Publications by authors named "Guillermo León"

78 Publications

Development and characterization of two equine formulations towards SARS-CoV-2 proteins for the potential treatment of COVID-19.

Sci Rep 2021 05 10;11(1):9825. Epub 2021 May 10.

Facultad de Microbiología, Instituto Clodomiro Picado, Universidad de Costa Rica, San José, Costa Rica.

In the current global emergency due to SARS-CoV-2 outbreak, passive immunotherapy emerges as a promising treatment for COVID-19. Among animal-derived products, equine formulations are still the cornerstone therapy for treating envenomations due to animal bites and stings. Therefore, drawing upon decades of experience in manufacturing snake antivenom, we developed and preclinically evaluated two anti-SARS-CoV-2 polyclonal equine formulations as potential alternative therapy for COVID-19. We immunized two groups of horses with either S1 (anti-S1) or a mixture of S1, N, and SEM mosaic (anti-Mix) viral recombinant proteins. Horses reached a maximum anti-viral antibody level at 7 weeks following priming, and showed no major adverse acute or chronic clinical alterations. Two whole-IgG formulations were prepared via hyperimmune plasma precipitation with caprylic acid and then formulated for parenteral use. Both preparations had similar physicochemical and microbiological quality and showed ELISA immunoreactivity towards S1 protein and the receptor binding domain (RBD). The anti-Mix formulation also presented immunoreactivity against N protein. Due to high anti-S1 and anti-RBD antibody content, final products exhibited high in vitro neutralizing capacity of SARS-CoV-2 infection, 80 times higher than a pool of human convalescent plasma. Pre-clinical quality profiles were similar among both products, but clinical efficacy and safety must be tested in clinical trials. The technological strategy we describe here can be adapted by other producers, particularly in low- and middle-income countries.
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http://dx.doi.org/10.1038/s41598-021-89242-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8110969PMC
May 2021

Comparative venomics and preclinical efficacy evaluation of a monospecific Hemachatus antivenom towards sub-Saharan Africa cobra venoms.

J Proteomics 2021 05 26;240:104196. Epub 2021 Mar 26.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.

Cobras are the most medically important elapid snakes in Africa. The African genera Naja and Hemachatus include snakes with neurotoxic and cytotoxic venoms, with shared biochemical, toxinological and antigenic characteristics. We have studied the antigenic cross-reactivity of four sub-Saharan Africa cobra venoms against an experimental monospecific Hemachatus haemachatus antivenom through comparative proteomics, preclinical assessment of neutralization, and third generation antivenomics. The venoms of H. haemachatus, N. annulifera, N. mossambica and N. nigricollis share an overall qualitative family toxin composition but depart in their proportions of three-finger toxin (3FTxs) classes, phospholipases A (PLAs), snake venom metalloproteinases (SVMPs), and cysteine-rich secretory proteins (CRISPs). A monospecific anti-Hemachatus antivenom produced by Costa Rican Instituto Clodomiro Picado neutralized the lethal activity of the homologous and heterologous neuro/cytotoxic (H. haemachatus) and cyto/cardiotoxic (N. mossambica and N. nigricollis) venoms of the three spitting cobras sampled, while it was ineffective against the lethal and toxic activities of the neurotoxic venom of the non-spitting snouted cobra N. annulifera. The ability of the anti-Hemachatus-ICP antivenom to neutralize toxic (dermonecrotic and anticoagulant) and enzymatic (PLA) activities of spitting cobra venoms suggested a closer kinship of H. haemachatus and Naja subgenus Afrocobra spitting cobras than to Naja subgenus Uraeus neurotoxic taxa. These results were confirmed by third generation antivenomics. BIOLOGICAL SIGNIFICANCE: African Naja species represent the most widespread medically important elapid snakes across Africa. To gain deeper insight into the spectrum of medically relevant toxins, we compared the proteome of three spitting cobras (Hemachatus haemachatus, Naja mossambica and N. nigricollis) and one non-spitting cobra (N. annulifera). Three finger toxins and phospholipases A are the two major protein families among the venoms analyzed. The development of antivenoms of broad species coverage is an urgent need in sub-Saharan Africa. An equine antivenom raised against H. haemachatus venom showed cross-reactivity with the venoms of H. haemachatus, N. mossambica and N. nigricollis, while having poor recognition of the venom of N. annulifera. This immunological information provides clues for the design of optimum venom mixtures for the preparation of broad spectrum antivenoms.
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http://dx.doi.org/10.1016/j.jprot.2021.104196DOI Listing
May 2021

Tests for Assessing the Neutralizing Ability of Snake Antivenoms: Toward the 3Rs Principles.

Front Immunol 2020 11;11:617429. Epub 2021 Jan 11.

Instituto Clodomiro Picado, Facultad de Microbiología, San José, Costa Rica.

There is an urgent need to strengthen the implementation of the 3Rs principle (Replacement, Reduction and Refinement) in the use of experimental animals in toxinological research and in the assessment of the neutralizing efficacy of snake antivenoms. This is a challenging task owing to the inherent complexity of snake venoms. The state of the art on this topic is hereby reviewed, with emphasis on the studies in which a correlation has been observed between toxicity tests and surrogate assays, particularly in the study of lethal activity of venoms and its neutralization. Correlations have been described with some venoms-antivenoms when using: (a) enzyme immunoassays, (b) hemagglutination, (c) enzyme assays (proteinase, phospholipase A), (d) coagulant effect on plasma, (e) cell culture assays for cytotoxicity, (f) functional assays for assessing neurotoxicity , (g) use of hens' eggs, and (h) antivenomics. Additionally, the routine introduction of analgesia in these assays and the design of more 'humane' protocols for the lethality test are being pursued. It is expected that the next years will witness a growing awareness of the relevance of the 3Rs principles in antivenom testing, and that new alternatives and more 'humane' experimental designs will emerge in this field.
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http://dx.doi.org/10.3389/fimmu.2020.617429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7829219PMC
January 2021

Measurable Residual Disease Assessment and Allogeneic Transplantation as Consolidation Therapy in Adult Acute Lymphoblastic Leukemia in Colombia.

Clin Lymphoma Myeloma Leuk 2021 Apr 14;21(4):e365-e372. Epub 2020 Nov 14.

