A comparison of in vitro methods for assessing the potency of therapeutic antisera against the venom of the coral snake Micrurus nigrocinctus

Therapeutic antisera against Micrurus nigrocinctus venom were tested for protection against lethality, as well as for inhibition of the nicotinic acetylcholine receptor (AchR)-binding and neutralization of phospholipase A2 (PLA2) activities of the homologous venom. Protection against venom lethality did not correlate with inhibition of AchR-binding activity, whereas there was a significant correlation between antisera potency and inhibition of PLA2 activity (r = 0.82, n = 10, P < 0.02). Inhibition of PLA2 activity could be useful in assessing the protective efficacy of M. nigrocinctus antisera during antivenom production. Micrurus nigrocinctus nigrocinctus venom proteins were fractionated by cation-exchange chromatography on Mono S FPLC and fractions assayed for lethality, AchR-binding and PLA2 activities. Antisera were titrated by enzyme-linked immunoassay (ELISA) against a crude M. n. nigrocinctus venom, two FPLC lethal fractions containing AchR-binding activity, and two toxins purified from M. n. nigrocinctus venom. No correlation was found between protective efficacy and the ELISA titer against any of these antigens. Compared to other elapid venoms that contain few toxins as major components, M. n. nigrocinctus venom appears to be more complex and its lethal effect is likely to be due to the combined effect of several neurotoxins.Universidad de Costa Rica/[74l-93-301]/UCR/Costa RicaInternational Foundation for Science/[F/0883-4]/IFS/SueciaSwedish Agency for Research Cooperation with Developing Countries//SAREC/SueciaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Isolation, characterization and molecular cloning of AnMIP, a new α-type phospholipase A2 myotoxin inhibitor from the plasma of the snake Atropoides nummifer (Viperidae: Crotalinae)

A new phospholipase A2 (PLA2)-inhibitory protein was isolated from the plasma of Atropoides nummifer, a crotaline snake from Central America. This inhibitor was named AnMIP, given its ability to neutralize the activity of basic PLA2 myotoxins of its own and related venoms. The cDNA of AnMIP was cloned and sequenced, showing that it belongs to the α group of phospholipase A2 inhibitors (PLIs). AnMIP appears as a homotrimer in the native state, held together by non-covalent forces, with a subunit molecular mass of 22,247–22,301 and an isoelectric point of 4.1–4.7. This trimeric structure is the first observed in a PLIα from American crotaline snakes, previously reported only in Asian species. Sequencing, mass spectrometry, and analytical isoelectrofocusing indicated the existence of isoforms, as reported for other PLIαs isolated from snake plasma. The inhibitory profile of AnMIP showed specificity towards group II PLA2s, either belonging to the catalytically-active (D49) or -inactive (K49) subtypes, exemplified in this study by Bothrops asper myotoxin I and A. nummifer myotoxin II, respectively. By phylogenetic analysis it was shown that AnMIP is closely related to CgMIP-II, previously isolated from the plasma of Cerrophidion godmani, showing 93% amino acid sequence identity.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Snake venomics and antivenomics of Middle and South America rattlesnakes.Identification of neurotoxin crotoxin as an adaptive trait during Crotalus Durissus invasion of South America

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Heterologous hyperimmune polyclonal antibodies against SARS-COV-2: A broad coverage, affordable, and scalable potential immunotherapy for Covid-19

The emergence and dissemination of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the resulting COVID-19 pandemic triggered a global public health crisis. Although several SARS-CoV-2 vaccines have been developed, demand far exceeds supply, access to them is inequitable, and thus, populations in low- and middle-income countries are unlikely to be protected soon (1). Furthermore, there are no specific therapies available, which is a challenge for COVID-19 patient care (2). Thus, the appearance of SARS-CoV-2 variants and reports of reinfections associated with immune escape (3, 4) highlight the urgent need for effective and broad coverage COVID-19 therapeutics. Intravenous administration of human or heterologous antibodies is a therapy successfully used in patients with viral respiratory diseases (5). Accordingly, formulations containing SARS-CoV-2 specific antibodies are an attractive therapeutic option for COVID-19 patients (6). SARS-CoV-2 specific antibodies could limit infection by direct virion neutralization and/or by targeting infected cells for elimination via complement or antibody-mediated cytotoxicity (6). Specific SARS-CoV-2 antibody-based therapeutics include convalescent plasma (CP), monoclonal antibodies (mAbs), human polyclonal IgG formulations purified from CP or transgenic animals, and heterologous hyperimmune polyclonal antibodies (pAbs) (6). Although the window for using antibody-based therapeutics varies, clinical data show that they are mainly effective if administered early after symptoms onset (6).Universidad de Costa Rica/[741-C0-198]/UCR/Costa RicaCaja Costarricense del Seguro Social/[]/CCSS/Costa RicaBanco Centroamericano de Integración Económica/[]/BCIE/Costa RicaGerman academic exchange services/[57592642]/DAAD/AlemaniaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de MedicinaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

