Single-Molecule Investigation of the Binding Interface Stability of SARS-CoV-2 Variants with ACE2.

The SARS-CoV-2 pandemic spurred numerous research endeavors to comprehend the virus and mitigate its global severity. Understanding the binding interface between the virus and human receptors is pivotal to these efforts and paramount to curbing infection and transmission. Here we employ atomic force microscopy and steered molecular dynamics simulation to explore SARS-CoV-2 receptor binding domain (RBD) variants and angiotensin-converting enzyme 2 (ACE2), examining the impact of mutations at key residues upon binding affinity. Our results show that the Omicron and Delta variants possess strengthened binding affinity in comparison to the Mu variant. Further, using sera from individuals either vaccinated or with acquired immunity following Delta strain infection, we assess the impact of immunity upon variant RBD/ACE2 complex formation. Single-molecule force spectroscopy analysis suggests that vaccination before infection may provide stronger protection across variants. These results underscore the need to monitor antigenic changes in order to continue developing innovative and effective SARS-CoV-2 abrogation strategies.

Lung Interstitial Macrophages Can Present Soluble Antigens and Induce Foxp3+ Regulatory T Cells.

Lung macrophages constitute a sophisticated surveillance and defense system that contributes to tissue homeostasis, host defense, and allows the host to cope with the myriad of insults and antigens to which the lung mucosa is exposed. As opposed to alveolar macrophages, lung interstitial macrophages express high levels of type 2 major histocompatibility complex (MHC-II), a hallmark of antigen-presenting cells. Here, we showed that lung IMs, like dendritic cells (DCs), possess the machinery to present soluble antigens in an MHC-II-restricted way. Using ex vivo ovalbumin (OVA)-specific T cell proliferation assays, we found that OVA-pulsed IMs could trigger OVA-specific CD4+ T cell proliferation and Foxp3 expression via MHC-II-, IL-10 and Tgfß-dependent mechanisms. Moreover, we showed that IMs efficiently captured locally instilled antigens in vivo, did not migrate to the draining lymph nodes and enhanced local interactions with CD4+ T cells in a model of OVA-induced allergic asthma. These results support that IMs can present antigens to CD4+ T cells and trigger regulatory T cells, which might attenuate lung immune responses and have functional consequences for lung immunity and T-cell-mediated disorders.

Nationwide quality assurance of high-throughput diagnostic molecular testing during the SARS-CoV-2 pandemic: role of the Belgian National Reference Centre.

Since the onset of the coronavirus disease (COVID-19) pandemic in Belgium, UZ/KU Leuven has played a crucial role as the National Reference Centre (NRC) for respiratory pathogens, to be the first Belgian laboratory to develop and implement laboratory developed diagnostic assays for SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and later to assess the quality of commercial kits. To meet the growing demand for decentralised testing, both clinical laboratories and government-supported high-throughput platforms were gradually deployed across Belgium. Consequently, the role of the NRC transitioned from a specialised testing laboratory to strengthening capacity and coordinating quality assurance. Here, we outline the measures taken by the NRC, the national public health institute Sciensano and the executing clinical laboratories to ensure effective quality management of molecular testing throughout the initial two years of the pandemic (March 2020 to March 2022).

Systematic Review of the Key Factors Influencing the Indoor Airborne Spread of SARS-CoV-2.

The COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has been plaguing the world since late 2019/early 2020 and has changed the way we function as a society, halting both economic and social activities worldwide. Classrooms, offices, restaurants, public transport, and other enclosed spaces that typically gather large groups of people indoors, and are considered focal points for the spread of the virus. For society to be able to go "back to normal", it is crucial to keep these places open and functioning. An understanding of the transmission modes occurring in these contexts is essential to set up effective infection control strategies. This understanding was made using a systematic review, according to the Preferred Reporting Items for Systematic reviews and Meta-Analyses statement (PRISMA) 2020 guidelines. We analyze the different parameters influencing airborne transmission indoors, the mathematical models proposed to understand it, and discuss how we can act on these parameters. Methods to judge infection risks through the analysis of the indoor air quality are described. Various mitigation measures are listed, and their efficiency, feasibility, and acceptability are ranked by a panel of experts in the field. Thus, effective ventilation procedures controlled by CO2-monitoring, continued mask wearing, and a strategic control of room occupancy, among other measures, are put forth to enable a safe return to these essential places.

