Viral interference between severe acute respiratory syndrome coronavirus 2 and influenza A viruses (preprint)

Some respiratory viruses can cause a viral interference through the activation of the interferon (IFN) pathway that reduces the replication of another virus. Epidemiological studies of coinfections between SARS-CoV-2 and other respiratory viruses have been hampered by non-pharmaceutical measures applied to mitigate the spread of SARS-CoV-2 during the COVID-19 pandemic. With the ease of these interventions, SARS-CoV-2 and influenza A viruses can now co-circulate. It is thus of prime importance to characterize their interactions. In this work, we investigated viral interference effects between an Omicron variant and a contemporary influenza A/H3N2 strain, in comparison with an ancestral SARS-CoV-2 strain and the 2009 pandemic influenza A/H1N1 virus. We infected nasal human airway epitheliums with SARS-CoV-2 and influenza, either simultaneously or 24 h apart. Viral load was measured by RT-qPCR and IFN-/{beta}/{lambda}1/{lambda}2 proteins were quantified by immunoassay. Expression of four interferon-stimulated genes (ISGs; OAS1/IFITM3/ISG15/MxA) was also measured by RT-droplet digital PCR. Additionally, susceptibility of each virus to IFN-/{beta}/{lambda}2 recombinant proteins was determined. Our results showed that influenza A, and especially A/H3N2, interfered with both SARS-CoV-2 viruses, but that SARS-CoV-2 only interfered with A/H1N1. Consistently with these results, influenza, and particularly the A/H3N2 strain, caused a higher production of IFN proteins and expression of ISGs than SARS-CoV-2. The IFN production induced by SARS-CoV-2 was marginal and its presence during coinfections with influenza was associated with a reduced IFN response. All viruses were susceptible to exogenous IFNs, with the ancestral SARS-CoV-2 and Omicron being less susceptible to type I and type III IFNs, respectively. Thus, influenza A causes a viral interference towards SARS-CoV-2 most likely through an IFN response. The opposite is not necessarily true, and a concurrent infection with both viruses leads to a lower IFN response. Taken together, these results help us to understand how SARS-CoV-2 interacts with another major respiratory pathogen.

Seroprevalence, seroconversion, and seroreversion of infection-induced SARS-CoV-2 antibodies among a cohort of children and adolescents in Montreal, Canada (preprint)

Importance: Repeated serological testing for SARS-CoV-2 allows the monitoring of antibody dynamics in populations, including detecting infections that are missed by RT-PCR or antigen testing. Understanding the factors associated with seroconversion and seroreversion as well as the duration of infection-induced antibodies can also inform public health recommendations regarding disease prevention and mitigation efforts. Objective: To use serological testing to assess the prevalence, seroconversion, and seroreversion of infection-induced SARS-CoV-2 antibodies in children and adolescents in Montreal, Canada. Design: This analysis reports on three rounds of data collection from a prospective cohort study (Enfants et COVID-19: Etude de seroprevalence [EnCORE]). The study rounds occurred as follows: Round 1 October 2020-March 2021, Round 2 May to July 2021, and Round 3 November 2021 to January 2022. Most Round 3 samples were collected prior to the spread of the Omicron BA.1 variant in Quebec. Setting: Population-based sample. Participants: Children and adolescents aged 2 to 17 years in Montreal, Canada. Exposure: Potential exposure to SARS-CoV-2. Main Outcomes and Measures: Participants provided dried blood spots (DBS) for antibody detection and parents completed online questionnaires for sociodemographics and COVID-19 symptoms and testing history. The serostatus of participants was determined by enzyme-linked immunosorbent assays (ELISAs) using the receptor-binding domain (RBD) from the spike protein and the nucleocapsid protein (N) as antigens. We estimated seroprevalence for each round of data collection and by participant and household characteristics. Seroconversion rates were calculated as were the likelihoods of remaining seropositive at six months and one year. Results: The study included DBS samples from 1 632, 936, and 723 participants in the first, second, and third rounds of data collection, respectively. The baseline seroprevalence was 5.8% (95% CI 4.8-7.1), which increased to 10.5% and 10.9% for the respective follow-ups (95% CI 8.6-12.7; 95% CI 8.8-13.5). The overall average crude rate of seroconversion over the study period was 12.7 per 100 person-years (95% CI 10.9-14.5). Adjusted hazard rates of seroconversion by child and household characteristics showed higher rates in children who were female, whose parent identified as a racial or ethnic minority, and in households with incomes less than 100K. The likelihood of remaining seropositive at six months was 67% (95% CI 59-76) and dropped to 19% (95% CI 11%-33%) at one year. Conclusions and Relevance: The data reported here provide estimates of pre-Omicron seroprevalence, seroconversion rates and time to seroreversion in a population-based cohort of children and adolescents. Serological studies continue to provide valuable contributions for infection prevalence estimates and help us better understand the dynamics of antibody levels following infection. Continued study of seroconversion and seroreversion can inform public health recommendations such as COVID-19 vaccination and booster schedules.

