Pan-American Lancehead Pit-Vipers: Coagulotoxic Venom Effects and Antivenom Neutralisation of Bothrops asper and B. atrox Geographical Variants.

The toxin composition of snake venoms and, thus, their functional activity, can vary between and within species. Intraspecific venom variation across a species' geographic range is a major concern for antivenom treatment of envenomations, particularly for countries like French Guiana that lack a locally produced antivenom. Bothrops asper and Bothrops atrox are the most medically significant species of snakes in Latin America, both producing a variety of clinical manifestations, including systemic bleeding. These pathophysiological actions are due to the activation by the venom of the blood clotting factors Factor X and prothrombin, thereby causing severe consumptive coagulopathy. Both species are extremely wide-ranging, and previous studies have shown their venoms to exhibit regional venom variation. In this study, we investigate the differential coagulotoxic effects on human plasma of six venoms (four B. asper and two B. atrox samples) from different geographic locations, spanning from Mexico to Peru. We assessed how the venom variation of these venom samples affects neutralisation by five regionally available antivenoms: Antivipmyn, Antivipmyn-Tri, PoliVal-ICP, Bothrofav, and Soro Antibotrópico (SAB). The results revealed both inter- and intraspecific variations in the clotting activity of the venoms. These variations in turn resulted in significant variation in antivenom efficacy against the coagulotoxic effects of these venoms. Due to variations in the venoms used in the antivenom production process, antivenoms differed in their species-specific or geographical neutralisation capacity. Some antivenoms (PoliVal-ICP, Bothrofav, and SAB) showed species-specific patterns of neutralisation, while another antivenom (Antivipmyn) showed geographic-specific patterns of neutralisation. This study adds to current knowledge of Bothrops venoms and also illustrates the importance of considering evolutionary biology when developing antivenoms. Therefore, these results have tangible, real-world implications by aiding evidence-based design of antivenoms for treatment of the envenomed patient. We stress that these in vitro studies must be backed by future in vivo studies and clinical trials before therapeutic guidelines are issued regarding specific antivenom use in a clinical setting.

Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama). / Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama).

OBJECTIVES: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. MATERIALS AND METHODS: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. RESULTS: The venom's LD50 was 3.96 µg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 µL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. CONCLUSIONS: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

OBJETIVOS: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. MATERIALES Y MÉTODOS: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. RESULTADOS: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. CONCLUSIONES: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

Neutralización de la actividad letal del veneno de serpiente Bothrops atrox por suero hiperinmune de llama (Lama glama) / Neutralization of the lethal activity from Bothrops atrox venom by hyperimune llama serum (Lama glama)

RESUMEN Objetivos: Evaluar la capacidad del suero hiperinmune de llama (Lama glama) para neutralizar la letalidad del veneno de la serpiente Bothrops atrox en ratones de laboratorio. Materiales y métodos: Se calculó la dosis letal media (DL50) de un pool de venenos de serpientes de Bothrops atrox de Perú, y se midieron los títulos de anticuerpos por ensayo ELISA; así como la potencia de neutralización del suero inmune por el cálculo de la dosis efectiva media (DE50) durante el periodo de inmunización. Resultados: La DL50 del veneno fue de 3,96 µg/g, similar a otros trabajos realizados en Bothrops atrox en Perú. Los títulos de anticuerpos contra el veneno se incrementan rápidamente en la llama mostrando una rápida respuesta inmune; sin embargo, la capacidad de neutralización se incrementa más lentamente y requiere de varias dosis y refuerzos de las inmunizaciones alcanzado una DE50 de 3,30 µL/g ratón y una potencia de neutralización 3,6 mg/mL después de 15 inmunizaciones. Conclusiones: El suero hiperinmune de llama es capaz de neutralizar la letalidad del veneno de la serpiente Bothrops atrox de Perú en ratones de laboratorio.