Department of Hematology, Clínica Reina Sofía, Bogotá, Colombia.

Introduction: Detectable minimal residual disease (MRD) after therapy for acute lymphoblastic leukemia (ALL) is the strongest predictor of hematologic relapse. The objective of the study was to assess disease-free survival (DFS) and overall survival (OS) of patients with ALL according with MRD status at the end of induction therapy in a Colombian population.

Patients And Methods: We assessed a retrospective cohort to compare DFS and OS in adults with de novo ALL according to MRD status at the end of induction chemotherapy, and the type of postinduction consolidation strategy used.

Results: A total of 165 adults with ALL were included in the MRD part of the study, 73 patients in the MRD-negative group and 92 in the MRD-positive group. Median DFS for the MRD-positive group was 11 months (95% confidence interval, 11.7-22.2) and was not reached for the MRD-negative group (P < .001). At 3 years, DFS was 18% and 55%, respectively (P < .001). The median OS for MRD-positive patients was 16 months (95% confidence interval, 8.8-23.15) and was not reached in the MRD-negative group. At 3 years, OS was 26% and 51% for the former and latter group, respectively. Among subjects who did not receive a transplant, median DFS was 21 months for MRD-negative patients and 9 months for MRD-positive patients (P < .001). The median DFS was not reached in either group, whereas 3-year DFS was 64% for MRD-negative and 70% for MRD-positive patients who underwent transplantation in first remission (P = .861).

Conclusion: MRD status at the end of induction is an independent prognostic factor for DFS and OS in adult ALL. Allogeneic transplantation in first remission could overcome the adverse prognostic impact of MRD.
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http://dx.doi.org/10.1016/j.clml.2020.11.010DOI Listing
April 2021

Toxicological profile of medically relevant Crotalus species from Mexico and their neutralization by a Crotalus basiliscus/Bothrops asper antivenom.

Toxicon 2020 May 18;179:92-100. Epub 2020 Mar 18.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

Specimens of the Crotalus genus represent a potential snakebite problem in Mexico, and despite the great number of species of Crotalus present in this country, only a few of them are relevant from a medical point of view. Crotalus envenomed patients can present a range of signs and symptoms, depending on the species involved, and their treatment is indistinctly with either of the anti-viperid antivenoms available in the Mexican Public Health System. One of these antivenoms is produced by immunization of horses with a mixture of only two venoms: Crotalus basiliscus and Bothrops asper venoms. In light of the high variability found in Crotalus species venom composition, it is important to demonstrate the cross-neutralization of this antivenom against other Crotalus species. Therefore, in this work the toxic variability of eight medically important Crotalus venoms from Mexico and its neutralization by the Crotalus basiliscus/Bothrops asper antivenom were assessed. The present study evidenced the variability of toxic and enzymatic activities among the following Crotalus venoms: (1) Crotalus atrox, (2) Crotalus basiliscus, (3) Crotalus culminatus, (4) Crotalus simus, (5) Crotalus tzabcan, (6) Crotalus scutulatus salvini, (7) Crotalus scutulatus scutulatus-A, and (8) Crotalus scutulatus scutulatus-B. All venoms studied possess lethal and hemorrhagic activity on a murine model, although there are important variations among the species; in contrast, the PLA activity was similar for all venoms. Interestingly, only C. simus venom exhibited coagulant activity on human plasma under 100 μg. The antivenom neutralized the lethality and all the other assessed activities for all venoms tested. However, the dose required varied depending on the venom and the evaluated activity. Our preclinical data support the recommendation of using this antivenom to clinically manage Crotalus snakebites produced by the species assessed in this study. Nonetheless, only clinical trials could categorically validate these results.
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http://dx.doi.org/10.1016/j.toxicon.2020.03.006DOI Listing
May 2020

Phylovenomics of Daboia russelii across the Indian subcontinent. Bioactivities and comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms against venoms from India, Bangladesh and Sri Lanka.

J Proteomics 2019 09 17;207:103443. Epub 2019 Jul 17.

Evolutionary and Translational Venomics Laboratory, Consejo Superior de Investigaciones Científicas (CSIC), Valencia, Spain. Electronic address:

Russell's viper (Daboia russelii) is, together with Naja naja, Bungarus caeruleus and Echis carinatus, a member of the medically important 'Big Four' species responsible for causing a large number of morbidity and mortality cases across the Indian subcontinent. Despite the wide distribution of Russell's viper and the well-documented ubiquity of the phenomenon of geographic variability of intraspecific snake venom composition, Indian polyvalent antivenoms against the "Big Four" venoms are raised against venoms sourced mainly from Chennai in the southeastern Indian state of Tamil Nadu. Biochemical and venomics investigations have consistently revealed notable compositional, functional, and immunological differences among geographic variants of Russell's viper venoms across the Indian subcontinent. However, these studies, carried out by different laboratories using different protocols and involving venoms from a single geographical region, make the comparison of the different venoms difficult. To bridge this gap, we have conducted bioactivities and proteomic analyses of D. russelii venoms from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and Tamil Nandu (India) and Sri Lanka, along with comparative in vivo neutralization and in vitro third-generation antivenomics of antivenoms used in India, Bangladesh and Sri Lanka. These analyses let us to propose two alternative routes of radiation for Russell's viper in the Indian subcontinent. Both radiations, towards the northeast of India and Bangladesh and towards south India and Sri Lanka, have a common origin in Pakistan, and provide a phylovenomics ground for rationalizing the geographic variability in venom composition and their distinct immunoreactivity against available antivenoms. BIOLOGICAL SIGNIFICANCE: Russell's viper (Daboia russelii), the Indian cobra (Naja naja), the common krait (Bungarus caeruleus), and the saw-scaled viper (Echis carinatus) constitute the 'Big Four' snake species responsible for most snakebite envenomings and deaths in the Indian subcontinent. Despite the medical relevance of Daboia russelii, and the well documented variations in the clinical manifestations of envenomings by this wide distributed species, which are doubtless functionally related to differences in venom composition of its geographic variants, antivenoms for the clinical treatment of envenomings by D. russelii across the Indian subcontinent are invariably raised using venom sourced mainly from the southeastern Indian state of Tamil Nadu. We have applied a phylovenomics approach to compare the venom proteomes of Russell's vipers from the three corners of the Indian subcontinent, Pakistan, Bangladesh, and South India/Sri Lanka, and have assessed the in vitro (third-generation antivenomics) and in vivo preclinical efficacy of a panel of homologous antivenoms. The identification of two dispersal routes of ancestral D. russelii into the Indian subcontinent provides the ground for rationalizing the variability in composition and immunoreactivity of the venoms of extant geographic variants of Russell's viper. Such knowledge is relevant for envisioning strategies to improve the clinical coverage of anti- D. russelii antivenoms.
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http://dx.doi.org/10.1016/j.jprot.2019.103443DOI Listing
September 2019

Nephrotoxicity induced by the venom of Hypnale hypnale from Sri Lanka: Studies on isolated perfused rat kidney and renal tubular cell lines.

