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 AddaS0₃^TM, 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.

Subgenomic RNAs (sgRNAs) are potential markers of active SARS-CoV-2 replication, serving as templates for the synthesis of structural and accessory proteins in infectious viral particles. This study aimed to use RT-qPCR to quantify sgRNA and negative RNA intermediates, assessing viral replication in virus samples inactivated by β-propiolactone (βPL). Inactivated viruses subjected to five blind serial passages (BSs) were amplified by RT-qPCR using primers to target the envelope (ENV) and nucleoproteins (N1 and N2) of genomic genes, subgenomic envelope RNA (sgENV), and intermediate envelope RNA (ENV-). All positive controls showed consistent viral titers across passages (10 log10 copies/mL in N1/N2 and 11 log10 copies/mL in ENV) during BSs. Inactivated viral samples for ENV and ENV- targets ranged from 11.34 log10 copies/mL in BS1 to 11.20 log10 copies/mL in BS5. The sgENV was no longer detected in the inactivated SARS-CoV-2 samples after the second passage, suggesting successful inactivation. Replication kinetics showed consistent profiles for N1/N2, ENV, and ENV- targets in the first three post-infection hours (pih) and maintained approximately 5 log10 copies/mL at 1 pih, 2 pih, and 3 pih. A sharp exponential increase in the viral titer was observed from 24 pih onwards, peaking at 11.64 log10 copies/mL at 48 pih. Transmission electron microscopy confirmed viral particles only in cells infected with active SARS-CoV-2. These results support the use of sgRNA as a reliable marker for SARS-CoV-2 replication, especially in distinguishing between active replication and non-viable particles and in the development of diagnostic and therapeutic strategies.

Background: CLEC5A is a C-type lectin expressed by monocytes and neutrophils, playing an important role in innate immunity. Although it has been shown to interact with the spike protein of SARS-CoV-2, its role during vaccination remains poorly understood. Methods: To address this question, we combined in vitro assays to characterize CLEC5A and spike expression and their impact on monocyte differentiation and T-cell activation; in vivo studies to evaluate CLEC5A expression, immune responses, and vaccine efficacy in a murine model; and in silico analyses to identify potential spike epitopes and CLEC5A interaction sites. Results: The Pfizer-BioNTech bivalent mRNA vaccine induced spike expression and CLEC5A upregulation in THP-1 monocytes, promoting M1-like differentiation and CD86⁺ activation. In PBMC co-cultures, CLEC5A⁺ monocytes acted as antigen-presenting cells, releasing inflammatory chemokines and activating both CD4⁺ and CD8⁺ T cells, thereby linking CLEC5A expression to adaptive immunity. In mice, CLEC5A expression was observed on inflammatory monocytes (CCR2⁺CX3CR1^low) within two days of vaccination. In vivo, CLEC5A expression increased during SARS-CoV-2 infection and after immunization, but declined following viral challenge in vaccinated animals. Consistently, robust humoral and cellular responses were detected post-immunization. In silico analysis further suggested differential CLEC5A binding across B- and T-cell epitopes within the spike glycoprotein. Conclusions: These findings suggest that CLEC5A may play a role in bridging innate and adaptive immune responses during SARS-CoV-2 vaccination. Although further studies with different vaccine platforms are necessary to confirm and expand these observations, our results provide preliminary evidence supporting the potential of CLEC5A as an exploratory biomarker of vaccine-induced immunity.

Objectives: The aim of this study is to evaluate some mechanisms of the immune response of people infected with SARS-CoV-2 in both acute infection and early and late convalescence phases. Methods: This is a cohort study of 70 cases of COVID-19, confirmed by RT-PCR, followed up to 60 days. Plasma Samples and clinical data were. Viral load, blood count, indicators inflammation were the parameters evaluated. Cellular immune response was evaluated by flow cytometry and Luminex immunoassays. Results: In the severe group, hypertension was the only reported comorbidity. Non severe patients have activated memory naive CD4+ T cells. Critically ill patients have central memory CD4+ T cell activation. Severe COVID-19 patients have both central memory and activated effector CD8+ T cells. Non-severe COVID-19 cases showed an increase in IL1β, IL-6, IL-10 and TNF and severely ill patients had higher levels of the cytokines IL-6, IL-10 and CXCL8. Conclusions: The present work showed that different cellular responses are observed according to the COVID-19 severity in patients from Brazil an epicenter the pandemic in South America. Also, we notice that some cytokines can be used as predictive markers for the disease outcome, possibility implementation of strategies effective by health managers.

Objectives: The aim of this study is to evaluate some mechanisms of the immune response of people infected with SARS-CoV-2 in both acute infection and early and late convalescence phases. Methods: This is a cohort study of 70 cases of COVID-19, confirmed by RT-PCR, followed up to 60 days. Plasma Samples and clinical data were. Viral load, blood count, indicators inflammation were the parameters evaluated. Cellular immune response was evaluated by flow cytometry and Luminex immunoassays. Results: In the severe group, hypertension was the only reported comorbidity. Non severe patients have activated memory naive CD4+ T cells. Critically ill patients have central memory CD4+ T cell activation. Severe COVIS-19 patients have both central memory TCD8+ T cells and activated effector TCD8+ cells. Non-severe COVID-19 cases showed an increase in IL1β, IL-6, IL-10 and TNF and severely ill patients had higher levels of the cytokines IL-6, IL-10 and CXCL8.Conclusion: The present work showed that different cellular responses are observed according to the COVID-19 severity in patients from Brazil an epicenter the pandemic in South America. Also, we notice that some cytokines can be used as predictive markers for the disease outcome, possibility implementation of strategies effective by health managers.