Exploring the Profile of Cell Populations and Soluble Immunological Mediators in Bothrops atrox Envenomations.

Bothrops atrox envenomations are common in the Brazilian Amazon. The venom of B. atrox is highly inflammatory, which results in severe local complications, including the formation of blisters. Moreover, there is little information on the immune mechanisms associated with this condition. Thus, a longitudinal study was carried out to characterize the profile of the cell populations and soluble immunological mediators in the peripheral blood and blisters in B. atrox patients s according to their clinical manifestations (mild and severe). A similar response in both B. atrox patient groups (MILD and SEV) was observed, with an increase in inflammatory monocytes, NKT, and T and B cells, as well as CCL2, CCL5, CXCL9, CXCL10, IL-1ß and IL-10, when compared with the group of healthy blood donors. After the administration of antivenom, the participation of patrolling monocytes and IL-10 in the MILD group was observed. In the SEV group, the participation of B cells was observed, with high levels of CCL2 and IL-6. In the blister exudate, a hyperinflammatory profile was observed. In conclusion, we revealed the involvement of cell populations and soluble mediators in the immune response to B. atrox envenomation at the local and peripheral level, which is related to the onset and extent of the inflammation/clinical manifestation.

Oral Tolerance Induction by Bothrops jararaca Venom in a Murine Model and Cross-Reactivity with Toxins of Other Snake Venoms.

Oral tolerance is defined as a specific suppression of cellular and humoral immune responses to a particular antigen through prior oral administration of an antigen. It has unique immunological importance since it is a natural and continuous event driven by external antigens. It is characterized by low levels of IgG in the serum of animals after immunization with the antigen. There is no report of induction of oral tolerance to Bothrops jararaca venom. Here, we induced oral tolerance to B. jararaca venom in BALB/c mice and evaluated the specific tolerance and cross-reactivity with the toxins of other Bothrops species after immunization with the snake venoms adsorbed to/encapsulated in nanostructured SBA-15 silica. Animals that received a high dose of B. jararaca venom (1.8 mg) orally responded by showing antibody titers similar to those of immunized animals. On the other hand, mice tolerized orally with three doses of 1 µg of B. jararaca venom showed low antibody titers. In animals that received a low dose of B. jararaca venom and were immunized with B. atrox or B. jararacussu venom, tolerance was null or only partial. Immunoblot analysis against the venom of different Bothrops species provided details about the main tolerogenic epitopes and clearly showed a difference compared to antiserum of immunized animals.

Association of a Network of Immunologic Response and Clinical Features With the Functional Recovery From Crotalinae Snakebite Envenoming.

Background: The immunologic pathways activated during snakebite envenoming (SBE) are poorly described, and their association with recovery is unclear. The immunologic response in SBE could inform a prognostic model to predict recovery. The purpose of this study was to develop pre- and post-antivenom prognostic models comprised of clinical features and immunologic cytokine data that are associated with recovery from SBE. Materials and Methods: We performed a prospective cohort study in an academic medical center emergency department. We enrolled consecutive patients with Crotalinae SBE and obtained serum samples based on previously described criteria for the Surgical Critical Care Initiative (SC2i)(ClinicalTrials.gov Identifier: NCT02182180). We assessed a standard set of clinical variables and measured 35 unique cytokines using Luminex Cytokine 35-Plex Human Panel pre- and post-antivenom administration. The Patient-Specific Functional Scale (PSFS), a well-validated patient-reported outcome of functional recovery, was assessed at 0, 7, 14, 21 and 28 days and the area under the patient curve (PSFS AUPC) determined. We performed Bayesian Belief Network (BBN) modeling to represent relationships with a diagram composed of nodes and arcs. Each node represents a cytokine or clinical feature and each arc represents a joint-probability distribution (JPD). Results: Twenty-eight SBE patients were enrolled. Preliminary results from 24 patients with clinical data, 9 patients with pre-antivenom and 11 patients with post-antivenom cytokine data are presented. The group was mostly female (82%) with a mean age of 38.1 (SD ± 9.8) years. In the pre-antivenom model, the variables most closely associated with the PSFS AUPC are predominantly clinical features. In the post-antivenom model, cytokines are more fully incorporated into the model. The variables most closely associated with the PSFS AUPC are age, antihistamines, white blood cell count (WBC), HGF, CCL5 and VEGF. The most influential variables are age, antihistamines and EGF. Both the pre- and post-antivenom models perform well with AUCs of 0.87 and 0.90 respectively. Discussion: Pre- and post-antivenom networks of cytokines and clinical features were associated with functional recovery measured by the PSFS AUPC over 28 days. With additional data, we can identify prognostic models using immunologic and clinical variables to predict recovery from SBE.

