The effect of dose-interval on antibody response to mRNA COVID-19 vaccines: a prospective cohort study.

Background: Vaccination against COVID-19 is highly effective in preventing severe disease and hospitalization, but primary COVID mRNA vaccination schedules often differed from those recommended by the manufacturers due to supply chain issues. We investigated the impact of delaying the second dose on antibody responses to COVID mRNA-vaccines in a prospective cohort of health-care workers in Quebec. Methods: We recruited participants from the McGill University Health Centre who provided serum or participant-collected dried blood samples (DBS) at 28-days, 3 months, and 6 months post-second dose and at 28-days after a third dose. IgG antibodies to SARS-CoV2 spike (S), the receptor-binding domain (RBD), nucleocapsid (N) and neutralizing antibodies to the ancestral strain were assessed by enzyme-linked immunosorbent assay (ELISA). We examined associations between long (≤89 days) versus short (<89 days) between-dose intervals and antibody response through multivariable mixed-effects models adjusted for age, sex, prior covid infection status, time since vaccine dose, and assay batch. Findings: The cohort included 328 participants who received up to three vaccine doses (>80% Pfizer-BioNTech). Weighted averages of the serum (n=744) and DBS (n=216) cohort results from the multivariable models showed that IgG anti-S was 31% higher (95% CI: 12% to 53%) and IgG anti-RBD was 37% higher (95% CI: 14% to 65%) in the long vs. short interval participants, across all time points. Interpretation: Our study indicates that extending the covid primary series between-dose interval beyond 89 days (approximately 3 months) provides stronger antibody responses than intervals less than 89 days. Our demonstration of a more robust antibody response with a longer between dose interval is reassuring as logistical and supply challenges are navigated in low-resource settings.

Continuous false positive results by SARS-CoV-2 rapid antigen testing: a case report.

Efficient and rapid identification of active SARS-CoV-2 infections has been key to monitoring and mitigating the spread of the virus. The implementation of nucleic acid testing (e.g., RT-PCR) was broadly adopted by most public health organizations at the national and community levels across the globe, which was followed by more accessible means of home testing including lateral flow immunochromatographic assay (LFA), also known as a rapid antigen test. Here we report the case of an adult female who repeatedly and consecutively tested positive by RAT (BTNX inc). This sustained false positive was not linked with an active SARS-CoV-2 infection, which was ruled out by RT-PCR and serological analyses. SARS-CoV-2 serology revealed no detectable levels of antibodies against the nucleocapsid suggesting no recent prior infection by SARS-CoV-2. This continuous false positive was limited to BTNX testing devices. This case report aims to describe that such continuous false positives can occur and describes alternative testing approaches that can be performed to confirm RAT results. In addition, broader awareness of such occurrences is warranted in the healthcare and public health community to avoid unnecessary negative impacts on individual's day to day life.

Results of the Stop the Spread Ottawa (SSO) cohort study: a Canadian urban-based prospective evaluation of antibody responses and neutralisation efficiency to SARS-CoV-2 infection and vaccination.

BACKGROUND: Predictors of COVID-19 vaccine immunogenicity and the influence of prior severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require elucidation. METHODS: Stop the Spread Ottawa is a prospective cohort of individuals at-risk for or who have been infected with SARS-CoV-2, initially enrolled for 10 months beginning October 2020. This cohort was enriched for public-facing workers. This analysis focuses on safety and immunogenicity of the initial two doses of COVID-19 vaccine. RESULTS: Post-vaccination data with blood specimens were available for 930 participants. 22.8% were SARS-CoV2 infected prior to the first vaccine dose. Cohort characteristics include: median age 44 (IQR: 22-56), 66.6% women, 89.0% white, 83.2% employed. 38.1% reported two or more comorbidities and 30.8% reported immune compromising condition(s). Over 95% had detectable IgG levels against the spike and receptor binding domain (RBD) 3 months post second vaccine dose. By multivariable analysis, increasing age and high-level immune compromise predicted diminishing IgG spike and RBD titres at month 3 post second dose. IgG spike and RBD titres were higher immediately post vaccination in those with SARS-CoV-2 infection prior to first vaccination and spike titres were higher at 6 months in those with wider time intervals between dose 1 and 2. IgG spike and RBD titres and neutralisation were generally similar by sex, weight and whether receiving homogeneous or heterogeneous combinations of vaccines. Common symptoms post dose 1 vaccine included fatigue (64.7%), injection site pain (47.5%), headache (27.2%), fever/chills (26.2%) and body aches (25.3%). These symptoms were similar with subsequent doses. CONCLUSION: The initial two COVID-19 vaccine doses are safe, well-tolerated and highly immunogenic across a broad spectrum of vaccine recipients including those working in public facing environments.