Tirap controls Mycobacterium tuberculosis phagosomal acidification.

Progression of tuberculosis is tightly linked to a disordered immune balance, resulting in inability of the host to restrict intracellular bacterial replication and its subsequent dissemination. The immune response is mainly characterized by an orchestrated recruitment of inflammatory cells secreting cytokines. This response results from the activation of innate immunity receptors that trigger downstream intracellular signaling pathways involving adaptor proteins such as the TIR-containing adaptor protein (Tirap). In humans, resistance to tuberculosis is associated with a loss-of-function in Tirap. Here, we explore how genetic deficiency in Tirap impacts resistance to Mycobacterium tuberculosis (Mtb) infection in a mouse model and ex vivo. Interestingly, compared to wild type littermates, Tirap heterozygous mice were more resistant to Mtb infection. Upon investigation at the cellular level, we observed that mycobacteria were not able to replicate in Tirap-deficient macrophages compared to wild type counterparts. We next showed that Mtb infection induced Tirap expression which prevented phagosomal acidification and rupture. We further demonstrate that the Tirap-mediated anti-tuberculosis effect occurs through a Cish-dependent signaling pathway. Our findings provide new molecular evidence about how Mtb manipulates innate immune signaling to enable intracellular replication and survival of the pathogen, thus paving the way for host-directed approaches to treat tuberculosis.

MafB-restricted local monocyte proliferation precedes lung interstitial macrophage differentiation.

Resident tissue macrophages (RTMs) are differentiated immune cells that populate distinct niches and exert important tissue-supportive functions. RTM maintenance is thought to rely either on differentiation from monocytes or on RTM self-renewal. Here, we used a mouse model of inducible lung interstitial macrophage (IM) niche depletion and refilling to investigate the development of IMs in vivo. Using time-course single-cell RNA-sequencing analyses, bone marrow chimeras and gene targeting, we found that engrafted Ly6C+ classical monocytes proliferated locally in a Csf1 receptor-dependent manner before differentiating into IMs. The transition from monocyte proliferation toward IM subset specification was controlled by the transcription factor MafB, while c-Maf specifically regulated the identity of the CD206+ IM subset. Our data provide evidence that, in the mononuclear phagocyte system, the ability to proliferate is not merely restricted to myeloid progenitor cells and mature RTMs but is also a tightly regulated capability of monocytes developing into RTMs in vivo.

The innate immune brakes of the lung.

Respiratory mucosal surfaces are continuously exposed to not only innocuous non-self antigens but also pathogen-associated molecular patterns (PAMPs) originating from environmental or symbiotic microbes. According to either "self/non-self" or "danger" models, this should systematically result in homeostasis breakdown and the development of immune responses directed to inhaled harmless antigens, such as T helper type (Th)2-mediated asthmatic reactions, which is fortunately not the case in most people. This discrepancy implies the existence, in the lung, of regulatory mechanisms that tightly control immune homeostasis. Although such mechanisms have been poorly investigated in comparison to the ones that trigger immune responses, a better understanding of them could be useful in the development of new therapeutic strategies against lung diseases (e.g., asthma). Here, we review current knowledge on innate immune cells that prevent the development of aberrant immune responses in the lung, thereby contributing to mucosal homeostasis.

SARS-CoV-2 Transmission in Belgian French-Speaking Primary Schools: An Epidemiological Pilot Study.

Schools have been a point of attention during the pandemic, and their closure one of the mitigating measures taken. A better understanding of the dynamics of the transmission of SARS-CoV-2 in elementary education is essential to advise decisionmakers. We conducted an uncontrolled non-interventional prospective study in Belgian French-speaking schools to describe the role of attending asymptomatic children and school staff in the spread of COVID-19 and to estimate the transmission to others. Each participant from selected schools was tested for SARS-CoV-2 using a polymerase chain reaction (PCR) analysis on saliva sample, on a weekly basis, during six consecutive visits. In accordance with recommendations in force at the time, symptomatic individuals were excluded from school, but per the study protocol, being that participants were blinded to PCR results, asymptomatic participants were maintained at school. Among 11 selected schools, 932 pupils and 242 school staff were included between January and May 2021. Overall, 6449 saliva samples were collected, of which 44 came back positive. Most positive samples came from isolated cases. We observed that asymptomatic positive children remaining at school did not lead to increasing numbers of cases or clusters. However, we conducted our study during a period of low prevalence in Belgium. It would be interesting to conduct the same analysis during a high prevalence period.

