Differential Adaptive Immune Responses Following SARS-CoV-2 Infection in Children Compared to Adults (preprint)

BackgroundSARS-CoV-2 infection elicits distinct clinical features in children and adults. Profiling the adaptive immune response following infection in children is essential to better understand and characterize these differences. MethodsHumoral and cell-mediated immune responses from unvaccinated pediatric and adult participants were analyzed following asymptomatic or mild non-Omicron SARS-CoV-2 infection. Levels of IgG and IgA targeting spike (S), receptor-binding domain (RBD), and nucleocapsid (N) proteins of SARS-CoV-2 were measured, while neutralizing antibody (nAb) titers were assessed against three viral strains (Wuhan, Omicron BA.1 and BA.4/BA.5). Specific T-cell memory responses were investigated by quantifying interferon-gamma (IFN-{gamma}) secreting cells after stimulation with ancestral and variant strains of SARS-CoV-2, and seasonal human {beta}- coronaviruses (HCoV)-OC43 and -HKU1. ResultsThe study comprised 28 children (3 to 17 [median=10] years old) and 28 adults (19 to 62 [median=42]). At a mean time of seven months ({+/-} 2.8 months) after SARS-CoV-2 infection, children and adults mounted comparable antibody levels against S and RBD, as well as similar neutralization capacity. However, children displayed a weaker cellular memory response to SARS- CoV-2 than adults, with a median of 88 [28-184] spot forming units per million of PBMCs in children compared to 208 [141-340] in adults (***, P < .001). In children, the level of IFN-{gamma} secreting cells in response to SARS-CoV-2 corresponds to that of seasonal coronaviruses. ConclusionLong-term memory T-cell responses to SARS-CoV-2 are enhanced in adults compared to children who demonstrate equivalent responses to SARS-CoV-2 and other HCoV. HIGHLIGHTSO_LIChildren infected with SARS-CoV-2 show comparable binding and neutralizing antibody levels as adults seven months after infection. C_LIO_LIThere are notable differences in the intensity of the T-cell response following SARS-CoV- 2 infection between children and adults. C_LIO_LIChildren have more pronounced T-cell immunodominance towards the spike versus non- spike proteins compared to adults at seven months post-infection C_LIO_LIIn contrast, T-cell responses to SARS-CoV-2 are globally reduced in children compared to adults but are alike to other seasonal {beta}-coronaviruses. C_LI

Longitudinal Study on Seroprevalence and Immune Response to SARS-CoV-2 in a Population of Food and Retail Workers Through Transformation of ELISA Datasets (preprint)

Since the onset of the global pandemic caused by the emergence and spread of SARS-CoV-2 in early 2020, numerous studies have been conducted worldwide to understand our immune response to the virus. This study investigates the humoral response elicited by vaccination and by SARS-CoV-2 infection in the poorly studied food and retail workers in the Quebec City area. The 1.5-year study period spans from early 2021, when vaccination became available in this region, to mid-2022, following waves of virulence due to the emergence of the first Omicron variants. Cross-correlated with data on workplace protective measures, pre-existing conditions, activities and other potentially relevant factors, this longitudinal study applies recently developed ELISA data transformation to our dataset to obtain normal distribution. This unlocked the possibility to use the ANOVA-Welsh method for statistical analysis to obtain a statistical perspective of the serological response. Our work allows the identification of factors contributing to statistically relevant differences in the humoral response of the cohort and strengthens the utility of the use of decentralized approaches to serological analysis.

Multi-variate statistical and machine learning reveals the interplay between sex and age in antibody responses to de novo SARS-CoV-2 infection and vaccination (preprint)

Prevention of negative COVID19 infection outcomes and infection/vaccine-acquired immunity is associated with the quality of antibody responses, whose variance by age and sex are poorly understood. Integrated, network approaches, identified sex and age effects in antibody responses and neutralization potential of de novo infection and vaccination throughout the Covid-19 pandemic. Cluster analysis found neutralization values followed SARS-CoV-2 specific receptor binding RIgG, spike SIgG and S and RIgA levels based on COVID19 status. Stochastic behavior tests and other analytical methods revealed sex differences only in persons <40y.o. Serum IgA antibody titers correlated with neutralization only in females 40-60y.o. Network analysis found males could improve IgA responses after vaccination dose 2, unlike >60 y.o. females. Complex correlation analyses found vaccination induced less antibody isotype switching and neutralization in older persons, especially in females. Sex dependent antibody & neutralization behavior decayed fastest in older males and with vaccination. Such sex and age characterization by machine learning can direct studies integrating cell mediated responses to define yet elusive correlates of protection and inform age and sex precision-focused vaccine design.

Seasonal Human Coronaviruses OC43, 229E, and NL63 Induce Cell Surface Modulation of Entry Receptors and Display Host Cell-Specific Viral Replication Kinetics (preprint)

The emergence of the COVID-19 pandemic prompted increased interest in seasonal human coronaviruses. 229E, OC43, NL63 and HKU1 are endemic seasonal coronaviruses that cause the common cold and are associated with generally mild respiratory symptoms. In this study, we identified cell lines that exhibited cytopathic effects (CPE) upon infection by three of these coronaviruses and characterized their viral replication kinetics and the effect of infection on host surface receptor expression. We found that NL63 produced CPE in LLC-MK2 cells, while OC43 produced CPE in MRC-5, HCT-8 and WI-38 cell lines, while 229E produced CPE in MRC-5 and WI-38 by day 3 post-infection. We observed a sharp increase in nucleocapsid and spike viral RNA (vRNA) from day 3 to day 5 post-infection for all viruses, however the abundance and the proportion of vRNAs copies measured in the supernatants and cell lysates of infected cells varied considerably depending on the virus-host cell pair. Importantly, we observed modulation of coronavirus entry and attachment receptors upon infection. Infection with 229E and OC43 led to a downregulation of CD13 and GD3, respectively. In contrast, infection with NL63, and also with OC43, lead to an increase in ACE2 expression. Attempts to block entry of NL63 using either soluble ACE2 or anti-ACE2 monoclonal antibodies demonstrated the potential of these strategies to greatly reduce infection. Overall, our results enable a better understanding of seasonal coronaviruses infection kinetics in permissive cell lines, and reveal entry receptor modulation that may have implications in facilitating co-infections with multiple coronaviruses in humans. IMPORTANCESeasonal human coronavirus are an important cause of the common cold associated with generally mild upper respiratory tract infections that can result in respiratory complications for some individuals. There are no vaccines available for these viruses, with only limited antiviral therapeutic options to treat the most severe cases. A better understanding of how these viruses interact with host cells is essential to identify new strategies to prevent infection-related complications. By analyzing viral replication kinetics in different permissive cell lines, we find that cell-dependent host factors influence how viral genes are expressed and virus particles released. We also analyzed entry receptor expression on infected cells and found that these can be up or down modulated depending on the infecting coronavirus. Our findings raise concerns over the possibility of infection enhancement upon co-infection by some coronaviruses, which may facilitate genetic recombination and the emergence of new variants and strains.

Serological markers and Post COVID-19 Condition (PCC): A rapid review of the evidence (preprint)

BackgroundPost COVID-19 Condition (PCC) is highly heterogeneous, often debilitating, and may last for years after infection. The etiology of PCC remains uncertain. Examination of potential serological markers of PCC, accounting for clinical covariates, may yield emergent pathophysiological insights. MethodsIn adherence to PRISMA guidelines, we carried out a rapid review of the literature. We searched Medline and Embase for primary observational studies that compared IgG response in individuals who experienced COVID-19 symptoms persisting [≥]12 weeks post-infection with those who did not. We examined relationships between serological markers and PCC status and investigated sources of inter-study variability, such as severity of acute illness, PCC symptoms assessed, and target antigen(s). ResultsOf 8,018 unique records, we identified 29 as being eligible for inclusion in synthesis. Definitions of PCC varied. In studies that reported anti-nucleocapsid (N) IgG (n=10 studies; n=989 participants in aggregate), full or partial anti-Spike IgG (i.e., the whole trimer, S1 or S2 subgroups, or receptor binding domain, n=19 studies; n=2606 participants), or neutralizing response (n=7 studies; n=1123 participants), we did not find strong evidence to support any difference in serological markers between groups with and without persisting symptoms. However, most studies did not account for severity or level of care required during acute illness, and other potential confounders. ConclusionsPooling of studies would enable more robust exploration of clinical and serological predictors among diverse populations. However, substantial inter-study variations hamper comparability. Standardized reporting practices would improve the quality, consistency, and comprehension of study findings.

Predictors of SARS-CoV-2 anti-Spike IgG antibody levels following two COVID-19 vaccine doses among children and adults in the Canadian CHILD Cohort (preprint)

Background: Vaccination helps prevent SARS-CoV-2 infection and severe COVID-19. However, vaccine-induced humoral immune responses vary among individuals and wane over time. We aimed to describe the SARS-CoV-2 anti-spike IgG antibody response to vaccination and identify health and demographic factors associated with this response among children and adults. Methods: We studied a subset of double-vaccinated children (n= 151; mean age: 12 {+/-}1.5 years, 46% female) and adults (n= 995; 44 {+/-}6.0 years, 60% female) from the Canadian CHILD Cohort. Dried blood spots were collected over two time periods (March 2021 to September 2021; October 2021 to January 2022). Antibody levels were quantified using automated chemiluminescent ELISAs. Demographic, vaccination, and health data were collected via online questionnaires. Associations were determined using multivariable regression. Results: Our cohort had SARS-CoV-2 anti-spike seropositivity rate of 97% following two COVID-19 vaccine doses. In both children and adults, the highest antibody levels were observed around three months post-vaccination and did not differ by biological sex. Higher antibody levels were associated with: prior SARS-CoV-2 infection ({beta}=0.15 scaled luminescence units, 95%CI, 0.06-0.24), age <18 years ({beta}=0.15, 95%CI 0.05-0.26) and receiving the Moderna mRNA ({beta}=0.23, 95%CI 0.11-0.34) or Pfizer-BioNTech mRNA vaccines ({beta}= 0.10, 95%CI, 0.02-0.18) vs. a combination of mRNA and Oxford-AstraZeneca viral vector vaccines. There were no differences in antibody levels when comparing a 3-8 vs. 9-16-week interval between vaccine doses. Interpretation: We identified key factors associated with post-vaccination antibody responses in children and adults, which could help improve future vaccine development and deployment among different population subgroups.

CLINICAL AND SEROLOGICAL PREDICTORS OF POST COVID-19 CONDITION: FINDINGS FROM A CANADIAN PROSPECTIVE COHORT STUDY (preprint)

Introduction: More than three years into the pandemic, there is persisting uncertainty as to the etiology, biomarkers, and risk factors of Post COVID-19 Condition (PCC). Serological research data remain a largely untapped resource. Few studies have investigated the potential relationships between post-acute serology and PCC, while accounting for clinical covariates. Methods: We compared clinical and serological predictors among COVID-19 survivors with (n=102 cases) and without (n=122 controls) persistent symptoms [≥] 12 weeks post-infection. We selected four primary serological predictors (anti-nucleocapsid (N), anti-Spike, and anti-receptor binding domain (RBD) IgG titres, and neutralization efficiency), and specified clinical covariates a priori. Results: Similar proportions of PCC-cases (66.7%, n=68) and infected-controls (71.3%, n=87) tested positive for anti-N IgG. More cases tested positive for anti-Spike (94.1%, n=96) and anti-RBD (95.1%, n=97) IgG, as compared with controls (anti-Spike: 89.3%, n=109; anti-RBD: 84.4%, n=103). Similar trends were observed among unvaccinated participants. Effects of IgG titres on PCC status were non-significant in univariate and multivariate analyses. Adjusting for age and sex, PCC-cases were more likely to be efficient neutralizers (OR 2.2, 95% CI 1.11 - 4.49), and odds was further increased among cases to report deterioration in quality of life (OR 3.4, 95% CI 1.64 - 7.31). Clinical covariates found to be significantly related to PCC included obesity (OR 2.3, p=0.02), number of months post COVID-19 (OR 1.1, p<0.01), allergies (OR 1.8, p=0.04), and need for medical support (OR 4.1, p<0.01). Conclusion: Despite past COVID-19 infection, approximately one third of PCC-cases and infected-controls were seronegative for anti-N IgG. Findings suggest higher neutralization efficiency among cases as compared with controls, and that this relationship is stronger among cases with more severe PCC. Cases also required more medical support for COVID-19 symptoms, and described complex, ongoing health sequelae. More data from larger cohorts are needed to substantiate results, permit subgroup analyses of IgG titres, and explore for differences between clusters of PCC symptoms. Future assessment of IgG subtypes may also elucidate new findings.

Investigating SARS-CoV-2 infection and the health and psychosocial impact of the COVID-19 pandemic in the Canadian CHILD Cohort study: methodology and cohort profile (preprint)

Background: The COVID-19 pandemic is affecting all Canadian families, with some impacted differently than others. Our study aims to: 1) determine the prevalence and transmission of SARS-CoV-2 infection among Canadian families, 2) identify predictors of infection susceptibility and severity of SARS-CoV-2 and 3) identify health and psychosocial impacts of the COVID-19 pandemic. Methods: This study builds upon the CHILD Cohort Study, an ongoing multi-ethnic general population prospective cohort consisting of 3454 Canadian families with children born in Vancouver, Edmonton, Manitoba, and Toronto between 2009-12. During the pandemic, 1462 CHILD households (5378 individuals) consented to participate in the CHILD COVID-19 Add-On Study involving: (1) brief biweekly surveys about COVID-19 symptoms and testing; (2) quarterly questionnaires assessing COVID-19 exposure, testing and vaccination status, physical and mental health, and pandemic-driven life changes; (3) in-home biological sampling kits to collect blood and stool. Mean ages were 9 years (range 0-17) for children and 43 years (range 18-85) for adults. Prevalence of SARS-CoV-2 infection will be estimated from survey data and confirmed through serology testing. We will combine these new data with a wealth of pre-pandemic CHILD data and use multivariate modelling and machine learning methods to identify risk and resilience factors for susceptibility and severity to the direct and indirect effects of the pandemic. Interpretation: Our short-term findings will inform key stakeholders and knowledge users to shape current and future pandemic responses. Additionally, this study provides a unique resource to study the long-term impacts of the pandemic as the CHILD Cohort Study continues.

Seasonal Respiratory Virus Circulation was Diminished during the COVID-19 Pandemic (preprint)

Background: Measures introduced during the COVID-19 pandemic intended to address the spread of SARS-CoV-2 may also influence the incidence of other common seasonal respiratory viruses (SRV). This evaluation reports laboratory confirmed cases of common SRV in a well-defined region of central Canada to address this issue. Methods: Surveillance data for common non-SARS-CoV-2 SRV in the Ottawa, Canada region was provided by the Eastern Ontario Regional Laboratory Association (EORLA) reference virology lab. Weekly reports of the number of positive tests and proportion that yielded positive results was analyzed from August 26, 2018 to January 2, 2022. Results: A drastic reduction in influenza and other common SRV was observed during the 2020-2021 influenza season in the Ottawa region. Influenza was virtually undetected post SARS-CoV-2 emergence. Rhinoviruses and enteroviruses were the only viruses that remained relatively unaffected during this period. Conclusions: We speculated that the introduction of non-pharmaceutical measures including masking to prevent SARS-CoV-2 transmission contributed to the near absence of SRV in the Ottawa region. These measures should remain a key component in addressing spikes in SRV activity and future pandemics.