Toxicon 2019 Jul 26;165:40-46. Epub 2019 Apr 26.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

The hump-nosed pit viper Hypnale hypnale is responsible for a high number of snakebite cases in southwestern India and Sri Lanka. Although most patients only develop local signs and symptoms of envenoming, there is a growing body of evidence indicating that these envenomings may be associated with systemic alterations, including acute kidney injury. In this study we evaluated the renal toxicity of H. hypnale venom by using a perfused isolated rat kidney system and by assessing cytotoxicity in two different renal tubular cell lines in culture. The venom caused alterations in several renal functional parameters, such as reduction on perfusion pressure, renal vascular resistance, and sodium and chloride tubular transport, whereas glomerular filtration rate and urinary flow initially decreased and then increased after venom perfusion. In addition, this venom was cytotoxic to proximal and distal renal tubular cells in culture, with predominance of necrosis over apoptosis. Moreover, the venom affected the mitochondrial membrane potential and induced an increment in reactive oxygen species in these cells. Taken together, our results demonstrate a nephrotoxic activity of H. hypnale venom in these experimental models, in agreement with clinical observations.
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http://dx.doi.org/10.1016/j.toxicon.2019.04.014DOI Listing
July 2019

Current technology for the industrial manufacture of snake antivenoms.

Toxicon 2018 Sep 28;151:63-73. Epub 2018 Jun 28.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.

Snake antivenoms are formulations of animal immunoglobulins used in the treatment of snakebite envenomation. The general scheme for producing snake antivenoms has undergone few changes since its development more than a century ago; however, technological innovations have been introduced in the manufacturing process. These medicines must comply with identity, purity, safety and efficacy profiles, as requested by the current Good Manufacturing Practices (GMPs) applied to modern biopharmaceutical drugs. Industrial production of snake antivenoms comprises several stages, such as: 1) production of reference venom pools, 2) production of hyperimmune plasma, 3) purification of the antivenom immunoglobulins, 4) formulation of the antivenom, 5) stabilization of the formulation, and 6) quality control of in-process and final products. In this work, a general review of the existing technology used for the industrial manufacture of snake antivenoms is presented.
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http://dx.doi.org/10.1016/j.toxicon.2018.06.084DOI Listing
September 2018

Effect of exposition to chlorpyrifos upon plasmatic cholinesterases, hematology and blood biochemistry values in Bothrops asper (Serpentes: Viperidae).

Chemosphere 2018 Aug 20;205:209-214. Epub 2018 Apr 20.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.

The terciopelo (Bothrops asper), is one of the most important venomous snakes in Costa Rica and common on agriculture where insecticides are frequently used for pest control. To assess the exposure to organophosphates on captive B. asper, an experiment using chlorpyrifos and butyrylcholinesterase (BChE), as a biomarker was conducted. In addition to BChE, hematology, aspartate aminotransferase (AST), total proteins (TP) and albumin were measured after exposure. Different concentrations of chlorpyrifos were used in Group A (0.1%) and B (1%), while the Control Group received distilled water; each group was composed of 5 snakes. Values of BChE, AST, TP, and albumin were measured before exposure, and at 6, 12, 24, 196, 360 and 528 h post-exposure. Hematology values were measured after 24 h post-exposure. As result, an important variation between subjects in all groups before exposure was obtained. Moreover, BChE activity showed 37% inhibition of Group A when compared to Control Group at 12 h post-exposure, and a higher inhibition of Group B (97%) related to Control Group, at 6 h post-exposure. Recovery of BChE occurred towards 528 h, never reaching initial values. Despite some variation in the rest of parameters used, a marked relative lymphopenia and monocytosis occurred at 24 h, assuming stress as the main cause.
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http://dx.doi.org/10.1016/j.chemosphere.2018.04.111DOI Listing
August 2018

Proteomic and toxinological characterization of the venom of the South African Ringhals cobra Hemachatus haemachatus.

J Proteomics 2018 06 12;181:104-117. Epub 2018 Apr 12.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

The protein composition and toxinological profile of the venom of the African spitting elapid Hemachatus haemachatus (Ringhals) were characterized by bottom-up proteomics and functional in vitro and in vivo assays. Venom is composed of abundant three-finger toxins (3FTxs; 63.3%), followed by phospholipases A (PLAs; 22.8%), snake venom metalloproteinases (SVMPs; 7.1%), cysteine-rich secretory proteins (CRISPs; 4.1%) and Kunitz type protease inhibitors (KTPIs; 1.5%). 3FTxs are the main responsible for lethality and myotoxicity in mice and in vitro anticoagulant activity. In contrast to closely related spitting species, whose venom 3FTxs induces dermonecrosis, the 3FTxs of H. haemachatus did not induce dermonecrotic activity. The venom showed in vitro PLA activity, and most likely PLAs contribute to some extent in venom lethality, as judged by partial reduction in toxicity after inhibition of their catalytic activity. Despite its relatively high content of SVMPs, compared to most elapids, the venom of H. haemachatus did not exert hemorrhagic effect, proteolytic activity on azocasein or defibrinogenating activity. Toxicovenomic characterization of H. haemachatus venom revealed that RP-HPLC fractions with higher abundance of 3FTxs presented lethal activity, while fractions with high content of PLAs did not, underscoring the role of 3FTxs in the pathophysiology caused by this venom.

