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Indisputably, the use of antivenoms for the treatment of snakebite envenoming is beneficial for the victims. However, there are few studies addressing the effect of long-term hyperimmunization in inoculated horses. It is known that the injection of snake venoms and adjuvants leads to local and systemic reactions in horses, but little is known about the response of inflammatory proteins. The aim of this study was to evaluate serum proteins and the electrophoretic profile of horses undergoing crotalid venom hyperimmunization. Twenty horses were divided into two groups: an inoculated group, comprising ten horses that were already being used for production of a Crotalus sp. antivenom, and a control group, comprising ten animals that had never been used for hyperimmunization. All animals were clinically healthy and without laboratory abnormalities. Total protein and albumin concentrations were measured in serum. Serum globulins were obtained by calculation. Plasma fibrinogen estimates were determined by the heat precipitation method. Serum proteinograms were obtained using agarose gel electrophoresis. The results revealed a significant increase in the concentrations of total serum proteins, globulins, and ß-globulins in the inoculated group, exceeding the reference values. There were slight increases in the α-1- and α-2-globulin subfractions in serum-producing horses, with no statistical significance. We also observed that horses used to produce hyperimmune plasma developed hypoalbuminemia, although the decrease in albumin production was not statistically significant. Our findings suggest that the continuous use of horses to produce crotalid antivenom may lead to a chronic inflammatory stimulus, with changes in plasma levels of inflammatory proteins.

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Resumen La transfusión de plasma hiperinmune o convaleciente en pacientes internados, es un problema en la actualidad porque existe desconocimiento de protocolos donde se determine la cuantificación de anticuerpos, tipo de donante, método de obtención y momento de administración. Objetivo. Elaborar un protocolo de administración de plasma hiperinmune en pacientes Covid-19, internados en el Hospital "Presidente Germán Busch" de Trinidad, gestión 2020. Materiales y métodos. Investigación descriptiva, se realizó una encuesta y revisión documental a población de 26 personales de salud y 25 pacientes respectivamente. Resultados. El desconocimiento de protocolos por parte del personal de salud fue de 73,1%, considerando que el 61% cuentan con postgrados. De los pacientes transfundidos el 60% fallecieron, siendo de la tercera edad, sexo masculino y en estado crítico. El 28% fueron dados de alta hospitalaria, tomando en cuenta que fueron transfundidos en estado moderado. Conclusiones. El porcentaje de desconocimiento de protocolo de administración de plasma hiperinmune y los fallecimientos de pacientes transfundidos son elevados, es por ello que se propone la elaboración de un protocolo de administración de plasma hiperinmune para pacientes Covid-19, que se caracterice por: estudio previo de los donantes, método de obtención, títulos de anticuerpos y momento de administración del hemocomponente.

Abstract Transfusion of hyperimmune or convalescent plasma in hospitalized patients is currently a problem because there is a lack of knowledge of protocols to determine the quantification of antibodies, type of donor, method of obtaining and time of administration. Objective. To develop a protocol for the administration of hyperimmune plasma in Covid-19 patients, admitted to the Hospital "Presidente Germán Busch" in Trinidad, management 2020. Materials and methods: Descriptive research, a survey and documentary review were carried out on a population of 26 health personnel and 25 patients respectively. Results. Lack of knowledge of protocols by health personnel was 73.1%, and 61% have postgraduate degrees. Of the transfused patients, 60% died, being elderly, male and in critical condition. 28% were discharged from hospital, taking into account that they were transfused in moderate condition. Conclusions. The percentage of ignorance of the hyperimmune plasma administration protocol and the deaths of transfused patients are high, which is why the development of a hyperimmune plasma administration protocol for Covid-19 patients is proposed, characterized by: study previous donor, method of obtaining, antibody titers and time of administration of the blood component.

