The Serological Sciences Network (SeroNet) for COVID-19: Depth and Breadth of Serology Assays and Plans for Assay Harmonization

BackgroundIn October 2020, the National Cancer Institute (NCI) Serological Sciences Network (SeroNet) was established to study the immune response to COVID-19, and "to develop, validate, improve, and implement serological testing and associated technologies." SeroNet is comprised of 25 participating research institutions partnering with the Frederick National Laboratory for Cancer Research (FNLCR) and the SeroNet Coordinating Center. Since its inception, SeroNet has supported collaborative development and sharing of COVID-19 serological assay procedures and has set forth plans for assay harmonization. MethodsTo facilitate collaboration and procedure sharing, a detailed survey was sent to collate comprehensive assay details and performance metrics on COVID-19 serological assays within SeroNet. In addition, FNLCR established a protocol to calibrate SeroNet serological assays to reference standards, such as the U.S. SARS-CoV-2 serology standard reference material and First WHO International Standard (IS) for anti-SARS-CoV-2 immunoglobulin (20/136), to facilitate harmonization of assay reporting units and cross-comparison of study data. ResultsSeroNet institutions reported development of a total of 27 ELISA methods, 13 multiplex assays, 9 neutralization assays, and use of 12 different commercial serological methods. FNLCR developed a standardized protocol for SeroNet institutions to calibrate these diverse serological assays to reference standards. ConclusionsSeroNet institutions have established a diverse array of COVID-19 serological assays to study the immune response to SARS-CoV-2 virus and vaccines. Calibration of SeroNet serological assays to harmonize results reporting will facilitate future pooled data analyses and study cross-comparisons.

Inactivation of SARS Coronavirus 2 and COVID-19 patient samples for contemporary immunology and metabolomics studies

In late 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged from Wuhan, China spurring the Coronavirus Disease-19 (COVID-19) pandemic that has resulted in over 219 million confirmed cases and nearly 4.6 million deaths worldwide. Intensive research efforts ensued to constrain SARS-CoV-2 and reduce COVID-19 disease burden. Due to the severity of this disease, the US Centers for Disease Control and Prevention (CDC) and World Health Organization (WHO) recommend that manipulation of active viral cultures of SARS-CoV-2 and respiratory secretions from COVID-19 patients be performed in biosafety level 3 (BSL3) containment laboratories. Therefore, it is imperative to develop viral inactivation procedures that permit samples to be transferred and manipulated at lower containment levels (i.e., BSL2), and maintain the fidelity of downstream assays to expedite the development of medical countermeasures (MCMs). We demonstrate optimal conditions for complete viral inactivation following fixation of infected cells with paraformaldehyde solution or other commonly-used branded reagents for flow cytometry, UVC inactivation in sera and respiratory secretions for protein and antibody detection assays, heat inactivation following cDNA amplification of single-cell emulsions for droplet-based single-cell mRNA sequencing applications, and extraction with an organic solvent for metabolomic studies. Thus, we provide a suite of protocols for viral inactivation of SARS-CoV-2 and COVID-19 patient samples for downstream contemporary immunology assays that facilitate sample transfer to BSL2, providing a conceptual framework for rapid initiation of high-fidelity research as the COVID-19 pandemic continues.

Evidence of Persisting Autoreactivity in Post-Acute Sequelae of SARS-CoV-2 Infection

While significant attention has been paid to the immunologic determinants of disease states associated with COVID-191,2, their contributions to post-acute sequelae of COVID-19 (PASC) remain less clear3-5. Due to the wide array of PASC presentations6, it is critical to understand if specific features of the disease are associated with discrete immune processes, and whether those processes may be therapeutically targeted. To this end, we performed wide immunologic and serological characterization of patients in the early recovery phase of COVID-19 across a breadth of symptomatic presentations. Using high-parameter proteomics screening and applied machine learning (ML), we identify clear signatures of immunologic activity between PASC patients and uncomplicated recovery, dominated by inflammatory cytokine signaling, neutrophil activity, and markers of cell death. Consistent with disease complexity, heterogeneity in plasma profiling reveals distinct PASC subsets with striking divergence in these ongoing inflammatory processes, here termed plasma quiescent (plaq) and inflammatory (infl) PASC. In addition to elevated inflammatory blood proteomics, inflPASC patients display positive clinical tests of acute inflammation including C-reactive protein and fibrinogen, increased B cell activity with extrafollicular involvement coupled with elevated targeting of viral nucleocapsid protein and clinical autoreactivity. Further, the unique plasma signatures of PASC patients allowed for the creation of refined models with high sensitivity and specificity for the positive identification of inflPASC with a streamlined assessment of 12 blood markers. Additionally, refined ML modeling highlights the unexpected significance of several markers of potential diagnostic or therapeutic use for PASC in general, including the peptide hormone, epiregulin. In all, this work identifies clear biological signatures of PASC with potential diagnostic and therapeutic potential and establishes clear disease subtypes that are both easily identifiable and highly relevant to ongoing efforts in both therapeutic targeting and epidemiological investigation of this highly complex disease.

Elevated SARS-CoV-2 Antibodies Distinguish Severe Disease in Early COVID-19 Infection

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.

Broadly-targeted autoreactivity is common in severe SARS-CoV-2 Infection

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.