In vitro Characterization of Anti-SARS-CoV-2 Intravenous Immunoglobulins (IVIg) Produced From Plasma of Donors Immunized With the BNT162b2 Vaccine and Its Comparison With a Similar Formulation Produced From Plasma of COVID-19 Convalescent Donors

Despite vaccines are the main strategy to control the ongoing global COVID-19 pandemic, their effectiveness could not be enough for individuals with immunosuppression. In these cases, as well as in patients with moderate/severe COVID-19, passive immunization with anti-SARS-CoV-2 immunoglobulins could be a therapeutic alternative. We used caprylic acid precipitation to prepare a pilot-scale batch of anti-SARS-CoV-2 intravenous immunoglobulins (IVIg) from plasma of donors immunized with the BNT162b2 (Pfizer-BioNTech) anti-COVID-19 vaccine (VP-IVIg) and compared their in vitro efficacy and safety with those of a similar formulation produced from plasma of COVID-19 convalescent donors (CP-IVIg). Both formulations showed immunological, physicochemical, biochemical, and microbiological characteristics that meet the specifications of IVIg formulations. Moreover, the concentration of anti-RBD and ACE2-RBD neutralizing antibodies was higher in VP-IVIg than in CP-IVIg. In concordance, plaque reduction neutralization tests showed inhibitory concentrations of 0.03–0.09 g/L in VP-IVIg and of 0.06–0.13 in CP-IVIg. Thus, VP-IVIg has in vitro efficacy and safety profiles that justify their evaluation as therapeutic alternative for Rojas-Jiménez et al. Anti-SARS-CoV-2 IVIg clinical cases of COVID-19. Precipitation with caprylic acid could be a simple, feasible, and affordable alternative to produce formulations of anti-SARS-CoV-2 IVIg to be used therapeutically or prophylactically to confront the COVID-19 pandemic in middle and low-income countries.Universidad de Costa Rica/[741-C0-198]/UCR/Costa RicaBanco Centroamericano de Integración Económica/[DI- 87/2020]/BCIE/Costa RicaMinisterio de Ciencia, Innovación, Tecnología y Telecomunicaciones de Costa Rica/[FV-0001- 20]/MICITT/Costa RicaGerman academic exchange services/[57592642]/DAAD/AlemaniaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Centro de Investigación en Enfermedades Tropicales (CIET

A cellular deficiency of gangliosides causes hypersensitivity to Clostridium perfringens phospholipase C

Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. Previously, a cellular UDP-Glc deficiency was related with a hypersensitivity to the cytotoxic effect of Cp-PLC. Because UDP-Glc is required in the synthesis of proteoglycans, N-linked glycoproteins, and glycosphingolipids, the role of these gly-coconjugates in the cellular sensitivity to Cp-PLC was studied. The cellular sensitivity to Cp-PLC was significantly enhanced by glycosphingolipid synthesis inhibitors, and a mutant cell line deficient in gangliosides was found to be hypersensitive to Cp-PLC. Gangliosides protected hypersensitive cells from the cytotoxic effect of Cp-PLC and prevented its membrane-disrupting effect on artificial membranes. Removal of sialic acids by C. perfringens sialidase increases the sensitivity of cultured cells to Cp-PLC and intramuscular co-injection of C. perfringens sialidase, and Cp-PLC in mice potentiates the myotoxic effect of the latter. This work demonstrated that a reduction in gangliosides renders cells more susceptible to the membrane damage caused by Cp-PLC and revealed a previously unrecognized synergism between Cp-PLC and C. perfringens sialidase, providing new insights toward understanding the pathogenesis of clostridial myonecrosis.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Immunochemical, biochemical and evolutionary studies on Micrurus nigrocinctus nigrocinctus venom toxins