Cohort profile: investigating SARS-CoV-2 infection and the health and psychosocial impact of the COVID-19 pandemic in the Canadian CHILD Cohort.

The coronavirus disease 2019 (COVID-19) pandemic has affected all Canadian families, with some impacted differently than others. Our study aims to: (1) determine the prevalence and transmission of severe acute respiratory syndrome coronavirus 2 (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. This study builds upon the CHILD Cohort Study, an ongoing multi-ethnic general population prospective cohort consisting of 3,454 Canadian families with children born in Vancouver, Edmonton, Manitoba, and Toronto between 2009 and 2012. During the pandemic, CHILD households were invited 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 and testing, vaccination status, physical and mental health, and pandemic-driven life changes; and (3) in-home biological sampling kits to collect blood and stool. In total, 1,462 households (5,378 participants) consented to the CHILD COVID-19 Add-On Study: 2,803 children (mean±standard deviation [SD], 9.0±2.7 years; range, 0-17 years) and 2,576 adults (mean±SD, 43.0±6.5 years; range, 18-85 years). We will leverage the wealth of pre-pandemic CHILD data to identify risk and resilience factors for susceptibility and severity to the direct and indirect pandemic effects. 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.

Antibody neutralization capacity after coronavirus disease 2019 vaccination in people with HIV in Canada.

OBJECTIVES: Many vaccines require higher/additional doses or adjuvants to provide adequate protection for people with HIV (PWH). Here, we compare coronavirus disease 2019 (COVID-19) vaccine-induced antibody neutralization capacity in PWH vs. HIV-negative individuals following two vaccine doses. DESIGN: In Canadian prospective observational cohorts, including a multicentre study of PWH receiving at least two COVID-19 vaccinations (mRNA or ChAdOx1-S), and a parallel study of HIV-negative controls (Stop the Spread Ottawa Cohort), we measured vaccine-induced neutralization capacity 3 months post dose 2 (±1 month). METHODS: COVID-19 neutralization efficiency was measured by calculating the half maximal inhibitory dilution (ID50) using a high-throughput protein-based neutralization assay for Ancestral (Wuhan), Delta and Omicron (BA.1) spike variants. Univariable and multivariable quantile regression were used to compare COVID-19-specific antibody neutralization capacity by HIV status. RESULTS: Neutralization assays were performed on 256 PWH and 256 controls based on specimen availability at the timepoint of interest, having received two vaccines and known date of vaccination. There was a significant interaction between HIV status and previous COVID-19 infection status in median ID50. There were no differences in median ID50 for HIV+ vs. HIV-negative persons without past COVID-19 infection. For participants with past COVID-19 infection, median ICD50 was significantly higher in controls than in PWH for ancestral SARS-CoV-2 and Omicron variants, with a trend for the Delta variant in the same direction. CONCLUSION: Vaccine-induced SARS-CoV-2 neutralization capacity was similar between PWH vs. HIV-negative persons without past COVID-19 infection, demonstrating favourable humoral-mediated immunogenicity. Both HIV+ and HIV-negative persons demonstrated hybrid immunity. TRIAL REGISTRATION: clinicaltrials.gov NCT04894448.

Cross-sectional Characterization of SARS-CoV-2 Antibody Levels and Decay Rates Following Infection of Unvaccinated Elderly Individuals.

Background: SARS-CoV-2 infections have disproportionally burdened elderly populations with excessive mortality. While several contributing factors exists, questions remain about the quality and duration of humoral antibody-mediated responses resulting from infections in unvaccinated elderly individuals. Methods: Residual serum/plasma samples were collected from individuals undergoing routine SARS-CoV-2 polymerase chain reaction testing in a community laboratory in Canada. The samples were collected in 2020, before vaccines became available. IgG, IgA, and IgM antibodies against SARS-CoV-2 nucleocapsid, trimeric spike, and its receptor-binding domain were quantified via a high-throughput chemiluminescent enzyme-linked immunosorbent assay. Neutralization efficiency was also quantified through a surrogate high-throughput protein-based neutralization assay. Results: This study analyzed SARS-CoV-2 antibody levels in a large cross-sectional cohort (N = 739), enriched for elderly individuals (median age, 82 years; 75% >65 years old), where 72% of samples tested positive for SARS-CoV-2 by polymerase chain reaction. The age group ≥90 years had higher levels of antibodies than that <65 years. Neutralization efficiency showed an age-dependent trend, where older persons had higher levels of neutralizing antibodies. Antibodies targeting the nucleocapsid had the fastest decline. IgG antibodies targeting the receptor-binding domain remained stable over time, potentially explaining the lack of neutralization decay observed in this cohort. Conclusions: Despite older individuals having the highest levels of antibodies postinfection, they are the cohort in which antibody decay was the fastest. Until a better understanding of correlates of protection is acquired, along with the protective role of nonneutralizing antibodies, booster vaccinations remain important in this demographic.