Compliance with Barrier Gestures during COVID-19 Pandemic as a Function of the Context: A Longitudinal Observational Survey at the University of Liège.

During the COVID-19 pandemic, barrier gestures such as mask wearing, physical distancing, greetings without contact, one-way circulation flow, and hand sanitization were major strategies to prevent the spread of SARS-CoV-2, but they were only useful if consistently applied. This survey was a follow-up of the first survey performed in 2020 at the University of Liège. We aim to evaluate the compliance with these gestures on campuses and examine differences in the extent of the compliance observed in different educational activities and contexts. During 3.5 months, the counting of compliant and non-compliant behaviors was performed each week in randomly selected rooms. Using data collected during both surveys (2020 and 2021), binomial negative regression models of compliance depending on periods (teaching periods and exam sessions), type of rooms, and campuses were conducted to evaluate prevalence ratios of compliance. The percentage of compliance in this second survey was the highest for mask wearing and physical distancing during educational activities (90% and 88%, respectively) and lowest for physical distancing outside educational activities and hand sanitization (45% and 52%, respectively). Multivariate analyses revealed that the compliance with most gestures was significantly higher in teaching rooms than in hallways and restaurants and during exam sessions. The compliance with physical distancing was significantly higher (from 66%) in auditoriums, where students had to remain seated, than during practical works that allowed or required free movement. Therefore, the compliance with barrier gestures was associated with contextual settings, which should be considered when communicating and managing barrier gestures. Further studies should specify and confirm the determining contextual characteristics regarding the compliance with barrier gestures in times of pandemic.

The Dynamic Relationship between the Intention and Final Decision for the COVID-19 Booster: A Study among Students and Staff at the University of Liège, Belgium.

While many studies have documented the intentions for the COVID-19 vaccine booster, few have explored the change from intention to final decision. This study explores the COVID-19 booster intentions and the change from intention to decision in a primo-vaccinated university population, with a distinction between staff members and students. It looks at the sociodemographic and medical characteristics, health literacy, personal COVID-19 infection and vaccination history, and attitudes/intentions regarding the booster, among the 1030 participants (64.4% staff members, 61.3% female, median age 36.0 years). Of the 8.7% who were initially hesitant, 72.7% ultimately got a booster and 27.3% did not. Another 84.2% intended to get a booster and 7.1% did not. Among the latter two groups, 88.9% maintained their intention and 11.1% changed their minds. The determinants associated with the intentions were health literacy and previous intentions regarding the COVID-19 primo-vaccination. The determinants associated with the change to non-vaccination were a previous COVID-19 infection, a past COVID-19 primo-vaccination intention, and a neutralizing antibody level. The results point to an opening for the support in decision-making, with a significant percentage of the study population potentially changing their mind between intention and final decision; this process should start early and be tailored to the individual's COVID-19 history. A personalized approach seems necessary in order to ensure that individuals make an informed choice.

Factors influencing the adoption and participation rate of nursing homes staff in a saliva testing screening programme for COVID-19.

Testing strategies are crucial to prevent and control the spread of covid-19 but suffer from a lack of investment in understanding the human factors that influence their implementation. The aim of this study was to understand the factors that encourage participation and the level of engagement of nursing homes staff in a routine saliva testing programme for COVID-19 In December 2020, nursing homes (n = 571) in Wallonia (Belgium) were invited to participate in a saliva testing programme for their staff. The directors were questioned by telephone at the end of a 3-week pilot phase. 445 nursing homes took part in the evaluation questionnaire, of which 36(8%) answered that they chose not to participate in the testing programme. The average participation rate of nursing staff was 49(±25)%. Perception of the justification of the efforts required for testing and perception of practicability of the procedure were significantly associated with the adoption of the system by the nursing homes directors (OR(95%CI): 5.96(1.97-18.0), p = 0.0016); OR(95%CI): 5.64(1.94-16.4), p = 0.0015 respectively). Staff support, incentives and meetings increased the level of engagement in testing (p<0.05). While the adoption of the programme confirmed the acceptability of salivary testing as a means of screening, the participation rate confirmed the need for studies to understand the factors that encourage health care staff to take part. The results suggested rethinking strategies to consider staff engagement from a health promotion perspective.