ABSTRACT Objectives: To evaluate the capacity of the hyperimmune llama serum (Lama glama) to neutralize the lethal activity of Bothrops atrox venom in laboratory mice. Materials and methods: Mean lethal dose (LD50) was calculated from a Bothrops atrox venom sample pool from Peru. The antibody titers were measured by ELISA assay; and the immune serum neutralization potency was measured by calculating the mean effective dose (ED50) during the immunization period. Results: The venom's LD50 was 3.96 μg/g; similar to what was found in other studies about Bothrops atrox carried out in Peru. The titers of antibodies against the venom increased rapidly in the llama, demonstrating a fast immune response; however, the neutralization capacity increased slowly and required several doses and immunization reinforcements, obtaining a ED50 of 3.30 μL/g mouse and a neutralization potency of 3.6 mg/mL after 15 immunizations. Conclusions: The hyperimmune llama serum is able to neutralize the lethality of the Bothrops atrox venom from Peru in laboratory mice.

Identification of cross-reactive human single-chain variable fragments against phospholipases A2 from Lachesis muta and Bothrops spp venoms.

Bushmasters (Lachesis spp) and lancehead vipers (Bothrops spp) are two of the most dangerous snakes found in Latin America. Victims of envenoming by these snakes require urgent administration of antivenom. Here, we report the identification of a small set of broadly neutralizing human monoclonal single-chain variable fragment (scFv) antibodies targeting key phospholipases A2 from Lachesis and Bothrops spp using phage display technology and demonstrate their in vitro efficacy using a hemolysis assay.

Identification of a linear B-cell epitope in the catalytic domain of bothropasin, a metalloproteinase from Bothrops jararaca snake venom.

Bothropasin is a hemorrhagic snake venom metalloproteinase (SVMP) from Bothrops jararaca venom, the snake responsible for most bites in Southeastern Brazil. SVMPs, such as bothropasin, are involved in the main bothropic envenoming symptoms, which include hemorrhage, inflammation, necrosis and blood coagulation deficiency. B-cell epitope mapping of SVMPs can lead to the identification of peptides capable of inducing neutralizing antibodies without causing toxic effects, therefore improving anti-venom production. Here, using the SPOT synthesis technique, we have identified an epitope located in the catalytic domain of bothropasin (202KARMYELANIVNEILRYLYMH222) which was synthesized and named BotEp1. The peptide was used to immunize Swiss mice and Anti-BotEp1 serum cross-reacted with bothropasin and crude venoms from B. jararaca and B. atrox venoms. Furthermore, Anti-BotEp1 antibodies were able to completely neutralize the hemorrhagic activity of a chromatographic fraction from B. jararaca venom, which contains hemorrhagic SVMPs. In addition, the coagulation activity of the hemorrhagic fraction showed to be diminished when tested in serum from rabbit immunized with BotEp1 (compared to serum from non-immunized animal). Our results show the identification of neutralizing epitopes in bothropasin and provide basis for the use of synthetic peptides to improve the production of immunotherapeutics.

Compositional and functional investigation of individual and pooled venoms from long-term captive and recently wild-caught Bothrops jararaca snakes.

Intraspecific venom variability has been extensively reported in a number of species and is documented to be the result of several factors. However, current evidence for snake venom variability related to captivity maintenance is controversial. Here we report a compositional and functional investigation of individual and pooled venoms from long-term captive (LTC) and recently wild-caught (RWC) B. jararaca snakes. The composition of individual venoms showed a remarkable variability in terms of relative abundance of toxins (evidenced by 1-DE and RP-HPLC), enzymatic activities (proteolytic, PLA2, and LAAO) and coagulant activity, even among captive specimens. Thus, no compositional and functional pattern could be established to assign each individual venom to a specific group. Conversely, pooled venom from LTC and RWC snakes showed no significant differences regarding protein composition (characterized by 1-DE and shotgun proteomics), enzymatic activities (proteolytic, PLA2 and LAAO) and biological function (coagulant, hemorrhagic and lethal activities), except for edematogenic activity, which was more prominent in RWC venom pool. Additionally, both pooled venoms displayed similar immunoreactivity with the bothropic antivenom produced by Instituto Butantan. Taken together, our results highlight the complexity and the high intraspecific variation of B. jararaca venom, that is not influenced at a discernible extent by captivity maintenance. BIOLOGICAL SIGNIFICANCE: Bothrops jararaca snakes are one of the main causes of snakebites in Southeastern Brazil. Due to its medical interest, the venom of this species is the most studied and characterized among Brazilian snakes and captive B. jararaca specimens are maintained for long periods of time in our venom production facility. However, knowledge on the influence of captivity maintenance on B. jararaca venom variability is scarce. In this report, we described a high compositional and functional variability of individual venoms from LTC and RWC B. jararaca snakes, which are not observed between LTC and RWC pooled venoms. This intraspecific variability is more likely to be due to genetic/populational differences rather than "captivity vs wild" conditions. In this regard, data generated by the present work support the use of venom from captive and wild snakes for antivenom production and scientific research. Moreover, the data generated by this study highlight the importance of analyzing individual venom samples in studies involving intraspecific venom variability.