COVID-19 vaccine immunogenicity in people with HIV.

OBJECTIVES: Many vaccines require higher/additional doses or adjuvants to provide adequate protection for people with HIV (PWH). Our objective was to compare COVID-19 vaccine immunogenicity in PWH to HIV-negative individuals. DESIGN: In a Canadian multi-center prospective, observational cohort of PWH receiving at least two COVID-19 vaccinations, we measured vaccine-induced immunity at 3 and 6 months post 2nd and 1-month post 3rd doses. METHODS: The primary outcome was the percentage of PWH mounting vaccine-induced immunity [co-positivity for anti-IgG against SARS-CoV2 Spike(S) and receptor-binding domain proteins] 6 months post 2nd dose. Univariable and multivariable logistic regressions were used to compare COVID-19-specific immune responses between groups and within subgroups. RESULTS: Data from 294 PWH and 267 controls were analyzed. Immunogenicity was achieved in over 90% at each time point in both groups. The proportions of participants achieving comparable anti-receptor-binding domain levels were similar between the group at each time point. Anti-S IgG levels were similar by group at month 3 post 2nd dose and 1-month post 3rd dose. A lower proportion of PWH vs. controls maintained vaccine-induced anti-S IgG immunity 6 months post 2nd dose [92% vs. 99%; odds ratio: 0.14 (95% confidence interval: 0.03, 0.80; P = 0.027)]. In multivariable analyses, neither age, immune non-response, multimorbidity, sex, vaccine type, or timing between doses were associated with reduced IgG response. CONCLUSION: Vaccine-induced IgG was elicited in the vast majority of PWH and was overall similar between groups. A slightly lower proportion of PWH vs. controls maintained vaccine-induced anti-S IgG immunity 6 months post 2nd dose demonstrating the importance of timely boosting in this population.

Seasonal respiratory virus circulation was diminished during the COVID-19 pandemic.

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 Ottawa, Canada, was provided by the Eastern Ontario Regional Laboratory Association (EORLA) reference virology lab. Weekly reports of the number of positive tests and the proportion that yielded positive results were 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 nonpharmaceutical 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.

Scalable agroinfiltration-based production of SARS-CoV-2 antigens for use in diagnostic assays and subunit vaccines.

Agroinfiltration is a method used in biopharming to support plant-based biosynthesis of therapeutic proteins such as antibodies and viral antigens involved in vaccines. Major advantages of generating proteins in plants is the low cost, massive scalability and the rapid yield of the technology. Herein, we report the agroinfiltration-based production of glycosylated SARS-CoV-2 Spike receptor-binding domain (RBD) protein. We show that it exhibits high-affinity binding to the SARS-CoV-2 receptor angiotensin-converting enzyme 2 (ACE2) and displays folding similar to antigen produced in mammalian expression systems. Moreover, our plant-expressed RBD was readily detected by IgM, IgA, and IgG antibodies from the serum of SARS-CoV-2 infected and vaccinated individuals. We further demonstrate that binding of plant-expressed RBD to ACE2 is efficiently neutralized by these antibodies. Collectively, these findings demonstrate that recombinant RBD produced via agroinfiltration exhibits suitable biochemical and antigenic features for use in serological and neutralization assays, and in subunit vaccine platforms.

SARS-CoV-2 Seroprevalence in Those Utilizing Public Transportation or Working in the Transportation Industry: A Rapid Review.

