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

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

Freeze-dried snake antivenoms formulated with sorbitol, sucrose or mannitol: Comparison of their stability in an accelerated test

Freeze-drying is used to improve the long term stability of pharmaceutical proteins. Sugars and polyols have been successfully used in the stabilization of proteins. However, their use in the development of freeze-dried antivenoms has not been documented. In this work, whole IgG snake antivenom, purified from equine plasma, was formulated with different concentrations of sorbitol, sucrose or mannitol. The glass transition temperatures of frozen formulations, determined by Differential Scanning Calorimetry (DSC), ranged between −13.5 °C and −41 °C. In order to evaluate the effectiveness of the different stabilizers, the freeze-dried samples were subjected to an accelerated stability test at 40 ± 2 °C and 75 ± 5% relative humidity. After six months of storage at 40 °C, all the formulations presented the same residual humidity, but significant differences were observed in turbidity, reconstitution time and electrophoretic pattern. Moreover, all formulations, except antivenoms freeze-dried with mannitol, exhibited the same potency for the neutralization of lethal effect of Bothrops asper venom. The 5% (w:v) sucrose formulation exhibited the best stability among the samples tested, while mannitol and sorbitol formulations turned brown. These results suggest that sucrose is a better stabilizer than mannitol and sorbitol in the formulation of freeze-dried antivenoms under the studied conditions.Universidad de Costa Rica/[741-B2-091]/UCR/Costa RicaPrograma Iberoamericano de Ciencia y Tecnología para el Desarrollo/[BIOTOX 212RT0467]/CYTED/EspañaUCR::Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP

Electrophoretic analysis of antivenoms.

<p>Non-reduced samples were loaded in a 7.5% polyacrylamide gel in the presence of SDS. After separation, proteins were stained with Coomassie Brilliant Blue R-250. Migration of molecular mass markers is depicted to the left.</p

Analysis of antivenoms by gel filtration.

<p>Aliquots of the antivenoms were separated by gel filtration in Superdex 200 10/300 GL column and elution was carried out with 150 mM NaCl, 20 mM Tris-HCl, pH 7.5 buffer. Both antivenoms showed a major peak, corresponding to either F(ab')₂ or IgG monomers, which comprise >90% of the total protein.</p

Physicochemical characteristics of antivenoms.

a<p>Monomer content is expressed as the percentage of antivenom protein present as either IgG or F(ab')₂ monomers, as analyzed by gel filtration (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001144#s3" target="_blank">Materials and Methods</a>).</p>b<p>Turbidity is expressed as Nephelometric Turbidity Units (see details in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001144#s3" target="_blank">Materials and Methods</a>).</p

Toxic activities of <i>O. scutellatus</i> venom and neutralisation by antivenoms.

a<p>Lethal activity was determined for the i.p. and i.v. routes in CD-1 mice. Median Lethal Dose (LD₅₀) is expressed as µg venom/mouse (mean ± S.D)</p>b<p>Coagulant activity, expressed as the Minimum Coagulant Concentration (MCC), either in citrated plasma or in citrated plasma to which CaCl₂ was added immediately before the test. Results are the mean ± S.D.</p>c<p>Myotoxic activity, expressed as the Minimum Myotoxic Dose (MMD).</p>d<p>Neutralisation is expressed as either ED₅₀ (lethality and PLA₂) or ED (coagulant and myotoxic effects) (see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0001144#s3" target="_blank">Materials and Methods</a> for details). Results are presented as mg venom neutralised per mL antivenom. For lethality, the 95% confidence limits are included in parenthesis. For the other effects, results are presented as mean ± S.D. (n = 4–6). Challenge doses of venom correspond to 4 LD₅₀s (lethality), 2 MCCs (coagulant), 1 MMD (myotoxicity), and 1.5 µg venom (PLA₂ activity).</p>e<p>CSL taipan antivenom is labelled as containing at least 12,000 Neutralising Units of antivenom, where 1 Unit = 0.01 mg venom neutralised (i.e.: 12,000 units neutralises 120 mg venom). Actual neutralising unit values for each antivenom are given here for comparison and are calculated from fill volumes of each product.</p>*<p>p<0.05 when the two antivenoms are compared.</p

Snake venomics and antivenomics of Protobothrops mucrosquamatus and Viridovipera stejnegeri from Taiwan: Keys to understand the variable immune response in horses

The proteomes of the venoms of the snakes Viridovipera stejnegeri and Protobothrops mucrosquamatus from Taiwan were characterized by N-terminal sequencing, MALDI-TOF mass fingerprinting, and collision-induced dissociation tandem mass spectrometry of in-gel generated tryptic peptides. Proteins belonging to the following toxin classes were identified: metalloproteinase, phospholipase A2 (PLA2), serine proteinase, C-type lectin-like, CRISP, l-amino acid oxidase, disintegrin, and peptides (vasoactive and inhibitors of SVMPs). Nine horses were immunized with a mixture of these venoms. All horses developed a satisfactory immune response against lethality of the venom of V. stejnegeri, whereas only three horses reached the accepted neutralizing potency against the venom of P. mucrosquamatus. Antivenoms were prepared from pools of ‘good responder’ (GR) and ‘poor responder’ (PR) horses and compared by antivenomics and neutralization tests. A similar neutralizing response was observed between the GR and PR antivenoms against the venom of V. stejnegeri, whereas antivenom from PR had a lower neutralizing activity against effects of P. mucrosquamatus venom than antivenom from GR. The low potency of the plasma of some horses against this venom is a consequence of the low immunogenicity of the neurotoxic PLA2 trimucrotoxin. Our results provide clues for innovating the immunization scheme to generate improved antivenoms.Ministerio de Economía y Competitividad/[BFU2010-17373]/MINECO/EspañaGeneralitat Valenciana/[PROMETEO/2010/005]//EspañaUniversidad de Costa Rica/[741-A9-003]/UCR/Costa RicaUCR::Vicerrectoría de Investigación::Unidades de Investigación::Ciencias de la Salud::Instituto Clodomiro Picado (ICP