Antiparasitic effects induced by polyclonal IgY antibodies anti-phospholipase A2 from Bothrops pauloensis venom.

Activities of phospholipases (PLAs) have been linked to pathogenesis in various microorganisms, and implicated in cell invasion and so the interest in these enzymes as potential targets that could contribute to the control of parasite survival and proliferation. Chicken eggs immunized with BnSP-7, a Lys49 phospholipase A2 (PLA2) homologue from Bothrops pauloensis snake venom, represent an excellent source of polyclonal antibodies with potential inhibitory activity on parasite PLAs. Herein, we report the production, characterization and anti-parasitic effect of IgY antibodies from egg yolks of hens immunized with BnSP-7. Produced antibodies presented increasing avidity and affinity for antigenic toxin epitopes throughout immunization, attaining a plateau after 4weeks. Pooled egg yolks-purified anti-BnSP-7 IgY antibodies were able to specifically recognize different PLA2s from Bothrops pauloensis and Bothrops jararacussu venom. Antibodies also neutralized BnSP-7 cytotoxic activity in C2C12 cells. Also, the antibodies recognized targets in Leishmania (Leishmania) amazonensis and Toxoplasma gondii extracts by ELISA and immunofluorescence assays. Anti-BnSP-7 IgY antibodies were cytotoxic to T. gondii tachyzoite and L. (L.) amazonensis promastigotes, and were able to decrease proliferation of both parasites treated before infection. These data suggest that the anti-BnSP-7 IgY is an important tool for discovering new parasite targets and blocking parasitic effects.

Use of EDTA in the treatment of local tissue damage caused by the Bothrops alternatus venom / Utilização do EDTA no tratamento da lesão tecidual local causado pelo veneno de Bothrops alternatus

Twelve adult rabbits were distributed in three groups and received on the femoral biceps region, via intradermal injection (ID), 25µg of Bothrops alternatus venom dissolved in NaCl 0.9% and diluted in 0.25mL of phosphate buffered saline (PBS). Thirty minutes later, the group G1 received 0.25mL of phosphate buffered saline (PBS) ID while to G2 and G3 25mg of ethylenediamine tetraacetic acid (EDTA) dissolved in 0.25mL of PBS were administered via intramuscular (IM) and intravenous (IV) injection, respectively. Evaluations included local lesion and blood profile of all animals, before (time zero) and at 1, 2, 3, 4, 5, 6, 12, 18 and 24h after venom administration. All animal treated with PBS (G1) and EDTA IV (G3) presented increase of nociceptive stimuli at the site of inoculation of the venom, followed by moderate edema that persisted for 24h. Animals treated with IM EDTA (G2) only manifested increase of nociceptive stimuli at the site of injection 1h after treatment with discrete local edema between 12 and 24h. In relation to the local hemorrhagic halo no differences were found amongst the studied groups. Blood profile revealed significant decrease of segmented neutrophils in all groups. There was also increase in triglycerides and decrease in total protein and albumin in all groups. The local lesion was not altered by the treatments.(AU)