Proximity and duration of social contact while working or using public transportation may increase users' risk of SARS-CoV-2 exposure. This review aims to assess evidence of an association between use of public transportation or work in the transportation industry and prevalence of SARS-CoV-2 antibodies as well as to identify factors associated with seropositivity in transit users. A literature search of major databases was conducted from December 2019 to January 2022 using key worlds including "seroprevalence", "SARS-CoV-2", and "public transit". A narrative review of included studies was completed for the following categories: those working in the transportation industry, healthcare workers relying on public transit, and population-based studies. The association between work in the transit industry and seroprevalence varied based on location, demographic characteristics, and test sensitivities. No association was found in healthcare workers. Several population-based studies indicated higher seroprevalence in those using public transit. Overall seroprevalence estimates varied based on geographic location, population demographics, study methodologies, and calendar date of assessment. However, seropositivity was consistently higher in racial minorities and low-income communities.

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

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.

Comparative performance data for multiplex SARS-CoV-2 serological assays from a large panel of dried blood spot specimens.

The extent of the COVID-19 pandemic will be better understood through serosurveys and SARS-CoV-2 antibody testing. Dried blood spot (DBS) samples will play a central role in large scale serosurveillance by simplifying biological specimen collection and transportation, especially in Canada. Direct comparative performance data on multiplex SARS-CoV-2 assays resulting from identical DBS samples are currently lacking. In our study, we aimed to provide performance data for the BioPlex 2200 SARS-CoV-2 IgG (Bio-Rad), V-PLEX SARS-CoV-2 Panel 2 IgG (MSD), and Elecsys Anti-SARS-CoV-2 (Roche) commercial assays, as well as for two highly scalable in-house assays (University of Ottawa and Mount Sinai Hospital protocols) to assess their suitability for DBS-based SARS-CoV-2 DBS serosurveillance. These assays were evaluated against identical panels of DBS samples collected from convalescent COVID-19 patients (n = 97) and individuals undergoing routine sexually transmitted and bloodborne infection (STBBI) testing prior to the COVID-19 pandemic (n = 90). Our findings suggest that several assays are suitable for serosurveillance (sensitivity >97% and specificity >98%). In contrast to other reports, we did not observe an improvement in performance using multiple antigen consensus-based rules to establish overall seropositivity. This may be due to our DBS panel which consisted of samples collected from convalescent COVID-19 patients with significant anti-spike, -receptor binding domain (RBD), and -nucleocapsid antibody titers. This study demonstrates that biological specimens collected as DBS coupled with one of several readily available assays are useful for large-scale COVID-19 serosurveillance.

Homogeneous surrogate virus neutralization assay to rapidly assess neutralization activity of anti-SARS-CoV-2 antibodies.

The COVID-19 pandemic triggered the development of numerous diagnostic tools to monitor infection and to determine immune response. Although assays to measure binding antibodies against SARS-CoV-2 are widely available, more specific tests measuring neutralization activities of antibodies are immediately needed to quantify the extent and duration of protection that results from infection or vaccination. We previously developed a 'Serological Assay based on a Tri-part split-NanoLuc® (SATiN)' to detect antibodies that bind to the spike (S) protein of SARS-CoV-2. Here, we expand on our previous work and describe a reconfigured version of the SATiN assay, called Neutralization SATiN (Neu-SATiN), which measures neutralization activity of antibodies directly from convalescent or vaccinated sera. The results obtained with our assay and other neutralization assays are comparable but with significantly shorter preparation and run time for Neu-SATiN. As the assay is modular, we further demonstrate that Neu-SATiN enables rapid assessment of the effectiveness of vaccines and level of protection against existing SARS-CoV-2 variants of concern and can therefore be readily adapted for emerging variants.

Prevalence of SARS-CoV-2 infection among obstetric patients in Ottawa, Canada: a descriptive study.

BACKGROUND: There is limited information on the prevalence of SARS-CoV-2 infection in obstetric settings in Canada, beyond the first wave of the COVID-19 pandemic (February to June 2020). We sought to describe the prevalence of SARS-CoV-2 infection in pregnant people admitted to triage units at a tertiary care hospital in Ottawa, Canada. METHODS: We conducted a descriptive study of pregnant people admitted to obstetric triage assessment units at The Ottawa Hospital between Oct. 19 and Nov. 27, 2020 (second local wave of the COVID-19 pandemic). Participants underwent SARS-CoV-2 polymerase chain reaction (PCR) (via naso- or oropharyngeal swabs) and serology testing upon admission. We excluded individuals younger than 18 years, those who did not speak English or French, those who enrolled in conflicting studies, those admitted for pregnancy termination and those triaged between 11:31 pm and 7:29 am. Swab and serology samples were analyzed using digital droplet PCR and enzyme-linked immunosorbent assays, respectively. We defined SARS-CoV-2 seropositivity as a positive result for immunoglobulin (Ig) G, either alone or in combination with IgM or IgA. RESULTS: Of the 632 eligible patients, 363 (57.4%) consented to participation and 362 collectively provided 284 swab and 352 blood samples eligible for analysis. Common reasons for declining participation included feeling overwhelmed or anxious, being worried about repercussions of testing, pain or discomfort with testing or disinterest in research. Participants were mostly multiparous (53.9%) and in their third trimester upon admission (88.4%). In all, 18 (4.9%) participants had evidence of SARS-CoV-2 exposure; 2 (0.7%) of 284 were positive for SARS-CoV-2 by PCR and 16 (4.5%) of 352 were positive for IgG antibodies to SARS-CoV-2. INTERPRETATION: During the second local wave of the COVID-19 pandemic, the prevalence of active SARS-CoV-2 infection among obstetric patients in Ottawa was 0.7% and seroprevalence was 4.5%. Our low participation rate highlights the need for improvements in patient education and public health messaging on the benefits of SARS-CoV-2 testing programs.