The changing landscape of respiratory viruses contributing to respiratory hospitalisations: results from a hospital-based surveillance in Quebec, Canada, 2012-13 to 2021-22 (preprint)

Background. A comprehensive description of the combined effect of SARS-CoV-2 and respiratory viruses (RV) other than SARS-CoV-2 (ORV) on hospitalisations is lacking. Aim. To compare viral etiology of acute respiratory infections (ARI) hospitalisations before and during two pandemic years from a surveillance network in Quebec, Canada. Method. We compared detection of ORV and SARS-CoV-2 during 2020-21 and 2021-22 to 8 pre-pandemic influenza seasons in patients hospitalised with ARI who were tested systematically by a multiplex PCR. Results. During pre-pandemic influenza seasons, overall RV detection was 92.7% (1,493) (48.3% respiratory syncytial virus (RSV)) in children and 62.8% (4,339) (40.1% influenza) in adults. Overall RV detection in 2020-21 was 58.6% (29) in children (all ORV) and 43.7% (333) in adults (3.4% ORV, 40.3% SARS-CoV2, both including coinfections). In 2021-22 overall RV detection was 91.0% (201) in children (82.8% ORV, 8.1% SARS-CoV-2, both including coinfections) and 55.5% (527) in adults (14.1% ORV, 41.4% SARS-CoV-2, both including coinfections). Virtually no influenza was detected in 2020-21 and in 2021-22 up to epi-week 2022-9 presented here; no RSV was detected in 2020-21. In 2021-22, detection of RSV was comparable to pre-pandemic years but with an unusually early season. There were significant differences in ORV and SARS-CoV-2 detection between time periods and age groups. Conclusion. Significant continuous shifts in age distribution and viral etiology of ARI hospitalisations occurred during two pandemic years. This reflects evolving RV epidemiology and underscores the need for increased scrutiny of ARI hospitalisation etiology to inform tailored public health recommendations.

Immunogenicity of Pfizer-BioNTech COVID-19 mRNA Primary Vaccination Series in Recovered Individuals Depends on Symptoms at Initial Infection (preprint)

ABSTRACT Importance Public health vaccination recommendations for COVID-19 primary series and boosters in previously infected individuals differ worldwide. As infection with SARS-CoV-2 is often asymptomatic, it remains to be determined if vaccine immunogenicity is comparable in all previously infected subjects. We present detailed immunological evidence to clarify the requirements for one-or two-dose primary vaccination series for naturally primed individuals. Objective Evaluate the immune response to COVID-19 mRNA vaccines in healthcare workers (HCWs) who recovered from a SARS-CoV-2 infection. Design Multicentric observational prospective cohort study of HCWs with a PCR-confirmed SARS-CoV-2 infection designed to evaluate the dynamics of T and B cells immune responses to primary infection and COVID-19 mRNA vaccination over 12 months. Participants Unvaccinated HCWs with PCR-confirmed SARS-CoV-2 infection were selected based on the presence or absence of symptoms at infection and serostatus at enrollment. Age- and sex-matched adults not infected with SARS-CoV-2 prior to vaccination were included as naïve controls. Exposure Vaccination with Pfizer BioNTech BNT162b2 mRNA vaccine. Main Outcome(s) and Measure(s) Immunity score (zero to three), before and after vaccination, based on anti-RBD IgG ratio, serum capacity to neutralize live virus and IFN-γ secretion capacity in response to SARS-CoV-2 peptide pools above the positivity threshold for each of the three assays. We compared the immunity score between groups based on subjects’ symptoms at diagnosis and/or serostatus prior to vaccination. Results None of the naïve participants (n=14) showed a maximal immunity score of three following one dose of vaccine compared to 84% of the previously infected participants (n=55). All recovered individuals who did not have an immunity score of three were seronegative prior to vaccination, and 67% had not reported symptoms resulting from their initial infection. Following one dose of vaccine, their immune responses were comparable to naïve individuals, with significantly weaker responses than those who were symptomatic during infection. Conclusions and Relevance Individuals who did not develop symptoms during their initial SARS-CoV-2 infection and were seronegative prior to vaccination present immune responses comparable to that of naïve individuals. These findings highlight the importance of administering the complete two-dose primary regimen and following boosters of mRNA vaccines to individuals who experienced asymptomatic SARS-CoV-2 infection. KEY POINTS Question Is a single dose of COVID-19 mRNA vaccine sufficient to induce robust immune responses in individuals with prior SARS-CoV-2 infection? Findings In this cohort of 55 health care workers previously infected with SARS-CoV-2, we show that the absence of symptoms during initial infection and negative serostatus prior to vaccination predict the strength of immune responses to COVID-19 mRNA vaccine. Lack of symptoms and a negative serostatus prior to vaccination leads to immune responses comparable to naïve individuals. Meaning Our results support a two-dose primary series requirement for any individual with prior history of asymptomatic SARS-CoV-2 infection.

In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance. (preprint)

In vitro selection of remdesivir-resistant SARS-CoV-2 revealed the emergence of a V166L substitution, located outside of the polymerase active site of the nsp12 protein, after 9 passages. V166L remained the only nsp12 substitution after 17 passages at a final concentration of 10 M RDV, conferring a 2.3-fold increase in EC50. When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to RDV resistance.

The REinfection in COVID-19 Estimation of Risk (RECOVER) study: Reinfection and serology dynamics in a cohort of Canadian healthcare workers. (preprint)

Background : Understanding the immune response to natural infection by SARS-CoV-2 is key to pandemic management, especially in the current context of emerging variants. Uncertainty remains regarding the efficacy and duration of natural immunity against reinfection. Method : We conducted an observational prospective cohort study in Canadian healthcare workers (HCWs) with a history of PCR-confirmed SARS-CoV-2 infection to : (i) measure the average incidence rate of reinfection and (ii), describe the serological immune response to the primary infection. Results : We detected 5 cases of reinfection over 14 months of follow-up, for a reinfection incidence rate of 3.3 per 100 person-years. Median duration of seropositivity was 420 days in symptomatics at primary infection compared to 213 days in asymptomatics (p<0.0001). Other variables associated with prolonged seropositivity for IgG against the spike protein included age 55 and above, obesity, and non-Caucasian ethnicity. Summary : Among healthcare workers, the incidence of reinfection with SARS-CoV-2 following a primary infection remained rare, although our analysis predates the circulation of the Omicron variant.

