Implementación de un método para la purificación de albúmina a partir de plasma equino

Proyecto de Graduación (Bachillerato en Ingeniería en Biotecnología) Instituto Tecnológico de Costa Rica, Escuela de Biología, 2006.La principal actividad del Instituto Clodomiro Picado de la Universidad de Costa Rica, es la producción de suero antiofídico para el tratamiento de personas que han sufrido mordeduras de serpientes. El suero se obtiene mediante el fraccionamiento de plasma para purificar la fracción de las inmunoglobulinas. En dicho proceso se deshecha la albúmina, una proteína plasmática de gran utilidad, tanto a nivel terapéutico como a nivel de reactivo de laboratorio. Con el fin de recuperar y aprovechar esta proteína, en el presente trabajo se evaluaron varias metodologías para la purificación de albúmina a partir de plasma equino. Las metodologías empleadas fueron el fraccionamiento alcohólico en frío, la termocoagulación selectiva con etanol y con polietilenglicol, y la cromatografía de intercambio iónico. Luego de evaluar el rendimiento y la pureza de las muestras obtenidas por cada método, se concluyó que la termocoagulación selectiva es más efectiva que el fraccionamiento alcohólico en frío, y que la cromatografía de intercambio iónico es una técnica útil como etapa final del proceso de purificación de la albúmina. Los resultados obtenidos en esta investigación, sirven de referencia para futuros trabajos relacionados con el desarrollo de un producto a partir de plasma equino o humano, que cumpla con las especificaciones internacionales y que sea útil para la sociedad

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

Stability of equine IgG antivenoms obtained by caprylic acid precipitation: Towards a liquid formulation stable at tropical room temperature

Liquid formulations of antivenom require a cold chain for their distribution and storage, especially in tropical countries characterized by high temperature and humidity (climatic zone IV). Since cold chain is often deficient in many regions, there is a need to develop novel formulations of liquid antivenoms of higher stability at room temperatures. The effect of addition of the polyols mannitol and sorbitol on the thermal stability of caprylic acid-fractionated equine whole IgG antivenoms was assessed in preparations having different concentrations of protein and phenol. Results evidenced that: (1) turbidity increases proportionally to phenol and protein concentration. (2) After one year of storage at 25 °C, caprylic acid-purified antivenoms, formulated with or without polyols, did not show evidences of instability. (3) Formulation of antivenoms with 2.0 M sorbitol prevents the appearance of turbidity after one year storage at 37 °C; however, there was a partial loss in neutralizing potency in these conditions. Results suggest that formulation based on sorbitol is an option to obtain liquid whole IgG antivenoms of higher stability at tropical room temperatures.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

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

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

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

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

Preclinical Evaluation of Caprylic Acid-Fractionated IgG Antivenom for the Treatment of Taipan (Oxyuranus scutellatus) Envenoming in Papua New Guinea

articulo (arbitrado) -- Universidad de Costa Rica, Instituto de Investigaciones Clodomiro Picado, 2011Background: Snake bite is a common medical emergency in Papua New Guinea (PNG). The taipan, Oxyuranus scutellatus, inflicts a large number of bites that, in the absence of antivenom therapy, result in high mortality. Parenteral administration of antivenoms manufactured in Australia is the current treatment of choice for these envenomings. However, the price of these products is high and has increased over the last 25 years; consequently the country can no longer afford all the antivenom it needs. This situation prompted an international collaborative project aimed at generating a new, low-cost antivenom against O. scutellatus for PNG. Methodology/Principal Findings: A new monospecific equine whole IgG antivenom, obtained by caprylic acid fractionation of plasma, was prepared by immunising horses with the venom of O. scutellatus from PNG. This antivenom was compared with the currently used F(ab’)2 monospecific taipan antivenom manufactured by CSL Limited, Australia. The comparison included physicochemical properties and the preclinical assessment of the neutralisation of lethal neurotoxicity and the myotoxic, coagulant and phospholipase A2 activities of the venom of O. scutellatus from PNG. The F(ab’)2 antivenom had a higher protein concentration than whole IgG antivenom. Both antivenoms effectively neutralised, and had similar potency, against the lethal neurotoxic effect (both by intraperitoneal and intravenous routes of injection), myotoxicity, and phospholipase A2 activity of O. scutellatus venom. However, the whole IgG antivenom showed a higher potency than the F(ab’)2 antivenom in the neutralisation of the coagulant activity of O. scutellatus venom from PNG. Conclusions/Significance: The new whole IgG taipan antivenom described in this study compares favourably with the currently used F(ab’)2 antivenom, both in terms of physicochemical characteristics and neutralising potency. Therefore, it should be considered as a promising low-cost candidate for the treatment of envenomings by O. scutellatus in PNG, and is ready to be tested in clinical trials.This study was supported by Vicerrectoría de Investigación, Universidad de Costa Rica (project 741-A9-003); the PNG Office of Higher Education, CTP Limited (Milne Bay Estates), and the Australian Venom Research Unit (University of Melbourne), which is funded by the Australian Government Department of Health and Ageing, the Australia Pacific Science Foundation and Snowy Nominees. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Minipool Caprylic Acid Fractionation of Plasma Using Disposable Equipment: A Practical Method to Enhance Immunoglobulin Supply in Developing Countries

