Inmunoterapia con anticuerpos F(ab')2 neutralizantes anti- SARS-CoV-2 de suero equino en el tratamiento de pacientes ambulatorios con neumonía bilateral por COVID-19 / Immunotherapy with anti-SARS-COV-2 neutralyzing F(ab´)2 antibodies from equine serum in the treatment of out-patients with bilateral COVID-19 pneumonia

Resumen Introducción: La inmunoterapia pasiva es una alternativa terapéutica para los pacientes con COVID-19. Métodos: Se tomó la decisión de realizar una base de datos prospectiva de los pacientes con diagnóstico de neumonía por SARS-CoV-2, no hipoxémica, tratados de forma ambulatoria en el Hospital de Bolívar, Dr. Miguel Capredoni, provincia de Buenos Aires, Argentina, con el objetivo de evaluar la eficacia del tratamiento con suero equino hiperinmune en la reducción de casos graves e internaciones. Realizamos un análisis retrospectivo del período comprendido entre el 26/05/2021 al 28/08/2021, se incluyeron 151 pacientes. Las opciones fueron meprednisona y colchicina asociadas a dos infusiones de suero equino (n = 92) o me prednisona y colchicina durante 10 días por vía oral (n = 59). Resultados: No se observaron diferencias en las características poblacionales y comorbilidades entre ambos grupos. El 46% (69) de los pacientes había recibido por lo menos una dosis de vacuna contra COVID-19. Durante el seguimiento el 23% (35) requirió internación, sin diferencias entre el grupo suero equino y el grupo control (p = 0.89). Se observó una tendencia no significa tiva al riesgo de internación prolongada del 15.7%. (Grupo suero equino 38.1% vs. grupo control 53.8%, Fisher Exact test p = 0.41). La mortalidad en el grupo suero equino fue del 3.97% (4), sin observarse diferencias entre ambos grupos. Se observaron diferencias entre los pacientes vacunados y no vacunados, en puntos duros como necesidad de ARM (0% vs. 8% p = 0.001) y muerte (0% vs. 8% p = 0.001). Discusión: Si bien las tasas de internación y muerte fueron menores de lo esperado, la utilización de suero equino hiperinmune en el escenario ambulatorio impresiona no aportar beneficio clínico.

Abstract Introduction: Passive immunotherapy is a therapeutic alternative for patients with COVID-19. Methods: The decision was made to create a prospective database of patients diagnosed with SARS-CoV-2 pneumonia, non-hypoxemic, treated on an outpatient basis at the Hospital de Bolívar, Dr. Miguel Capredoni, province of Buenos Aires, Argentina, with the aim of evaluating the efficacy in reducing severe cases and hospitalizations of treatment with hyperimmune equine serum in this subgroup of patients. We performed a retrospective analysis of the period from 05/26/2021 to 08/28/2021, where a total of 151 patients were included. The options were meprednisone plus colchicine associated with two equine serum infusions (n = 92) or oral meprednisone and colchicine for 10 days (59). Results: No differences were observed between the population characteristics and comorbidities between both groups. A 46% (69) of the patients had received at least one dose of vaccine against COVID-19. During follow-up, 23% (35) required hospitalization, with no differences between the equine serum group and the control group (p = 0.89). A non-significant trend of 15.7% was observed for the risk of prolonged hospitalization. (Equine serum group 38.1% vs. control group 53.8%, Fisher Exact test p = 0.41). Mortality between the equine serum group was 3.97% (4), with no differences between the two groups. Differences were observed between vaccinated and unvaccinated patients in hard points such as the need for MRA (0% vs. 8% p = 0.001) and death (0% vs. 8% p = 0.001). Discussion: Although the rate of hospitalization and death were lower than expected, the use of hyperimmune equine serum in the outpatient setting impresses as not providing clinical benefit.

Action of Bromelain and Ficin on horse anti Bothrops sp venom Antibodies

Abstract The treatment with hyperimmune sera constitute the only specific and effective therapy available against snakebite envenomation, most common in developing countries. Serum quality is an important factor on patient recovery time and in the incidence of death and permanent disability. To date, most sera consist of pepsin digested IgG antibodies harvested from hyperimmune animals. The use of animal derived enzymes, such as pepsin, to digest IgG, constitute a source of adventitious agents and contaminants, such as porcine circovirus. The present study aims to evaluate the use of the plant derived enzymes bromelain and ficin, as an alternative to pepsin. To this purpose, horse serum immunized against Bothrops venoms was purified with caprylic acid and digested with bromelain or ficin. SDS-PAGE results evidence the formation of F(ab)'2 fragments and suggest that a digestion time superior to 8 hours may be required to completely digest the antibodies with bromelain or ficin. F(ab)'2 fragments obtained by digestion with either bromelain or ficin digestion preserved the ability to recognize Bothrops sp. venom in western blotting assays. Therefore, both enzymes are suitable for use in large-scale production, minimizing contamination risks and increasing safety and efficiency of serotherapy treatments.