Impact of temperature on the affinity of SARS-CoV-2 Spike for ACE2 (preprint)

The seasonal nature in the outbreaks of respiratory viral infections with increased transmission during low temperatures has been well established. The current COVID-19 pandemic makes no exception, and temperature has been suggested to play a role on the viability and transmissibility of SARS-CoV-2. The receptor binding domain (RBD) of the Spike glycoprotein binds to the angiotensin-converting enzyme 2 (ACE2) to initiate viral fusion. Studying the effect of temperature on the receptor-Spike interaction, we observed a significant and stepwise increase in RBD-ACE2 affinity at low temperatures, resulting in slower dissociation kinetics. This translated into enhanced interaction of the full Spike to ACE2 receptor and higher viral attachment at low temperatures. Interestingly, the RBD N501Y mutation, present in emerging variants of concern (VOCs) that are fueling the pandemic worldwide, bypassed this requirement. This data suggests that the acquisition of N501Y reflects an adaptation to warmer climates, a hypothesis that remains to be tested.

Seroprevalence of Anti-SARS-CoV-2 Antibodies Among School and Daycare Children: Baseline Results From a Cohort Study in Montreal, Canada (preprint)

Background: Montreal was one of the highest COVID-19 burdened cities in Canada during the first and second waves of the pandemic. We estimated the seroprevalence of SARS-CoV-2 in children and teenagers in four neighbourhoods of Montreal, Canada.Methods: All children attending selected schools and daycares within the four neighbourhoods were invited to participate in the study. Study participation included an online questionnaire that parents completed, followed by at-home dried blood spot (DBS) collection. Serological results were analyzed using a research-based ELISA assay. Statistical analyses included multivariable logistic regression models to calculate average marginal effects and robust standard errors to account for clustering by school or daycare. Several different sociodemographic differences were examined between the seronegative and seropositive children.Findings: There were 30 daycares, 22 primary schools, and 11 secondary schools that participated in the study with 1,632 participants having provided a DBS sample that was of sufficient quality for the serological analysis. The average seroprevalence was 5·8% (95%CI 4·6 to 7·0) but increased over time from 3·2% (95% CI 0·7 to 5·8) in October-November 2020 to 8.4% (95% CI 4·4 to 12·4) in March-April 2021. The children of visible minority parents were nearly twice as likely to be seropositive as children of non-visible minority parents (1·93, 95%CI 1·11 to 2·75). Interpretation: Our results provide a benchmark of the seroprevalence status in Canadian children and provide further evidence of COVID-19 inequities. It will be important to continue monitoring the serological status of children, particularly in the context of new COVID-19 variants of concern and in the absence of mass vaccination campaigns targeting young children.Funding Information: Public Health Agency of Canada through the COVID-19 Immunity Task Force.Declaration of Interests: All authors declare no competing interests.Ethics Approval Statement: All participants provided informed consent for the survey and ethics approval was received from the research ethics boards of the Université de Montréal and the Centre Hospitalier Universitaire Sainte-Justine.

Older Age and Frailty Are Associated With Higher Mortality but Lower Intensive Care Unit Admission Among Adults Admitted to Canadian Hospitals With COVID-19: A Report From the Canadian Immunization Research Network (CIRN) Serious Outcomes Surveillance Network (preprint)

