Anti-SARS-CoV-2 equine F (Ab')2 immunoglobulin as a possible therapy for COVID-19.

The new outbreak of coronavirus disease 2019 (COVID-19) has infected and caused the death of millions of people worldwide. Intensive efforts are underway around the world to establish effective treatments. Immunoglobulin from immunized animals or plasma from convalescent patients might constitute a specific treatment to guarantee the neutralization of the virus in the early stages of infection, especially in patients with risk factors and a high probability of progressing to severe disease. Worldwide, a few clinical trials using anti-SARS-CoV-2 immunoglobulins from horses immunized with the entire spike protein or fragments of it in the treatment of patients with COVID-19 are underway. Here, we describe the development of an anti-SARS-CoV-2 equine F(ab')2 immunoglobulin using a newly developed SARS-CoV-2 viral antigen that was purified and inactivated by radiation. Cell-based and preclinical assays showed that the F(ab')2 immunoglobulin successfully neutralizes the virus, is safe in animal models, and reduces the severity of the disease in a hamster model of SARS-CoV-2 infection and disease.

Validation of a virus neutralization potency test in BHK-21 cells for rabies immunoglobulins in a two-center study.

A rabies virus neutralization potency test (VNPT), adapted to microplates from the rapid fluorescent focus inhibition test (RFFIT) for rabies therapeutic immunoglobulin potency evaluation, was standardized and validated in a two-center study in Brazil. The two institutes involved in the study were: Instituto Nacional de Controle de Qualidade em Saúde (Fundação Oswaldo Cruz) and Instituto Butantan. Two equine rabies immunoglobulin samples, all diluted to 1IU/ml, were tested against the WHO 2nd Rabies Human Ig International Standard. Four dilutions of the samples and standards were tested with the VNPT. The potency of the samples was calculated in IU/ml using the probit method; linearity, accuracy, repeatability (intra-assay variation), intermediate precision (inter-assay variation) and reproducibility (inter-laboratory variation) were assessed to evaluate the reliability of the VNPT. Laboratories were arbitrarily coded as Laboratory A and Laboratory B. The following results were obtained with the International Standard: (a) linearity, the overall coefficient of correlation of the dose-response curve was -0.97; (b) accuracy, % error of -0.70 (IU/ml); (c) repeatability, 17.06% (Laboratory A) and 11.61% (Laboratory B); (d) intermediate precision, 16.99% (Laboratory A) and 22.05% (Laboratory B); (e) reproducibility, 14.5%. The final conclusion was that VNPT presents satisfactory linearity, accuracy, repeatability, intermediate precision and reproducibility and is a reliable and suitable method by which to evaluate rabies immunoglobulin potency.

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies.

Dengue nonstructural protein 1 (NS1) is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2), which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV) protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

Hyperimmune antirabies sera titration by standard mouse neutralization and counterimmunoelectrophoresis tests, comparing results of different laboratories

Para a determinacao do nivel de anticorpos de vinte quatro amostras de soros equinos hiperimunes contra a Raiva, foram utilizadas as tecnicas de soroneutralizacao em camundongos (SN) e contraimunoeletroforese (CIE). As provas foram realizadas nos Institutos Butantan (IB) e Panamericano de Proteccion de Alimentos y Zoonosis (INPPAZ). A analise estatistica mostrou que para o SN e CIE feitas no IB, a correlacao foi de r=0,9317, enquanto que no INPPAZ foi de r=0,974. Comparando-se os dados obtidos pelo CIE nos dois laboratorios observou-se uma correlacao de r=0,845. A tecnica de CIE demonstrou ser tao sensivel e eficiente quanto a SN na titulacao de soros anti-rabicos hiperimunes de origem equina. Assim, com base nos resultados de CIE, que e uma tecnica simples, rapida e economica, pode-se estimar os titulos de anticorpos dos soros em SN.

Termoestabilidade da vacina contra a raiva, tipo Fuenzalida & Palacios, uso humano / Thermostability of rabies vaccine, type Fuenzalida & Palacios, for human use

Ten lots of Fuenzalida & Palacios type antirabies vaccine for human use, produced at the Instituto Butantan (São Paulo, Brazil) were stored at temperatures of 45, 37, 28 and 2-8 degrees C. The potency of each lot was determined in samples taken at varied time intervals using the NIH method and lots presenting antigenic values > or = 0,3 were considered satisfactory for use. After 2 hours at 45 degrees C the antigenic value of one out of 10 lots tested was found to be less than the minimum required value. At 37 degrees C all lots maintained satisfactory antigenic values until the third day of storage, whilst at 28 and 2-8 degrees C the potency was fully maintained, respectively for 10 and 360 days. At the ideal temperature of 2-8 degrees C, 100 of the tested vaccines maintained the minimum required antigenicity for a longer period (16 months) than the expiration time of 6-12 months usually recommended for this type of biological produced in Latin American and Caribbean countries. Thus, the obtained data suggested that in countries still producing Fuenzalida & Palacios type vaccine, the expiration tim could be extended to 16 months, what could prevent the unnecessary discarding of products still in useful condition.