Protocolo para la administración de plasma hiperinmune a pacientes covid-19

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.

El plasma de convalecientes como un tratamiento potencial en pacientes con COVID-19 / Convalescent plasma as potential treatment for COVID-19 patients

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)

Development of a hyperimmune equine serum therapy for COVID-19 in Argentina

The disease named COVID-19, caused by the SARS-CoV-2 coronavirus, is currently generating a global pandemic. Vaccine development is no doubt the best long-term immunological approach, but in the current epidemiologic and health emergency there is a need for rapid and effective solutions. Convalescent plasma is the only antibody-based therapy available for COVID-19 patients to date. Equine polyclonal antibodies (EpAbs) put forward a sound alternative. The new generation of processed and purified EpAbs containing highly purified F(ab’)2 fragments demonstrated to be safe and well tolerated. EpAbs are easy to manufacture allowing a fast development and scaling up for a treatment. Based on these ideas, we present a new therapeutic product obtained after immunization of horses with the receptor-binding domain of the viral Spike glycoprotein. Our product shows around 50 times more potency in in vitro seroneutralization assays than the average of convalescent plasma. This result may allow us to test the safety and efficacy of this product in a phase 2/3 clinical trial to be conducted in July 2020 in the metropolitan area of Buenos Aires, Argentina.

La enfermedad denominada COVID-19 es causada por el coronavirus SARS-CoV-2 y es actualmente considerada una pandemia a nivel global. El desarrollo de vacunas es sin duda la mejor estrategia a largo plazo, pero debido a la emergencia sanitaria, existe una necesidad urgente de encontrar soluciones rápidas y efectivas para el tratamiento de la enfermedad. Hasta la fecha, el uso de plasma de convalecientes es la única inmunoterapia disponible para pacientes hospitalizados con COVID-19. El uso de anticuerpos policlonales equinos (EpAbs) es otra alternativa terapéutica interesante. La nueva generación de EpAbs incluyen el procesamiento y purificación de los mismos y la obtención de fragmentos F(ab’)2 con alta pureza y un excelente perfil de seguridad en humanos. Los EpAbs son fáciles de producir, lo cual permite el desarrollo rápido y la elaboración a gran escala de un producto terapéutico. En este trabajo mostramos el desarrollo de un suero terapéutico obtenido luego de la inmunización de caballos utilizando el receptor-binding domain de la glicoproteína Spike del virus. Nuestro producto mostró ser alrededor de 50 veces más potente en ensayos de seroneutralización in vitro que el promedio de los plasmas de convalecientes. Estos resultados nos permitirían testear la seguridad y eficacia de nuestro producto en ensayos clínicos de fase 2/3 a realizarse a partir de julio de 2020 en la zona metropolitana de Buenos Aires, Argentina.

Respuesta anticlases mayores de anticuerpos humanos séricos en antisueros hiperinmunes obtenidos en carneros / Characterization of hyperimmune anti-human serum produced in sheep

Introducción: La inmunoelectroforesis es una técnica de precipitación que permite la caracterización de muestras biológicas complejas. En el Departamento de Inmunología del Instituto de Ciencias Básicas y Preclínicas Victoria de Girón se cuenta con un antisuero hiperinmune obtenido por inmunizaciones de carneros contra proteínas totales séricas humanas y con otro antisuero anti IgA de calostro humano. Objetivo: Identificar IgG, IgM e IgA en suero humano y determinar respuesta anti IgM humana en el antisuero anti IgA de calostro humano obtenido en carnero. Material y Métodos: Se realizó un estudio observacional, descriptivo y transversal desde noviembre de 2017 hasta junio de 2018. Se desarrolló una inmunoelectroforesis de suero humano normal empleando el antisuero hiperinmune. Resultados: Se identificaron IgG, IgM e IgA además de albúmina y otras fracciones proteicas y se determinó respuesta anti IgM humana en el antisuero anti IgA de calostro humano obtenido en carnero. Conclusiones: Este trabajo permitió identificar y determinar la respuesta anticlases mayores de inmunoglobulinas en la muestra de estudio(AU)

