Plasma proteome of Long-COVID patients indicates HIF-mediated vasculo-proliferative disease with impact on brain and heart function.

AIMS: Long-COVID occurs after SARS-CoV-2 infection and results in diverse, prolonged symptoms. The present study aimed to unveil potential mechanisms, and to inform prognosis and treatment. METHODS: Plasma proteome from Long-COVID outpatients was analyzed in comparison to matched acutely ill COVID-19 (mild and severe) inpatients and healthy control subjects. The expression of 3072 protein biomarkers was determined with proximity extension assays and then deconvoluted with multiple bioinformatics tools into both cell types and signaling mechanisms, as well as organ specificity. RESULTS: Compared to age- and sex-matched acutely ill COVID-19 inpatients and healthy control subjects, Long-COVID outpatients showed natural killer cell redistribution with a dominant resting phenotype, as opposed to active, and neutrophils that formed extracellular traps. This potential resetting of cell phenotypes was reflected in prospective vascular events mediated by both angiopoietin-1 (ANGPT1) and vascular-endothelial growth factor-A (VEGFA). Several markers (ANGPT1, VEGFA, CCR7, CD56, citrullinated histone 3, elastase) were validated by serological methods in additional patient cohorts. Signaling of transforming growth factor-ß1 with probable connections to elevated EP/p300 suggested vascular inflammation and tumor necrosis factor-α driven pathways. In addition, a vascular proliferative state associated with hypoxia inducible factor 1 pathway suggested progression from acute COVID-19 to Long-COVID. The vasculo-proliferative process predicted in Long-COVID might contribute to changes in the organ-specific proteome reflective of neurologic and cardiometabolic dysfunction. CONCLUSIONS: Taken together, our findings point to a vasculo-proliferative process in Long-COVID that is likely initiated either prior hypoxia (localized or systemic) and/or stimulatory factors (i.e., cytokines, chemokines, growth factors, angiotensin, etc). Analyses of the plasma proteome, used as a surrogate for cellular signaling, unveiled potential organ-specific prognostic biomarkers and therapeutic targets.

Organ and cell-specific biomarkers of Long-COVID identified with targeted proteomics and machine learning.

BACKGROUND: Survivors of acute COVID-19 often suffer prolonged, diffuse symptoms post-infection, referred to as "Long-COVID". A lack of Long-COVID biomarkers and pathophysiological mechanisms limits effective diagnosis, treatment and disease surveillance. We performed targeted proteomics and machine learning analyses to identify novel blood biomarkers of Long-COVID. METHODS: A case-control study comparing the expression of 2925 unique blood proteins in Long-COVID outpatients versus COVID-19 inpatients and healthy control subjects. Targeted proteomics was accomplished with proximity extension assays, and machine learning was used to identify the most important proteins for identifying Long-COVID patients. Organ system and cell type expression patterns were identified with Natural Language Processing (NLP) of the UniProt Knowledgebase. RESULTS: Machine learning analysis identified 119 relevant proteins for differentiating Long-COVID outpatients (Bonferonni corrected P < 0.01). Protein combinations were narrowed down to two optimal models, with nine and five proteins each, and with both having excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, F1 = 1.00). NLP expression analysis highlighted the diffuse organ system involvement in Long-COVID, as well as the involved cell types, including leukocytes and platelets, as key components associated with Long-COVID. CONCLUSIONS: Proteomic analysis of plasma from Long-COVID patients identified 119 highly relevant proteins and two optimal models with nine and five proteins, respectively. The identified proteins reflected widespread organ and cell type expression. Optimal protein models, as well as individual proteins, hold the potential for accurate diagnosis of Long-COVID and targeted therapeutics.

Elevated vascular transformation blood biomarkers in Long-COVID indicate angiogenesis as a key pathophysiological mechanism.

