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

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.Funding: This research was supported by the SPRI I+D COVID-19 fund (Basque Government, bG-COVID-19), BIOEF EITB Maratoia (BIO21/COV/037 to AP), the European Research Council (ERC) (ERC-2018-StG 804236-NEXTGEN-IO to AP), the Instituto de Salud Carlos iii (ISCiii, DTS21/00094 to AP and DTS20/00138 to MM-C), Ministerio de Ciencia, Innovación y Universidades (MICINN, PID2019-107956RA-I00 and TED2021-129433B-C21 to AP; PID2020-117116RB-I00 and RTC2019-007125-1 to MM-C) and the FERO Foundation to AP. Personal fellowships: EP-F (Juan de la Cierva-Formación, FJC2018-035449-I), ABo (AECC Bizkaia Scientific Foundation, PRDVZ19003BOSC), AG (Programa Bikaintek from the Basque Government, 48-AF-W1-2019-00012), AA-V (La Caixa Inphinit, LCF/BQ/DR20/11790022), BJ-L (Basque Government, PRE_2019_1_0320), ABl (AECC Bizkaia Scientific Foundation, PRDVZ21640DEBL), PV-B (Proyectos I +D+I, PRE2020-092342) and AP (Ramón y Cajal, RYC2018- 024183-I; and Ikerbasque Research Associate). Acknowledgments: The plasmids for the generation of pseudotyped lentiviral particles were kindly provided by Dr Jesse D. Bloom (Fred Hutchinson Cancer Research Center) and Dr Jean-Philippe Julien (The Hospital for Sick Children). HEK293T-ACE2 cells were kindly provided by Dr. June Ereño-Orbea (CIC bioGUNE) and Dr. Jean-Philippe Julien (The Hospital for Sick Children Research Institute, Toronto)

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.Acknowledgements: This work was supported by the Ministry of Economy, Industry and Competitiveness PID2020-117116RB-I0, PID2019-107956RA-I00 integrado en el Plan Estatal de Investigación Cientifica y Técnica y Innovación, cofinanciado con Fondos FEDER to (MLM-C, AP); Subprograma Retos Colaboración RTC2019-007125-1 to MLM-C; La Caixa Foundation Program HR17-00601 (to MLM-CH), Proyectos Investigacion en Salud DTS20/00138 (to MLM-CH) and DTS21/00094 (to AP), Departamento de Industria del Gobierno Vasco (to MLM-CH); Ciberehd_ISCIII_ is funded by the Instituto de Salud Carlos III. This work was partially supported by the Cantabrian Government (grant number 2020UIC22-PUB-001), and by Instituto de Salud Carlos III (grant number COV20/00170 to ML-H). ERC Starting grant 804236 NEXTGEN-IO (to AP), Ayuda RYC2018-024183-I financiada por MCIN/AEI416 /10.13039/501100011033 y por El FSE invierte en tu futuro (to AP). PhD fellowship from Asociación Española contra el Cáncer (PRDVZ172010SERR and PRDVZ19003BOSC) awarded to MS-M and AB. PhD fellowship from Gobierno Vasco awarded to NG-U, SL, and BJ-L (PRE_2020_2_0154). Finally, we would like to acknowledge Begoña Rodríguez Iruretagoyena for the technical support provided