Kinetics and Persistence of the Cellular and Humoral Immune Responses to BNT162b2 mRNA Vaccine in SARS-CoV-2-Naive and -Experienced Subjects: Impact of Booster Dose and Breakthrough Infections.

Background: Understanding and measuring the individual level of immune protection and its persistence at both humoral and cellular levels after SARS-CoV-2 vaccination is mandatory for the management of the vaccination booster campaign. Our prospective study was designed to assess the immunogenicity of the BNT162b2 mRNA vaccine in triggering the cellular and humoral immune response in healthcare workers up to 12 months after the initial vaccination, with one additional boosting dose between 6 and 12 months. Methods: This prospective study enrolled 208 healthcare workers (HCWs) from the Liège University Hospital (CHU) of Liège in Belgium. Participants received two doses of BioNTech/Pfizer COVID-19 vaccine (BNT162b2) and a booster dose 6-12 months later. Fifty participants were SARS-CoV-2 experienced and 158 were naïve before the vaccination. Blood sampling was performed at the day of the first (T0) and second (T1) vaccine doses administration, then at 2 weeks (T2), 4 weeks (T3), 6 months (T4) and 12 months (T5) after the second dose. Between T4 and T5, participants also got the third boosting vaccine dose. A total of 1145 blood samples were collected. All samples were tested for the presence of anti-Spike antibodies, using the DiaSorin LIAISON SARS-CoV-2 Trimeric S IgG assay, and for anti-Nucleocapsid antibodies, using Elecsys anti-SARS-CoV-2 assay​​. Neutralizing antibodies against the SARS-CoV-2 Wuhan-like variant strain were quantified in all samples using a Vero E6 cell-based neutralization assay. Cell-mediated immune response was evaluated at T4 and T5 on 80 and 55 participants, respectively, by measuring the secretion of IFN-γ on peripheral blood lymphocytes using the QuantiFERON Human IFN-γ SARS-CoV-2, from Qiagen. We analyzed separately the naïve and experienced participants. Findings: We found that anti-spike antibodies and neutralization capacity levels were significantly higher in SARS-CoV-2 experienced HCWs compared to naïve HCWs at all time points analyzed except the one after boosting dose. Cellular immune response was also higher in experienced HCWs six months following vaccination. Besides the impact of SARS-CoV-2 infection history on immune response to BNT162b2 mRNA vaccine, we observed a significant negative association between age and persistence of humoral response. The booster dose induced an increase in humoral and cellular immune responses, particularly in naive individuals. Breakthrough infections resulted in higher cellular and humoral responses after the booster dose. Conclusions: Our data strengthen previous findings demonstrating that immunization through vaccination combined with natural infection is better than 2 vaccine doses immunization or natural infection alone. The benefit of the booster dose was greater in naive individuals. It may have implications for personalizing mRNA vaccination regimens used to prevent severe COVID-19 and reduce the impact of the pandemic on the healthcare system. More specifically, it may help prioritizing vaccination, including for the deployment of booster doses.

A 2-month field cohort study of SARS-CoV-2 in saliva of BNT162b2 vaccinated nursing home workers.