Avaliaçäo das condiçöes de estocagem de vacinas vivas, atenuadas contra o sarampo, em postos de vacinaçäo credenciados e em centros de saúde do Estado de Säo Paulo (Brasil) / Evaluation of storage conditions of live, attenuated vaccines against measles, in authorized vaccination centers and health services of the State of Säo Paulo (Brazil)

Para avaliar as condiçöes de estocagem de vacinas vivas, atenuadas contra o sarampo, da rede de vacinaçäo do Estado de Säo Paulo (Brasil), foram visitados 71 Postos de Vacinaçäo Credenciados particulares (PVC), assim como 117 Centros de Saúde oficiais (CS), sobre os quais interessava saber a respeito da qualidade da estocagem a frio. Os parâmetros adotados foram: a) temperatura das geladeiras de uso (+2 a +8-C) e de estoque (< +8-C); b) validade do produto; c) título das vacinas conservadas nestas geladeiras, avaliado pela inoculaçäo de diluiçöes das amostras de vacinas em células Vero; d) proteçäo à luz. dos CS pesquisados, 85,33% apresentaram geladeiras com temperatura de acordo com a recomendada e 100% das vacinas neles estocadas com título e validade satisfatórios. Nos PVC foram encontrados, com maior frequência, lotes de vacina fora do prazo de validade (14,49%), com títulos abaixo do mínimo requerido (3,53%) e geladeiras de uso e de estoque com temperaturas inadequadas (33,80%). Necessário se faz que as condiçöes de estocagem das vacinas contra o sarampo (temperatura e proteçäo à luz), prevalentes no momento, sejam melhoradas e que as bulas passem a acompanhar o produto a eles entregue, para que os responsáveis pela vacinaçäo obedeçam as recomendaçöes do laboratório producto com relaçäo às condiçöes de estocagem, validade e administraçäo do imunobiológico, uma vez que a pesquisa revelou que estas näo säo observadas com o rigor necessário

Seringas hipodérmicas descartáveis versus reutilizáveis: estudo de possíveis efeitos sobre o vírus da vacina viva, atenuada contra o sarampo / Disposable vs. reusable hypodermic syringes: study of feasible effects upon the virus of the live, attenuated measles vaccine

Objetivou-se verificar entre seringas hipodérmicas descartáveis e reutilizáveis qual interfere mais com o vírus vivo presente na vacina contra o sarampo. Vacinas pertencentes a dois lotes foram reconstituídas com os dois tipos de seringas, de modo a formarem dois pools distintos, mantidos à temperatura de +2 a+8-C e protegidos da luz. De cada lote foram realizadas, no mínimo, seis titulaçöes em paralelo, com amostragem a cada hora, de zero a seis horas após reconstituiçäo. A análise estatística dos resultados obtidos nas titulaçöes, feita pelo sistema de retas de regressäo demonstrou que embora as vacinas manipuladas com ambos os tipos de seringas apresentassem um decréscimo de título estatisticamente significativo com o decorrer das horas, ele foi bem mais acentuado para as vacinas reconstituídas com seringas reutilizáveis. A menor interferência das descartáveis no título da vacina viva, atenuada contra o sarampo, demonstrou que a preconizaçäo e uso desse tipo de seringas pela Secretaria de Estado da Saúde de Säo Paulo é o ideal e recomendável, por preservar mais a vacina desde a reconstituiçäo até sua administraçäo e, conseqüentemente, a sua eficácia na prevençäo dessa infecçäo

Teste de tumorigenicidade em células vero / Tumorigenicity testing of vero cells

Six-week-old athymic nude mices were used to test the capacity of Vero cell line to induce the tumor formation. KB cell line, obtained from human oral epidermoid carcinoma, was used as a positive test control. KB cell has proved to be a good positive control for tumorigenicity tests, since all the animals inoculated with this cell developed tumorous mass. These tumors started appearing in mice between 4th and 8 th days after inocullation, gradually progressed until reaching the maximal axes of 18 to 21 mm in the end of the test. None of the animals inoculated with Vero cells developed tumors in the test, demonstrating that way, that the Vero line at passages 145, 149 and 151 is not carcinogenic