Longitudinal profiles of plasma gelsolin, cytokines and antibody expression predict COVID-19 severity and hospitalization outcomes (preprint)

Background Prognostic markers for COVID-19 disease outcome are currently lacking. Plasma gelsolin (pGSN) is an actin-binding protein and an innate immune marker involved in disease pathogenesis and viral infections. Here, we demonstrate the utility of pGSN as a prognostic marker for COVID-19 disease outcome; a test performance that is significantly improved when combined with cytokines and antibodies compared to other conventional markers such as CRP and ferritin. Methods Blood samples were longitudinally collected from hospitalized COVID-19 patients as well as COVID-19 negative controls and the levels of pGSN in μg/mL, cytokines and anti-SARS-CoV-2 spike protein antibodies assayed. Mean±SEM values were correlated with clinical parameters to develop a prognostic platform. Results pGSN levels were significantly reduced in COVID-19 patients compared to healthy individuals. Additionally, pGSN levels combined with plasma IL-6, IP-10 and M-CSF significantly distinguished COVID-19 patients from healthy individuals. While pGSN and anti-spike IgG titers together strongly predict COVID-19 severity and death, the combination of pGSN and IL-6 was a significant predictor of milder disease and favorable outcomes. Conclusion Taken together, these findings suggest that multi-parameter analysis of pGSN, cytokines and antibodies could predict COVID-19 hospitalization outcomes with greater certainty compared with conventional clinical laboratory markers such as CRP and ferritin. This research will inform and improve clinical management and health system interventions in response to SARS-CoV-2 infection. Trial Registration N/A Funding The Ottawa Hospital Department of Medicine - Special Pandemic Agile Research Competition

What factors converged to create a COVID-19 hot-spot? Lessons from the South Asian community in Ontario. (preprint)

Background: South Asians represent the largest non-white ethnic group in Canada. The Greater Toronto Area (GTA), home to a high proportion of South Asians, emerged as a COVID-19 hot spot. Early in the pandemic, the South Asian community was identified as having risk factors for exposure and specific barriers to accessing testing and reliable health information, rendering them uniquely vulnerable to SARS-CoV-2 infection. Objectives: To investigate the burden of SARS-CoV-2 infection among South Asians in the GTA, and to determine which demographic characteristics were most closely aligned with seropositivity, in this cross-sectional analysis of a prospective cohort study. Methods: Participants from the GTA were enrolled between April and July 2021. Seropositivity for anti-spike and anti-nucleocapsid antibodies was determined from dried blood spots, and age and sex standardized to the Ontario South Asian population. Demographics, risk perceptions, and sources of COVID-19 information were collected via questionnaire in a subset. Results: Among the 916 South Asians enrolled, mean age 41 years, the age and sex standardized seropositivity was 23.6% (95% CI: 20.8%-26.4%). Approximately one-third identified as essential workers, and 19% reported living in a multi-generational household. Over half perceived high COVID-19 risk due to their geographic location, and 36% due to their type of employment. The top three most trusted sources of COVID-related information included healthcare providers/public health, traditional media sources, and social media. Conclusion: By the third wave of the COVID-19 pandemic, approximately one-quarter of a sample of South Asians in Ontario had serologic evidence of prior SARS-CoV-2 infection. Insight into factors that render certain populations at risk can help future pandemic planning and disease control efforts.

Arsenal of Nanobodies for Broad-Spectrum Countermeasures against Current and Future SARS-CoV-2 Variants of Concerns (preprint)

Nanobodies offer several potential advantages over mAbs for the control of SARS-CoV-2. Their ability to access cryptic epitopes conserved across SARS-CoV-2 variants of concern (VoCs) and feasibility to engineer modular, multimeric designs, make these antibody fragments ideal candidates for developing broad-spectrum therapeutics against current and continually emerging SARS-CoV-2 VoCs. Here we describe a diverse collection of 37 anti-SARS-CoV-2 spike glycoprotein nanobodies extensively characterized as both monovalent and IgG Fc-fused bivalent modalities. The panel of nanobodies were shown to have high intrinsic affinity; high thermal, thermodynamic and aerosolization stability; broad subunit/domain specificity and cross-reactivity across many VoCs; wide-ranging epitopic and mechanistic diversity; high and broad in vitro neutralization potencies; and high neutralization efficacies in hamster models of SARS-CoV-2 infection, reducing viral burden by up to six orders of magnitude to below detectable levels. In vivo protection was demonstrated with anti-RBD and previously unreported anti-NTD and anti-S2 nanobodies. This collection of nanobodies provides a therapeutic toolbox from which various cocktails or multi-paratopic formats could be built to tackle current and future SARS-CoV-2 variants and SARS-related viruses. Furthermore, the high aerosol-ability of nanobodies provides the option for effective needle-free delivery through inhalation.