Background: Nursing home (NH) residents have been severely affected during the COVID-19 pandemic because of their age and underlying comorbidities. Infection and outbreaks in NHs are most likely triggered by infected workers. Screening for asymptomatic NH workers can prevent risky contact and viral transmission to the residents. This study examined the effect of the BNT162b2 mRNA COVID­19 (Comirnaty®; BioNTech and Pfizer) vaccination on the saliva excretion of SARS-CoV-2 among NH workers, through weekly saliva RT-qPCR testing. Methods: A 2-month cohort study was conducted among 99 NHs in the Walloon region (Belgium), at the start of February 2021. Three groups of workers, i.e., non-vaccinated (n = 1618), one-dosed vaccinated (n = 1454), and two-dosed vaccinated (n = 2379) of BNT162b2 mRNA COVID­19 vaccine, were followed-up weekly. Their saliva samples were used to monitor the shedding of SARS-CoV-2. All positive samples were sequenced and genotyped to identify the circulating wild-type virus or variants of concern. Results: The protection fraction against the excretion of the SARS-CoV-2 in the saliva samples of the workers after the second dose is estimated at 0.90 (95% CI: 0.18; 0.99) at 1 week and 0.83 (95% CI: 0.54; 0.95) at 8 weeks. We observe more circulating SARS-CoV-2 and a greater variability of viral loads in the unvaccinated group compared to those of the vaccinated group. Conclusions: This field cohort study advances our knowledge of the efficacy of the mRNA BNT162b2 COVID-19 vaccine on the viral shedding in the saliva specimens of vaccinated NH workers, contributing to better decision-making in public health interventions and management.

Airway Macrophages Encompass Transcriptionally and Functionally Distinct Subsets Altered by Smoking.

Alveolar macrophages (AMs) are functionally important innate cells involved in lung homeostasis and immunity and whose diversity in health and disease is a subject of intense investigations. Yet, it remains unclear to what extent conditions like smoking or chronic obstructive pulmonary disease (COPD) trigger changes in the AM compartment. Here, we aimed to explore heterogeneity of human AMs isolated from healthy nonsmokers, smokers without COPD, and smokers with COPD by analyzing BAL fluid cells by flow cytometry and bulk and single-cell RNA sequencing. We found that subpopulations of BAL fluid CD206+ macrophages could be distinguished based on their degree of autofluorescence in each subject analyzed. CD206+ autofluorescenthigh AMs were identified as classical, self-proliferative AM, whereas autofluorescentlow AMs were expressing both monocyte and classical AM-related genes, supportive of a monocytic origin. Of note, monocyte-derived autofluorescentlow AMs exhibited a functionally distinct immunoregulatory profile, including the ability to secrete the immunosuppressive cytokine IL-10. Interestingly, single-cell RNA-sequencing analyses showed that transcriptionally distinct clusters of classical and monocyte-derived AM were uniquely enriched in smokers with and without COPD as compared with healthy nonsmokers. Of note, such smoking-associated clusters exhibited gene signatures enriched in detoxification, oxidative stress, and proinflammatory responses. Our study independently confirms previous reports supporting that monocyte-derived macrophages coexist with classical AM in the airways of healthy subjects and patients with COPD and identifies smoking-associated changes in the AM compartment that may favor COPD initiation or progression.

Acceptability of Community Saliva Testing in Controlling the COVID-19 Pandemic: Lessons Learned from Two Case Studies in Nursing Homes and Schools.

Current public health debate centers on COVID-19 testing methods and strategies. In some communities, high transmission risk may justify routine testing, and this requires test methods that are safe and efficient for both patients and the administrative or health-care workers administering them. Saliva testing appears to satisfy those criteria. There is, however, little documentation on the acceptability of this method among beneficiaries. This article presents the lessons learned from a pilot study on the use of saliva testing for routine screening of nursing home and secondary school personnel in Wallonia (the French-speaking part of Belgium), conducted in December 2020 to April 2021, respectively. Administrators at the facilities in question seemed to think highly of saliva testing and wished to continue it after the pilot study was over. This result reinforces the criteria (the noninvasive aspect, in particular) supporting a key role for saliva testing in monitoring community spread of the virus. Nevertheless, wider-scale deployment of this particular method will only be possible if the testing strategy as a whole takes a health promotion approach.

Decision-based interactive model to determine re-opening conditions of a large university campus in Belgium during the first COVID-19 wave.