Plasma-derived immunoglobulin G (IgG) is on WHO’s Essential Medicines List, yet developing countries face severe shortages of this critical treatment. Infusion of IgG prepared from locally-collected plasma provides an advantageous mix of antibodies to viral and bacterial pathogens found in the living environment, and this can reduce recurrent infections in immune-deficient patients. We developed a simple manufacturing process using disposable equipment (blood bags, hemodialyzer, and filters) to isolate immunoglobulins from minipools of 20 plasma donations. This process yields a ca. 90% pure virally-inactivated immunoglobulin fraction at 50–60% recovery. Anti-hepatitis B and anti-rubella immunoglobulins were enriched fourfold to sixfold. The product was free of in-vitro thrombogenic and proteolytic activity, confirming its expected clinical safety profile. Virus validations showed caprylic acid treatment robustly inactivated or removed infectivity of lipid-enveloped viruses, including human immunodeficiency virus (HIV) and hepatitis C virus model. This simple and cost-effective process is implemented in Egypt to prepare experimental batches for clinical evaluation. It can enhance immunoglobulin supplies to treat immunodeficient patients through passive transmission of antibodies directed against local pathogens. The method requires minimal training and reasonable infrastructure, and is a practical means to prepare convalescent hyperimmune IgG during infectious outbreaks such as the current Ebola episode.UCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP)UCR::Vicerrectoría de Docencia::Salud::Facultad de Microbiologí

ESTUDIO DE VALIDACIÓN PRECLÍNICA DEL USO TRADICIONAL ANTI-TUMORAL DE UNCARIA TOMENTOSA (UÑA DE GATO)

Uncaria tomentosa has been widely used in several countries as an anticancer remedy, but due to its pharmacological effects this plant has been extensively harvested and faces extinction.     Regardless of its traditional anti-tumoral use, root bark water extract was the lesser active extract. Hydroalcoholic preparation of the root bark showed LD50 ranging from 150 to 500 μg/mL and leaf extracts (water and hydroalcoholic) showed LD50 as low as 180.1μg/mL.  Selectivity index showed that except for astrocytoma and leukemia, all tumor cells were 2-8 times more sensitive to the extracts than to non tumor cells.  Oxindole pentacyclic alkaloids (mitraphylline, isomitraphylline, uncarine C and uncarine E) are present in the extracts and they probably play a role in the cytotoxic effect on tumor cell lines.  Our study suggets that Uncaria tomentosa roots could be replaced by the leaves in order to obtain good pharmacological results, protecting the plant from extinction.Uncaria tomentosa ha sido usada en varios países como una medicina anti-inflamatoria y anti-tumoral, pero debido a sus efectos farmacológicos esta planta ha sido colectada extensivamente y enfrenta la extinción.   A pesar de su uso tradicional anti-tumoral, el extracto acuoso de la corteza de la activo. La preparación hidroalcohólica de la corteza de la raíz mostró una LD50 de entre 150 y 500 μg/mL y los extractos de las hojas (acuoso e hidroalcohólico) mostraron LD50 tan bajas como 180.1 µg/mL.  El índice de selectividad mostró que excepto por las células de astrocitoma y leucémicas, todas las líneas tumorales fueron de 2-8 veces más sensibles a los extractos que las células no tumorales.   Alcaloides pentaciclicosoxindólicos (mitrafilina, isomitrafilina, uncarina C y uncarina E) están presentes en los extractos y probablemente juegan un papel en los efectos citotóxicos sobre las líneas celulares tumorales.  Nuestro estudio sugiere que las raíces de Uncaria tomentosa podrían ser reemplazadas por las hojas para obtener buenos resultados farmacológicos, protegiendo así la planta de la extinción