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

BACKGROUND: Testing for antibodies 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 "Made-in-Canada" solution that can 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 a set of 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 the nucleocapsid (N) protein. These antigens are complemented by a detection antibody (human anti-IgG fused to horseradish peroxidase (HRP)) and a positive control reference antibody (recombinant IgG against the RBD), permitting intra- and inter-laboratory comparisons. Using this toolkit and commercial reagents, we optimized automated ELISAs on two different high throughput platforms to measure antibody responses to SARS-CoV-2 antigens. The assays were calibrated to a reference standard from the World Health Organization. We also automated a surrogate neutralization (sn)ELISA that measures inhibition of ACE2-Spike or -RBD interactions by antibodies using biotinylated ACE2. RESULTS: Our individual IgG-based ELISAs measure antibody levels in single-point measurements in reference to a standard antibody curve to accurately distinguish non-infected and infected individuals (area under the curve > 0.96 for each assay). Positivity thresholds can be established in individual assays using precision-recall analysis (e.g., by fixing the false positive rate), or more stringently, by scoring against the distribution of the means of negative samples across multiple assays performed over several months. For seroprevalence assessment (in a non-vaccinated cohort), classifying a sample as positive if antibodies were detected for at least 2 of the 3 antigens provided the highest specificity. In vaccinated cohorts, increases in anti-spike and -RBD (but not -N) antibodies are observed. Here, we present detailed protocols to perform these assays using either serum/plasma or dried blood spots both manually and on two automated platforms, and to express the results in international units to facilitate data harmonization and inter-study comparisons. We also demonstrate that the snELISA can be performed automatically at single points, increasing the scalability of this functional assay for large seroprevalence studies. INTERPRETATION: The ability to measure antibodies to three viral antigens and identify neutralizing antibodies capable of disrupting spike-ACE2 interactions in high-throughput assays enables large-scale analyses of humoral immune responses to SARS-CoV-2 infection and vaccination. The "Made-in-Canada" set of protein reagents, produced at the National Research Council of Canada are publicly available to enable the up-scaling of standardized serological assays, permitting nationwide data comparison and aggregation.

Plant-based production of SARS-CoV-2 antigens for use in a subunit vaccine (preprint)

The COVID-19 pandemic has brought to the forefront an urgent need for the rapid development of highly efficacious vaccines, particularly in light of the ongoing emergence of multiple variants of concern. Plant-based recombinant protein platforms are emerging as cost-effective and highly scalable alternatives to conventional protein production. Viral glycoproteins, however, are historically challenging to produce in plants. Herein, we report the production of plant-expressed wild-type glycosylated SARS-CoV-2 Spike RBD (receptor-binding domain) protein that is recognized by anti-RBD antibodies and exhibits high-affinity binding to the SARS-CoV-2 receptor ACE2 (angiotensin-converting enzyme 2). Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from naturally infected convalescent, vaccinated, or convalescent and vaccinated individuals. We further demonstrate that RBD binding to the ACE2 receptor was efficiently neutralized by antibodies from sera of SARS-CoV-2 convalescent and partially and fully vaccinated individuals. Collectively, these findings demonstrate that recombinant RBD produced in planta exhibits suitable biochemical and antigenic features for use in a subunit vaccine platform.

