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BackgroundSARS-CoV-2 has caused over 36,000,000 cases and 1,000,000 deaths globally. Comprehensive assessment of the multifaceted anti-viral antibody response is critical for diagnosis, differentiation of severe disease, and characterization of long-term immunity. Initial observations suggest that severe disease is associated with higher antibody levels and greater B cell/plasmablast responses. A multi-antigen immunoassay to define the complex serological landscape and clinical associations is essential. MethodsWe developed a multiplex immunoassay and evaluated serum/plasma from adults with RT-PCR-confirmed SARS-CoV-2 infections during acute illness (N=52) and convalescence (N=69); and pre-pandemic (N=106) and post-pandemic (N=137) healthy adults. We measured IgA, IgG, and/or IgM against SARS-CoV-2 Nucleocapsid (N), Spike domain 1 (S1), receptor binding domain (S1-RBD) and S1-N-terminal domain (S1-NTD). ResultsTo diagnose infection, the combined [IgA+IgG+IgM] or IgG for N, S1, and S1-RBD yielded AUC values -0.90 by ROC curves. From days 6-30 post-symptom onset, the levels of antigen-specific IgG, IgA or [IgA+IgG+IgM] were higher in patients with severe/critical compared to mild/moderate infections. Consistent with excessive concentrations of antibodies, a strong prozone effect was observed in sera from severe/critical patients. Notably, mild/moderate patients displayed a slower rise and lower peak in anti-N and anti-S1 IgG levels compared to severe/critical patients, but anti-RBD IgG and neutralization responses reached similar levels at 2-4 months. ConclusionThis SARS-CoV-2 multiplex immunoassay measures the magnitude, complexity and kinetics of the antibody response against multiple viral antigens. The IgG and combined-isotype SARS-CoV-2 multiplex assay is highly diagnostic of acute and convalescent disease and may prognosticate severity early in illness. One Sentence SummaryIn contrast to patients with moderate infections, those with severe COVID-19 develop prominent, early antibody responses to S1 and N proteins.
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An emerging feature of COVID-19 is the identification of autoreactivity in patients with severe disease that may contribute to disease pathology, however the origin and resolution of these responses remain unclear. Previously, we identified strong extrafollicular B cell activation as a shared immune response feature between both severe COVID-19 and patients with advanced rheumatic disease. In autoimmune settings, this pathway is associated with relaxed peripheral tolerance in the antibody secreting cell compartment and the generation of de novo autoreactive responses. Investigating these responses in COVID-19, we performed single-cell repertoire analysis on 7 patients with severe disease. In these patients, we identify the expansion of a low-mutation IgG1 fraction of the antibody secreting cell compartment that are not memory derived, display low levels of selective pressure, and are enriched for autoreactivity-prone IGHV4-34 expression. Within this compartment, we identify B cell lineages that display specificity to both SARS-CoV-2 and autoantigens, including pathogenic autoantibodies against glomerular basement membrane, and describe progressive, broad, clinically relevant autoreactivity within these patients correlated with disease severity. Importantly, we identify anti-carbamylated protein responses as a common hallmark and candidate biomarker of broken peripheral tolerance in severe COVID-19. Finally, we identify the contraction of this pathway upon recovery, and re-establishment of tolerance standards coupled with a concomitant loss of acute-derived ASCs irrespective of antigen specificity. In total, this study reveals the origins, breadth, and resolution of acute-phase autoreactivity in severe COVID-19, with significant implications in both early interventions and potential treatment of patients with post-COVID sequelae.
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Antibodies are a principal determinant of immunity for most RNA viruses and have promise to reduce infection or disease during major epidemics. The novel coronavirus SARS-CoV-2 has caused a global pandemic with millions of infections and hundreds of thousands of deaths to date1,2. In response, we used a rapid antibody discovery platform to isolate hundreds of human monoclonal antibodies (mAbs) against the SARS-CoV-2 spike (S) protein. We stratify these mAbs into five major classes based on their reactivity to subdomains of S protein as well as their cross-reactivity to SARS-CoV. Many of these mAbs inhibit infection of authentic SARS-CoV-2 virus, with most neutralizing mAbs recognizing the receptor-binding domain (RBD) of S. This work defines sites of vulnerability on SARS-CoV-2 S and demonstrates the speed and robustness of new antibody discovery methodologies.