Antibody Seronegativity in COVID-19 RT-PCR-Positive Children.

This substudy of a prospective case-ascertained household transmission study investigated severe acute respiratory syndrome coronavirus 2 reverse transcription polymerase chain reaction-positive individuals without antibody development and factors associated with nonseroconversion. Approximately 1 of 8 individuals with coronavirus disease 2019 did not seroconvert. Children, particularly the youngest, were approximately half as likely to seroconvert compared with adults. Apart from the absence of fever/chills, individual symptoms did not strongly predict nonseroconversion.

Household transmission of SARS-CoV-2 from unvaccinated asymptomatic and symptomatic household members with confirmed SARS-CoV-2 infection: an antibody-surveillance study.

BACKGROUND: Household transmission contributes to SARS-CoV-2 spread, but the role of children in transmission is unclear. We conducted a study that included symptomatic and asymptomatic children and adults exposed to SARS-CoV-2 in their households with the objective of determining how SARS-CoV-2 is transmitted within households. METHODS: In this case-ascertained antibody-surveillance study, we enrolled households in Ottawa, Ontario, in which at least 1 household member had tested positive for SARS-CoV-2 on reverse transcription polymerase chain reaction testing. The enrolment period was September 2020 to March 2021. Potentially eligible participants were identified if they had tested positive for SARS-CoV-2 at an academic emergency department or affiliated testing centre; people who learned about the study through the media could also self-identify for participation. At least 2 participants were required for a household to be eligible for study participation, and at least 1 enrolled participant per household had to be a child (age < 18 yr). Enzyme-linked immunosorbent assays were used to evaluate SARS-CoV-2-specific IgA, IgM and IgG against the spike-trimer and nucleocapsid protein. The primary outcome was household secondary attack rate, defined as the proportion of household contacts positive for SARS-CoV-2 antibody among the total number of household contacts participating in the study. We performed descriptive statistics at both the individual and household levels. To estimate and compare outcomes between patient subgroups, and to examine predictors of household transmission, we fitted a series of multivariable logistic regression with robust standard errors to account for clustering of individuals within households. RESULTS: We enrolled 695 participants from 180 households: 180 index participants (74 children, 106 adults) and 515 of their household contacts (266 children, 249 adults). A total of 487 household contacts (94.6%) (246 children, 241 adults) had SARS-CoV-2 antibody testing, of whom 239 had a positive result (secondary attack rate 49.1%, 95% confidence interval [CI] 42.9%-55.3%). Eighty-eight (36.8%, 95% CI 29.3%-43.2%) of the 239 were asymptomatic; asymptomatic rates were similar for children (51/130 [39.2%, 95% CI 30.7%-48.5%]) and adults (37/115 [32.2%, 95% CI 24.2%-41.4%]) (odds ratio [OR] 1.3, 95% CI 0.8-2.1). Adults were more likely than children to transmit SARS-CoV-2 (OR 2.2, 95% CI 1.3-3.6). The odds of transmission from asymptomatic (OR 0.6, 95% CI 0.2-1.4) versus symptomatic (OR 0.9, 95% CI 0.6-1.4) index participants to household contacts was uncertain. Predictors of household transmission included household density (number of people per bedroom), relationship to index participant and number of cases in the household. INTERPRETATION: The rate of SARS-CoV-2 transmission within households was nearly 50% during the study period, and children were an important source of spread. The findings suggest that children are an important driver of the COVID-19 pandemic; this should inform public health policy.

Platelet activation by SARS-CoV-2 implicates the release of active tissue factor by infected cells.

