Viral and Immune Factors in Acute Respiratory Virus Infections: Evaluating the Role of SARS-CoV-2 Nucleocapsid Antigenemia in the Diagnosis and Pathogenesis of SARS-CoV-2 Infection Open Access

Verkerke, Hans (Summer 2022)

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In the first months of the SARS-CoV-2 pandemic, we undertook several translational studies as part of the COVID-19 response at Emory to evaluate early biomarkers of disease severity and investigate the use of serological tests as adjunctive diagnostic tools. As part of these efforts, we aided in the development, validation, and implementation of the FDA authorized SARS-CoV-2 IgG test, still the primary serological test used in the Emory system for COVID-19. We then employed versions of this test in studies evaluating the antibody components of therapeutic COVID-19 convalescent plasma units, finding significant heterogeneity in levels of SARS-CoV-2 IgG, IgA, and IgM. Finally, we have spearheaded the development of novel, high-throughput testing platforms for SARS-CoV-2 neutralizing antibody activity, a primary correlate of vaccine efficacy and protection after natural infection.

While developing these tools, we conducted several clinical studies to uncover biomarkers of COVID-19 severity. In these studies, we found that viral nucleocapsid antigenemia could reliably be measured in blood samples taken during the acute phase of infection. This observation led us to test whether nucleocapsid antigenemia may be a specific diagnostic biomarker of acute SARS-CoV-2 infection. To this end, we conducted a large retrospective serological survey of COVID-19 patients, quantifying antigenemia in 1860 specimens from 1607 patients. In this cohort, antigenemia exhibited 85.8% sensitivity and 98.6% specificity in diagnosing acute COVID-19 cases, suggesting its possible utility as a screening tool for SARS-CoV-2 infection in blood samples.

Infection with SARS-CoV-2 results in a broad spectrum of clinical manifestations from asymptomatic carriage to severe respiratory failure and death. Notably, many patients with severe COVID-19 suffer from systemic disease involving microvascular inflammation, thromboembolism, and multi-system organ failure. However, the viral and immune factors driving these complications remain incompletely understood. Our work on viral antigenemia as a diagnostic tool led us to also evaluate its utility as a prognostic biomarker and potential driver of systemic pathology. We found that early nucleocapsid antigenemia levels predict disease severity and mortality and are associated with specific patterns of antiviral cytokine response. Furthermore, SARS-CoV-2 nucleocapsid itself can stimulate endothelial cells to elicit antiviral cytokine secretion and upregulation of leukocyte adhesion molecules. Together these studies provide a novel framework for understanding nucleocapsid antigenemia as both a diagnostic marker of acute infection and a clinical biomarker of severe COVID-19 that may itself contribute to endothelial dysfunction and systemic disease.

Table of Contents

Table of Contents

Chapter 1     SARS-CoV-2 diagnosis and pathogenesis


SARS-CoV-2 Diagnostics

Detection of SARS-CoV-2 Infection

SARS-CoV-2 Infection and infectiousness

Viral Life Cycle

Spike Protein

Nucleocapsid Protein



Accessory Proteins


Innate Response

Adaptive Response

Humoral Response

Autoantibody formation

Cytokine storm

COVID-19 Coagulopathy

Conclusions and outlook

Thesis Objectives

Figures for Chapter 1

Chapter 2     Nucleocapsid Antigenemia in the diagnosis of SARS-CoV-2 Infection





Experimental Procedures

Author Contributions

Tables Chapter 2

Figures Chapter 2

Chapter 3





Experimental Methods

Chapter 3 Tables

Figures for Chapter 3

Chapter 4     SARS-CoV-2 antibody class-specific serology: applications in diagnostics and characterization of COVID-19 convalescent plasma





Experimental Methods

Chapter 4 Tables

Chapter 4 Figures

Chapter 5     Rapid, automated detection of SARS-CoV-2 neutralizing antibodies against native-like vaccine and delta variant spike trimers





Experimental Methods

Figures for Chapter 5

Chapter 6     Conclusions and Outlook

SARS-CoV-2 Serological Tests: Then and Now

Viral antigenemia: Implications for diagnosis

Viral Antigenemia: implications for COVID-19 pathology

Appendix 1      Supplementary materials to Chapter 2

Supplementary Experimental Methods

Supplementary Figures and Tables

Appendix 2      Galectins, An Ancient Family of Carbohydrate Binding Proteins with Modern Functions



The Evolution of Carbohydrate Recognition

Galectin Evolution and Biochemistry

Galectin Evolution

The Galectin CRD

Synthesis of Galectin Carbohydrate Ligands

Mammalian Galectin Expression and Localization

Nuclear and Cytoplasmic Galectins

Galectin Secretion

Modern Galectin Functions

Regulation of Pre-mRNA Splicing and mRNA Stability

Intracellular Signaling: Proliferation and Apoptosis

The Galectin Lattice



Roles in the Mammalian Immune System

Engagement and Crosslinking of Surface Receptors

Regulation of Immune Cell Turnover

Modulation of Inflammatory Intracellular Signaling Pathways

Intracellular Pattern Recognition and Damage Sensing

Galectin-Pathogen Interactions

Summary and Conclusions

Appendix 3      Evaluating the Role of Galectin-1 during Influenza A Virus Infection





Experimental Procedures

Figures for Appendix 3


*Author contributions are included in chapters that include co-first authored materials

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