Resumo A transfusão de plasma hiperimune ou convalescente em pacientes hospitalizados é atualmente um problema, pois há falta de conhecimento de protocolos para determinar a quantificação de anticorpos, tipo de doador, método de obtenção e tempo de administração. Objetivo: Desenvolver um protocolo para a administração de plasma hiperimune em pacientes Covid-19, internados no Hospital "Presidente Germán Busch" em Trinidad, gestão 2020. Materiais e métodos. Pesquisa descritiva, levantamento e revisão documental foram realizados em uma população de 26 profissionais de saúde e 25 pacientes, respectivamente. Resultados. O desconhecimento dos protocolos por parte dos profissionais de saúde foi de 73,1%, e 61% possuem pós-graduação. Dos pacientes transfundidos, 60% morreram, sendo idosos, do sexo masculino e em estado crítico. 28% tiveram alta hospitalar, levando-se em consideração que foram transfundidos em estado moderado. Conclusões. O percentual de desconhecimento do protocolo de administração de plasma hiperimune e os óbitos de pacientes transfundidos são elevados, razão pela qual é proposto o desenvolvimento de um protocolo de administração de plasma hiperimune para pacientes com Covid-19, caracterizado por: estudo de doador prévio, método de obtenção, títulos de anticorpos e tempo de administração do hemocomponente.

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Introducción: El plasma de convalecientes es una inmunoterapia pasiva que se ha usado para el tratamiento y prevención de muchas enfermedades infecciosas por más de un siglo. Dada la falta de tratamiento específico para el nuevo coronavirus SARS-CoV-2, el plasma de convalecientes es una alternativa terapéutica potencial contra la COVID-19. Objetivo: Realizar una revisión del empleo del plasma de convalecientes como alternativa terapéutica a la COVID-19. Desarrollo: Se empleó la estrategia de búsqueda del tema; consultando las bases de datos Pubmed, SciELO, Lilacs, Cochrane Library y Web of Science. El plasma de convalecientes ha mostrado efectividad en el tratamiento de varias enfermedades virales. Así, la evidencia sobre su uso en los pacientes con COVID-19 es escasa, aunque se han obtenido resultados alentadores, pero no concluyentes por falta de un número mayor ensayos clínicos. Al mismo tiempo, Cuba incluye en sus protocolos de actuación contra la COVID-19 este tratamiento. Conclusiones: Esta alternativa resulta una herramienta inmunoterapéutica en los pacientes con la COVID-19, ya que mejora el estado clínico y disminuir la tasa de letalidad. Sin embargo, se necesitan más ensayos clínicos controlados y aleatorizados que afirmen su efectividad y seguridad(AU)

Introduction: Convalescent plasma is a form of passive immunotherapy which has been used for the treatment and prevention of many infectious diseases for more than one century. Given the absence of a specific treatment for the novel coronavirus SARS-CoV-2, convalescent plasma is a potential therapeutic alternative against COVID-19. Objective: Carry out a review about the use of convalescent plasma as a therapeutic alternative against COVID-19. Discussion: A search was conducted about the topic in the databases Pubmed, SciELO, Lilacs, Cochrane Library and Web of Science. Convalescent plasma has been shown to be effective in the treatment of several viral diseases. However, evidence of its use in COVID-19 patients is scant. Promising results have been obtained, though, but they are not conclusive due to the need of a larger number of clinical trials. In Cuba this treatment is included among the clinical management protocols for COVID-19. Conclusions: This alternative is an immunotherapeutic tool for the treatment of COVID-19 patients, since it improves their clinical status and reduces lethality rates. However, more controlled and randomized clinical trials are required confirming its effectiveness and safety(AU)

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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 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.

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BACKGROUND: Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. METHODS: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. RESULTS: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. CONCLUSION: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.

RESUMO

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)

