Snake Venomics and Antivenomics of Bothrops diporus, a Medically Important Pitviper in Northeastern Argentina

Snake species within genus Bothrops are responsible for more than 80% of the snakebites occurring in South America. The species that cause most envenomings in Argentina, B. diporus, is widely distributed throughout the country, but principally found in the Northeast, the region with the highest rates of snakebites. The venom proteome of this medically relevant snake was unveiled using a venomic approach. It comprises toxins belonging to fourteen protein families,be ing dominated by PI- and PIII-SVMPs, PLA2 molecules, BPP-like peptides, L-amino acid oxidase and serine proteinases. This toxin profile largely explains the characteristic pathophysiological effects of bothropic snakebites observed in patients envenomed by B. diporus. Antivenomic analysis of the SAB antivenom (Instituto Vital Brazil) against the venom of B. diporus showed that this pentabothropic antivenom efficiently recognized all the venom proteins and exhibited poor affinity towards the small peptide (BPPs and tripeptide inhibitors of PIII-SVMPs) components of the venom.Fil: Gay, Claudia Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Nordeste. Instituto de Química Básica y Aplicada del Nordeste Argentino. Universidad Nacional del Nordeste. Facultad de Ciencias Exactas Naturales y Agrimensura. Instituto de Química Básica y Aplicada del Nordeste Argentino; ArgentinaFil: Sanz, Libia. Instituto de Biomedicina de Valencia; EspañaFil: Calvete, Juan J.. Instituto de Biomedicina de Valencia; EspañaFil: Pla, Davinia. Instituto de Biomedicina de Valencia; Españ

Antivenomics and in vivo preclinical efficacy of six Latin American antivenoms towards south-western Colombian Bothrops asper lineage venoms

Snakebite envenoming is an important occupational health problem, particularly in rural areas of developing countries. The timely administration of an effective antivenom remains the mainstay of snakebite management. However, the use of antivenoms is often limited by non-availability due to high cost or by lack of effectiveness. Antivenom shortage can be addressed through the generation of novel polyspecific antivenoms of wide clinical efficacy against the venoms of the medically-relevant snake species within the geographical range where these antivenoms are intended to be deployed, but also by optimizing the paraspecific use of current antivenoms. In Colombia, antivenoms are supplied by two manufacturers, one public, the Instituto Nacional de Salud (INS), and one private, Laboratorios Probiol (PROBIOL). However, the antivenom supply in Colombia has traditionally been insufficient, a circumstance that has led the Colombian Ministerio de Salud y Protección Social to issue several resolutions and decrees to announce this health emergency in the country, and to import antivenoms produced in México and Costa Rica. Contrary to these countries, where B. asper represents the only species of the genus, in SW Colombia three close phylogenetically related B. asper lineages, B. asper (sensu stricto), B. rhombeatus, and B. ayerbei, are responsible for most severe cases of snakebite accidents and exhibit remarkable differences in the physiopathological profile of their envenomings. This work aimed to assess the immunorecognition characteristics of a panel of antivenoms manufactured in Colombia, Costa Rica, Argentina, Perú and Venezuela towards the venoms of the three SW Colombian B. asper lineages. Additionally, combined quantitative in vitro and in vivo data show that the homologous antivenoms produced in Colombia (INS-COL, PROBIOL) and Costa Rica (ICP) effectively neutralize the lethality and the major toxic activities tested of the three SW Colombian B. asper lineage venoms. Heterologous Argentinian (BIOL), Venezuelan (UCV) and Peruvian (INS-PERU) antivenoms also showed comparable, even higher, effective immunocapturing ability towards the venom proteomes of SW Colombian B. asper (sensu stricto), B. rhombeatus, and B. ayerbei, than the Colombian and Costa Rican antivenoms. These results are in line with previous studies highlighting the notable conservation of paraspecific antigenic determinants across the phylogeny of genus Bothrops, and advocate for considering the heterologous Argentinian, Venezuelan and Peruvian antivenoms as further therapeutic alternatives for the treatment of B. asper spp. snakebites in Colombia.Ministerio de Ciencia e Innovación/[BFU2017-89103-P]//EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Omics Meets Biology: Application to the Design and Preclinical Assessment of Antivenoms

