Assembly reactions of SARS-CoV-2 nucleocapsid protein with nucleic acid (preprint)

The viral genome of SARS-CoV-2 is packaged by the nucleocapsid (N-) protein into ribonucleoprotein particles (RNPs), 38{+/-}10 of which are contained in each virion. Their architecture has remained unclear due to the pleomorphism of RNPs, the high flexibility of N-protein intrinsically disordered regions, and highly multivalent interactions between viral RNA and N-protein binding sites in both N-terminal (NTD) and C-terminal domain (CTD). Here we explore critical interaction motifs of RNPs by applying a combination of biophysical techniques to mutant proteins binding different nucleic acids in an in vitro assay for RNP formation, and by examining mutant proteins in a viral assembly assay. We find that nucleic acid-bound N-protein dimers oligomerize via a recently described protein-protein interface presented by a transient helix in its long disordered linker region between NTD and CTD. The resulting hexameric complexes are stabilized by multi-valent protein-nucleic acid interactions that establish crosslinks between dimeric subunits. Assemblies are stabilized by the dimeric CTD of N-protein offering more than one binding site for stem-loop RNA. Our study suggests a model for RNP assembly where N- protein scaffolding at high density on viral RNA is followed by cooperative multimerization through protein-protein interactions in the disordered linker.

Dynamics of SARS-CoV-2 Seroprevalence in a Large US population Over a Period of 12 Months (preprint)

Due to a combination of asymptomatic or undiagnosed infections, the proportion of the United States population infected with SARS-CoV-2 was unclear from the beginning of the pandemic. We previously established a platform to screen for SARS-CoV-2 positivity across a representative proportion of the US population, from which we reported that almost 17 million Americans were estimated to have had undocumented infections in the Spring of 2020. Since then, vaccine rollout and prevalence of different SARS-CoV-2 variants have further altered seropositivity trends within the United States population. To explore the longitudinal impacts of the pandemic and vaccine responses on seropositivity, we re-enrolled participants from our baseline study in a 6- and 12- month follow-up study to develop a longitudinal antibody profile capable of representing seropositivity within the United States during a critical period just prior to and during the initiation of vaccine rollout. Initial measurements showed that, since July 2020, seropositivity elevated within this population from 4.8% at baseline to 36.2% and 89.3% at 6 and 12 months, respectively. We also evaluated nucleocapsid seropositivity and compared to spike seropositivity to identify trends in infection versus vaccination relative to baseline. These data serve as a window into a critical timeframe within the COVID-19 pandemic response and serve as a resource that could be used in subsequent respiratory illness outbreaks.

Camel nanobodies broadly neutralize SARS-CoV-2 variants (preprint)

With the emergence of SARS-CoV-2 variants, there is urgent need to develop broadly neutralizing antibodies. Here, we isolate two VHH nanobodies (7A3 and 8A2) from dromedary camels by phage display, which have high affinity for the receptor-binding domain (RBD) and broad neutralization activities against SARS-CoV-2 and its emerging variants. Cryo-EM complex structures reveal that 8A2 binds the RBD in its up mode and 7A3 inhibits receptor binding by uniquely targeting a highly conserved and deeply buried site in the spike regardless of the RBD conformational state. 7A3 at a dose of [≥]5 mg/kg efficiently protects K18-hACE2 transgenic mice from the lethal challenge of B.1.351 or B.1.617.2, suggesting that the nanobody has promising therapeutic potentials to curb the COVID-19 surge with emerging SARS-CoV-2 variants.

