The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models’ useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.
Successful SARS-CoV-2 inactivation allows its safe use in Biosafety Level 2 facilities, and the use of the whole viral particle helps in the development of analytical methods and a more reliable immune response, contributing to the development and improvement of in vitro and in vivo assays. In order to obtain a functional product, we evaluated several inactivation protocols and observed that 0.03% beta-propiolactone for 24 h was the best condition tested, as it promoted SARS-CoV-2 inactivation above 99.99% and no cytopathic effect was visualized after five serial passages. Moreover, RT-qPCR and transmission electron microscopy revealed that RNA quantification and viral structure integrity were preserved. The antigenicity of inactivated SARS-CoV-2 was confirmed by ELISA using different Spike-neutralizing monoclonal antibodies. K18-hACE2 mice immunized with inactivated SARS-CoV-2, formulated in AddaS03TM, presented high neutralizing antibody titers, no significant weight loss, and longer survival than controls from a lethal challenge, despite RNA detection in the oropharyngeal swab, lung, and brain. This work emphasizes the importance of using different techniques to confirm viral inactivation and avoid potentially disastrous contamination. We believe that an efficiently inactivated product can be used in several applications, including the development and improvement of molecular diagnostic kits, as an antigen for antibody production as well as a control for non-clinical trials.
Introduction:The expanded interest in studying SARS-CoV-2 to address the current pandemic requires that many laboratories acquire the capacity to work with the virus. However, safety is one of the main limiting factors in a SARS-CoV-2 study due to the high risk of transmission and exposure of healthcare professionals and scientists. Therefore, the infectious virus must be handled in a BSL3 laboratory or higher and it is necessary the development of methods to safely inactivate the virus and to allow a set of studies to be carried out at lower levels of biocontainment. Successful inactivation of the virus allows the material transfer from a BSL3 to a BSL2 environment, enabling its safe use in applications such as standards to challenge diagnostic kits, ELISA and development of monoclonal antibodies.Objective: In this study, different methodologies for inactivation of SARS-CoV-2 were evaluated in order to produce a batch of inactivated viruses. The criteria for selecting the best condition(s) include: inactivation capacity greater than 99.9% of the virus; cost, execution time, scale up capacity and integrity of the viral particle and genome.Methodology: 44 different conditions were tested between chemical and physical agents (Ascorbic acid, Guanidine, Glutaraldehyde, Beta-propiolactone -BPL, and high temperatures). For screening, TCID 50 and RT-qPCR assays were performed to assess the inactivation profile and the maintenance of SARS-CoV-2 RNA copies, respectively. The best results were subjected to new screening, including Transmission Electron Microscopy (MET) and serial passages to ensure viral inactivation. Complete inactivation was indicated by absence of CPE in all sub cultured flasks and by quantifying the absence of viral replication by qPCR in the culture supernatants. Results:The chemical agents BPL, Glutaraldehyde and Guanidine showed equivalent inactivation efficiency (> 99.99%) compared to Ascorbic acid (70%). The detection of viral RNA by RT-qPCR showed that BPL and Guanidine were more efficient in maintaining RNA quantification (<0.5 Log), when compared to Glutaraldehyde (> 1 Log). MET images suggest that BPL was the only chemical agent to preserve the structure of the viral particle, which is an important feature for selecting the inactivation methodology. Temperature inactivation is apparently dependent on the sample volume and makes it difficult to scale up the process. Conclusion:After the characterization steps, BPL inactivation was selected for the production of the inactivated SARS-CoV-2 bank, which will serve as an input to assist LATEV partners in diagnostic tests and quality control of Bio-Manguinhos kits, in addition to attending tests functional for the detection and selection of antibodies. The next step is to establish methodologies for purifying inactivated material to increase the specificity of antibody selection and recognition.
Objetivos: O presente trabalho tem por objetivo compreender os principais sintomas e dificuldades enfrentadas por portadores de Gonartrose, para que deste modo seja possível apresentar os benefícios trazidos pela equiparação dessa doença como deficiência física a pretexto de permitir melhor tratamento médico após a redução da jornada de trabalho do servidor, permitida atualmente pela legislação apenas para deficiências físicas. Método: Exploratório, de abordagem qualitativa, método dialético e procedimentos bibliográfico e documental. Resultados: A equiparação da Gonartrose à deficiência física ao garantir o direito à redução da jornada de trabalho garante também a possibilidade de realização de várias das atividades necessárias para a diminuição dos impactos negativos trazidos pela doença como fisioterapia, hidroginástica e acupuntura, diminuindo os problemas com a locomoção, o próprio desempenho das atividades laborativas e atividades consideradas normais, como andar, sentar, abaixar-se, dirigir ou subir escadas. Deste modo, essa benesse garante a possibilidade de tratamento, que associado a mudanças de estilo de vida, em um processo de cuidado contínuo, que nem sempre leva à cura, pode caracterizar significativa melhora na qualidade de vida do servidor. Conclusões: A Gonartrose deve ser equiparada a deficiência física para fins de redução de jornada de trabalho para que o servidor possa utilizar o tempo disponível para realizar o tratamento necessário para diminuição dos sintomas.
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