Metabolomics analysis identifies glutamic acid and cystine imbalances in COVID-19 patients without comorbid conditions. Implications on redox homeostasis and COVID-19 pathophysiology.

It is well known that the presence of comorbidities and age-related health issues may hide biochemical and metabolic features triggered by SARS-CoV-2 infection and other diseases associated to hypoxia, as they are by themselves chronic inflammatory conditions that may potentially disturb metabolic homeostasis and thereby negatively impact on COVID-19 progression. To unveil the metabolic abnormalities inherent to hypoxemia caused by COVID-19, we here applied gas chromatography coupled to mass spectrometry to analyze the main metabolic changes exhibited by a population of male patients less than 50 years of age with mild/moderate and severe COVID-19 without pre-existing comorbidities known to predispose to life-threatening complications from this infection. Several differences in serum levels of particular metabolites between normal controls and patients with COVID-19 as well as between mild/moderate and severe COVID-19 were identified. These included increased glutamic acid and reduced glutamine, cystine, threonic acid, and proline levels. In particular, using the entire metabolomic fingerprint obtained, we observed that glutamine/glutamate metabolism was associated with disease severity as patients in the severe COVID-19 group presented the lowest and higher serum levels of these amino acids, respectively. These data highlight the hypoxia-derived metabolic alterations provoked by SARS-CoV-2 infection in the absence of pre-existing co-morbidities as well as the value of amino acid metabolism in determining reactive oxygen species recycling pathways, which when impaired may lead to increased oxidation of proteins and cell damage. They also provide insights on new supportive therapies for COVID-19 and other disorders that involve altered redox homeostasis and lower oxygen levels that may lead to better outcomes of disease severity.

Circulating B10 regulatory cells are decreased in severe and critical COVID-19.

The contribution of B cells in COVID-19 pathogenesis, beyond the production of specific antibodies against SARS-CoV-2, is still not well understood. Since one of their most relevant functional roles includes their immune-suppressive mechanisms, we decided to evaluate one of the most recognized human B regulatory subpopulations: the IL-10+ B10 cells, during COVID-19 onset. After stimulation of PBMCs for IL-10 induction, we employed multiparametric flow cytometry to determine B10 frequencies in severe and critical COVID-19 patients and then correlated those with clinical and laboratory parameters. Compared with healthy individuals, we detected a significant reduction in the B10 subset in both patient groups, which correlates with some inflammatory parameters that define the disease severity. This evidence suggests an aberrant role of B10 cells in immune responses against SARS-CoV-2 that needs to be further explained.

Severity of SARS-CoV-2 infection is linked to double-negative (CD27- IgD-) B cell subset numbers.

OBJECTIVES: The role of B cells in COVID-19, beyond the production of specific antibodies against SARS-CoV-2, is still not well understood. Here, we describe the novel landscape of circulating double-negative (DN) CD27- IgD- B cells in COVID-19 patients, representing a group of atypical and neglected subpopulations of this cell lineage. METHODS: Using multiparametric flow cytometry, we determined DN B cell subset amounts from 91 COVID-19 patients, correlated those with cytokines, clinical and laboratory parameters, and segregated them by principal components analysis. RESULTS: We detected significant increments in the DN2 and DN3 B cell subsets, while we found a relevant decrease in the DN1 B cell subpopulation, according to disease severity and patient outcomes. These DN cell numbers also appeared to correlate with pro- or anti-inflammatory signatures, respectively, and contributed to the segregation of the patients into disease severity groups. CONCLUSION: This study provides insights into DN B cell subsets' potential role in immune responses against SARS-CoV-2, particularly linked to the severity of COVID-19.

Recombinant Protein Expression and Purification of N, S1, and RBD of SARS-CoV-2 from Mammalian Cells and Their Potential Applications.