The humanized nanobody RBD-1-2G tolerates the spike N501Y mutation to neutralize SARS-CoV-2 (preprint)

Neutralizing antibodies targeting the SARS-CoV-2 spike protein have shown a great preventative/therapeutic potential. Here, we report a rapid and efficient strategy for the development and design of SARS-CoV-2 neutralizing humanized nanobody constructs with sub-nanomolar affinities and nanomolar potencies. CryoEM-based structural analysis of the nanobodies in complex with spike revealed two distinct binding modes. The most potent nanobody, RBD-1-2G(NCATS-BL8125), tolerates the N501Y RBD mutation and remains capable of neutralizing the B.1.1.7 (Alpha) variant. Molecular dynamics simulations provide a structural basis for understanding the neutralization process of nanobodies exclusively focused on the spike-ACE2 interface with and without the N501Y mutation on RBD. A primary human airway air-lung interface (ALI) ex vivo model showed that RBD-1-2G-Fc antibody treatment was effective at reducing viral burden following WA1 and B.1.1.7 SARS-CoV-2 infections. Therefore, this presented strategy will serve as a tool to mitigate the threat of emerging SARS-CoV-2 variants.

Pre-pandemic SARS-CoV-2 serological reactivity in rural malaria-experienced Cambodians (preprint)

Greater Mekong inhabitants are exposed to pathogens, zoonotic and otherwise, that may influence SARS-CoV-2 seroreactivity. A pre-pandemic (2005 to 2011) serosurvey of from 528 malaria-experienced Cambodians demonstrated higher-than-expected (up to 13.8 %) positivity of non-neutralizing IgG to SARS-CoV-2 spike and RBD antigens. These findings have implications for interpreting large-scale serosurveys. Article Summary LineIn the pre-COVID19 pandemic years of 2005 to 2011, malaria experienced Cambodians from rural settings had higher-than-expected seroreactivity to SARS-CoV-2 spike and receptor binding domain proteins.

SARS-CoV-2 Seroprevalence and Drug Use in Trauma Patients from Six Sites in the United States (preprint)

In comparison to the general patient population, trauma patients show higher level detections of bloodborne infectious diseases, such as Hepatitis and Human Immunodeficiency Virus. In comparison to bloodborne pathogens, the prevalence of respiratory infections such as SARS-CoV-2 and how that relates with other variables, such as drug usage and trauma type, is currently unknown in trauma populations. Here, we evaluated SARS-CoV-2 seropositivity and antibody isotype profile in 2,542 trauma patients from six Level-1 trauma centers between April and October of 2020 during the first wave of the COVID-19 pandemic. We found that the seroprevalence in trauma victims 18-44 years old (9.79%, 95% confidence interval/CI: 8.33 11.47) was much higher in comparison to older patients (45-69 years old: 6.03%, 4.59-5.88; 70+ years old: 4.33%, 2.54 - 7.20). Black/African American (9.54%, 7.77 - 11.65) and Hispanic/Latino patients (14.95%, 11.80 - 18.75) also had higher seroprevalence in comparison, respectively, to White (5.72%, 4.62 7.05) and Non-Latino patients (6.55%, 5.57 - 7.69). More than half (55.54%) of those tested for drug toxicology had at least one drug present in their system. Those that tested positive for narcotics or sedatives had a significant negative correlation with seropositivity, while those on anti-depressants trended positive. These findings represent an important consideration for both the patients and first responders that treat trauma patients facing potential risk of respiratory infectious diseases like SARS-CoV-2.

mRNA Vaccine-Induced Antibodies More Effective than Natural Immunity in Neutralizing SARS-CoV-2 and its High Affinity Variants (preprint)

Several variants of SARS-CoV-2 have emerged. Those with mutations in the angiotensin-converting enzyme (ACE2) receptor binding domain (RBD) are associated with increased transmission and severity. In this study, we developed both antibody quantification and functional assays. Analyses of both COVID-19 convalescent and diagnostic cohorts strongly support the use of RBD antibody levels as an excellent surrogate to biochemical neutralization activities. Data further revealed that the samples from mRNA vaccinated individuals had a median of 17 times higher RBD antibody levels and a similar degree of increased neutralization activities against RBD-ACE2 binding than those from natural infections. Our data showed that N501Y RBD had 5-fold higher ACE2 binding than the original variant. While antisera from naturally infected subjects had substantially reduced neutralization ability against N501Y RBD, all blood samples from vaccinated individuals were highly effective in neutralizing it. Thus, our data indicates that mRNA vaccination is far more effective than natural immunity in generating highly effective neutralizing antibodies. It further suggests a potential need to maintain high RBD antibody levels to control the more infectious SARS-CoV-2 variants.