Biological Significance: The proteomic composition and toxinological profile of the venom of Ringhals snake, Hemachatus haemachatus, a cobra-like spitting snake endemic to southern Africa, were investigated. In vitro, Ringhals venom showed anticoagulant and phospholipase A activities, but was devoid of proteolytic activity on azocasein. In mice, venom induced lethality and myotoxicity, but no local hemorrhage or dermonecrosis. The lack of dermonecrotic activity is in sharp contrast to venoms of closely related spitting cobras which present a similar relative abundance of 3FTxs but are potently dermonecrotic. 3FTxs, the most abundant protein family in the venom, are predominantly responsible for toxic effects. PLA enzyme inactivation experiments suggest that H. haemachatus venom lethality is not dependent on PLAs, but instead is more related to neurotoxic or cardiotoxic 3FTxs. The characterization of this venom, based on proteomic and toxicovenomic approaches, is useful for more in depth studies associated with biogeography, phylogeny, toxinology and antivenom efficacy towards the venom of this species, and its association with related elapids.
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http://dx.doi.org/10.1016/j.jprot.2018.04.007DOI Listing
June 2018

Preclinical efficacy against toxic activities of medically relevant Bothrops sp. (Serpentes: Viperidae) snake venoms by a polyspecific antivenom produced in Mexico.

Rev Biol Trop 2017 Mar;65(1):345-50

The assessment of the preclinical neutralizing ability of antivenoms in Latin America is necessary to determine their scope of efficacy. This study was aimed at analyzing the neutralizing efficacy of a polyspecific bothropic-crotalic antivenom manufactured by BIRMEX in Mexico against lethal, hemorrhagic, defibrinogenating and in vitro coagulant activities of the venoms of Bothrops jararaca (Brazil), B. atrox (Perú and Colombia), B. diporus (Argentina), B. mattogrossensis (Bolivia), and B. asper (Costa Rica). Standard laboratory tests to determine these activities were used. In agreement with previous studies with bothropic antivenoms in Latin America, a pattern of cross-neutralization of heterologous venoms was observed. However, the antivenom had low neutralizing potency against defibrinogenating effect of the venoms of B. atrox (Colombia) and B. asper (Costa Rica), and failed to neutralize the in vitro coagulant activity of the venom of B. asper (Costa Rica) at the highest antivenom/venom ratio tested. It is concluded that, with the exception of coagulant and defibrinogenating activities of B. asper (Costa Rica) venom, this antivenom neutralizes toxic effects of various Bothrops sp venoms. Future studies are necessary to assess the efficacy of this antivenom against other viperid venoms.
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http://dx.doi.org/10.15517/rbt.v65i1.18908DOI Listing
March 2017

Contributions of the snake venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys to the paraspecificity of the Central American polyspecific antivenom (PoliVal-ICP).

Toxicon 2018 Mar 4;144:1-6. Epub 2018 Feb 4.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

PoliVal-ICP antivenom is produced from plasma of horses immunized toward the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys. The antibody response induced by these venoms confers PoliVal-ICP the capacity to neutralize the venoms of the most important Central American viperids, including not only homologous venoms (i.e., venoms used as immunogen), but many heterologous venoms (i.e., venoms not used as immunogen). In this work, the individual contributions of homologous venoms to the paraspecificity of PoliVal-ICP were inferred from the capacity of experimental monospecific antivenoms toward venoms of B. asper (anti-Ba), C. simus (anti-Cs) and L. stenophrys (anti-Ls), and an experimental polyspecific antivenom (anti-Ba/Cs/Ls) to neutralize the lethality induced by different venoms in mice. It was found that all antivenoms neutralized their corresponding homologous venoms. Moreover, the anti-Ba antivenom cross-neutralized the venoms of Agkistrodon howardgloydi, Atropoides picadoi, Bothriechis lateralis, Bothriechis supraciliaris and Porthidium ophryomegas; the anti-Cs antivenom cross-neutralized the venoms of B. lateralis, B. supraciliaris, Cerrophidion sasai and Porthidium nasutum; and the anti-Ls antivenom cross-neutralized the venoms of B. lateralis, B. supraciliaris, C. sasai and Lachesis melanocephala. All venoms neutralized by any monospecific antivenom were also neutralized by the anti-Ba/Cs/Ls antivenom. Venoms of Atropoides mexicanus, Bothriechis nigroviridis and Bothriechis schlegelii were not neutralized by any experimental antivenom, thus explaining the limitations of PoliVal-ICP to neutralize these venoms. Consequently, an enlargement of the neutralization scope of PoliVal-ICP could be achieved by including these venoms in the group of those used as immunogens.
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http://dx.doi.org/10.1016/j.toxicon.2018.01.016DOI Listing
March 2018

An improved technique for the assessment of venom-induced haemorrhage in a murine model.

Toxicon 2017 Dec 9;139:87-93. Epub 2017 Oct 9.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

Haemorrhage is a common clinical manifestation in envenomings caused by bites from snakes of the family Viperidae. Therefore, knowing the haemorrhagic potential of venoms and the capacity of antivenoms to neutralise this effect are of paramount relevance in toxinology. The most widely used method for quantifying haemorrhage involves the intradermal injection of venom (or a mixture of venom/antivenom) in mice, and the assessment of the resulting haemorrhagic area in the inner side of the skin. Although this method allows a straightforward assessment of the haemorrhagic activity of a venom, it does not account for haemorrhagic lesions having a similar area but differing in the depth and intensity of haemorrhage. We have developed an approach that allows the assessment of both area and intensity of a venom-induced haemorrhagic lesion using computational tools and propose a unit to represent the combination of these two factors as a measure of haemorrhage intensity, namely haemorrhagic unit (HaU). A strong correlation was observed between haemoglobin extracted from a haemorrhagic lesion and the associated HaUs. The method was used to determine the haemorrhagic activity of the venoms of Bothrops asper, Echis ocellatus and Crotalus basiliscus and the haemorrhage neutralising capabilities of the three associated antivenoms. Overall, the ease of use, as well as the time involved in this new method, makes its implementation very feasible in the determination of haemorrhagic activity of venoms and its neutralisation by antivenoms in the murine model.
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http://dx.doi.org/10.1016/j.toxicon.2017.10.005DOI Listing
December 2017

Cross-reactivity and cross-immunomodulation between venoms of the snakes Bothrops asper, Crotalus simus and Lachesis stenophrys, and its effect in the production of polyspecific antivenom for Central America.

