A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants.

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

A flow cytometry-based neutralization assay for simultaneous evaluation of blocking antibodies against SARS-CoV-2 variants

Vaccines against SARS-CoV-2 have alleviated infection rates, hospitalization and deaths associated with COVID-19. In order to monitor humoral immunity, several serology tests have been developed, but the recent emergence of variants of concern has revealed the need for assays that predict the neutralizing capacity of antibodies in a fast and adaptable manner. Sensitive and fast neutralization assays would allow a timely evaluation of immunity against emerging variants and support drug and vaccine discovery efforts. Here we describe a simple, fast, and cell-free multiplexed flow cytometry assay to interrogate the ability of antibodies to prevent the interaction of Angiotensin-converting enzyme 2 (ACE2) and the receptor binding domain (RBD) of the original Wuhan-1 SARS-CoV-2 strain and emerging variants simultaneously, as a surrogate neutralization assay. Using this method, we demonstrate that serum antibodies collected from representative individuals at different time-points during the pandemic present variable neutralizing activity against emerging variants, such as Omicron BA.1 and South African B.1.351. Importantly, antibodies present in samples collected during 2021, before the third dose of the vaccine was administered, do not confer complete neutralization against Omicron BA.1, as opposed to samples collected in 2022 which show significant neutralizing activity. The proposed approach has a comparable performance to other established surrogate methods such as cell-based assays using pseudotyped lentiviral particles expressing the spike of SARS-CoV-2, as demonstrated by the assessment of the blocking activity of therapeutic antibodies (i.e. Imdevimab) and serum samples. This method offers a scalable, cost effective and adaptable platform for the dynamic evaluation of antibody protection in affected populations against variants of SARS-CoV-2.

An NMR-Based Model to Investigate the Metabolic Phenoreversion of COVID-19 Patients throughout a Longitudinal Study.

After SARS-CoV-2 infection, the molecular phenoreversion of the immunological response and its associated metabolic dysregulation are required for a full recovery of the patient. This process is patient-dependent due to the manifold possibilities induced by virus severity, its phylogenic evolution and the vaccination status of the population. We have here investigated the natural history of COVID-19 disease at the molecular level, characterizing the metabolic and immunological phenoreversion over time in large cohorts of hospitalized severe patients (n = 886) and non-hospitalized recovered patients that self-reported having passed the disease (n = 513). Non-hospitalized recovered patients do not show any metabolic fingerprint associated with the disease or immune alterations. Acute patients are characterized by the metabolic and lipidomic dysregulation that accompanies the exacerbated immunological response, resulting in a slow recovery time with a maximum probability of around 62 days. As a manifestation of the heterogeneity in the metabolic phenoreversion, age and severity become factors that modulate their normalization time which, in turn, correlates with changes in the atherogenesis-associated chemokine MCP-1. Our results are consistent with a model where the slow metabolic normalization in acute patients results in enhanced atherosclerotic risk, in line with the recent observation of an elevated number of cardiovascular episodes found in post-COVID-19 cohorts.

The spike of SARS-CoV-2 promotes metabolic rewiring in hepatocytes.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes a multi-organ damage that includes hepatic dysfunction, which has been observed in over 50% of COVID-19 patients. Liver injury in COVID-19 could be attributed to the cytopathic effects, exacerbated immune responses or treatment-associated drug toxicity. Herein we demonstrate that hepatocytes are susceptible to infection in different models: primary hepatocytes derived from humanized angiotensin-converting enzyme-2 mice (hACE2) and primary human hepatocytes. Pseudotyped viral particles expressing the full-length spike of SARS-CoV-2 and recombinant receptor binding domain (RBD) bind to ACE2 expressed by hepatocytes, promoting metabolic reprogramming towards glycolysis but also impaired mitochondrial activity. Human and hACE2 primary hepatocytes, where steatosis and inflammation were induced by methionine and choline deprivation, are more vulnerable to infection. Inhibition of the renin-angiotensin system increases the susceptibility of primary hepatocytes to infection with pseudotyped viral particles. Metformin, a common therapeutic option for hyperglycemia in type 2 diabetes patients known to partially attenuate fatty liver, reduces the infection of human and hACE2 hepatocytes. In summary, we provide evidence that hepatocytes are amenable to infection with SARS-CoV-2 pseudovirus, and we propose that metformin could be a therapeutic option to attenuate infection by SARS-CoV-2 in patients with fatty liver.