Introduction: Immunoelectrophoresis is a precipitation technique that allows the characterization of complex biological samples. The Immunology Department of the Institute of Basic and Pre-Clinical Sciences Victoria de Girón has a hyperimmune antiserum obtained by immunization of sheep against human serum total proteins and it also has an anti-human IgA antiserum obtained from human colostrum. Objective: The aim of this study was to identify IgG, IgM and IgA in human serum and to determine response to anti-human IgM in human colostral IgA with antiserum obtained in sheep. Material and Methods: An observational descriptive cross-sectional study was conducted from November 2017 to June 2018. Immunoelectrophoresis of normal human serum was performed using hyperimmune antiserum. Results: These procedures allowed to identify IgG, IgM and IgA in addition to albumin and other protein fractions and to determine response to anti-human IgM in human colostral IgA with antiserum obtained in sheep. Conclusions: This work allowed us to identify and determine significant anti-class responses of immunoglobulins in the sample studied(AU)

Streamlined downstream process for efficient and sustainable (Fab)2 antivenom preparation

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)

Monocyte Activation Test (MAT) as a possibility of replacement for the rabbit pyrogen test in hyperimmune sera

ABSTRACT The use of a commercial kit for the monocyte-activation test (MAT) was evaluated for assessing pyrogenic contamination of hyperimmune sera . Three batches of sera, two pyrogen free and one pyrogenic, were tested. Endotoxin spike recover indicated that sample dilutions from 1/2 to 1/10 are suitable. Kit transport and storage conditions were also evaluated, proving that an adequate cold chain must be assured to achieve good results. Furthermore, the commercial MAT kit seemed suitable to replace the rabbit pyrogen test (RPT) for pyrogen testing of hyperimmune sera, although further tests are needed to a full validation.

Raising of Hyper-immune Serum Against FMD Virus Type “O” Prevailing in Punjab, Pakistan

Introduction: Foot and mouth disease (FMD) is supposed to be an imperative disease of domestic and wild ruminants which is a vast reason of high mortality in young animals and production losses in adults. The supreme prevailing stains of FMD in Asia are “O”, “A “and “Asia-I”, which are supposed to be a big threat to economy and commonly not properly diagnosed. For appropriate diagnoses, hyper-immune serum is required. Methods: A study was conducted to produce hyper-immune serum in rabbits which were divided into three groups including Group-I, Group-II and a control group. Results: First two groups were weekly inoculated with FMD virus Serotype “O” for six weeks and confirmation of the infection was done with the help of compliment fixation test (CFT), while antibody titer was measured by using ager gel precipitation test (AGPT). Group-II consisting of female rabbits showed earlier and higher titer (Log27) than group-I (Male rabbits) having lower titer (Log25). Conclusion: The study recommended the use of females rabbits to raise hyper-immune serum to attain higher titer.

Anticuerpos antileptospirales en Intacglobín nueva posibilidad terapéutica para tratar la leptospirosis / Antileptospiral antibodies in Intacglobin, a new therapeutic possibility for treating leptospirosis

Introducción: el Intaglobin es un producto biológico obtenido en la Planta de Hemoderivados de La Habana, contiene inmunoglobulinas policlonales de las clases IgG, IgM e IgA, empleadas actualmente como complemento inmunológico en diversas afecciones. Objetivo: demostrar la presencia y efectividad de anticuerpos de inmunoglobulinas antileptospirales en el Intacglobín para el tratamiento específico de la leptospirosis. Material y método: se realizó un estudio en 13 lotes de Intaglobin para demostrar la presencia de inmunoglobulinas antileptospirales en este producto. Se emplearon dos técnicas de laboratorio, la técnica de microaglutinación con 10 serogrupos de leptospiras (técnica de referencia de la OMS), y la técnica UMELISA, con un Ag desarrollado por la Universidad de Ciencias Médicas. Las muestras de Intaglobin fueron diluidas a partir de 1:50 hasta 1.3200 para la técnica de microaglutinación y de 1:21 para UMELISA. Se consideró positiva la prueba cuando aglutinaba el 50% o más de los antígenos para técnica de microaglutinación y positiva según corte establecido para UMELISA. Resultados: en siete de los 13 lotes estudiados se detectaron reacciones serológicas frente a las dos técnicas empleadas. Hasta 1:1600, específicamente en el caso del serogrupo L. Hebdomadis 1:400, en el de L. Icterohaemorrhagiae, Canícola, Pomona y Australis 1:200, y Ballum 1:100. Conclusiones: se abre una nueva posibilidad de tratamiento para la leptospirosis, aumentando el arsenal terapéutico con este producto el cual adquiere ahora un valor agregado.