Rapidly increasing SARS-CoV-2 seroprevalence and limited clinical disease in three Malian communities: a prospective cohort study (preprint)

Background The extent of SARS-CoV-2 exposure and transmission in Mali and the surrounding region is not well understood, although infection has been confirmed in nearly 14,000 symptomatic individuals and their contacts since the first case in March 2020. We aimed to estimate the cumulative incidence of SARS-CoV-2 in three Malian communities, and understand factors associated with infection. Methods Between 27 July 2020 and 29 January 2021, we collected blood samples along with demographic, social, medical and self-reported symptoms information from residents aged 6 months and older in three study communities at two study visits. SARS-CoV-2 antibodies were measured using a highly specific two-antigen ELISA optimized for use in Mali. We calculated cumulative adjusted seroprevalence for each site and evaluated factors associated with serostatus at each visit by univariate and multivariate analysis. Findings Overall, 94.8% (2533/2672) of participants completed both study visits. A total of 50.3% (1343/2672) of participants were male, and 31.3% (837/2672) were aged <10 years, 27.6% (737/2672) were aged 10-17 years, and 41.1% (1098/2572) were aged >/=18 years. The cumulative SARS-CoV-2 exposure rate was 58.5% (95% CI: 47.5 to 69.4). This varied between sites and was 73.4% (95% CI: 59.2 to 87.5) in the urban community of Sotuba, 53.2% (95% CI: 42.8 to 63.6) in the rural town of Bancoumana, and 37.1% (95% CI: 29.6 to 44.5) in the rural village of Doneguebougou. This equates to an infection rate of approximately 1% of the population every three days in the study communities between visits. Increased age and study site were associated with serostatus at both study visits. There was minimal difference in reported symptoms based on serostatus. Interpretation The true extent of SARS-CoV-2 exposure in Mali is greater than previously reported and now approaches hypothetical herd immunity in urban areas. The epidemiology of the pandemic in the region may be primarily subclinical and within background illness rates. In this setting, ongoing surveillance and augmentation of diagnostics to characterize locally circulating variants will be critical to implement effective mitigation strategies like vaccines. Funding This project was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, National Institute of Biomedical Imaging and Bioengineering, and National Cancer Institute.

SARS-CoV-2 seroassay optimization and performance in a population with high background reactivity in Mali (preprint)

ABSTRACT Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa. From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of an ELISA developed in the US, using two-antigen SARS-CoV-2 spike protein and receptor-binding domain. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative). Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43.4% (135/311) for spike protein, 22.8% (71/312) for RBD, and 33.9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73.9% (51.6-89.8), Specificity: 99.4% (97.7-99.9)]. In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.

SARS-CoV-2 Seroassay Optimization and Performance in a Population with High Background Reactivity in Mali (preprint)