Neddylation tunes peripheral blood mononuclear cells immune response in COVID-19 patients.

The COVID-19 pandemic caused by SARS-CoV-2 has reached 5.5 million deaths worldwide, generating a huge impact globally. This highly contagious viral infection produces a severe acute respiratory syndrome that includes cough, mucus, fever and pneumonia. Likewise, many hospitalized patients develop severe pneumonia associated with acute respiratory distress syndrome (ARDS), along an exacerbated and uncontrolled systemic inflammation that in some cases induces a fatal cytokine storm. Although vaccines clearly have had a beneficial effect, there is still a high percentage of unprotected patients that develop the pathology, due to an ineffective immune response. Therefore, a thorough understanding of the modulatory mechanisms that regulate the response to SARS-CoV-2 is crucial to find effective therapeutic alternatives. Previous studies describe the relevance of Neddylation in the activation of the immune system and its implications in viral infection. In this context, the present study postulates Neddylation, a reversible ubiquitin-like post-translational modification of proteins that control their stability, localization and activity, as a key regulator in the immune response against SARS-CoV-2. For the first time, we describe an increase in global neddylation levels in COVID-19 in the serum of patients, which is particularly associated with the early response to infection. In addition, the results showed that overactivation of neddylation controls activation, proliferation, and response of peripheral blood mononuclear cells (PBMCs) isolated from COVID-19 patients. Inhibition of neddylation, and the subsequent avoidance of activated PBMCs, reduces cytokine production, mainly IL-6 and MCP-1 and induce proteome modulation, being a critical mechanism and a potential approach to immunomodulate COVID-19 patients.

Assessing the Mobility of Severe Acute Respiratory Syndrome Coronavirus-2 Spike Protein Glycans by Structural and Computational Methods.

Two years after its emergence, the coronavirus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) remains difficult to control despite the availability of several vaccines. The extensively glycosylated SARS-CoV-2 spike (S) protein, which mediates host cell entry by binding to the angiotensin converting enzyme 2 (ACE2) through its receptor binding domain (RBD), is the major target of neutralizing antibodies. Like to many other viral fusion proteins, the SARS-CoV-2 spike protein utilizes a glycan shield to thwart the host immune response. To grasp the influence of chemical signatures on carbohydrate mobility and reconcile the cryo-EM density of specific glycans we combined our cryo-EM map of the S ectodomain to 4.1 Å resolution, reconstructed from a limited number of particles, and all-atom molecular dynamics simulations. Chemical modifications modeled on representative glycans (defucosylation, sialylation and addition of terminal LacNAc units) show no significant influence on either protein shielding or glycan flexibility. By estimating at selected sites the local correlation between the full density map and atomic model-based maps derived from molecular dynamics simulations, we provide insight into the geometries of the α-Man-(1→3)-[α-Man-(1→6)-]-ß-Man-(1→4)-ß-GlcNAc(1→4)-ß-GlcNAc core common to all N-glycosylation sites.

Hypoxia reduces cell attachment of SARS-CoV-2 spike protein by modulating the expression of ACE2, neuropilin-1, syndecan-1 and cellular heparan sulfate.

