Process development and characterization of recombinant nucleocapsid protein for its application on COVID-19 diagnosis.

COVID-19 pandemic was caused by the severe acute respiratory syndrome coronavirus 2 (Sars-CoV-2). The nucleocapsid (N) protein from Sars-CoV-2 is a highly immunogenic antigen and responsible for genome packing. Serological assays are important tools to detect previous exposure to SARS-CoV-2, complement epidemiological studies, vaccine evaluation and also in COVID-19 surveillance. SARS-CoV-2 N (r2N) protein was produced in Escherichia coli, characterized, and the immunological performance was evaluated by enzyme-linked immunosorbent assay (ELISA) and beads-based array immunoassay. r2N protein oligomers were evidenced when it is associated to nucleic acid. Benzonase treatment reduced host nucleic acid associated to r2N protein, but crosslinking assay still demonstrates the presence of higher-order oligomers. Nevertheless, after RNase treatment the higher-order oligomers reduced, and dimer form increased, suggesting RNA contributes to the oligomer formation. Structural analysis revealed nucleic acid did not interfere with the thermal stability of the recombinant protein. Interestingly, nucleic acid was able to prevent r2N protein aggregation even with increasing temperature while the protein benzonase treated begin aggregation process above 55 °C. In immunological characterization, ELISA performed with 233 serum samples presented a sensitivity of 97.44% (95% Confidence Interval, CI, 91.04%, 99.69%) and a specificity of 98.71% (95% CI, 95.42%, 99.84%) while beads-based array immunoassay carried out with 217 samples showed 100% sensitivity and 98.6% specificity. The results exhibited an excellent immunological performance of r2N protein in serologic assays showing that, even in presence of nucleic acid, it can be used as a component of an immunoassay for the sensitive and specific detection of SARS-CoV-2 antibodies.

Serum biomarkers associated with SARS-CoV-2 severity.

Immunity with SARS-CoV-2 infection during the acute phase is not sufficiently well understood to differentiate mild from severe cases and identify prognostic markers. We evaluated the immune response profile using a total of 71 biomarkers in sera from patients with SARS-CoV-2 infection, confirmed by RT-PCR and controls. We correlated biological marker levels with negative control (C) asymptomatic (A), nonhospitalized (mild cases-M), and hospitalized (severe cases-S) groups. Among angiogenesis markers, we identified biomarkers that were more frequently elevated in severe cases when compared to the other groups (C, A, and M). Among cardiovascular diseases, there were biomarkers with differences between the groups, with D-dimer, GDF-15, and sICAM-1 higher in the S group. The levels of the biomarkers Myoglobin and P-Selectin were lower among patients in group M compared to those in groups S and A. Important differences in cytokines and chemokines according to the clinical course were identified. Severe cases presented altered levels when compared to group C. This study helps to characterize biological markers related to angiogenesis, growth factors, heart disease, and cytokine/chemokine production in individuals infected with SARS-CoV-2, offering prognostic signatures and a basis for understanding the biological factors in disease severity.

A clinical scoring system to predict long-term arthralgia in Chikungunya disease: A cohort study.

BACKGROUND: Chikungunya virus (CHIKV) has caused worldwide epidemics that impose a major burden on health systems. Approximately half of infected individuals develop chronic debilitating arthralgia, affecting their quality of life. Here, we identified the relevant clinical and demographic variables in the acute phase of CHIKV infection prospectively linked to chronic arthralgia to elaborate a prognostic scoring system. METHODS: Acute CHIKV infection cases (n = 134) confirmed by serology or molecular test were examined <10 days of disease onset and followed for one year to evaluate for disease progression. Potential risk factors for chronic arthralgia were evaluated by multivariate analysis to develop a prognostic scoring system, which was subsequently tested in an independent validation cohort consisting of 42 individuals. RESULTS: A total of 107 out of 134 (80%) acute CHIKV-confirmed cases from the derivation cohort were re-examined one year after enrollment. Chronic arthralgia post-CHIKV infection was diagnosed in 64 (60%). Five of the 12 parameters evaluated in the acute phase were statistically associated with persistent arthralgia and were further tested by Bayesian analysis. These variables were weighted to yield a prognosis score denominated SHERA (Sex, Hypertension, Edema, Retroocular pain, Age), which exhibited 81.3% accuracy in predicting long-term arthralgia post-CHIKV infection in the derivation cohort, and 76.5% accuracy in the validation cohort. CONCLUSIONS: The simplified and externally validated prognostic scoring system, SHERA, is a useful method to screen acutely CHIKV-infected patients at elevated risk of chronic arthralgia who will benefit from specific interventions. This tool could guide public health policies, particularly in resource-constrained settings.