Doze coelhos adultos, distribuídos em três grupos, receberam, na região de bíceps femoral, por via intradérmica (ID), 25µg de veneno de Bothrops alternatus, dissolvidos em NaCl 09%, diluído em 0,25mL de tampão salina fosfato (PBS). Trinta minutos após o desafio, o grupo G1 recebeu 0,25mL de (PBS) ID, e os grupos G2 e G3 receberam 25mg de ácido etilenodiamino tetra-acético (EDTA), dissolvidos em mL de PBS por via intramuscular (IM) e intravenosa (IV), respectivamente. Foram avaliados lesão local e perfil sanguíneo de todos os animais, antes - tempo zero, e à uma, às duas, três, quatro, cinco, seis, 12, 18 e 24 horas após a injeção do veneno. Tanto os animais tratados com PBS (G1) como os animais tratados com EDTA IV (G3) apresentaram aumento do estímulo nociceptivo no local da administração do veneno, seguido por moderado edema, que perdurou por 24h. Os animais tratados com EDTA IM (G2) somente manifestaram aumento do estímulo nociceptivo local uma hora após tratamento e discreto edema local entre 12 e 24 horas. Em relação ao halo hemorrágico, não houve diferença entre os três grupos estudados. No perfil hematológico, observou-se diminuição significativa dos neutrófilos segmentados nos três grupos estudados. Da mesma forma, houve aumento dos triglicerídeos e diminuição da proteína total e albumina em todos os grupos. Conclui-se que a lesão local não foi alterada pelos tratamentos.(AU)

Cross-reactivity and inhibition myotoxic effects induced by Bothrops snake venoms using specific polyclonal anti-BnSP7 antibodies.

Polyclonal antibodies raised in Balb-c mice against BnSP-7, a Lys-49 phospholipase A2, were used to measure cross reactivity against other snake venoms. Using ELISA, these antibodies were able to recognize PLA2s isoforms present in venoms of botropic snakes at 1:6400, 1:3200 and 1:100 ratios (w/w). These antibodies highly recognized proteins of low molecular weight (∼14,000) from crude snake venom Bp and Bm by Western Blotting. PLA2 these venoms, by alignment of primary structures demonstrated high identity with BnSP-7 PLA2, especially in the C-terminal region. However, the crude snake venom Bd and Bj, showed low recognition. The PLA2 activity of Bothrops pauloensis, Bothrops moojeni venoms or BpPLA2-TXI was inhibited significantly when anti-BnSP-7 antibodies were incubated at 1:10 and 1:20 ratios (venoms or toxin:anti-BnSP-7, w/w), respectively. The myotoxic effect induced by the same venoms was also reduced significantly at 1:1, 1:10 and 1:20 ratios, by decreased creatine kinase levels. The anti-PLA2 polyclonal antibodies effectively recognized PLA2s from Bothrops pauloensis and Bothrops moojeni venoms, and neutralized specific catalytic and myotoxic activity.

Active immunization of cattle with a bothropic toxoid does not abrogate envenomation by Bothrops asper venom, but increases the likelihood of survival.

This study assessed the protective effect of active immunization of cattle to prevent the envenomation induced by B. asper venom. Two groups of oxen were immunized with a bothropic toxoid and challenged by an intramuscular injection of either 10 or 50 mg B. asper venom, to induce moderate or severe envenomations, respectively. Non-immunized oxen were used as controls. It was found that immunized oxen developed local edema similar to those observed in non-immunized animals. However, systemic effects were totally prevented in immunized oxen challenged with 10 mg venom, and therefore antivenom treatment was not required. When immunized oxen were challenged with 50 mg venom, coagulopathy was manifested 3-16 h later than in non-immunized oxen, demonstrating a delay in the onset of systemic envenomation. In these animals, active immunization did not eliminate the need for antivenom treatment, but increased the time lapse in which antivenom administration is still effective. All experimentally envenomed oxen completely recovered after a week following venom injection. Our results suggest that immunization of cattle with a bothropic toxoid prevents the development of systemic effects in moderate envenomations by B. asper, but does not abrogate these effects in severe envenomation.

Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.

In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA2, proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2µg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30µg/site) and significantly inhibited by both ratios. Venom (10-300µg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai.