artículo -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado. 2014Snakebite envenoming represents a neglected tropical disease that has a heavy public health impact worldwide, mostly affecting poor people involved in agricultural activities in Africa, Asia, Latin America and Oceania. A key issue that complicates the treatment of snakebite envenomings is the poor availability of the only validated treatment for this disease, antivenoms. Antivenoms can be an efficacious treatment for snakebite envenoming, provided they are safe, effective, affordable, accessible and administered appropriately. The shortage of antivenoms in various regions, particularly in Sub-Saharan Africa and some parts of Asia, can be significantly alleviated by optimizing the use of current antivenoms and by the generation of novel polyspecific antivenoms having a wide spectrum of efficacy. Complementing preclinical testing of antivenom efficacy using in vivo and in vitro functional neutralization assays, developments in venomics and antivenomics are likely to revolutionize the design and preclinical assessment of antivenoms by being able to test new antivenom preparations and to predict their paraspecific neutralization to the level of species-specific toxinsUniversidad de Costa Rica. CSIC.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Dagestan blunt-nosed viper, Macrovipera lebetina obtusa (Dwigubsky, 1832), venom. Venomics, antivenomics, and neutralization assays of the lethal and toxic venom activities by anti-Macrovipera lebetina turanica and anti-Vipera berus berus antivenoms

10 páginas 2 figuras, 3 tablasWe have applied a combination of venomics, in vivo neutralization assays, and in vitro third-generation antivenomics analysis to assess the preclinical efficacy of the monospecific anti-Macrovipera lebetina turanica (anti-Mlt) antivenom manufactured by Uzbiopharm® (Uzbekistan) and the monospecific anti-Vipera berus berus antivenom from Microgen® (Russia) against the venom of Dagestan blunt-nosed viper, Macrovipera lebetina obtusa (Mlo). Despite their low content of homologous (anti-Mlt, 5-10%) or para-specific (anti-Vbb, 4-9%) F(ab')2 antibody fragments against M. l. obtusa venom toxins, both antivenoms efficiently recognized most components of the complex venom proteome's arsenal, which is made up of toxins derived from 11 different gene families and neutralized, albeit at different doses, key toxic effects of M. l. obtusa venom, i.e., in vivo lethal and hemorrhagic effects in a murine model, and in vitro phospholipase A2, proteolytic and coagulant activities. The calculated lethality neutralization potencies for Uzbiopharm® anti-Mlt and anti-Vbb Microgen® antivenoms were 1.46 and 1.77 mg/mL, indicating that 1 mL of Uzbiopharm® and Microgen® antivenoms may protect mice from 41 to 50 LD50s of Mlo venom, respectively. The remarkable degree of conservation of immunogenic determinants between species of the clades of European and Oriental viper, which evolved geographically segregated since the early Miocene, suggests an eventual window of opportunity for the treatment of envenomings by Eurasian snakes. Clearly, the rational use of heterologous antivenoms requires establishing their para-specificity landscapes. This paper illustrates the analytical power of combining in vitro and in vivo preclinical quantitative assays toward this goalThis research was partly funded by grant BFU2017-89103-P (Ministerio de Ciencia, Innovación y Universidades, Madrid, Spain), by Vicerrectoría de Investigación, Universidad de Costa Rica (741-A0-804), and by LLC “Innova plus” (Saint-Petersburg, Russia).Peer reviewe

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

11 páginas, 3 figuras, 3 tablasCobras 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 A2 (PLA2s), 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 (PLA2) 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 A2 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.This study was partly supported by grant BFU2017-89103-P from the Ministerio de Ciencia e Innovaci ́on, Madrid (Spain), Project 741-B7-108 from the Vicerrectoría de Investigaci ́on of Universidad de Costa Rica, and Beca del Mill ́on, SEP-484-2019, from the Sistema de Estudios de Posgrado, Universidad de Costa Rica. The collaboration of our col- leagues at Instituto Clodomiro Picado and Laboratorio de Ven ́omica Evolutiva y Traslacional, Instituto de Biomedicina de Valencia, CSIC, is gratefully acknowledged. This work was carried out in partial fulfill- ment of the requirements for the M.Sc. degree for A. S ́anchez at the University of Costa RicaPeer reviewe

Preclinical evaluation of the efficacy of antivenoms for snakebite envenoming: State-of-the-art and challenges ahead

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 immunotherapeuticsMinisterio de Economía y Competitividad/[BFU2013-42833-P]//EspañaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