Real-World Serologic Responses to Extended-Interval and Heterologous COVID-19 mRNA Vaccination in Frail Elderly - Interim Report from a Prospective Observational Cohort Study (preprint)

Background: The Coronavirus disease 2019 (Covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted accelerated vaccines development. Their use was prioritized to protect the most vulnerable, notably, the elderly. Because of fluctuations in vaccine availability, strategies such as delayed second dose and heterologous prime-boost have been employed. The effectiveness of these strategies in the frail elderly are unknown. Methods: In this real-world vaccination study, under a government-decreed rationing strategy, elderly adults residing in long-term care facilities, with or without previously-documented SARS-CoV-2 infection, were administered homologous or heterologous mRNA vaccines, with an extended 16-week interval between doses. Clinical data and blood were serially collected during and after this interval period. Sera were tested for SARS-CoV-2-specific IgG antibodies (to trimeric S; RBD; nucleocapsid) by automated chemiluminescent ELISA. Findings: After a significant increase 4 weeks post-prime dose, there was a significant decline in anti-RBD and anti-S IgG levels until the boost dose, followed by an increase 4 weeks later. Previously uninfected individuals exhibited lower antibody responses up to 16 weeks post-prime dose, but achieved comparable levels to previously infected counterparts by 4 weeks post-second dose. Individuals primed with BNT162b2 exhibited larger decrease in anti-RBD and anti-S IgG levels with 16-week interval between doses, compared to those who received mRNA-1273. No differences in antibody levels 4 weeks after the second dose were noted between the two vaccines, in either homologous or heterologous combinations. Interpretations: These interim results of this ongoing longitudinal study show that, among frail elderly, neither age, sex, nor comorbidity affect antigenicity of mRNA-based COVID vaccines, but previous SARS-CoV-2 infection and type of mRNA vaccine influenced antibody responses when used with a 16-week interval between doses. Homologous/heterologous use of mRNA vaccines was not associated with significant differences in antibody responses 4 weeks following second dose, supporting their interchangeability.Funding: This project was supported by funding from the Public Health Agency of Canada, through the Vaccine Surveillance Reference group and the COVID-19 Immunity Task Force (CITF).Declaration of Interest: None to declare.Ethical Approval: This study was conducted in 12 LTC facilities of the Montréal Centre-Sud – Integrated University Health and Social Services Centre (CIUSSS du Centre-Sud-de-l'Île-de-Montréal) and was approved by its research ethics board (Comité d'éthique de la rechercheVieillissement-Neuroimagerie, protocol number 20-21-36 MP).

Real-world serologic responses to Extended-interval and Heterologous COVID-19 mRNA vaccination in Frail Elderly - Interim report from a prospective observational cohort study (preprint)

BackgroundThe Coronavirus disease 2019 (Covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has prompted accelerated vaccines development. Their use was prioritized to protect the most vulnerable, notably, the elderly. Because of fluctuations in vaccine availability, strategies such as delayed second dose and heterologous prime-boost have been employed. The effectiveness of these strategies in the frail elderly are unknown. MethodsIn this real-world vaccination study, under a government-decreed rationing strategy, elderly adults residing in long-term care facilities, with or without previously-documented SARS-CoV-2 infection, were administered homologous or heterologous mRNA vaccines, with an extended 16-week interval between doses. Clinical data and blood were serially collected during and after this interval period. Sera were tested for SARS-CoV-2-specific IgG antibodies (to trimeric S; RBD; nucleocapsid) by automated chemiluminescent ELISA. FindingsAfter a significant increase 4 weeks post-prime dose, there was a significant decline in anti-RBD and anti-S IgG levels until the boost dose, followed by an increase 4 weeks later. Previously uninfected individuals exhibited lower antibody responses up to 16 weeks post-prime dose, but achieved comparable levels to previously infected counterparts by 4 weeks post-second dose. Individuals primed with BNT162b2 exhibited larger decrease in anti-RBD and anti-S IgG levels with 16-week interval between doses, compared to those who received mRNA-1273. No differences in antibody levels 4 weeks after the second dose were noted between the two vaccines, in either homologous or heterologous combinations. InterpretationsThese interim results of this ongoing longitudinal study show that, among frail elderly, neither age, sex, nor comorbidity affect antigenicity of mRNA-based COVID vaccines, but previous SARS-CoV-2 infection and type of mRNA vaccine influenced antibody responses when used with a 16-week interval between doses. Homologous/heterologous use of mRNA vaccines was not associated with significant differences in antibody responses 4 weeks following second dose, supporting their interchangeability. FundingThis project was supported by funding from the Public Health Agency of Canada, through the Vaccine Surveillance Reference group and the COVID-19 Immunity Task Force (CITF).