Background: We report characteristics and outcomes of adults admitted to Canadian Immunization Research Network (CIRN) Serious Outcomes Surveillance (SOS) Network hospitals with COVID-19 in 2020. Methods: Adult patients with laboratory-confirmed COVID-19 admitted to eleven sites in Ontario, Quebec, Alberta and Nova Scotia up to December 31, 2020 were enrolled in this prospective observational cohort study. Age, sex, demographics, housing, exposure characteristics, Clinical Frailty Scale, comorbidities, and outcomes including length of stay, intensive care unit (ICU) admission, mechanical ventilation, and survival were conducted. Descriptive analyses and multivariable logistic regressions were conducted. Findings: Among 2011 patients, mean age was 71·0 (range 19-105) years. 45·7% were women and 74·0% were white. 21·5% were admitted from Assisted Living facilities, 8·2% from long term care, and 2·1% from homeless shelters. The full spectrum of frailty was represented in both younger and older age groups. The majority (61·7% of adults <65 and 91·2% of those >=65) had at least one underlying comorbidity and 27·2% had obesity. Mortality was 14·3% among those not admitted to ICU, and 24·6% for those admitted to ICU. Older age and higher frailty were associated with reduced ICU admission but increased mortality. Obesity was independently associated with ICU admission but not with death. Associations between underlying comorbidities and adverse outcomes were attenuated but persisted when adjusting for frailty.Interpretation: Frailty and age were independent predictors of lower ICU use and higher mortality; when accounting for frailty, obesity was not an independent predictor of mortality, and associations of comorbidities with mortality were weakened.Funding Statement: Funding was provided by the Public Health Agency of Canada and the Canadian Institutes of Health Research.Declaration of Interests: MKA reports grant funding from the Public Health Association of Canada, CIHR, Canadian Frailty Network, Sanofi Pasteur and GSK group of companies, and payments from Pfizer, Sanofi Pasteur and Seqirus outside the submitted work. AM reports payments from GSK, Seqirus and Sanofi Pasteur, outside the submitted work. JEM reports payments from RestorBio, Sanofi, GSK, Merck and Medicago outside of the submitted work. TFH reports grants from Pfizer and GSK. ML reports payments from Sanofi, Medicago, Sequirus, and Pfizer outside the submitted work. SAM reports grants and payments from Pfizer, GSK, Merck, Novartis and Sanofi, outside the submitted work. JG, JJL, GB, LV, ME, DM-C, AA, KW, ST, SS, AMc and KK report no conflicts of interest.Ethics Approval Statement: The protocol for active COVID-19 surveillance has been approved by each local site’s Research Ethics Board.

Seroprevalence of anti-SARS-CoV-2 antibodies among school and daycare children and personnel: Protocol for a cohort study in Montreal, Canada (preprint)

Background: Further evidence is needed to understand the contribution of schools and daycares to the spread of COVID-19 in the context of diverse transmission dynamics and continually evolving public health interventions. The Enfants et COVID-19: Etude de seroprevalence (EnCORE) study will estimate the seroprevalence and seroconversion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among school and daycare children and personnel. In addition, the study will examine associations between seroprevalence and socio-demographic characteristics and reported COVID-19 symptoms and tests, and investigates changes in health, lifestyle and well-being outcomes. Methods: This study includes children and personnel from 62 schools and daycares in four neighbourhoods in Montreal, Canada. All children age 2-17 years attending one of the participating schools or daycares and their parents are invited to participate, as well as a sample of personnel members. Participants respond to brief questionnaires and provide blood samples, collected via dried blood spot (DBS), at baseline (October 2020-March 2021) and follow-up (May-June 2021). Questionnaires include socio-demographic and household characteristics, reported COVID-19 symptoms and tests, potential COVID-19 risk factors and prevention efforts, and health and lifestyle information. Logistic regression using generalized estimating equations will be used to estimate seroprevalence and seroconversion, accounting for school-level clustering. Discussion: The results of the EnCORE study will contribute to our knowledge about SARS-CoV-2 transmission in schools and daycares, which is critical for decisions regarding school attendance and the management of school outbreaks through the remainder of this school year and beyond. Keywords SARS-CoV-2; COVID-19; Children; School; Serology; Protocol; Canada

Genetics of symptom remission in outpatients with COVID-19 (preprint)

We conducted a genome-wide association study of time to remission of COVID-19 symptoms in 1723 outpatients with at least one risk factor for disease severity from the COLCORONA clinical trial. We found a significant association at 5p13.3 (rs1173773; P = 4.94 x 10-8) near the natriuretic peptide receptor 3 gene (NPR3). By day 15 of the study, 44%, 54% and 59% of participants with 0, 1, or 2 copies of the effect allele respectively, had symptom remission. In 851 participants not treated with colchicine (placebo), there was a significant association at 9q33.1 (rs62575331; P = 2.95 x 10-8) in interaction with colchicine (P = 1.19 x 10-5) without impact on risk of hospitalisations, highlighting a possibly shared mechanistic pathway. By day 15 of the study, 46%, 62% and 64% of those with 0, 1, or 2 copies of the effect allele respectively, had symptom remission. The findings need to be replicated and could contribute to the biological understanding of COVID-19 symptom remission.