Examination of the Efficacy and Cross-Reactivity of a Novel Polyclonal Antibody Targeting the Disintegrin Domain in SVMPs to Neutralize Snake Venom.

Snake envenomation can result in hemorrhage, local necrosis, swelling, and if not treated properly can lead to adverse systemic effects such as coagulopathy, nephrotoxicity, neurotoxicity, and cardiotoxicity, which can result in death. As such, snake venom metalloproteinases (SVMPs) and disintegrins are two toxic components that contribute to hemorrhage and interfere with the hemostatic system. Administration of a commercial antivenom is the common antidote to treat snake envenomation, but the high-cost, lack of efficacy, side effects, and limited availability, necessitates the development of new strategies and approaches for therapeutic treatments. Herein, we describe the neutralization ability of anti-disintegrin polyclonal antibody on the activities of isolated disintegrins, P-II/P-III SVMPs, and crude venoms. Our results show disintegrin activity on platelet aggregation in whole blood and the migration of the SK-Mel-28 cells that can be neutralized with anti-disintegrin polyclonal antibody. We characterized a SVMP and found that anti-disintegrin was also able to inhibit its activity in an in vitro proteolytic assay. Moreover, we found that anti-disintegrin could neutralize the proteolytic and hemorrhagic activities from crude Crotalus atrox venom. Our results suggest that anti-disintegrin polyclonal antibodies have the potential for a targeted approach to neutralize SVMPs in the treatment of snakebite envenomations.

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.

Prevalence of Acute Hypersensitivity Reactions in Pediatric Patients Receiving Crotalidae Polyvalent Immune Fab.

INTRODUCTION: Studies of acute hypersensitivity reactions in pediatric populations receiving Crotalidae Polyvalent Immune Fab (CPIF) are complicated by small size, wide age ranges, and diverse definitions of such reactions. METHODS: This is a retrospective chart review of patients aged 13 years or younger treated with CPIF for Crotalid envenomation from November 2006 to 2016. The primary outcome was the presence of an acute hypersensitivity reaction to CPIF and was defined as the development of any of the following symptoms within 3 hours of initiation of CPIF infusion: urticaria, wheezing or respiratory distress, angioedema, hypotension, nausea, and/or vomiting. Demographics, CPIF dose to control and total dose, bite location, level of care, and length of stay were also recorded. RESULTS: Thirty-four patients were ultimately treated with CPIF. Ages ranged from 10 months to 13 years. Twenty-one patients (60%) were male, 24 (70.6%) were admitted to the ICU, and the median length of stay was 2 days with a range of 1-11 days. Zero patients developed an acute hypersensitivity reaction to CPIF. CONCLUSION: Acute hypersensitivity reactions to CPIF did not occur in this cohort. Such reactions are rare with the use of CPIF in pediatric patients.

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.

Correlating Fibrinogen Consumption and Profiles of Inflammatory Molecules in Human Envenomation's by Bothrops atrox in the Brazilian Amazon.

Snakebites are considered a major public health problem worldwide. In the Amazon region of Brazil, the snake Bothrops atrox (B. atrox) is responsible for 90% of the bites. These bites may cause local and systemic signs from acute inflammatory reaction and hemostatic changes, and present common hemorrhagic disorders. These alterations occur due the action of hemostatically active and immunogenic toxins which are capable of triggering a wide range of hemostatic and inflammatory events. However, the crosstalk between coagulation disorders and inflammatory reaction still has gaps in snakebites. Thus, the goal of this study was to describe the relationship between the consumption of fibrinogen and the profile of inflammatory molecules (chemokines and cytokines) in evenomations by B. atrox snakebites. A prospective study was carried out with individuals who had suffered B. atrox snakebites and presented different levels of fibrinogen consumption (normal fibrinogen [NF] and hypofibrinogenemia [HF]). Seventeen patients with NF and 55 patients with HF were eligible for the study, in addition to 50 healthy controls (CG). The molecules CXCL-8, CCL-5, CXCL-9, CCL-2, CXCL-10, IL-6, TNF, IL-2, IL-10, IFN-γ, IL-4, and IL-17A were quantified in plasma using the CBA technique at three different times (pre-antivenom therapy [T0], 24 h [T1], and 48 h [T2] after antivenom therapy). The profile of the circulating inflammatory response is different between the groups studied, with HF patients having higher concentrations of CCL-5 and lower IFN-γ. In addition, antivenom therapy seems to have a positive effect, leading to a profile of circulating inflammatory response similar in quantification of T1 and T2 on both groups. Furthermore, these results suggest that a number of interactions of CXCL-8, CXCL-9, CCL-2, IL-6, and IFN-γ in HF patients are directly affected by fibrinogen levels, which may be related to the inflammatory response and coagulation mutual relationship induced by B. atrox venom. The present study is the first report on inflammation-coagulation crosstalk involving snakebite patients and supports the better understanding of envenomation's pathophysiology mechanisms and guides in the search for novel biomarkers and prospective therapies.