Relative Ratios of Human Seasonal Coronavirus Antibodies Predict the Efficiency of Cross-Neutralization of SARS-CoV-2 Spike Binding to ACE2 (preprint)

Antibodies raised against highly prevalent human seasonal coronaviruses (sCoVs), which are responsible for the common cold, are known to cross-react with SARS-CoV-2 antigens. This cross-reactivity prompts questions about their protective role against SARS-CoV-2 infections and COVID-19 disease severity. However, the relationship between sCoV exposure and SARS-CoV-2 correlates of protection have not been clearly identified. Here we performed a cross-sectional analysis of cross-reactivity and cross-neutralization to three SARS-CoV-2 antigens using pre-pandemic serum from four different groups: pediatrics and adolescents (<21 yrs of age), persons 21 to 70 yrs of age, persons older than 70 yrs of age, and persons living with HCV or HIV. We find that antibody cross-reactivity to SARS-CoV-2 antigens varied between 1.6% and 15.3% depending on the cohort and the isotype-antigen pair analyzed. We also demonstrate a broad range of neutralizing activity (0-45%) in pre-pandemic serum that interferes with SARS-CoV-2 spike attachment to ACE2. While the abundance of sCoV antibodies did not directly correlate with neutralization efficiency, by using machine learning methodologies, we show that neutralizing activity is rather dependent on the latent variables related to the pattern ratios of sCoVs antibodies presented by each person. These were independent of age or sex, and could be accurately predicted by comparing the relative ratios of IgGs in sera directed to NL63, 229E, HKU-1, and OC43 spike proteins. More specifically, we identified antibodies to NL63 and OC43 as being the two most important predictors of latent variables responsible for protection, and 229E as being the least weighted. Our data support that exposure to sCoVs triggers various cellular and immune responses that influence the efficiency of SARS-CoV-2 spike binding to ACE2, and may impact COVID-19 disease severity through various other latent variables.

Dried blood spot specimens for SARS-CoV-2 antibody testing: A multi-site, multi-assay comparison (preprint)

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.

Meta-Analysis of the Dynamics of the Emergence of Mutations and Variants of SARS-CoV-2 (preprint)

The novel Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) emerged in late December 2019 in Wuhan, China, and is the causative agent for the worldwide COVID-19 pandemic. SARS-CoV-2 is a 29,811 nucleotides positive-sense single-stranded RNA virus belonging to the betacoronavirus genus. Due to inefficient proofreading ability of the viral RNA-dependent polymerase complex, coronaviruses are known to acquire new mutations following replication, which constitutes one of the main factors driving the evolution of its genome and the emergence of new genetic variants. In the last few months, the identification of new B.1.1.7 (UK), B.1.351 (South Africa) and P.1 (Brazil) variants of concern (VOC) highlighted the importance of tracking the emergence of mutations in the SARS-CoV-2 genome and their impact on transmissibility, infectivity, and neutralizing antibody escape capabilities. These VOC demonstrate increased transmissibility and antibody escape, and reduce current vaccine efficacy. Here we analyzed the appearance and prevalence trajectory of mutations that appeared in all SARS-CoV-2 genes from December 2019 to January 2021. Our goals were to identify which modifications are the most frequent, study the dynamics of their spread, their incorporation into the consensus sequence, and their impact on virus biology. We also analyzed the structural properties of the spike glycoprotein of the B.1.1.7, B.1.351 and P.1 variants. This study offers an integrative view of the emergence, disappearance, and consensus sequence integration of successful mutations that constitute new SARS-CoV-2 variants and their impact on neutralizing antibody therapeutics and vaccines.