Toxicon 2017 Nov 9;138:43-48. Epub 2017 Aug 9.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

A mixture of the venoms of Bothrops asper, Crotalus simus and Lachesis stenophrys is used as immunogen to produce the polyspecific Central American antivenom (PoliVal-ICP). In this work, we studied the ability of each of these venoms to modulate the antibody response induced by the other two venoms included in the immunization mixture. For that, equine monospecific, bispecific and polyspecific antivenoms were prepared and compared regarding their ability to neutralize the phospholipase A, coagulant and lethal activities of each venom, and their anti-venom antibodies concentration. Results indicate that there is low cross-reactivity and cross-neutralization between venoms of B. asper, C. simus and L. stenophrys, hence justifying the use of all of them as immunogens for the production of the Central American antivenom. It was also found that the venom of B. asper reduces the anti-crotalic response while the venom of C. simus does not affect the anti-bothropic response. On the other hand, the venoms of B. asper and C. simus increase the anti-lachesic response, and L. stenoprhys venom reduced both the anti-bothropic and anti-crotalic responses. On the basis of these results, the immunization strategy can be adjusted by preventing or taking advantage of cross-immunomodulation between venoms, in order to maximize the antibody response towards all venoms. Immune responses can be improved by injecting horses with several immunogen mixtures, composed by one or two of the three venoms, and administering them at different times during the immunization, eventually generating a high titer against the three venoms. Our results suggest that addressing the issue of immunomodulation by venoms might improve antivenom manufacture worldwide.
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http://dx.doi.org/10.1016/j.toxicon.2017.08.009DOI Listing
November 2017

Preclinical Evaluation of the Efficacy of Antivenoms for Snakebite Envenoming: State-of-the-Art and Challenges Ahead.

Toxins (Basel) 2017 05 13;9(5). Epub 2017 May 13.

Instituto de Biomedicina de Valencia, CSIC, Valencia 46010, Spain.

Animal-derived antivenoms constitute the mainstay in the therapy of snakebite envenoming. The efficacy of antivenoms to neutralize toxicity of medically-relevant snake venoms has to be demonstrated through meticulous preclinical testing before their introduction into the clinical setting. The gold standard in the preclinical assessment and quality control of antivenoms is the neutralization of venom-induced lethality. In addition, depending on the pathophysiological profile of snake venoms, the neutralization of other toxic activities has to be evaluated, such as hemorrhagic, myotoxic, edema-forming, dermonecrotic, in vitro coagulant, and defibrinogenating effects. There is a need to develop laboratory assays to evaluate neutralization of other relevant venom activities. The concept of the 3Rs (Replacement, Reduction, and Refinement) in Toxinology is of utmost importance, and some advances have been performed in their implementation. A significant leap forward in the study of the immunological reactivity of antivenoms against venoms has been the development of "antivenomics", which brings the analytical power of mass spectrometry to the evaluation of antivenoms. International partnerships are required to assess the preclinical efficacy of antivenoms against snake venoms in different regions of the world in order to have a detailed knowledge on the neutralizing profile of these immunotherapeutics.
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http://dx.doi.org/10.3390/toxins9050163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5450711PMC
May 2017

Freeze-dried EchiTAb+ICP antivenom formulated with sucrose is more resistant to thermal stress than the liquid formulation stabilized with sorbitol.

Toxicon 2017 Jul 3;133:123-126. Epub 2017 May 3.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

EchiTAb + ICP is a pan-African antivenom used for the treatment of snakebite envenomation in rural sub-Saharan African communities, where the cold chain can be difficult to maintain. To develop a formulation of EchiTAb + ICP that can be distributed and stored without refrigeration, we submitted three different formulations of EchiTAb + ICP: control (i.e. liquid antivenom formulated without stabilizer), liquid antivenom stabilized with sorbitol, and freeze-dried antivenom formulated with sucrose, to an accelerated stability study (i.e. 38 ± 2 °C and 75% relative humidity for 6 months). We analyzed changes in color, residual humidity, reconstitution time (for freeze-dried preparation), pH, osmolality, total protein concentration, antibody monomers content, turbidity, bacterial endotoxins, and pre-clinical neutralizing efficacy of the lethal effect of Echis ocellatus venom at 0, 3 and 6 months. In the control formulation, instability was evidenced by the development of a yellow coloration and an increment in aggregation and turbidity, without change in its neutralizing activity. The sorbitol-stabilized formulation did not develop marked aggregation or turbidity, but instability was evidenced by the development of yellow coloration and a drop in the neutralizing potency. The freeze-dried formulation maintained its neutralizing potency and did not show marked signs of instability, thus indicating that freeze-drying could confer EchiTAb + ICP with improved thermal stability required for distribution and storage at room temperature in sub-Saharan Africa.
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http://dx.doi.org/10.1016/j.toxicon.2017.05.006DOI Listing
July 2017

Effect of premedication with subcutaneous adrenaline on the pharmacokinetics and immunogenicity of equine whole IgG antivenom in a rabbit model.

Biomed Pharmacother 2017 Jun 15;90:740-743. Epub 2017 Apr 15.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

Subcutaneous administration of a low dose of adrenaline is used to prevent the early adverse reactions (EARs) induced by snake antivenoms. We used a rabbit model to study the effect of premedication with adrenaline on the potential of antivenoms to exert therapeutic effects and to induce late adverse reactions. We found that premedication with adrenaline did not change the heart rate or blood pressure of normal rabbits, but reduced the rise in temperature in rabbits previously sensitized with antivenom. Pharmacokinetic studies suggest that premedication with adrenaline does not affect the ability of the antivenom to exert the initial control of envenomation nor the susceptibility of rabbits to develop recurrence of antigenemia and envenomation. Our results also indicate that it is unlikely that premedication with adrenaline decreases the incidence of late reactions induced by the antivenom administration, although it reduces the extent of early reactions.
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http://dx.doi.org/10.1016/j.biopha.2017.04.039DOI Listing
June 2017

Proteomic, toxicological and immunogenic characterization of Mexican west-coast rattlesnake (Crotalus basiliscus) venom and its immunological relatedness with the venom of Central American rattlesnake (Crotalus simus).