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.

Development of Nanobodies Against Hemorrhagic and Myotoxic Components of Bothrops atrox Snake Venom.

Snake envenoming is a globally neglected public health problem. Antivenoms produced using animal hyperimmune plasma remain the standard therapy for snakebites. Although effective against systemic effects, conventional antivenoms have limited efficacy against local tissue damage. In addition, potential hypersensitivity reactions, high costs for animal maintenance, and difficulties in obtaining batch-to-batch homogeneity are some of the factors that have motivated the search for innovative and improved therapeutic products against such envenoming. In this study, we have developed a set of nanobodies (recombinant single-domain antigen-binding fragments from camelid heavy chain-only antibodies) against Bothrops atrox snake venom hemorrhagic and myotoxic components. An immune library was constructed after immunizing a Lama glama with whole venom of B. atrox, from which nanobodies were selected by phage display using partially purified hemorrhagic and myotoxic proteins. Biopanning selections retrieved 18 and eight different nanobodies against the hemorrhagic and the myotoxic proteins, respectively. In vivo assays in mice showed that five nanobodies inhibited the hemorrhagic activity of the proteins; three neutralized the hemorrhagic activity of whole B. atrox venom, while four nanobodies inhibited the myotoxic protein. A mixture of the anti-hemorrhagic and anti-myotoxic nanobodies neutralized the local tissue hemorrhage and myonecrosis induced by the whole venom, although the nanobody mixture failed to prevent the venom lethality. Nevertheless, our results demonstrate the efficacy and usefulness of these nanobodies to neutralize important pathologies of the venom, highlighting their potential as innovative therapeutic agents against envenoming by B. atrox, a viperid species causing many casualties in South America.

Thromboelastography with Platelet Studies (TEG® with PlateletMapping®) After Rattlesnake Envenomation in the Southwestern United States Demonstrates Inhibition of ADP-Induced Platelet Activation As Well As Clot Lysis.

INTRODUCTION: Hematologic effects of North American rattlesnake envenomation can include fibrinogenolysis and thrombocytopenia, depending on species, geography, and other variables. During treatment, these effects are routinely monitored through assessment of fibrinogen concentrations and platelet counts. However, these tests provide no information about fibrinolysis or platelet dysfunction, both of which can also occur with venom from some species. METHODS: This was a retrospective chart review of patients admitted to a quaternary care academic hospital (Banner - University Medical Center Phoenix) in the southwestern United States for treatment of rattlesnake envenomation, over an approximately 1-year period from March 2017 through April 2018. Patients who had thromboelastography with platelet studies (TEG® with PlateletMapping®) during their care were included. RESULTS: Twelve patients were identified for this study. Four patients exhibited inhibition of ADP-induced platelet activation: one had normal fibrinogen and platelet count, two had concurrent hypofibrinogenemia, and one had concurrent thrombocytopenia. Crotalidae polyvalent immune Fab (ovine) reversed platelet inhibition in the single patient for whom serial thromboelastographs were available. Fibrinolysis was present in seven patients and resolved in the two patients with serial thromboelastographs. CONCLUSIONS: Inhibition of ADP-induced platelet aggregation and fibrinolysis occurred independent of hypofibrinogenemia and thrombocytopenia, indicating fibrinogen concentration (or protime) and platelet count monitoring alone is insufficient to assess the extent of hematologic toxicity in rattlesnake envenomation. Crotalidae polyvalent immune Fab (ovine) reversed platelet inhibition in one case, suggesting platelet inhibition could also be used in treatment decisions. Fibrinolysis could also be reversed, although the timing to antivenom administration was less clear.