Background: Serological tests are an indispensable tool to understand the epidemiology of the SARS-CoV-2 pandemic, particularly in areas where molecular diagnostics are limited. Poor assay performance may hinder the utility of these tests, including high rates of false-positivity previously reported in sub-Saharan Africa.Methods: From 312 Malian samples collected prior to 2020, we measured antibodies to the commonly tested SARS-CoV-2 antigens and four other betacoronaviruses by ELISA, and assessed functional cross-reactivity in a subset by SARS-CoV-2 pseudovirus neutralization assay. We then evaluated the performance of a two-antigen test developed in the US, using SARS-CoV-2 spike protein and receptor-binding domain ELISA measurements. To optimize test performance, we compared single and two-antigen approaches using existing assay cutoffs and population-specific cutoffs for Malian control samples (positive and negative).Findings: Background reactivity to SARS-CoV-2 antigens was common in pre-pandemic samples compared to US controls (43·4% (135/311) for spike protein, 22·8% (71/312) for RBD, and 33·9% (79/233) for nucleocapsid protein). SARS-CoV-2 reactivity correlated weakly with other betacoronavirus reactivity, varied between Malian communities, and increased with age. No pre-pandemic samples demonstrated functional activity. Regardless of the cutoffs applied, specificity improved using a two-antigen approach. Test performance was optimal using a two-antigen assay with population-specific cutoffs derived from ROC curve analysis [Sensitivity: 73·9% (51·6-89·8), Specificity: 99·4% (97·7-99·9)].Interpretation: In the setting of high background reactivity, such as sub-Saharan Africa, SARS-CoV-2 serological assays need careful qualification is to characterize the epidemiology of disease, prevent unnecessary harm, and allocate resources for targeted control measures.Funding: This project was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases, as well as the National Cancer Institute, National Institutes of Health.Declaration of Interests: The authors declare no conflicts of interest.Ethics Approval Statement: All pre-pandemic samples were collected during NIH-sponsored studies that were approved by the Malian USTTB FMPOS human research ethics committee and the NIAID/NIH institutional review board.Convalescent PCR-confirmed COVID-19 US positive control samples (n=10) were provided by the Adventist Hospital, Maryland. De-identified residual clinical samples for non-human subject research were obtained in accordance with 45 CFR 46.Convalescent samples were collected as part of a Public Health surveillance activity in collaboration with the Malian Ministry of Health COVID-19 Coordination Unit and with the approval of the USTTB FMOS-FAPH ethics committee (No2020/114/CE/FMOS/FAPH).

Towards Quantitative and Standardized Serological and Neutralization Assays for COVID-19 (preprint)

Quantitative and robust serology assays are critical measurements underpinning global COVID-19 response to diagnostic, surveillance, and vaccine development. Here we report the development of quantitative, multiplexed flow cytometry based serological and neutralization assays. The serology assays test the IgG and IgM against both the full-length spike antigens and the receptor binding domain (RBD) of the spike antigen. Benchmarking against an RBD-specific SARS-CoV IgG reference standard, the anti-SARS-CoV-2 RBD antibody titer was quantified in the range of 37.6 µg/mL to 31.0 ng/mL. The quantitative assays are highly specific with no correlative cross-reactivity with the spike proteins of MERS, SARS1, OC43 and HKU1 viruses. We further demonstrated good correlation between anti-RBD antibody titers and neutralizing antibody titers. The suite of serology and neutralization assays help to improve measurement confidence and are complementary and foundational for clinical and epidemiologic studies.Funding: The work was conducted as part of official duty of employee withfederal funds from the National Institute of Standards and Technology.Conflict of Interest: The authors declare no competing interests.Ethical Approval: The study was approved by the institutional research protection office of NIST.

Mapping a Pandemic: SARS-CoV-2 Seropositivity in the United States (preprint)

Asymptomatic SARS-CoV-2 infection and delayed implementation of diagnostics have led to poorly defined viral prevalence rates. To address this, we analyzed seropositivity in US adults who have not previously been diagnosed with COVID-19. Individuals with characteristics that reflect the US population (n = 11,382) and who had not previously been diagnosed with COVID-19 were selected by quota sampling from 241,424 volunteers (ClinicalTrials.gov NCT04334954). Enrolled participants provided medical, geographic, demographic, and socioeconomic information and 9,028 blood samples. The majority (88.7%) of samples were collected between May 10th and July 31st, 2020. Samples were analyzed via ELISA for anti-Spike and anti-RBD antibodies. Estimation of seroprevalence was performed by using a weighted analysis to reflect the US population. We detected an undiagnosed seropositivity rate of 4.6% (95% CI: 2.6 - 6.5%). There was distinct regional variability, with heightened seropositivity in locations of early outbreaks. Subgroup analysis demonstrated that the highest estimated undiagnosed seropositivity within groups was detected in younger participants (ages 18-45, 5.9%), females (5.5%), Black/African American (14.2%), Hispanic (6.1%), and Urban residents (5.3%), and lower undiagnosed seropositivity in those with chronic diseases. During the first wave of infection over the spring/summer of 2020 an estimate of 4.6% of adults had a prior undiagnosed SARS-CoV-2 infection. These data indicate that there were 4.8 (95% CI: 2.8-6.8) undiagnosed cases for every diagnosed case of COVID-19 during this same time period in the United States, and an estimated 16.8 million undiagnosed cases by mid-July 2020.