Two color morphs of the pelagic yellow-bellied sea snake, pelamis platura, from different locations of Costa Rica: snake venomics, toxicity, and neutralization by antivenom

artículo (preprint) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado, 2014The yellow-bellied sea snake, Pelamis platura, is the most broadly distributed snake species. Despite being endowed with a highly lethal venom, a proteomic analysis of its toxin composition was unavailable. The venoms of specimens collected in Golfo de Papagayo and Golfo Dulce (Costa Rica), where two distinctive color morphs occur, were chromatographically compared. The latter inhabits a fjord-like gulf where the transit of oceanic sea snakes into and from the basin is restricted, thus possibly affecting gene flow. RP-HPLC evidenced a conserved venom protein profile in both populations, despite their divergent color phenotypes. Following a trend observed in other sea snakes, P. platura venom is relatively simple, being composed of proteins of the three-finger toxin (3FTx), phospholipase A2 (PLA2), cysteine-rich secretory protein (CRISP), 5′-nucleotidase, and metalloproteinase families. The first three groups represent 49.9%, 32.9%, and 9.1% of total venom protein, respectively. The most abundant component (~ 26%) is pelamitoxin (P62388), a short-chain 3FTx, followed by a major basic PLA2 (~ 20%) and a group of three isoforms of CRISPs (~ 9%). Whereas isolated pelamitoxin was highly lethal to mice, neither the PLA2 nor the CRISP fraction caused death. However, the PLA2 rapidly increased plasma creatine kinase activity after intramuscular injection, indicating its myotoxic action. Differing from myotoxic PLA2s of viperids, this PLA2 was not cytolytic to murine myogenic cells in vitro, suggesting possible differences in its mechanism of action. The median lethal dose (LD50) estimates for P. platura crude venom in mice and in three species of fishes did not differ significantly. The sea snake antivenom manufactured by CSL Ltd. (Australia), which uses Enhydrina schistosa as immunogen, cross-recognized the three major components of P. platura venom and, accordingly, neutralized the lethal activity of crude venom and pelamitoxin, therefore being of potential usefulness in the treatment of envenomations by this species.Funded by grants from Ministerio de Economía y Competitividad, Madrid, BFU2010-17373; PROMETEO/2010/005 from the Generalitat Valenciana, Spain; Vicerrectoría de Investigación, Universidad de Costa Rica (741-B2-652 and 741-B3-760; Network for proteomic characterization of snake venoms of medical and biological relevance in Latin America), and CYTED (project BioTox, P211RT0412).UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Identificación y caracterización funcional de la interacción entre el proto-oncogen c-Fos y la proteína centrosomal CEP2/C-NAP1

196 p. : graf. ; 25 cmLa autora ha disfrutado de una beca de Formación de Personal Investigador del Ministerio de Educación y Ciencia. Este trabajo de Tesis Doctoral se ha realizado gracias a las siguientes ayudas de los siguientes organismos: RETIC en Enfermedades Cardiovasculares (C03/01 y RD06/0014/0021), Ministerio de Educación y Ciencia (SAF2004-03057), Ministerio de Ciencia e Innovación (SAF2007-62110) y Generalitat Valenciana (GVPRE/2008/163).Peer reviewe

Third Generation Antivenomics: Pushing the Limits of the In Vitro Preclinical Assessment of Antivenoms.

12 páginas, 5 figuras.Second generation antivenomics is a translational venomics approach designed to complement in vivo preclinical neutralization assays. It provides qualitative and quantitative information on the set of homologous and heterologous venom proteins presenting antivenom-recognized epitopes and those exhibiting impaired immunoreactivity. In a situation of worrying antivenom shortage in many tropical and sub-tropical regions with high snakebite mortality and morbidity rates, such knowledge has the potential to facilitate the optimal deployment of currently existing antivenoms and to aid in the rational design of novel broad specificity antidotes. The aim of the present work was to expand the analytical capability of the immunoaffinity second-generation antivenomics platform, endowing it with the ability to determine the maximal binding capacity of an antivenom toward the different toxins present in a venom, and to quantify the fraction of venom-specific antibodies present in a given antivenom. The application of this new platform, termed third generation (3G) antivenomics, in the preclinical evaluation of antivenoms is illustrated in this paper for the case of antivenom EchiTAb-Plus-ICP® reactivity towards the toxins of homologous (B. arietans) and heterologous (N. melanoleuca) venoms.This study was supported by Grant BFU2013-42833-P from the Ministerio de Economía y Competitividad, Madrid, Spain.Peer reviewe

Inducción temprana de componentes del complemento en la pared arterial en respuesta a hipercolesterolemia y posible papel patológico

1 página.-- Trabajo presentado por Claudia Castro como comunicación oral a la "4ª Reunión de la Red Valenciana de Genómica y Proteómica", sesión IV: "Proteómica" celebrada en Valencia (España) el 27 de Oct. del 2005.Peer reviewe