Immunochemical, biochemical and evolutionary studies on Micrurus nigrocinctus nigrocinctus venom toxins Monoclonal antibodies (MAbs) were produced against venom proteins of M. nigrocinctus nigrocinctus, the medically most important elapid of Central America. Affinity purified polyclonal antibodies and some of the MAbs were used to develop immunoassays for the quantitative deterrnination of M. n. nigrocinctus venom antigens in biological fluids. It was shown that these assays readily detect venoms from the the most important coral snakes of North and Central America at the level of nanograms. An ELISA-based non-radioactive assay was used to detect the nicotinic acetylcholine receptor (AchR) binding activity of M. n. nigrocinctus venom alpha-neurotoxins. Four alpha-neurotoxins were isolated and found to be antigenically and structurally related to short- and long-chain alpha-neurotoxins from cobras. A therapeutic antivenom was found to contain antibodies which readily inhibited the binding of the M n. nigrocinctus alpha-neurotoxins to AchR and also antibodies which reverse the binding of toxin(s) already complexed with the receptor. One MAb was used for the immunoaffinity isolation of a group of closely related PLA2 isozymes, called nigroxins A, B, Cl, C2, and C3, which are antigenically similar and constitute a new subclass of PLA2s. The myotoxic activities of nigroxins A and B were characterized, their primary structures were established and a molecular model of nigroxin A was built. The structures of nigroxins A and B were compared with those of 19 other PLA2s, whose myotoxic activity has been previously studied. The combination of amino acid residues Arg-17, Ala-100, Asn-108 and a hydrophobic residue at position 109 was found to be present exclusively among class I myotoxic PLA2s. These residues cluster on the protein surface and have been conserved for a long time of divergent evolution (> 25 million years) in venom PLA2s from different elapid subfamilies. Based on structural and phylogenetic analysis, we reconstructed the evolutionary pathway in which elapid PLA2s gained the myotoxic activity in the scaffold of their pancreatic PLA2-like precursor. In addition, we reconstructed the evolution of potassium channel blockers in the scaffold of the pancreatic trypsin inhibitor. Thus, the isolation and study of the nigroxins allowed to clarify that during the evolution of elapid venom glands, existing structures were repeatedly recruited and adapted to serve toxic functions. Key words: Monoclonal antibody, Phospholipase A2, neurotoxin, myotoxin, protein evolutio

Membrane-damaging and cytotoxic sphingomyelinases and phospholipases

This chapter presents an overview of the classification, structure, and main physiopathological activities of bacterial sphingomyelinases and phospholipases, providing examples of their roles as virulence factors in several human and animal diseases. Bacterial sphingomyelinases (SMases) and phospholipases (PLases) constitute a heterogeneous group of surface-associated or secreted esterases produced by a variety of intracellular and extracellular pathogens. These enzymes might favor in different ways tissue colonization establishment and progression of the infection, or evasion of the immune response. In several cases, mutant bacterial strains lacking a sphingomyelinase or a phospholipase encoding gene have impaired virulence in experimental animals, demonstrating the role of these enzymes in pathogenicity. However, PLases contribute also to other aspects of bacterial lifestyle, including survival in different environments, and competition with other microorganisms; thus, the multifunctional nature of these enzymes reflects the remarkable adaptability of some bacteria.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de MicrobiologíaUCR::Vicerrectoría de Docencia::Salud::Facultad de Medicina::Escuela de Medicin

Antibody-mediated neutralization and binding-reversal studies on α-neurotoxins from micrurus nigrocinctus nigrocinctus (coral snake) venom

An ELISA based, non-radioactive acetylcholine receptor (AchR) binding assay was used to detect the alpha-neurotoxins present in Micrurus nigrocinctus nigrocinctus venom. Sera from horses hyperimmunized against M. nigrocinctus venom contain antibodies which inhibit the binding of M. n. nigrocinctus alpha-neurotoxins to AchR and reverse the binding of toxins already complexed with the receptor. This result supports the importance of using antivenom therapeutically in M. n. nigrocinctus envenomations even after the onset of neurological symptoms. M. nigrocinctus antivenoms cross-reacted in an ELISA with several elapid alpha-neurotoxins and inhibited the binding of Bungarus multicinctus alpha-bungarotoxin and Naja naja oxiana neurotoxin II to AchR in vitro, suggesting the presence of short-chain and long-chain alpha-neurotoxins in M. nigrocinctus venom. In vivo neutralization experiments with M. nigrocinctus antivenom demonstrate that M. nigrocinctus venom contains short-chain alpha-neurotoxin(s) which share common neutralizing epitope(s) with Naja naja oxiana neurotoxin II.Swedish Agency for Research Cooperation with Developing Countries//SAREC/SueciaUniversidad de Costa Rica/[741-93-301]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP