic cross-reactivity and neutralization by different antisera. Antisera against JSU-5 and MOO-1 reacted equally with both myotoxins in ELISA assays. The specificity of these antisera was also similar, recognizing the same 14,000-18,000 mol. wt components in the venoms of Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi and Bothrops pradoi. Using creatine kinase assays, JSU-5 myotoxicity was completely neutralized by B. jararacussu antivenom or anti-JSU-5 antibodies and partially neutralized by B. moojeni antivenom or anti-MOO-1 antibodies. MOO-1 myotoxicity was completely neutralized by antisera against JSU-5 and MOO-1 and B. jararacussu antivenom, and only partially neutralized by B. moojeni antivenom. B. jararacussu venom induced high titres of antibodies against purified myotoxins. This antiserum completely inhibited the myotoxicity of the homologous venom and significantly reduced the myotoxicity of the remaining myotoxin-containing venoms. It is suggested that B. jararacussu venom is a good immunogen to induce antibodies against myotoxins present in the venoms of the different species of Bothrops.

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A comparative study was performed on some enzymatic and toxic activities of venoms collected from newborn, one-year old, two-years old and adult (more than five-years old) specimens of Lachesis muta stenophrys. There was an increase in lethal, hemorrhagic, edema-forming, myotoxic, proteolytic and phospholipase A2 activities of venoms as snakes aged. The venom of newborn specimens was almost devoid of toxicity. On the other hand, venom from newborn specimens showed the highest coagulant effect of human plasma. Electrophoretic and immunochemical results demonstrated conspicuous differences between venoms of different ages. Observations on the feeding behavior indicated that specimens of L. muta of different ages displayed a similar pattern, characterized by rapid strike and bite, holding the prey until they stopped their movements and swallowing them afterwards. It is concluded that venom of newborn L. muta has very low toxic and proteolytic activities and that it undergoes conspicuous changes during the first year of life.

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En este trabajo se revisan y discuten una serie de aspectos relativos a la produccion, estabilidad y capacidad neutralizante en el suero antiofidico polivalente producido en el Instituto Clodomiro Picado. Este suero hiperinmune, en su forma liquida, es estable por tres años en refrigeracion en tanto que la forma liofilizada es estable por 5 años, sin requerir refrigeracion. Los diferentes lotes de suero son uniformes en lo que respecta a su capacidad neutralizante contra las actividades toxicas de los venenos. La capacidad neutralizante de este suero a sido investigada mediante dos tipos de experimento:(a.aquellos a los que el suero se incuba con el veneno previo a su inoculacion en animales de experimentacion; y (b) aquellos en que el suero se administra por la via intravenosa a diferentes intervalos de tiempo despues de la inoculacion del veneno. Los resultados obtenidos señalan que el suero es eficiente en la neutralizacion de los efectos toxicos y enzimaticos inducidos por las serpientes costarricenses de la familia viperidae, cuando suero y veneno se incuban previo a su inoculacion. Esta neutralizacion tambien se ha demostrado contra venenos de serpientes de otros paises de centro y Sudaamerica. Por otra parte, cuando el suero se administra despues de innoculado el veneno, los efectos locales (mionecrosis, hemorragia local y edema) son neutralizados solo parcialmente, en tanto que los efectos letal y defibrinante se neutralizan con mayor eficacia estas observaciones se explican por el hecho de que los efectos locales se desencadenan con una rapidez tal que, cuando se administra el suero, ya se ha iniciado el desarrollo de estas alteraciones locales. Se concluye que el suero polivalente contiene anticuerpos eficaces en la neutralizacion de las actividades toxicas y enzimaticas de estos venenos; sin embargo, la neutralizacion de los efectos locales, en la practica, se hace dificil siendo necesaria una rapida administracion de un volumen suficiente de suero antiofidico

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Sera obtained from 18 Bothrops alternatus and 2 Phylodrias burmeisteri were analyzed for the presence of antileptospiral antibodies using a microscopic agglutination test. Out of 18 Bothrops sera, 13 (72%) gave positive results. Snake serum reacted with patoc, andamana, wolffi, tarassovi, pomona, pyrogenes and shermani serovars. It can be concluded that Leptospiral antibodies are present in snake sera in Argentina, suggesting that the circulating antibodies may have an important role in the epizoothiology of the disease.

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Proteins antigenically related to CoF, the anticomplementary protein of the venom of the Indian hooded cobra (Naja naja), were found in a variety of elapid and viper venoms but not in the venom of Brazilian crotalids . In keeping with this finding was the weak ability of Brazilian snake venom to convert C3 in human serum. All snake serums tested, including Brazilian crotalids , contained a beta-globulin antigenically related to CoF. This serum protein in Brazilian snake serum had a number of characteristics of mammalian C3, including conversion on storage or on incubation of this serum with endotoxin, zymosan or mammalian antigen-antibody precipitates. The serum protein did not, however, convert on incubation with hydrazine. Brazilian crotalid serum did not, as did cobra serum, have the ability to inactivate CoF's ability to activate complement in normal human serum. The crotalid serum had hemolytic activity for rabbit antibody-sensitized and unsensitized sheep red blood cells that was active in the presence of Ca++ and Mg++ or Mg++ alone but greater with Ca++ present, suggesting the presence of both classical and alternative pathways of complement activation. This activity was maximal at 37 degrees C, but was destroyed or inactive after heating at 50 degrees C for 1 hr, incubation with hydrazine or by addition of EDTA. A marked reduction of hemolytic activity of Bothrops serum occurred after removal of the CoF-like protein. These findings suggest that Brazilian snake venom has little CoF-like material, but its serum contains a CoF-like protein with many characteristics of C3.(ABSTRACT TRUNCATED AT 250 WORDS)

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