The coronavirus disease 2019 (COVID-19) pandemic has reached an unprecedented level. There is a strong demand for diagnostic and serological supplies worldwide, making it necessary for countries to establish their own technologies to produce high-quality biomolecules. The two main viral antigens used for the diagnostics for severe acute respiratory syndrome coronavirus (SARS-CoV-2) are the structural proteins spike (S) protein and nucleocapsid (N) protein. The spike protein of SARS-CoV-2 is cleaved into S1 and S2, in which the S1 subunit has the receptor-binding domain (RBD), which induces the production of neutralizing antibodies, whereas nucleocapsid is an ideal target for viral antigen-based detection. In this study, we designed plasmids, pcDNA3.1/S1 and pcDNA3.1/N, and optimized their expression of the recombinant S1 and N proteins from SARS-CoV-2 in a mammalian system. The RBD was used as a control. The antigens were successfully purified from Expi293 cells, with high yields of the S1, N, and RBD proteins. The immunogenic abilities of these proteins were demonstrated in a mouse model. Further, enzyme-linked immunosorbent assays with human serum samples showed that the SARS-CoV-2 antigens are a suitable alternative for serological assays to identify patients infected with COVID-19.

Severe COVID-19 is marked by dysregulated serum levels of carboxypeptidase A3 and serotonin.

The immune response plays a critical role in the pathophysiology of SARS-CoV-2 infection ranging from protection to tissue damage and all occur in the development of acute respiratory distress syndrome (ARDS). ARDS patients display elevated levels of inflammatory cytokines and innate immune cells, and T and B cell lymphocytes have been implicated in this dysregulated immune response. Mast cells are abundant resident cells of the respiratory tract and are able to release different inflammatory mediators rapidly following stimulation. Recently, mast cells have been associated with tissue damage during viral infections, but their role in SARS-CoV-2 infection remains unclear. In this study, we examined the profile of mast cell activation markers in the serum of COVID-19 patients. We noticed that SARS-CoV-2-infected patients showed increased carboxypeptidase A3 (CPA3) and decreased serotonin levels in their serum when compared with symptomatic SARS-CoV-2-negative patients. CPA3 levels correlated with C-reactive protein, the number of circulating neutrophils, and quick SOFA. CPA3 in serum was a good biomarker for identifying severe COVID-19 patients, whereas serotonin was a good predictor of SARS-CoV-2 infection. In summary, our results show that serum CPA3 and serotonin levels are relevant biomarkers during SARS-CoV-2 infection. This suggests that mast cells and basophils are relevant players in the inflammatory response in COVID-19 and may represent targets for therapeutic intervention.

Metabolomics analysis reveals a modified amino acid metabolism that correlates with altered oxygen homeostasis in COVID-19 patients.

We identified the main changes in serum metabolites associated with severe (n = 46) and mild (n = 19) COVID-19 patients by gas chromatography coupled to mass spectrometry. The modified metabolic profiles were associated to an altered amino acid catabolism in hypoxic conditions. Noteworthy, three α-hydroxyl acids of amino acid origin increased with disease severity and correlated with altered oxygen saturation levels and clinical markers of lung damage. We hypothesize that the enzymatic conversion of α-keto-acids to α- hydroxyl-acids helps to maintain NAD recycling in patients with altered oxygen levels, highlighting the potential relevance of amino acid supplementation during SARS-CoV-2 infection.

B Cell Subsets as Severity-Associated Signatures in COVID-19 Patients.

Background: SARS-CoV-2 infection represents a global health problem that has affected millions of people. The fine host immune response and its association with the disease course have not yet been fully elucidated. Consequently, we analyze circulating B cell subsets and their possible relationship with COVID-19 features and severity. Methods: Using a multiparametric flow cytometric approach, we determined B cell subsets frequencies from 52 COVID-19 patients, grouped them by hierarchical cluster analysis, and correlated their values with clinical data. Results: The frequency of CD19+ B cells is increased in severe COVID-19 compared to mild cases. Specific subset frequencies such as transitional B cell subsets increase in mild/moderate cases but decrease with the severity of the disease. Memory B compartment decreased in severe and critical cases, and antibody-secreting cells are increased according to the severity of the disease. Other non-typical subsets such as double-negative B cells also showed significant changes according to disease severity. Globally, these differences allow us to identify severity-associated patient clusters with specific altered subsets. Finally, respiratory parameters, biomarkers of inflammation, and clinical scores exhibited correlations with some of these subpopulations. Conclusions: The severity of COVID-19 is accompanied by changes in the B cell subpopulations, either immature or terminally differentiated. Furthermore, the existing relationship of B cell subset frequencies with clinical and laboratory parameters suggest that these lymphocytes could serve as potential biomarkers and even active participants in the adaptive antiviral response mounted against SARS-CoV-2.