BACKGROUND: Long-COVID is characterized by prolonged, diffuse symptoms months after acute COVID-19. Accurate diagnosis and targeted therapies for Long-COVID are lacking. We investigated vascular transformation biomarkers in Long-COVID patients. METHODS: A case-control study utilizing Long-COVID patients, one to six months (median 98.5 days) post-infection, with multiplex immunoassay measurement of sixteen blood biomarkers of vascular transformation, including ANG-1, P-SEL, MMP-1, VE-Cad, Syn-1, Endoglin, PECAM-1, VEGF-A, ICAM-1, VLA-4, E-SEL, thrombomodulin, VEGF-R2, VEGF-R3, VCAM-1 and VEGF-D. RESULTS: Fourteen vasculature transformation blood biomarkers were significantly elevated in Long-COVID outpatients, versus acutely ill COVID-19 inpatients and healthy controls subjects (P < 0.05). A unique two biomarker profile consisting of ANG-1/P-SEL was developed with machine learning, providing a classification accuracy for Long-COVID status of 96%. Individually, ANG-1 and P-SEL had excellent sensitivity and specificity for Long-COVID status (AUC = 1.00, P < 0.0001; validated in a secondary cohort). Specific to Long-COVID, ANG-1 levels were associated with female sex and a lack of disease interventions at follow-up (P < 0.05). CONCLUSIONS: Long-COVID patients suffer prolonged, diffuse symptoms and poorer health. Vascular transformation blood biomarkers were significantly elevated in Long-COVID, with angiogenesis markers (ANG-1/P-SEL) providing classification accuracy of 96%. Vascular transformation blood biomarkers hold potential for diagnostics, and modulators of angiogenesis may have therapeutic efficacy.

Epitope-specific antibody responses differentiate COVID-19 outcomes and variants of concern.

BACKGROUNDThe role of humoral immunity in COVID-19 is not fully understood, owing, in large part, to the complexity of antibodies produced in response to the SARS-CoV-2 infection. There is a pressing need for serology tests to assess patient-specific antibody response and predict clinical outcome.METHODSUsing SARS-CoV-2 proteome and peptide microarrays, we screened 146 COVID-19 patients' plasma samples to identify antigens and epitopes. This enabled us to develop a master epitope array and an epitope-specific agglutination assay to gauge antibody responses systematically and with high resolution.RESULTSWe identified linear epitopes from the spike (S) and nucleocapsid (N) proteins and showed that the epitopes enabled higher resolution antibody profiling than the S or N protein antigen. Specifically, we found that antibody responses to the S-811-825, S-881-895, and N-156-170 epitopes negatively or positively correlated with clinical severity or patient survival. Moreover, we found that the P681H and S235F mutations associated with the coronavirus variant of concern B.1.1.7 altered the specificity of the corresponding epitopes.CONCLUSIONEpitope-resolved antibody testing not only affords a high-resolution alternative to conventional immunoassays to delineate the complex humoral immunity to SARS-CoV-2 and differentiate between neutralizing and non-neutralizing antibodies, but it also may potentially be used to predict clinical outcome. The epitope peptides can be readily modified to detect antibodies against variants of concern in both the peptide array and latex agglutination formats.FUNDINGOntario Research Fund (ORF) COVID-19 Rapid Research Fund, Toronto COVID-19 Action Fund, Western University, Lawson Health Research Institute, London Health Sciences Foundation, and Academic Medical Organization of Southwestern Ontario (AMOSO) Innovation Fund.

Rapid COVID-19 testing: Speed, quality and cost. Can you have all three?

KEY MESSAGES.

Rapid and accurate agglutination-based testing for SARS-CoV-2 antibodies.

We have developed a rapid, accurate, and cost-effective serologic test for SARS-CoV-2 virus, which caused the COVID-19 pandemic, on the basis of antibody-dependent agglutination of antigen-coated latex particles. When validated using plasma samples that are positive or negative for SARS-CoV-2, the agglutination assay detected antibodies against the receptor-binding domain of the spike (S-RBD) or the nucleocapsid protein of SARS-CoV-2 with 100% specificity and ∼98% sensitivity. Furthermore, we found that the strength of the S-RBD antibody response measured by the agglutination assay correlated with the efficiency of the plasma in blocking RBD binding to the angiotensin-converting enzyme 2 in a surrogate neutralization assay, suggesting that the agglutination assay might be used to identify individuals with virus-neutralizing antibodies. Intriguingly, we found that >92% of patients had detectable antibodies on the day of a positive viral RNA test, suggesting that the agglutination antibody test might complement RNA testing for the diagnosis of SARS-CoV-2 infection.