Recovery from Copperhead Snake Envenomation: Role of Age, Sex, Bite Location, Severity, and Treatment.

INTRODUCTION: Few data exist to understand the recovery phase of pit viper envenomation. A recently published placebo-controlled clinical trial affords this opportunity. The purpose of this study is to examine the time course of recovery from copperhead snake (Agkistrodon contortrix) envenomation patients managed with and without the use of antivenom, stratified by age, sex, anatomic site of envenomation, initial severity of envenomation, and geographic region. METHODS: This is a post-hoc subgroup analysis of data from a multi-center double-blinded clinical trial of Fab antivenom (FabAV) vs. placebo. Outcomes were the Patient-Specific Functional Scale (PSFS) score at 3, 7, 10, and 14 days after envenomation. Least-squares mean PSFS score curves were calculated for each subgroup, and repeated measures ANOVA was used to estimate between-group comparisons. RESULTS: Seventy-two subjects were included, of whom 44 received FabAV. Males demonstrated better overall recovery than females (model predicted PSFS score 6.18 vs 4.99; difference 1.19; 95% CI 0.12 to 2.25; p = 0.029). No sex difference was found in response to FabAV. Overall recovery and effect of FabAV were similar in adult vs adolescent patients, patients with upper vs lower extremity envenomation, and patients with initially mild vs moderate envenomation signs. Analysis by geographic location was not successful due to ANOVA mode instability. CONCLUSIONS: Male victims of copperhead snake envenomation demonstrate slightly better recovery than females, but response to Fab antivenom overall is similar across all subgroups studied.

TFPR1 acts as an immune regulator and an efficient adjuvant for proteins and peptides by activating immune cells, primarily through TLR2.

Triflin, a non-toxic protein found in the venom of the Habu snake, belongs to the CRISP (cysteine-rich secretory protein) family, which comprises two domains: a C-terminal cysteine-rich domain (CRD) and an N-terminal pathogenesis-related-1 (PR-1) domain. The function of the highly structurally conserved PR-1 domain is unknown. Here, we successfully expressed the PR-1 domain of triflin (hereafter called TFPR1) in E. coli. Animal experiments showed that TFPR1 augmented Th1-biased antibody- and cell-mediated immune responses in mice immunized with two protein antigens (OVA and HBsAg) or a peptide antigen (HIV-1 pep). A flow cytometry-based binding assay and in vitro stimulation with TFPR1 showed that it triggered Th1-biased proinflammatory and immunoregulatory cytokine secretion primarily by binding to B cells and macrophages within the mouse splenocyte population. Quantitative RT-PCR, antibody blocking assays using a specific anti-mTLR2 antibody, and stimulatory experiments in vitro using splenocytes from TLR2-KO mice demonstrated that TFPR1 activated murine immune cells, primarily by stimulating toll-like receptor 2 (TLR2). These results suggest that TFPR1 acts as a novel immune modulator and potent adjuvant primarily by activating TLR2. Thus, the PR-1-based core domain might play a role in immune regulation.

Immunogenic Properties of Recombinant Enzymes from Bothrops Ammodytoides Towards the Generation of Neutralizing Antibodies against Its Own Venom.

Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a Bothrops ammodytoides venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR®2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from B. ammodytoides and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of B. ammodytoides venom.

Clinical implications of differential antivenom efficacy in neutralising coagulotoxicity produced by venoms from species within the arboreal viperid snake genus Trimeresurus.