J Proteomics 2017 03 24;158:62-72. Epub 2017 Feb 24.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

The venom of the Mexican west-coast rattlesnake (Crotalus basiliscus) was characterized for its protein composition, toxicological profile and immunogenic properties. This venom is composed of 68% Zn-dependent metalloproteinases (SVMPs), 14% phospholipases A (PLAs), 11% serine proteinases, 4% SVMPs-inhibitor tripeptides (SVMP-ITs), 2% bradykinin-potentiating peptides (BPPs), 0.6% cysteine-rich secretory proteins (CRISPs), and 0.2% l-amino acid oxidases (LAAOs). SVMPs present in the venom are responsible for azocasein hydrolysis and hemorrhagic activity, but their contribution to the lethal activity of the venom in mice is masked by the neurotoxic activity of PLAs, which in addition are also responsible for myotoxic activity. Despite its relatively high content of serine proteinases, the venom of C. basiliscus did not exert in vitro coagulant or in vivo defibrinogenating activities. The ability of antivenoms raised against the venoms of C. basiliscus and C. simus (from Costa Rica) to neutralize homologous and heterologous venoms revealed antigenic similarities between toxins of both venoms. Preclinical evaluation of an antivenom produced by using the venom of C. basiliscus as immunogen demonstrated that it is able to neutralize not only the most relevant toxic activities of C. basiliscus venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities.

Biological Significance: The Central American rattlesnake (Crotalus simus) is widely distributed from Mexico to west central Costa Rica, and induces an important number of envenomations in this region. On the other hand, the immunogenic mixture used by Laboratorios de Biológicos y Reactivos de Mexico S.A. (Birmex) to produce the snake antivenom more frequently used in Mexico does not include the venom of C. simus. This immunogenic mixture is composed by the venoms of the Fer-de-lance (Bothrops asper) and the Mexican west-coast rattlesnake (Crotalus basiliscus). We studied the protein composition, toxicological profile and immunogenic properties of the venom of C. basiliscus, and evaluated the ability of the Birmex antivenom to neutralize the venom of C. basiliscus and whether it cross-neutralizes the venom of C. simus from Costa Rica. Using proteomics analysis, in combination with in vitro and mouse tests, we determined that the venom of C. basiliscus is mainly composed by SVMPs, which confer proteolytic and hemorrhagic activities to the venom. Other major components of the venom of C. basiliscus are PLAs, which are responsible for the myotoxic activity and are the main contributors to the lethal activity. Non-clotting SVSPs correspond to 11% of the venom. Minor components include SVMP-ITs, BPPs, CRISPs and LAAOs, which have not been associated with toxicity. The antibodies induced in horses by the venom of C. basiliscus are able to neutralize not only the most relevant toxic activities of the homologous venom, but also those exerted by Costa Rican C. simus venom, including coagulant and defibrinogenating activities. Our preclinical evaluation suggests that Birmex antivenom can be used to treat envenomations by Costa Rican adult C. simus snakebites, despite this venom not being included in the immunizing mixture.
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http://dx.doi.org/10.1016/j.jprot.2017.02.015DOI Listing
March 2017

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

Biologicals 2017 Mar 22;46:1-5. Epub 2017 Jan 22.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

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.
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http://dx.doi.org/10.1016/j.biologicals.2016.10.008DOI Listing
March 2017

Comparison of the adjuvant activity of emulsions with different physicochemical properties on the antibody response towards the venom of West African carpet viper (Echis ocellatus).

Toxicon 2017 Mar 12;127:106-111. Epub 2017 Jan 12.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

Adjuvant emulsions are widely used to enhance the antibody response of the animals used as immunoglobulin source for producing antivenoms. Usually, the adjuvant activity of emulsions is attributed both to their ability to trigger "danger" signals from cells in which they induce death, and to form depots from which immunogens are slowly released. However, there is contradictory evidence suggesting that adjuvant activity of emulsions is independent of the dispersion type and the rate of immunogen release. In order to test how physical properties of emulsions, composed of mineral oil and water, affect their ability to enhance the antibody response towards snake venoms, we compared water-in-oil (W/O) emulsions prepared at volume ratios of 70/30, 50/50 or 30/70, a 50/50 oil-in-water (O/W) emulsion, and a water-in-oil-in-water (W/O/W) multiple emulsion. Comparison included their droplet-size, viscosity, rate of immunogen release and ability to enhance the antibody response of mice immunized with the venom of the African viperid snake Echis ocellatus. It was found that all emulsions released a low amount of venom, and that the 50/50 (W/O) and the multiple emulsion (W/O/W) were those that induced the higher anti-venom antibody response. Our results suggest that the ability of emulsions to enhance the anti-venom response is not associated to their ability to form depots from which the venom is slowly released.
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http://dx.doi.org/10.1016/j.toxicon.2017.01.011DOI Listing
March 2017

Physicochemical characterization of commercial freeze-dried snake antivenoms.

Toxicon 2017 Feb 10;126:32-37. Epub 2016 Dec 10.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica.

Freeze-drying is a process used to improve the stability of pharmaceutical proteins, including snake antivenoms. This additional step confers these with a higher stability in comparison to liquid formulations, especially in tropical regions where high temperatures could affect the activity of immunoglobulins. Currently, the knowledge about freeze-drying process conditions for snake antivenoms is very limited. Some of the scarce scientific works on this subject reported reconstitution times up to 90 min for these preparations, which could imply a delay in the beginning of the antivenom therapy at the clinical setting. Therefore there is a reasonable concern about whether freeze-dried antivenoms exhibit the desired attributes for solid pharmaceutical proteins. In this work, a physicochemical characterization of seven commercial freeze-dried snake antivenoms was performed based on tests recommended by the World Health Organization (WHO). No significant differences were observed between the products regarding macroscopic appearance of the solid cakes, reconstitution times, residual humidity and monomers content. On the other hand, total protein concentration, turbidity and electrophoretic profile were different among samples. Microscopic analysis by scanning electron microscopy showed no collapsed structure and, instead, most of the samples showed a characteristic protein morphology composed of smooth plates and channels. All the parameters tested in this study were according to literature recommendations and evidenced that, in spite of slight variations found for some products, formulation and freeze-drying conditions chosen by manufacturers are adequate to prevent aggregation and generate, in physicochemical terms, freeze-dried antivenoms of acceptable quality.
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http://dx.doi.org/10.1016/j.toxicon.2016.12.004DOI Listing
February 2017

Expanding the neutralization scope of the EchiTAb-plus-ICP antivenom to include venoms of elapids from Southern Africa.