A main clinical parameter of COVID-19 pathophysiology is hypoxia. Here we show that hypoxia decreases the attachment of the receptor-binding domain (RBD) and the S1 subunit (S1) of the spike protein of SARS-CoV-2 to epithelial cells. In Vero E6 cells, hypoxia reduces the protein levels of ACE2 and neuropilin-1 (NRP1), which might in part explain the observed reduction of the infection rate. In addition, hypoxia inhibits the binding of the spike to NCI-H460 human lung epithelial cells by decreasing the cell surface levels of heparan sulfate (HS), a known attachment receptor of SARS-CoV-2. This interaction is also reduced by lactoferrin, a glycoprotein that blocks HS moieties on the cell surface. The expression of syndecan-1, an HS-containing proteoglycan expressed in lung, is inhibited by hypoxia on a HIF-1α-dependent manner. Hypoxia or deletion of syndecan-1 results in reduced binding of the RBD to host cells. Our study indicates that hypoxia acts to prevent SARS-CoV-2 infection, suggesting that the hypoxia signalling pathway might offer therapeutic opportunities for the treatment of COVID-19.

Sensitive detection of SARS-CoV-2 seroconversion by flow cytometry reveals the presence of nucleoprotein-reactive antibodies in unexposed individuals.

There is an ongoing need of developing sensitive and specific methods for the determination of SARS-CoV-2 seroconversion. For this purpose, we have developed a multiplexed flow cytometric bead array (C19BA) that allows the identification of IgG and IgM antibodies against three immunogenic proteins simultaneously: the spike receptor-binding domain (RBD), the spike protein subunit 1 (S1) and the nucleoprotein (N). Using different cohorts of samples collected before and after the pandemic, we show that this assay is more sensitive than ELISAs performed in our laboratory. The combination of three viral antigens allows for the interrogation of full seroconversion. Importantly, we have detected N-reactive antibodies in COVID-19-negative individuals. Here we present an immunoassay that can be easily implemented and has superior potential to detect low antibody titers compared to current gold standard serology methods.

SARS-CoV-2 Infection Dysregulates the Metabolomic and Lipidomic Profiles of Serum.

COVID-19 is a systemic infection that exerts significant impact on the metabolism. Yet, there is little information on how SARS-CoV-2 affects metabolism. Using NMR spectroscopy, we measured the metabolomic and lipidomic serum profile from 263 (training cohort) + 135 (validation cohort) symptomatic patients hospitalized after positive PCR testing for SARS-CoV-2 infection. We also established the profiles of 280 persons collected before the coronavirus pandemic started. Principal-component analysis discriminated both cohorts, highlighting the impact that the infection has on overall metabolism. The lipidomic analysis unraveled a pathogenic redistribution of the lipoprotein particle size and composition to increase the atherosclerotic risk. In turn, metabolomic analysis reveals abnormally high levels of ketone bodies (acetoacetic acid, 3-hydroxybutyric acid, and acetone) and 2-hydroxybutyric acid, a readout of hepatic glutathione synthesis and marker of oxidative stress. Our results are consistent with a model in which SARS-CoV-2 infection induces liver damage associated with dyslipidemia and oxidative stress.

Structural Characterization of N-Linked Glycans in the Receptor Binding Domain of the SARS-CoV-2 Spike Protein and their Interactions with Human Lectins.

The glycan structures of the receptor binding domain of the SARS-CoV2 spike glycoprotein expressed in human HEK293F cells have been studied by using NMR. The different possible interacting epitopes have been deeply analysed and characterized, providing evidence of the presence of glycan structures not found in previous MS-based analyses. The interaction of the RBD 13 C-labelled glycans with different human lectins, which are expressed in different organs and tissues that may be affected during the infection process, has also been evaluated by NMR. In particular, 15 N-labelled galectins (galectins-3, -7 and -8 N-terminal), Siglecs (Siglec-8, Siglec-10), and C-type lectins (DC-SIGN, MGL) have been employed. Complementary experiments from the glycoprotein perspective or from the lectin's point of view have permitted to disentangle the specific interacting epitopes in each case. Based on these findings, 3D models of the interacting complexes have been proposed.