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii (preprint)

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas. RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated, lacking the amino terminus. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

SARS-CoV-2 binding and neutralizing antibody levels after vaccination with Ad26.COV2.S predict durable protection in rhesus macaques (preprint)

The first COVID-19 vaccines have recently gained authorization for emergency use.1,2 At this moment, limited knowledge on duration of immunity and efficacy of these vaccines is available. Data on other coronaviruses after natural infection suggest that immunity to SARS-CoV-2 might be short lived,3,4 and preliminary evidence indicates waning antibody titers following SARS-CoV-2 infection.5 Here we model the relationship between immunogenicity and protective efficacy of a series of Ad26 vectors encoding stabilized variants of the SARS-CoV-2 Spike (S) protein in rhesus macaques6,7,8 and validate the analyses by challenging macaques 6 months after immunization with the Ad26.COV2.S vaccine candidate that has been selected for clinical development. We find that Ad26.COV2.S confers durable protection against replication of SARS-CoV-2 in the lungs that is predicted by the levels of S-binding and neutralizing antibodies. These results suggest that Ad26.COV2.S could confer durable protection in humans and that immunological correlates of protection may enable the prediction of durability of protection.

A single dose of replication-competent VSV-vectored vaccine expressing SARS-CoV-2 S1 protects against virus replication in a hamster model of severe COVID-19 (preprint)

The development of effective countermeasures against Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the agent responsible for the COVID-19 pandemic, is a priority. We designed and produced ConVac, a replication-competent vesicular stomatitis virus (VSV) vaccine vector that expresses the S1 subunit of SARS-CoV-2 spike protein. We used golden Syrian hamsters as animal model of severe COVID-19 to test the efficacy of the ConVac vaccine. A single vaccine dose elicited high levels of SARS-CoV-2 specific binding and neutralizing antibodies; following intranasal challenge with SARS-CoV-2, animals were protected from weight loss and viral replication in the lungs. No enhanced pathology was observed in vaccinated animals upon challenge, but some inflammation was still detected. The data indicate rapid control of SARS-CoV-2 replication by the S1-based VSV-vectored SARS-CoV-2 ConVac vaccine.

Improved production of SARS-CoV-2 spike receptor-binding domain (RBD) for serology assays (preprint)

The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein is a commonly used antigen for serology assays critical to determining the extent of SARS-CoV-2 exposure in the population. Different versions of the RBD protein have been developed and utilized in assays, with higher sensitivity attributed to particular forms of the protein. To improve the yield of these high-sensitivity forms of RBD and support the increased demand for this antigen in serology assays, we investigated several protein expression variables including DNA elements such as promoters and signal peptides, cell culture expression parameters, and purification processes. Through this investigation, we developed a simplified and robust purification strategy that consistently resulted in high levels of the high-sensitivity form of RBD and demonstrated that a carboxyterminal tag is responsible for the increased sensitivity in the ELISA. These improved reagents and processes produce high-quality proteins which are functional in serology assays and can be used to investigate seropositivity to SARS-CoV-2 infection. Highlights: O_LIImproved yields of SARS-CoV-2 spike RBD through modification of DNA constructs and purification parameters C_LIO_LITwo versions of RBD show different sensitivity in serology assays C_LIO_LIYields of greater than 50 mg/l obtained under optimal conditions C_LIO_LIMagnetic bead purification technology improves throughput of protein production C_LI

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and antibody cross reactions (preprint)

Sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we developed and applied a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies that incorporates spike protein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human betacoronaviruses, HCoV-HKU1 and HCoV-OC43, that enables measurement of off-target pre-existing cross-reactive antibodies. The MMIA performances characteristics are: 98% sensitive and 100% specific for human subject samples collected as early as 10 days from symptom onset. The MMIA permitted the simultaneous identification of SARS-CoV-2 seroconversion and the induction of SARS-CoV-2 IgG antibody cross reactions to SARS-CoV-1 and MERS-CoV. Further, synchronous increases of HCoV-OC43 IgG antibody levels was detected with SARS-CoV-2 seroconversion in a subset of subjects for whom early infection sera were available prior to their SARS-CoV-2 seroconversion, suggestive of an HCoV-OC43 memory response triggered by SARS-CoV-2 infection.