Toxicon 2017 Jan 24;125:59-64. Epub 2016 Nov 24.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

EchiTAb-plus-ICP is an antivenom prepared from plasma of horses hyperimmunized with the venoms of the carpet viper (Echis ocellatus), the puff adder (Bitis arietans) and the black-necked spitting cobra (Naja nigricollis). Therefore, the use of this antivenom has been limited to Western Africa. In order to expand the neutralization scope of EchiTAb-plus-ICP, we supplemented the immunogenic mixture with the venoms of B. arietans, the black mamba (Dendroaspis polylepis), the Mozambique spitting cobra (Naja mossambica), the snouted cobra (N. annulifera), and the rinkhals (Hemachatus haemachatus) from Swaziland. The ability of the expanded-scope antivenom, hereby named EchiTAb + ICP, to neutralize the venoms of B. arietans, D. polylepis, N. mossambica and H. haemachatus was similar to those of FAV Afrique and the SVA African antivenoms. In comparison to the SAIMR antivenom, the expanded-scope EchiTAb + ICP had lower ability to neutralize the venom of B. arietans, but similar ability to neutralize the venoms of D. polylepis, N. mossambica and H. haemachatus. Owing to its low protein concentration, the expanded-scope EchiTAb + ICP had lower ability to neutralize the venom of N. annulifera than FAV Afrique and the SAIMR antivenoms. However, when formulated at a protein concentration as high as FAV Afrique and SAIMR antivenoms, the expanded-scope EchiTAb + ICP showed similar capacity to neutralize this poorly immunogenic venom. Our results encourage the transition to the new EchiTAb + ICP antivenom, with an expanded neutralization scope that includes venoms of some of the most medically important elapids from Southern Africa. Clinical trials are required to determine the minimum effective-safe dose of the new EchiTAb + ICP for each type of envenomation.
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http://dx.doi.org/10.1016/j.toxicon.2016.11.259DOI Listing
January 2017

Development of a new polyspecific antivenom for snakebite envenoming in Sri Lanka: Analysis of its preclinical efficacy as compared to a currently available antivenom.

Toxicon 2016 Nov 6;122:152-159. Epub 2016 Oct 6.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

A new whole IgG, freeze-dried, polyspecific antivenom was prepared from the plasma of horses immunized with the venoms of the snakes Daboia russelii, Echis carinatus, Hypnale hypnale, and Naja naja from Sri Lanka. The preclinical neutralizing ability of this antivenom against several toxic and enzymatic activities of these four venoms was analyzed, and compared with that of a batch of VINS antivenom (India) being currently used in Sri Lanka. The activities tested were: lethality, hemorrhagic, in vitro coagulant, proteinase and phospholipase A. Both antivenoms neutralized, to a different extent, these activities of the venom of D. russelii, E. carinatus, and N. naja. In general, the polyspecific Sri Lankan antivenom was more effective than the Indian antivenom in the neutralization of the venoms of D. russelii and E. carinatus, whereas the Indian antivenom showed a higher efficacy against the venom of N. naja. Regarding H. hypnale, the new Sri Lankan antivenom was effective in the neutralization of all activities tested, whereas the Indian antivenom neutralized lethality but not hemorrhagic, coagulant, proteinase and PLA activities, in agreement with the fact that this venom is not included in the immunization mixture for this antivenom. Results suggest that the new polyspecific Sri Lankan antivenom has a satisfactory preclinical neutralizing profile and compares favorably with the Indian antivenom. This is ready to be tested in a clinical trial to evaluate its efficacy and safety in human victims of snakebite envenomings by D. russelii, E. carinatus and H. hypnale in Sri Lanka.
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http://dx.doi.org/10.1016/j.toxicon.2016.10.007DOI Listing
November 2016

Development of a chicken-derived antivenom against the taipan snake (Oxyuranus scutellatus) venom and comparison with an equine antivenom.

Toxicon 2016 Sep 29;120:1-8. Epub 2016 Jun 29.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

A chicken-derived antivenom (ChDAv) towards taipan snake (Oxyuranus scutellatus) venom was produced by purifying anti-taipan IgY from egg yolks of hens immunized with taipan venom. The productivity, antivenomic profile, neutralization ability, pharmacokinetic properties and immunogenicity of the ChDAv were compared with those of an antivenom produced in horses (EDAv). We found that 382 eggs are required to produce the mass of anti-taipan antibodies contained in one liter of equine hyperimmune plasma, and that 63 chickens would be needed to generate the amount of anti-taipan antibodies annually produced by one horse. It was estimated that, in Costa Rica, the production of anti-taipan antibodies could be 40% cheaper if chickens were used as immunoglobulin source, instead of horses. During antivenomic assessment, ChDAv showed lower ability to immunocapture the α subunit of taipoxin, the most important neurotoxin in the venom. ChDAv showed a lower ability to neutralize the coagulant and lethal activities of taipan venom. ChDAv was more immunogenic in rabbits than EDAv, probably due to the fact that chickens are phylogenetically more distant to rabbits than horses. This finding may explain why clearance from rabbit bloodstream was faster for chicken-IgY than for equine-IgG in a pharmacokinetic study. In conclusion, the production of anti-taipan antivenom was less effective when chicken egg yolks were used as source of immunoglobulins instead of horses.
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http://dx.doi.org/10.1016/j.toxicon.2016.06.018DOI Listing
September 2016

Pathogenesis of dermonecrosis induced by venom of the spitting cobra, Naja nigricollis: An experimental study in mice.