Introduction: Intacglobin is biological product obtained in the Havana Hemoderivatives Plant, contains polyclonal immunoglobulins of the classes IgG, IgM and IgA, presently used as immunologic complement in diverse conditions. Objective: to show the presence and effectiveness of antileptospiral immunoglobulins antibodies in Intacglobin for the specific treatment of leptospirosis. Material and method: a study was carried out in thirteen lots of Intacglobin, in order to show the presence of antileptospiral immunoglobulins in this product. Two laboratory techniques were used: microagglutination with 10 serogroups of leptospira (WHO reference technique), and UMELISA, with an Ag developed the University of Medical Sciences. The Intacglobin samples were diluted from 1:50 to 1.3200 for the microagglutination and from 1:21 for UMELISA. The test was considered positive when agglutination of the antigens was 50% or higher for the microaaglutination technique and positive according to range stablished for UMELISA. Results: in seven of the thirteen studied lots serologic reactions were detected before the two used techniques. Up to 1:1600, specifically in the case of the serogroup L. Hebdomadis 1:400, in the L. Icterohaemorrhagiae, Canicola, Pomona y Australis 1:200, and Ballum 1:100. Conclusions: a new possibility of treating leptospirosis, increasing the therapeutic stock with this product, which acquires now an added value.

Characterization of a Hepatitis B virus strain in southwestern Paraná, Brazil, presenting mutations previously associated with anti-HBs Resistance / Caracterização de uma cepa de hepatite por vírus B no sudoeste do Paraná, Brasil, apresentando mutações previamente associadas à resistência anti-HBs

The present study investigated if hepatitis B virus (HBV) mutants circulate in the southwestern region of the State of Paraná, Brazil, by analyzing samples from children who received immunoprophylaxis but were born to HBV carrier mothers. Samples from 25 children were screened for HBV serum markers and for HBV DNA by PCR. Only one sample was positive for HBsAg, anti-HBs and HBV DNA, although the child had been vaccinated. Analysis of the S gene sequence of this sample showed the presence of a proline at position 105, a serine at position 114, three threonines at positions 115, 116 and 140, and a glutamine at position 129. The presence of these amino acids, except for serine at position 114, has been related to monoclonal or polyclonal therapy with anti-HBs after liver transplantation, whereas the presence of threonine at position 116 has been described in immunized children from Singapore. This finding demonstrates the possible circulation of HBV strains resistant to hepatitis B immunoprophylaxis in southwestern Paraná, Brazil. The genotype of the sample was identified as genotype D, which is frequently found in the region studied. Since 36 percent of the children had received incomplete or no immunoprophylaxis, more extensive follow-up of children born to HBsAg-positive mothers is needed.

O presente estudo investigou se mutantes do vírus da hepatite B (HBV) circulam na região Sudoeste do Estado do Paraná, Brasil, analisando amostras de crianças que receberam a imunoprofilaxia por terem nascido de mães portadoras do HBV. Amostras de 25 crianças foram analisadas para os marcadores sorológicos do HBV e para o DNA-HBV por PCR. Somente uma amostra foi positiva para AgHBs, anti-HBs e DNA-HBV, apesar da criança ter sido vacinada. Análises da seqüência do gene S desta amostra mostrou a presença de uma prolina na posição 105, uma serina na posição 114, três treoninas nas posições 115, 116 e 140, e uma glutamina na posição 129. A presença destes aminoácidos, exceto para Serina na posição 114, tem sido relacionada a terapia monoclonal ou policlonal com anti-HBs após transplante de fígado, enquanto a presença da treonina na posição 116 tem sido descrita em crianças imunizadas de Singapura. Este achado demonstra a possível circulação de cepas do HBV resistentes a imunoprofilaxia para hepatite B no Sudoeste do Paraná, Brasil. O genótipo da amostra foi identificado como genótipo D, o qual é frequentemente encontrado na região estudada. Desde que 36 por cento das crianças tinham recebido incompleta ou nenhuma imunoprofilaxia, um seguimento mais intensivo das crianças nascidas de mães AgHBs positivo é necessário.