Assuntos

RESUMO

Antivenoms are the only validated treatment against snakebite envenoming. Numerous drawbacks pertaining to their availability, safety and efficacy are becoming increasingly evident due to low sustainability of current productions. Technological innovation of procedures generating therapeutics of higher purity and better physicochemical characteristics at acceptable cost is necessary. The objective was to develop at laboratory scale a compact, feasible and economically viable platform for preparation of equine F(ab')2 antivenom against Vipera ammodytes ammodytes venom and to support it with efficiency data, to enable estimation of the process cost-effectiveness. Methods: The principle of simultaneous caprylic acid precipitation and pepsin digestion has been implemented into plasma downstream processing. Balance between incomplete IgG breakdown, F(ab')2 over-digestion and loss of the active drug's protective efficacy was achieved by adjusting pepsin to a 1:30 substrate ratio (w/w) and setting pH at 3.2. Precipitation and digestion co-performance required 2 h-long incubation at 21 °C. Final polishing was accomplished by a combination of diafiltration and flow-through chromatography. In vivo neutralization potency of the F(ab')2 product against the venom's lethal toxicity was determined. Results: Only three consecutive steps, performed under finely tuned conditions, were sufficient for preservation of the highest process recovery with the overall yield of 74%, comparing favorably to others. At the same time, regulatory requirements were met. Final product was aggregate- and pepsin-free. Its composition profile was analyzed by mass spectrometry as a quality control check. Impurities, present in minor traces, were identified mostly as IgG/IgM fragments, contributing to active drug. Specific activity of the F(ab')2 preparation with respect to the plasma was increased 3.9-fold. Conclusion: A highly streamlined mode for production of equine F(ab')2 antivenom was engineered. In addition to preservation of the highest process yield and fulfillment of the regulatory demands, performance simplicity and rapidity in the laboratory setting were demonstrated. Suitability for large-scale manufacturing appears promising.(AU)

Assuntos

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Hyperimmune plasma is the raw material for the production of heterologous serum, which corresponds to the main specific treatment against toxins from microorganisms and venomous animals. This work aims to perform a comparative analysis of the plasma collected in the three production bleeds, subjectively analyzing the production of immunoglobulins through the amount of total protein and measuring the levels of pH, glucose, leukocytes, density and fibrinogen. Samples were collected from 67 horses, divided into three groups according to the antigen to which they were immunized, being: Group 1 (G1) - immunized against tetanus toxoid, Group 2 (G2) - immunized against crotalic antigen and Group 3 ( G3) - immunized against scorpionic antigen. Samples were obtained during production bleeding procedures, by collecting whole blood in vacuum tubes with EDTA. The analyzes were performed using a refractometer to quantify total protein, density and fibrinogen and reagent strips for measuring pH, leukocytes and glucose. The results demonstrate significant variations between the three services studied, mainly in relation to total protein, where in G1 its peak production occurred in the second bleed, in G2 it remained stable in the first two bleeds with a slight drop in the third, and in G3 with peak in the first production bleed. Through the rapid tests proposed in this study it is possible to have an overview of the divergence of parameters between the three production bleeds, quickly and at a low cost, but to obtain more reliable results, especially with regard to immunoglobulins, indicated the adoption of more specific analysis techniques.

O plasma hiperimune é a matéria prima para produção de soro heterólogo, que corresponde ao principal tratamento específico contra toxinas oriundas de micro-organismos e animais peçonhentos. Este trabalho tem por objetivo realizar uma análise comparativa do plasma coletado nas três sangrias de produção, analisando subjetivamente a produção de imunoglobulinas através da quantidade de proteína total e dosar os níveis de pH, glicose, leucócitos, densidade e fibrinogênio. Foram coletadas amostras de 67 equinos, divididos em três grupos de acordo com o antígeno ao qual foram imunizados, sendo: Grupo 1 (G1) – imunizados contra o toxóide tetânico, Grupo 2 (G2) – imunizados contra o antígeno crotálico e Grupo 3 (G3) – imunizados contra o antígeno escorpiônico. As amostras foram obtidas durante os procedimentos de sangrias de produção, através da coleta de sangue total em tubos à vácuo com EDTA. As análises foram realizadas através do uso de refratômetro para quantificação de proteína total, densidade e fibrinogênio e de tiras reagentes para dosagem de pH, leucócitos e glicose. Os resultados demonstram significativas variações entre os três serviços estudados, principalmente em relação a proteína total, onde em G1 seu pico de produção ocorreu na segunda sangria, em G2 permaneceu estável nas duas primeiras sangrias com ligeira queda na terceira, e em G3 com pico na primeira sangria de produção. Através dos testes rápidos propostos nesse estudo é possível ter uma visão geral das divergências de parâmetros entre as três sangrias de produção, de forma rápida e com baixo custo, porém para se obter resultados mais fidedignos, principalmente no que se refere as imunoglobulinas, indica-se a adoção de técnicas mais específicas de análises.