Toxicon 2016 Sep 8;119:171-9. Epub 2016 Jun 8.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

The pathogenesis of dermonecrosis induced by the venom of the African spitting cobra Naja nigricollis was investigated in a mouse model. Intradermal injection of venom induced a macroscopic necrotic lesion. Histological examination revealed early edema of the dermis, followed by blistering, loss of skin appendages and reduction in cellularity. By 24 h, necrosis of the dermis was evident, sections of epidermis were lost, and a fibrinoid hyaline material filled the damaged areas. Abundant inflammatory infiltrate was present in the hypodermis and basal dermis, and there was an increment in the expression of matrix metalloproteinases (MMPs). Thrombi were observed in blood vessels. Abundant cells were present in the dermis by 7 days. By 14 and 28 days, re-epithelization had occurred, collagen was widespread in the dermis, and few skin appendages were present. The RP-HPLC fractions that reproduced the necrotic activity were composed of low molecular mass cytotoxins of the three-finger toxin family and, to a lesser extent, of phospholipases A2 (PLA2). Inhibition of PLA2 of venom by p-bromophenacyl bromide did not reduce the area of necrosis, but modified the appearance of necrotic regions. Depletion of neutrophils and inhibition of venom metalloproteinases and tissue MMPs did not affect dermonecrosis. IgG and F(ab')2 antivenoms were effective in the neutralization of dermonecrosis when incubated with venom prior to injection. However, when antivenoms were administered immediately after venom injection, dermonecrosis was reduced only to a partial extent, underscoring the difficulties in neutralizing this effect with antivenoms.
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http://dx.doi.org/10.1016/j.toxicon.2016.06.006DOI Listing
September 2016

Neutralization of the neuromuscular inhibition of venom and taipoxin from the taipan (Oxyuranus scutellatus) by F(ab')2 and whole IgG antivenoms.

Toxicol Lett 2016 Jan 24;241:175-83. Epub 2015 Nov 24.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

The neuromuscular junction activity of Oxyuranus scutellatus venom and its presynaptic neurotoxin, taipoxin, and their neutralization by two antivenoms were examined in mouse phrenic nerve-diaphragm preparations. The action of taipoxin was also studied at 21°C. The efficacy of the antivenoms was also assessed in an in vivo mouse model. Both antivenoms were effective in neutralizing the neuromuscular blocking activity in preincubation-type experiments. In experiments involving independent addition of venom and antivenoms, neutralization depended on the time interval between venom addition and antivenom application. When taipoxin was incubated for 5, 10 or 20min at 21°C, and antivenom added and temperature increased to 37°C, neutralization was achieved only when the toxin was incubated for 5 or 10min. The neutralization by the two antivenoms in an in vivo model showed that both whole IgG and F(ab')2 antivenoms were effective in neutralizing lethality. Our findings highlight the very rapid action of taipan venom at the nerve terminal, and the poor capacity of antivenoms to revert neurotoxicity as the time interval between venom or taipoxin application and antivenom addition increased. Additionally the disparity between molecular masses of the active substances of the two antivenoms did not result in differences in neutralization.
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http://dx.doi.org/10.1016/j.toxlet.2015.11.020DOI Listing
January 2016

Effect of geographical variation of Echis ocellatus, Naja nigricollis and Bitis arietans venoms on their neutralization by homologous and heterologous antivenoms.

Toxicon 2015 Dec 9;108:80-3. Epub 2015 Oct 9.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

Two antivenoms prepared by using Echis ocellatus, Bitis arietans and Naja nigricollis venoms from different locations in sub-Saharan Africa were compared for their neutralizing ability. Both antivenoms were similarly effective in the neutralization of the venoms of the three species from different locations. However in the case of E. ocellatus venom, antivenom prepared using venom from Nigerian specimens was more effective than antivenom prepared with venom from Cameroon specimens in the neutralization of coagulant activity.
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http://dx.doi.org/10.1016/j.toxicon.2015.10.001DOI Listing
December 2015

Assessing endotoxins in equine-derived snake antivenoms: Comparison of the USP pyrogen test and the Limulus Amoebocyte Lysate assay (LAL).

Toxicon 2015 Oct 29;105:13-8. Epub 2015 Aug 29.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

Snake antivenoms are parenterally administered; therefore, endotoxin content must be strictly controlled. Following international indications to calculate endotoxin limits, it was determined that antivenom doses between 20 mL and 120 mL should not exceed 17.5 Endotoxin Units per milliliter (EU/mL) and 2.9 EU/mL, respectively. The rabbit pyrogen test (RPT) has been used to evaluate endotoxin contamination in antivenoms, but some laboratories have recently implemented the LAL assay. We compared the capability of both tests to evaluate endotoxin contamination in antivenoms, and we found that both methods can detect all endotoxin concentrations in the range of the antivenom specifications. The acceptance criteria of RPT and LAL must be harmonized by calculating the endotoxin limit as the quotient of the threshold pyrogenic dose and the therapeutic dose and the dose administered to rabbits as the quotient of the threshold pyrogenic dose and the endotoxin limit. Since endotoxins from Gram-negative bacteria exert different pyrogenicity, if contamination occurred, antivenom batches that induce pyrogenic reactions may be found in spite of passing LAL specifications. Although LAL assay can be used to assess endotoxin content throughout the antivenom manufacturing process, we recommend that the release of final products be based on the results of both methods.
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http://dx.doi.org/10.1016/j.toxicon.2015.08.015DOI Listing
October 2015

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

Toxicon 2015 Sep 19;103:99-105. Epub 2015 Jun 19.

Instituto Clodomiro Picado, Facultad de Microbiología, Universidad de Costa Rica, San José, Costa Rica. Electronic address:

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.
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http://dx.doi.org/10.1016/j.toxicon.2015.06.016DOI Listing
September 2015

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease.

Phytopathology 2015 Jul 29;105(7):1013-25. Epub 2015 Jun 29.

First and eleventh authors: University of Florida, IFAS, Plant Pathology Department, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL; second and fourth authors: U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), Molecular Plant Pathology Laboratory, Beltsville, MD; first and third author: USDA-ARS, Foreign Disease-Weed Science Research Unit, Fort Detrick, MD; fifth author: Centro de Investigación La Libertad, CORPOICA, Villavicencio, Colombia; sixth author: Plant and Environmental Protection Sciences, University of Hawaii, 3190 Maile Way, St. John 205, Honolulu 96822; seventh author: Queensland Museum, South Brisbane, Queensland 4101, Australia; eighth author: Colegio de Postgraduados, Campus Montecillo, Texcoco, Edo. De Mex., CP 56230, México; ninth author: Electron and Confocal Microscopy Unit, USDA-ARS, Beltsville, MD; and tenth author: Systematic Entomology Laboratory, USDA-ARS, Beltsville, MD.

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.
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http://dx.doi.org/10.1094/PHYTO-12-14-0375-FIDOI Listing
July 2015