A betacoronavirus multiplex microsphere immunoassay detects early SARS-CoV-2 seroconversion and controls for pre-existing seasonal human coronavirus antibody cross-reactivity (preprint)

With growing concern of persistent or multiple waves of SARS-CoV-2 in the United States, sensitive and specific SARS-CoV-2 antibody assays remain critical for community and hospital-based SARS-CoV-2 surveillance. Here, we describe the development and application of a multiplex microsphere-based immunoassay (MMIA) for COVD-19 antibody studies, utilizing serum samples from non-human primate SARS-CoV-2 infection models, an archived human sera bank and subjects enrolled at five U.S. military hospitals. The MMIA incorporates prefusion stabilized spike glycoprotein trimers of SARS-CoV-2, SARS-CoV-1, MERS-CoV, and the seasonal human coronaviruses HCoV-HKU1 and HCoV-OC43, into a multiplexing system that enables simultaneous measurement of off-target pre-existing cross-reactive antibodies. We report the sensitivity and specificity performances for this assay strategy at 98% sensitivity and 100% specificity for subject samples collected as early as 10 days after the onset of symptoms. In archival sera collected prior to 2019 and serum samples from subjects PCR negative for SARS-CoV-2, we detected seroprevalence of 72% and 98% for HCoV-HKU1 and HCoV-0C43, respectively. Requiring only 1.25 uL of sera, this approach permitted the simultaneous identification of SARS-CoV-2 seroconversion and polyclonal SARS-CoV-2 IgG antibody responses to SARS-CoV-1 and MERS-CoV, further demonstrating the presence of conserved epitopes in the spike glycoprotein of zoonotic betacoronaviruses. Application of this serology assay in observational studies with serum samples collected from subjects before and after SARS-CoV-2 infection will permit an investigation of the influences of HCoV-induced antibodies on COVID-19 clinical outcomes.

Antibody reactivity to SARS-CoV-2 in adults from the Vancouver metropolitan area, Canada (preprint)

Background: Quantifying antibody reactivity to SARS-CoV-2 antigens may help understand its effect on COVID-19 severity at the population level. This antibody reactivity may be particularly prevalent among childcare providers, including pediatric health care workers (HCW) who may be more exposed to circulating coronaviruses. Methods: Cross-sectional study that included adults in the Vancouver area in British Columbia (BC), Canada, between May 17 and June 19, 2020. A novel 10-plex antibody assay (IgG) was used to measure antibody reactivity against the spike protein from circulating coronaviruses (229E, NL63, OC43, and HKU1), SARS-CoV, and four SARS-CoV-2 antigens. Seroreactivity from previous viral exposure was ascertained using this assay, and by measuring total SARS-CoV-2 IgG/M/A antibodies against a recombinant spike (S1) protein using a commercial CLIA assay. Findings: Among 276 participants (71% HCW), three showed evidence of direct viral exposure, yielding an adjusted seroprevalence of 0.6% [95%CI 0.2 to 3.1%], with no difference between HCW and non-HCW, or between paediatric and adult HCW. Among the remaining 273 unexposed individuals, 7.3% [95%CI 4.5% to 11.1%], 48.7 [95%CI 42.7% to 54.8%] and 82.4% [95%CI 77.4% to 86.7%] showed antibody reactivity against SARS-CoV-2 RBD, N or Spike proteins, respectively. This reactivity was evenly distributed as a function of age, sex or between paediatric and adult HCW, and partly correlated with reactivity to circulating coronaviruses (Spearman; range: 0.147 to 0.513 for significant correlation after false-discovery rate adjustment at 5%). Interpretation: A substantial proportion of individuals in this population showed antibody reactivity against SARS-CoV-2 antigens despite low serological evidence of SARS-CoV-2 exposure.