BACKGROUND: The role played by large-scale repetitive SARS-CoV-2 screening programs within university populations interacting continuously with an urban environment, is unknown. Our objective was to develop a model capable of predicting the dispersion of viral contamination among university populations dividing their time between social and academic environments. METHODS: Data was collected through real, large-scale testing developed at the University of Liège, Belgium, during the period Sept. 28th-Oct. 29th 2020. The screening, offered to students and staff (n = 30,000), began 2 weeks after the re-opening of the campus but had to be halted after 5 weeks due to an imposed general lockdown. The data was then used to feed a two-population model (University + surrounding environment) implementing a generalized susceptible-exposed-infected-removed compartmental modeling framework. RESULTS: The considered two-population model was sufficiently versatile to capture the known dynamics of the pandemic. The reproduction number was estimated to be significantly larger on campus than in the urban population, with a net difference of 0.5 in the most severe conditions. The low adhesion rate for screening (22.6% on average) and the large reproduction number meant the pandemic could not be contained. However, the weekly screening could have prevented 1393 cases (i.e. 4.6% of the university population; 95% CI: 4.4-4.8%) compared to a modeled situation without testing. CONCLUSION: In a real life setting in a University campus, periodic screening could contribute to limiting the SARS-CoV-2 pandemic cycle but is highly dependent on its environment.

Reliable and Scalable SARS-CoV-2 qPCR Testing at a High Sample Throughput: Lessons Learned from the Belgian Initiative.

We present our approach to rapidly establishing a standardized, multi-site, nation-wide COVID-19 screening program in Belgium. Under auspices of a federal government Task Force responsible for upscaling the country's testing capacity, we were able to set up a national testing initiative with readily available resources, putting in place a robust, validated, high-throughput, and decentralized qPCR molecular testing platform with embedded proficiency testing. We demonstrate how during an acute scarcity of equipment, kits, reagents, personnel, protective equipment, and sterile plastic supplies, we introduced an approach to rapidly build a reliable, validated, high-volume, high-confidence workflow based on heterogeneous instrumentation and diverse assays, assay components, and protocols. The workflow was set up with continuous quality control monitoring, tied together through a clinical-grade information management platform for automated data analysis, real-time result reporting across different participating sites, qc monitoring, and making result data available to the requesting physician and the patient. In this overview, we address challenges in optimizing high-throughput cross-laboratory workflows with minimal manual intervention through software, instrument and assay validation and standardization, and a process for harmonized result reporting and nation-level infection statistics monitoring across the disparate testing methodologies and workflows, necessitated by a rapid scale-up as a response to the pandemic.

University population-based prospective cohort study of SARS-CoV-2 infection and immunity (SARSSURV-ULiège): a study protocol.

INTRODUCTION: For a safe and sustainable return to normal functioning of academic activities in higher education, objective-driven testing strategies that are flexible and rapidly adaptable are essential to effectively monitor and respond to new developments of the COVID-19 pandemic. To date, prospective longitudinal research on SARS-CoV-2 antibody testing in saliva and seroprevalence in higher education contexts is substantially lacking, limiting our understanding of COVID-19 prevalence, incidence and nature of the immune response to SARS-CoV-2 at various stages of the infection and vaccination. To address this lack of evidence, a prospective population-based cohort study (SARSSURV-ULiège) has recently been started. METHODS AND ANALYSIS: Students (n=1396) and staff members (n=1143) of the University of Liège are followed up over more than 1 year. All participants are required to complete anamnestic, clinical and vaccine hesitancy questionnaires for medical histories and undertaken treatments. Previous proven or suspected SARS-CoV-2 infection is also registered. In phase 1, weekly saliva samples to perform RT-qPCR to detect SARS-CoV-2 and monthly COVID-19 serological rapid test results are collected. Once being positive to either saliva RT-qPCR assay for SARS-CoV-2 presence or to serological test, the participant is invited to enter phase 2. If participants get vaccinated during the study period, they are invited to phase 2. In this second phase, besides weekly saliva self-test, depending on the participants' profiles, both gargle and blood samples are collected to obtain various biological data to measure the presence of neutralising antibodies against SARS-CoV-2, determine the magnitude and the duration of antibody responses over time. ETHICS AND DISSEMINATION: The study has received the approval from the University Hospital of Liège Ethics Committee (reference number 2021/96, dated 26 March 2021). Potential protocol amendments will be presented to the Research Ethics Committee. The findings of the present study will be presented at scientific conferences and the results published in peer-review publications. Weekly reports will be submitted to the risk assessment group and the risk management group against COVID-19 of the university to enable a timely public health action if necessary.