Snake envenomation globally is attributed to an ever-increasing human population encroaching into snake territories. Responsible for many bites in Asia is the widespread genus Trimeresurus. While bites lead to haemorrhage, only a few species have had their venoms examined in detail. We found that Trimeresurus venom causes haemorrhaging by cleaving fibrinogen in a pseudo-procoagulation manner to produce weak, unstable, short-lived fibrin clots ultimately resulting in an overall anticoagulant effect due to fibrinogen depletion. The monovalent antivenom 'Thai Red Cross Green Pit Viper antivenin', varied in efficacy ranging from excellent neutralisation of T. albolabris venom through to T. gumprechti and T. mcgregori being poorly neutralised and T. hageni being unrecognised by the antivenom. While the results showing excellent neutralisation of some non-T. albolabris venoms (such as T. flavomaculaturs, T. fucatus, and T. macrops) needs to be confirmed with in vivo tests, conversely the antivenom failure T. hageni, and the very poor results against T. gumprechti and T. mcgregori, despite being conducted in the ideal scenario of preincubation of antivenom:venom, indicates that the likelihood of clinically relevant cross-reactivity for these species is low (T. gumprechti and T. mcgregori) to non-existent (T. hageni). These same latter three species were also not inhibited by the serine protease inhibitor AEBSF, suggesting that the toxins leading to a coagulotoxic effect in these species are non-serine proteases while in contrast T. albolabris coagulotoxicity was completely impeded by AEBSF, and thus driven by kallikrein-type serine proteases. There was a conspicuous lack of phylogenetic pattern in venom variation, with the most potent venoms (T. albolabris and T. hageni) being distant to each other on the organismal tree, and with the three most divergent and poorly neutralised venoms (T. gumprechti, T. hageni, and T. mcgregori) were also not each others closest relatives. This reinforces the paradigm that the fundamental dynamic evolution of venom results in organismal phylogeny being a poor predictor of venom potency or antivenom efficacy. This study provides a robust investigation on the differential venom effects from a wide range of Trimeresurus species on coagulation, highlighting differential fibrinogenolytic effects, while also investigating the relative antivenom neutralisation capabilities of the widely available Thai Red Cross Green Pit Viper antivenom. These results therefore have immediate, real-world implications for patients envenomed by Trimeresurus species.

Development of a cell-based in vitro assay as a possible alternative for determining bothropic antivenom potency.

Accidents with venomous snakes are a major health hazard in tropical countries. Bothrops genus is responsible for almost 80% of snakebites in Brazil. Immunotherapy is the only approved specific treatment against snake toxins and the production of therapeutic antivenoms requires quality control tests to determine their neutralizing potency. Currently, these controls are performed by in vivo lethality neutralization, however, the inhibition of particular events produced by bothropic venoms such as coagulopathy, hemorrhage, edema or cytotoxic effects are also required. The aim of this work is to develop an in vitro alternative assay for antivenom pre-clinical evaluation. In this sense, we designed a cell viability assay using different amounts (0.2-10 µL/well) of low and high potency anti-bothropic sera, previously classified by the traditional in vivo test, for assessing the antivenom capacity to protect the cells against B. jararaca venom cytotoxicity (5xEC50 = 58.95 µg/mL). We found that high potency sera are more effective in neutralizing B. jararaca venom cytotoxicity when compared to low potency sera, which is in accordance to their pre-determined in vivo potency. Considering sera in vitro inhibitory concentration able to prevent 50% cell death (IC50) and their known in vivo potency, a cut-off point was determined to discriminate low and high potency sera. Our data provide insights for the development of an in vitro method which can determine the anti-bothropic antivenom potency during its production.

Comparative proteomes, immunoreactivities and neutralization of procoagulant activities of Calloselasma rhodostoma (Malayan pit viper) venoms from four regions in Southeast Asia.

The intraspecific geographical venom variations of Calloselasma rhodostoma from Malaysia (CR-M), Indonesia (CR-I), Thailand (CR-T) and Vietnam (CR-V) were investigated through 1D SDS-PAGE and nano-ESI-LCMS/MS. The venom antigenicity, procoagulant activities and neutralization using Thai C. rhodostoma Monovalent Antivenom (CRMAV) were also investigated. SDS-PAGE patterns of the venoms were relatively similar with minor variations. Proteomic analysis revealed that snake venom metalloproteinases (SVMPs, particularly P-I class), serine proteases (SVSPs) and snaclecs dominated the venom protein composition (68.96-81.80%), followed by L-amino acid oxidase (LAAO) and phospholipase A2 (PLA2) (7.37-11.08% and 5.18-13.81%, respectively), corroborating C. rhodostoma envenoming effects (hemorrhage, consumptive coagulopathy, thrombocytopenia and local tissue necrosis). Other proteins of lower abundances (2.82-9.13%) identified include cysteine-rich secretory proteins (CRISP), phospholipase B, phosphodiesterase, nerve growth factor, 5'-nucleotidase, aminopeptidase and hyaluronidase. All four venoms exhibited strong procoagulant effects which were neutralized by CRMAV to different extents. CRMAV immunoreactivity was high toward venoms of CR-M, CR-I and CR-T but relatively low for CR-V venom. Among the venom samples from different locales, CR-V venom proteome has the smallest SVMP composition while SVSP, PLA2 and phosphodiesterase were more abundant in the venom. These variations in C. rhodostoma venom protein composition could partly explain the differences seen in immunoreactivity. (198 words).

