A "Made-in-Canada" serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination

OBJECTIVESAntibody testing against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been instrumental in detecting previous exposures and analyzing vaccine-elicited immune responses. Here, we describe a scalable solution to detect and quantify SARS-CoV-2 antibodies, discriminate between natural infection- and vaccination-induced responses, and assess antibody-mediated inhibition of the spike-angiotensin converting enzyme 2 (ACE2) interaction. METHODSWe developed methods and reagents to detect SARS-CoV-2 antibodies by enzyme-linked immunosorbent assay (ELISA). The main assays focus on the parallel detection of immunoglobulin (Ig)Gs against the spike trimer, its receptor binding domain (RBD), and nucleocapsid (N). We automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-spike or -RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. RESULTSOur single-point IgG-based ELISAs accurately distinguished non-infected and infected individuals. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for [≥] 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. We present detailed protocols for serum/plasma or dried blood spots analysis performed manually and on automated platforms. The snELISA can be performed automatically at single points, increasing its scalability. CONCLUSIONSMeasuring antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The reagents are available to enable scaling up of standardized serological assays, permitting inter-laboratory data comparison and aggregation.

A Multiplexed, Next Generation Sequencing Platform for High-Throughput Detection of SARS-CoV-2

Population scale sweeps of viral pathogens, such as SARS-CoV-2, that incorporate large numbers of asymptomatic or mild symptom patients present unique challenges for public health agencies trying to manage both travel and local spread. Physical distancing is the current major strategy to suppress spread of the disease, but with enormous socio-economic costs. However, modelling and studies in isolated jurisdictions suggest that active population surveillance through systematic molecular diagnostics, combined with contact tracing and focused quarantining can significantly suppress disease spread1-3 and has significantly impacted disease transmission rates, the number of infected people, and prevented saturation of the healthcare system4-7. However, reliable systems allowing for parallel testing of 10-100,000s of patients in larger urban environments have not yet been employed. Here we describe "COVID-19 screening using Systematic Parallel Analysis of RNA coupled to Sequencing" (C19-SPAR-Seq), a scalable, multiplexed, readily automated next generation sequencing (NGS) platform8 that is capable of analyzing tens of thousands of COVID-19 patient samples in a single instrument run. To address the strict requirements in clinical diagnostics for control of assay parameters and output, we employed a control-based Precision-Recall and predictive Receiver Operator Characteristics (coPR) analysis to assign run-specific quality control metrics. C19-SPAR-Seq coupled to coPR on a trial cohort of over 600 patients performed with a specificity of 100% and sensitivity of 91% on samples with low viral loads and a sensitivity of > 95% on high viral loads associated with disease onset and peak transmissibility. Our study thus establishes the feasibility of employing C19-SPAR-Seq for the large-scale monitoring of SARS-CoV-2 and other pathogens.

Evidence for sustained mucosal and systemic antibody responses to SARS-CoV-2 antigens in COVID-19 patients

While the antibody response to SARS-CoV-2 has been extensively studied in blood, relatively little is known about the mucosal immune response and its relationship to systemic antibody levels. Since SARS-CoV-2 initially replicates in the upper airway, the antibody response in the oral cavity is likely an important parameter that influences the course of infection, but how it correlates to the antibody response in serum is not known. Here, we profile by enzyme linked immunosorbent assays (ELISAs) IgG, IgA and IgM responses to the SARS-CoV-2 spike protein (full length trimer) and its receptor binding domain (RBD) in serum (n=496) and saliva (n=90) of acute and convalescent patients with laboratory-diagnosed COVID-19 ranging from 3-115 days post-symptom onset (PSO), compared to negative controls. Anti-CoV-2 antibody responses were readily detected in serum and saliva, with peak IgG levels attained by 16-30 days PSO. Whereas anti-CoV-2 IgA and IgM antibodies rapidly decayed, IgG antibodies remained relatively stable up to 105 days PSO in both biofluids. In a surrogate neutralization ELISA (snELISA), neutralization activity peaks by 31-45 days PSO and slowly declines, though a clear drop is detected at the last blood draw (105-115 days PSO). Lastly, IgG, IgM and to a lesser extent IgA responses to spike and RBD in the serum positively correlated with matched saliva samples. This study confirms that systemic and mucosal humoral IgG antibodies are maintained in the majority of COVID-19 patients for at least 3 months PSO. Based on their correlation with each other, IgG responses in saliva may serve as a surrogate measure of systemic immunity. One Sentence SummaryIn this manuscript, we report evidence for sustained SARS-CoV-2-specific IgG and transient IgA and IgM responses both at the site of infection (mucosae) and systemically in COVID-19 patients over 3 months and suggest that saliva could be used as an alternative biofluid for monitoring IgG to SARS-CoV-2 spike and RBD antigens.