Platelets are hyperactivated in coronavirus disease 2019 (COVID-19). However, the mechanisms promoting platelet activation by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are not well understood. This may be due to inherent challenges in discriminating the contribution of viral vs host components produced by infected cells. This is particularly true for enveloped viruses and extracellular vesicles (EVs), 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 αIIbß3, degranulation, aggregation under flow conditions, and the release of EVs. We find that neither SARS-CoV-2 nor purified spike activates platelets. In contrast, tissue factor (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 EVs were identified by electron microscopy. Through size-exclusion chromatography, TF activity was found to be associated with a virus or EVs, which were indistinguishable. Increased TF messenger RNA (mRNA) expression and activity were also found in lungs in a murine model of COVID-19 and 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 with platelet activation and the coagulation characteristic of COVID-19.

A scalable serology solution for profiling humoral immune responses to SARS-CoV-2 infection and vaccination.

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.

Cohort profile: Stop the Spread Ottawa (SSO)-a community-based prospective cohort study on antibody responses, antibody neutralisation efficiency and cellular immunity to SARS-CoV-2 infection and vaccination.

PURPOSE: To investigate the robustness and longevity of SARS-CoV-2 immune responses conferred by natural infection and vaccination among priority populations such as immunocompromised individuals and people with post-acute sequelae of COVID-19 in a prospective cohort study (Stop the Spread Ottawa-SSO) in adults living in the Ottawa region. In this paper, we describe the study design, ongoing data collection and baseline characteristics of participants. PARTICIPANTS: Since October 2020, participants who tested positive for COVID-19 (convalescents) or at high risk of exposure to the virus (under surveillance) have provided monthly blood and saliva samples over a 10-month period. As of 2 November 2021, 1026 adults had completed the baseline survey and 976 had attended baseline bloodwork. 300 participants will continue to provide bimonthly blood samples for 24 additional months (ie, total follow-up of 34 months). FINDINGS TO DATE: The median age of the baseline sample was 44 (IQR 23, range: 18-79) and just over two-thirds (n=688; 67.1%) were female. 255 participants (24.9%) had a history of COVID-19 infection confirmed by PCR and/or serology. Over 600 participants (60.0%) work in high-risk occupations (eg, healthcare, teaching and transportation). 108 participants (10.5%) reported immunocompromising conditions or treatments at baseline (eg, cancer, HIV, other immune deficiency, and/or use of immunosuppressants). FUTURE PLANS: SSO continues to yield rich research potential, given the collection of pre-vaccine baseline data and samples from the majority of participants, recruitment of diverse subgroups of interest, and a high level of participant retention and compliance with monthly sampling. The 24-month study extension will maximise opportunities to track SARS-CoV-2 immunity and vaccine efficacy, detect and characterise emerging variants, and compare subgroup humoral and cellular response robustness and persistence.

Characterization of Systemic and Mucosal Humoral Immune Responses to an Adjuvanted Intranasal SARS-CoV-2 Protein Subunit Vaccine Candidate in Mice.

Continuous viral evolution of SARS-CoV-2 has resulted in variants capable of immune evasion, vaccine breakthrough infections and increased transmissibility. New vaccines that invoke mucosal immunity may provide a solution to reducing virus transmission. Here, we evaluated the immunogenicity of intranasally administered subunit protein vaccines composed of a stabilized SARS-CoV-2 spike trimer or the receptor binding domain (RBD) adjuvanted with either cholera toxin (CT) or an archaeal lipid mucosal adjuvant (AMVAD). We show robust induction of immunoglobulin (Ig) G and IgA responses in plasma, nasal wash and bronchoalveolar lavage in mice only when adjuvant is used in the vaccine formulation. While the AMVAD adjuvant was more effective at inducing systemic antibodies against the RBD antigen than CT, CT was generally more effective at inducing overall higher IgA and IgG titers against the spike antigen in both systemic and mucosal compartments. Furthermore, vaccination with adjuvanted spike led to superior mucosal IgA responses than with the RBD antigen and produced broadly targeting neutralizing plasma antibodies against ancestral, Delta and Omicron variants in vitro; whereas adjuvanted RBD elicited a narrower antibody response with neutralizing activity only against ancestral and Delta variants. Our study demonstrates that intranasal administration of an adjuvanted protein subunit vaccine in immunologically naïve mice induced both systemic and mucosal neutralizing antibody responses that were most effective at neutralizing SARS-CoV-2 variants when the trimeric spike was used as an antigen compared to RBD.