Lachesis stenophrys venom reduces the equine antibody response towards Bothrops asper venom used as co-immunogen in the production of polyspecific snake antivenom.

The anti-bothropic activity of an antivenom prepared from the plasma of horses immunized with Bothrops asper venom (anti-B antivenom) was compared with a similar formulation produced from the plasma of horses immunized with a mixture of B. asper and Lachesis stenophrys venoms (anti-BL antivenom). Likewise, a comparison between the anti-lachesic activity of the anti-BL antivenom and a similar formulation prepared from horses immunized only with L. stenophrys venom (anti-L antivenom) was performed. The anti-BL antivenom had lower concentration of anti-bothropic antibodies than the anti-B antivenom. This difference was associated to a lower response towards all components of B. asper venom, but particularly towards some D49-phospholipases A2 (PLA2s) and PIII-metalloproteinases. Consequently, the anti-BL antivenom was less effective neutralizing lethal, coagulant, defibrinogenating, PLA2, and myotoxic activities of B. asper venom. On the other hand, anti-BL and anti-L antivenoms showed similar concentration of anti-lachesic antibodies, and similar capacity to recognize the HPLC fractions of L. stenophrys venom and to neutralize lethal, coagulant, proteolytic, hemorrhagic, PLA2 and myotoxic activities induced by this venom. It is concluded that, when used as co-immunogens, the venom of L. stenophrys reduces the antibody response towards B. asper venom, whereas the latter does not affect the anti-lachesic response.

Alkylation of histidine residues of Bothrops jararacussu venom proteins and isolated phospholipases A2: a biotechnological tool to improve the production of antibodies.

Crude venom of Bothrops jararacussu and isolated phospholipases A2 (PLA2) of this toxin (BthTX-I and BthTX-II) were chemically modified (alkylation) by p-bromophenacyl bromide (BPB) in order to study antibody production capacity in function of the structure-function relationship of these substances (crude venom and PLA2 native and alkylated). BthTX-II showed enzymatic activity, while BthTX-I did not. Alkylation reduced BthTX-II activity by 50% while this process abolished the catalytic and myotoxic activities of BthTX-I, while reducing its edema-inducing activity by about 50%. Antibody production against the native and alkylated forms of BthTX-I and -II and the cross-reactivity of antibodies to native and alkylated toxins did not show any apparent differences and these observations were reinforced by surface plasmon resonance (SPR) data. Histopathological analysis of mouse gastrocnemius muscle sections after injection of PBS, BthTX-I, BthTX-II, or both myotoxins previously incubated with neutralizing antibody showed inhibition of the toxin-induced myotoxicity. These results reveal that the chemical modification of the phospholipases A2 (PLA2) diminished their toxicity but did not alter their antigenicity. This observation indicates that the modified PLA2 may provide a biotechnological tool to attenuate the toxicity of the crude venom, by improving the production of antibodies and decreasing the local toxic effects of this poisonous substance in animals used to produce antivenom.

Comparison of the adjuvant activity of aluminum hydroxide and calcium phosphate on the antibody response towards Bothrops asper snake venom.

The adjuvanticity of aluminum hydroxide and calcium phosphate on the antibody response in mice towards the venom of the snake Bothrops asper was studied. It was found that, in vitro, most of the venom proteins are similarly adsorbed by both mineral salts, with the exception of some basic phospholipases A2, which are better adsorbed by calcium phosphate. After injection, the adjuvants promoted a slow release of the venom, as judged by the lack of acute toxicity when lethal doses of venom were administered to mice. Leukocyte recruitment induced by the venom was enhanced when it was adsorbed on both mineral salts; however, venom adsorbed on calcium phosphate induced a higher antibody response towards all tested HPLC fractions of the venom. On the other hand, co-precipitation of venom with calcium phosphate was the best strategy for increasing: (1) the capacity of the salt to couple venom proteins in vitro; (2) the venom ability to induce leukocyte recruitment; (3) phagocytosis by macrophages; and (4) a host antibody response. These findings suggest that the chemical nature is not the only one determining factor of the adjuvant activity of mineral salts.

