In December 2019, the novel betacoronavirus Severe Acute Respiratory Disease Coronavirus 2 (SARS-CoV-2) was first detected in Wuhan, China. SARS-CoV-2 has since become a pandemic virus resulting in hundreds of thousands of deaths and deep socioeconomic implications worldwide. In recent months, efforts have been directed towards detecting, tracking, and better understanding human humoral responses to SARS-CoV-2 infection. It has become critical to develop robust and reliable serological assays to characterize the abundance, neutralization efficiency, and duration of antibodies in virus-exposed individuals. Here we review the latest knowledge on humoral immune responses to SARS-CoV-2 infection, along with the benefits and limitations of currently available commercial and laboratory-based serological assays. We also highlight important serological considerations, such as antibody expression levels, stability and neutralization dynamics, as well as cross-reactivity and possible immunological back-boosting by seasonal coronaviruses. The ability to accurately detect, measure and characterize the various antibodies specific to SARS-CoV-2 is necessary for vaccine development, manage risk and exposure for healthcare and at-risk workers, and for monitoring reinfections with genetic variants and new strains of the virus. Having a thorough understanding of the benefits and cautions of standardized serological testing at a community level remains critically important in the design and implementation of future vaccination campaigns, epidemiological models of immunity, and public health measures that rely heavily on up-to-date knowledge of transmission dynamics.

Objectives Antibody 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. Methods We 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 neutralisation (sn)ELISA that measures inhibition of ACE2‐spike or ‐RBD interactions by antibodies. The assays were calibrated to a World Health Organization reference standard. Results Our 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. Conclusions Measuring antibodies to three viral antigens and identify neutralising 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 standardised serological assays, permitting inter‐laboratory data comparison and aggregation.

Platelets are hyper-activated in COVID-19. However, the mechanisms promoting platelet activation by SARS-CoV-2 are not well understood. This may be due to inherent challenges at discriminating the contribution of viral versus host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles, as they are concomitantly released during infection and share biophysical properties. To study this, we evaluated whether SARS-CoV-2 itself, or components derived from SARS-CoV-2-infected human lung epithelial cells, could activate isolated platelets from healthy donors. Activation was measured by the surface expression of P-selectin and the activated conformation of integrin αIIbβ3, degranulation, aggregation under flow conditions and the release of extracellular vesicles. We find that neither SARS-CoV-2 nor purified Spike activate platelets. In contrast, TF produced by infected cells was highly potent at activating platelets. This required trace amounts of plasma containing the coagulation factors FX, FII and FVII. Robust platelet activation involved thrombin and the activation of protease-activated receptor (PAR)-1 and -4 expressed by platelets. Virions and extracellular vesicles were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with virus or extracellular vesicles, which were indistinguishable. Increased TF mRNA expression and activity were also found in lungs in a murine model of COVID-19 and in plasma of severe COVID-19 patients, respectively. In summary, TF activity from SARS-CoV-2-infected cells activates thrombin, which signals to PARs on platelets. Blockade of molecules in this pathway may interfere in platelet activation and coagulation that is characteristic of COVID-19.

No abstract

The true severity of infection due to COVID-19 is under-represented because it is based on only those who are tested. Although nucleic acid amplifications tests (NAAT) are the gold standard for COVID-19 diagnostic testing, serological assays provide better population-level SARS-CoV-2 prevalence estimates. Implementing large sero-surveys present several logistical challenges within Canada due its unique geography including rural and remote communities. Dried blood spot (DBS) sampling is a practical solution but comparative performance data on SARS-CoV-2 serological tests using DBS is currently lacking. Here we present test performance data from a well-characterized SARS-CoV-2 DBS panel sent to laboratories across Canada representing 10 commercial and 2 in-house developed tests for SARS-CoV-2 antibodies. Three commercial assays identified all positive and negative DBS correctly corresponding to a sensitivity, specificity, positive predictive value, and negative predictive value of 100% (95% CI = 72.2, 100). Two in-house assays also performed equally well. In contrast, several commercial assays could not achieve a sensitivity greater than 40% or a negative predictive value greater than 60%. Our findings represent the foundation for future validation studies on DBS specimens that will play a central role in strengthening Canada’s public health policy in response to COVID-19.