Efficacy of Colchicine in Non-Hospitalized Patients with COVID-19 (preprint)

Background Evidence suggests the role of an inflammatory storm in COVID-19 complications. Colchicine is an orally administered, anti-inflammatory medication beneficial in gout, pericarditis and coronary disease. Methods We performed a randomized, double-blind trial involving non-hospitalized patients with COVID-19 diagnosed by polymerase chain reaction (PCR) testing or clinical criteria. The patients were randomly assigned to receive colchicine (0.5 mg twice daily for 3 days and once daily thereafter) or placebo for 30 days. The primary efficacy endpoint was the composite of death or hospitalization for COVID-19. Results A total of 4488 patients were enrolled. The primary endpoint occurred in 4.7% of the patients in the colchicine group and 5.8% of those in the placebo group (odds ratio, 0.79; 95.1% confidence interval (CI), 0.61 to 1.03; P=0.08). Among the 4159 patients with PCR-confirmed COVID-19, the primary endpoint occurred in 4.6% and 6.0% of patients in the colchicine and placebo groups, respectively (odds ratio, 0.75; 95% CI, 0.57 to 0.99; P=0.04). In these patients with PCR-confirmed COVID-19, the odds ratios were 0.75 (95% CI, 0.57 to 0.99) for hospitalization due to COVID-19, 0.50 (95% CI, 0.23 to 1.07) for mechanical ventilation, and 0.56 (95% CI, 0.19 to 1.66) for death. Serious adverse events were reported in 4.9% and 6.3% in the colchicine and placebo groups (P=0.05); pneumonia occurred in 2.9% and 4.1% of patients (P=0.02). Diarrhea was reported in 13.7% and 7.3% in the colchicine and placebo groups (P<0.0001). Conclusion Among non-hospitalized patients with COVID-19, colchicine reduces the composite rate of death or hospitalization. (COLCORONA ClinicalTrials.gov number: NCT04322682)

SARS-CoV-2 emerging complexity (preprint)

The novel SARS_CoV-2 virus, prone to variation when interacting with spatially extended ecosystems and within hosts1 can be considered a complex dynamic system2. Therefore, it behaves creating several space-time manifestations of its dynamics. However, these physical manifestations in nature have not yet been fully disclosed or understood. Here we show 4-3 and 2-D space-time patterns of rate of infected individuals on a global scale, giving quantitative measures of transitions between different dynamical behaviour. By slicing the spatio-temporal patterns, we found manifestations of the virus behaviour such as cluster formation and bifurcations. Furthermore, by analysing the morphogenesis processes by entropy, we have been able to detect the virus phase transitions, typical of adaptive biological systems3. Our results for the first time describe the virus patterning behaviour processes all over the world, giving for them quantitative measures. We know that the outcomes of this work are still partial and more advanced analyses of the virus behaviour in nature are necessary. However, we think that the set of methods implemented can provide significant advantages to better analyse the viral behaviour in the approach of system biology4, thus expanding knowledge and improving pandemic problem solving.

Neutralization of spike 69/70 deletion, E484K, and N501Y SARS-CoV-2 by BNT162b2 vaccine-elicited sera (preprint)

We engineered three SARS-CoV-2 viruses containing key spike mutations from the newly emerged United Kingdom (UK) and South African (SA) variants: N501Y from UK and SA; 69/70-deletion+N501Y+D614G from UK; and E484K+N501Y+D614G from SA. Neutralization geometric mean titers (GMTs) of twenty BTN162b2 vaccine-elicited human sera against the three mutant viruses were 0.81- to 1.46-fold of the GMTs against parental virus, indicating small effects of these mutations on neutralization by sera elicited by two BNT162b2 doses.