BnSP-7 toxin, a basic phospholipase A2 from Bothrops pauloensis snake venom, interferes with proliferation, ultrastructure and infectivity of Leishmania (Leishmania) amazonensis.

This paper reports the effects of BnSP-7 toxin, a catalytically inactive phospholipase A2 from Bothrops pauloensis snake venom, on Leishmania (Leishmania) amazonensis. BnSP-7 presented activity against promastigote parasite forms both in the MTT assay, with IC50 of 58.7 µg mL(-1) of toxin, and a growth curve, inhibiting parasite proliferation 60-70% at concentrations of 50-200 µg mL(-1) of toxin 96 h after treatment. Also, the toxin presented effects on amastigotes, reducing parasite viability by 50% at 28.1 µg mL(-1) and delaying the amastigote-promastigote differentiation process. Ultrastructural studies showed that BnSP-7 caused severe morphological changes in promastigotes such as mitochondrial swelling, nuclear alteration, vacuolization, acidocalcisomes, multiflagellar aspects and a blebbing effect in the plasma membrane. Finally, BnSP-7 interfered with the infective capacity of promastigotes in murine peritoneal macrophages, causing statistically significant infectivity-index reductions (P < 0.05) of 20-35%. These data suggest that the BnSP-7 toxin is an important tool for the discovery of new parasite targets that can be exploited to develop new drugs for treating leishmaniasis.

Human antibody fragments specific for Bothrops jararacussu venom reduce the toxicity of other Bothrops sp. venoms.

Approximately 20,000 snakebites are registered each year in Brazil. The classical treatment for venomous snakebite involves the administration of sera obtained from immunized horses. Moreover, the production and care of horses is costly, and the use of heterologous sera can cause hypersensitivity reactions. The production of human antibody fragments by phage display technology is seen as a means of overcoming some of these disadvantages. The studies here attempted to test human monoclonal antibodies specific to Bothrops jararacussu against other Bothrops sp. venoms, using the Griffin.1 library of human single-chain fragment-variable (scFv) phage antibodies. Using the Griffin.1 phage antibody library, this laboratory previously produced scFvs capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom. The structural and functional similarities of the various forms of phospholipase A2 (PLA2) in Bothrops venom served as the basis for the present study wherein the effectiveness of those same scFvs were evaluated against B. jararaca, B. neuwiedi, and B. moojeni venoms. Each clone was found to recognize all three Bothrops venoms, and purified scFvs partially inhibited their in vitro phospholipase activity. In vivo assays demonstrated that the scFv clone P2B7 reduced myotoxicity and increased the survival of animals that received the test venoms. The results here indicate that the scFv P2B7 is a candidate for inclusion in a mixture of specific antibodies to produce a human anti-bothropic sera. This data demonstrates that the human scFv P2B7 represents an alternative therapeutic approach to heterologous anti-bothropic sera available today.

Venom of Bothrops asper from Mexico and Costa Rica: intraspecific variation and cross-neutralization by antivenoms.

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.

Neutralization of Bothrops alternatus regional venom pools and individual venoms by antivenom: a systematic comparison.

In this study we report that variation in lethality, hemorrhagic potency and procoagulation between individual samples of Bothrops alternatus venom from a single region, and variation between regional pools at the national level are comparable in range. Furthermore, the range of relative neutralization potencies of individual venoms within a region overlaps, and sometimes exceeds, the range of neutralization of regional venom pools throughout the country. Thus, the potency of neutralization of a national venom pool is poorly predictive of the potencies of neutralization of its constituent regional venom pools and, furthermore, the potency of neutralization of a regional venom pool is poorly predictive of the potencies of neutralization of its individual venom constituents. The efficiencies of neutralization of each of these effects (lethality, hemorrhage and procoagulation) were not significantly related to each other and did not correlate to the corresponding toxic potency of each venom or venom pool. Some implications of these findings are discussed in the context of the distinction between experimental quantitation of antivenom potency and the amount of antivenom that might be actually required to successfully treat two apparently comparable B. alternatus envenomations.