Proposed reduction of the in vivo pyrogen test by the in vitro LAL assay for the quality control of anticrotallic, antiscorpion, antirabies and antitetanus sera.

The rationale for a formal study of the LAL (kinetic chromogenic method) assay for anticrotallic (SAC), antirabies (SAR), antitetanus (SAT) and antiscorpion (SAE) sera involved the determination of parameters required by the Brazilian National Health Surveillance Agency (ANVISA), the USA Food and Drug Administration (FDA), USP (United States Pharmacopeia, 39) and ICH (International Conference on Harmonization). The curve correlation coefficients obtained with the standard endotoxin control ranged from -0.980 to -1.000 in all experiments. Endotoxin recovery added to the SAC, SAR, SAT and SAE samples, at the working dilutions (1:10, 1:10, 1:10 and 1, 100 respectively), met the criteria required by the FDA, USP and Brazilian ANVISA for the Inhibition-Potentiation test. The applied methodology for the four analyzed sera fulfilled the required criteria for all performance parameters. Thus, the present study demonstrated that the in vivo pyrogen test can be potentially replaced by the LAL assay for all assessed sera samples displaying higher sensitivity and following the 3 Rs principle, in addition to maintaining quality control in Sanitary Surveillance.

Proteomic profiling, functional characterization, and immunoneutralization of the venom of Porthidium porrasi, a pitviper endemic to Costa Rica.

The genus Porthidium includes nine pitviper species inhabiting Mexico, Central America, and northern South America. Porthidium porrasi is a species endemic to the Southwest of Costa Rica, for which no information on its venom was available. In this study, the proteomic composition and functional activities of P. porrasi venom are described. The most abundant venom proteins were identified as metalloproteinases (36.5%). In descending order of abundance, proteins belonging to the disintegrin, phospholipase A2, serine proteinase, C-type lectin/lectin-like, vascular endothelial growth factor, Cysteine-rich secretory protein, L-amino acid oxidase, phospholipase B, and phosphodiesterase families were also identified. P. porrasi venom showed a weak lethal potency in mice (10 µg/g body weight by intraperitoneal route), induced marked hemorrhage and edema, and weak myotoxic effect. These in vivo activities, as well as those assayed in vitro (proteolytic and phospholipase A2 activities) correlated with compositional data. A comparison of P. porrasi venom with those of three other Porthidium species studied to date reveals a generally conserved compositional and functional pattern in this pitviper genus. Importantly, the lethal effect of P. porrasi venom in mice was adequately cross-neutralized by a heterospecific polyvalent antivenom, supporting its use in the treatment of eventual envenomings by this species.

IgY-based antivenom against Bothrops alternatus: Production and neutralization efficacy.

Antivenom for the treatment of bothropic snakebite is a priority for public health institutions from Latin America. An alternative to the conventional antivenom production is based on the use of egg yolk antibodies - IgY-technology - by immunizing laying hens. In this study, we produced, characterized and assessed the efficacy of IgY-based antivenoms against B. alternatus venom. Immunochemical studies (reactivity, avidity and antigen recognition pattern) as well as antivenom efficacy assays were performed. After the 3rd immunization, levels of specific IgY reached a maximum that was maintained throughout the observation period, while avidity indexes of the extracts increased after the successive immunizations. Furthermore, IgY against B. alternatus recognized protein complexes of the venom with high (>40 kDa), medium (20-40 kDa) and low (<20 kDa) molecular weights. IgY antivenoms obtained after 8 immunizations neutralized 35.65 µg of B. alternatus venom per mg of antivenom, while specific activities values ranged from 0.28 to 0.42. In conclusion, we produced and characterized IgY antivenoms capable of neutralizing the lethal activity of B. alternatus venom at a preclinical level. Thus, IgY-technology may allow the production of effective and affordable antivenoms fulfilling the urgent needs of many countries where conventional manufacture is unable to provide enough availability of antivenoms.