RECAST: Study protocol for an observational study for the understanding of the increased REsilience of Children compared to Adults in SARS-CoV-2 infecTion.

INTRODUCTION: The SARS-CoV-2 pandemic remains a threat to public health. Soon after its outbreak, it became apparent that children are less severely affected. Indeed, opposing clinical manifestations between children and adults are observed for other infections. The SARS-CoV-2 outbreak provides the unique opportunity to study the underlying mechanisms. This protocol describes the methods of an observational study that aims to characterise age dependent differences in immune responses to primary respiratory infections using SARS-CoV-2 as a model virus and to assess age differences in clinical outcomes including lung function. METHODS AND ANALYSIS: The study aims to recruit at least 120 children and 60 adults that are infected with SARS-CoV-2 and collect specimen for a multiomics analysis, including single cell RNA sequencing of nasal epithelial cells and peripheral blood mononuclear cells, mass cytometry of whole blood samples and nasal cells, mass spectrometry-based serum and plasma proteomics, nasal epithelial cultures with functional in vitro analyses, SARS-CoV-2 antibody testing, sequencing of the viral genome and lung function testing. Data obtained from this multiomics approach are correlated with medical history and clinical data. Recruitment started in October 2020 and is ongoing. ETHICS AND DISSEMINATION: The study was reviewed and approved by the Ethics Committee of Charité - Universitätsmedizin Berlin (EA2/066/20). All collected specimens are stored in the central biobank of Charité - Universitätsmedizin Berlin and are made available to all participating researchers and on request. TRIAL REGISTRATION NUMBER: DRKS00025715, pre-results publication.

Generalized anxiety disorder in Berlin school children after the third COVID-19 wave in Germany: a cohort study between June and September 2021.

BACKGROUND: During the COVID-19 pandemic, children and adolescents worldwide have disproportionally been affected in their psychological health and wellbeing. We conducted a cohort study among German school children, aiming at assessing levels of general anxiety disorder (GAD) and identifying associated factors in the second pandemic year. METHODS: A cohort of 660 students from 24 Berlin schools was recruited to fill in questionnaires including the GAD-7 tool on anxiety symptoms at three time points between June and September 2021. To adjust for non-random attrition, we applied inverse probability weighting. We describe reported GAD levels stratified by time point, sex, and school type and report odds ratios from univariate logistic regression. RESULTS: In total, 551 participants (83%) filled in at least one questionnaire at any time point. At the first time point in June 2021, 25% of the children and adolescents reported anxiety symptoms with a GAD-7 score ≥ 5, decreasing to 16% in August 2021 directly after the summer holidays and rising again to 26% in September 2021. The majority of reported anxiety levels belonged to the least severe category. Being female, attending secondary school, coming from a household with lower education or with lower income level, and being vaccinated against COVID-19 were significantly linked with reporting anxiety symptoms. Preceding COVID-19 infection and anxiety were negatively associated. CONCLUSION: Overall, anxiety in school children was lower in mid-2021 than in the first pandemic year, but still double compared to pre-pandemic data. Reporting of anxiety symptoms during the second pandemic year was especially high in females and in secondary school students. Policy makers should pay additional attention to the mental health status of school children, even as the pandemic situation might stabilize.

SARS-CoV-2 variant Alpha has a spike-dependent replication advantage over the ancestral B.1 strain in human cells with low ACE2 expression.

Epidemiological data demonstrate that Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha and Delta are more transmissible, infectious, and pathogenic than previous variants. Phenotypic properties of VOC remain understudied. Here, we provide an extensive functional study of VOC Alpha replication and cell entry phenotypes assisted by reverse genetics, mutational mapping of spike in lentiviral pseudotypes, viral and cellular gene expression studies, and infectivity stability assays in an enhanced range of cell and epithelial culture models. In almost all models, VOC Alpha spread less or equally efficiently as ancestral (B.1) SARS-CoV-2. B.1. and VOC Alpha shared similar susceptibility to serum neutralization. Despite increased relative abundance of specific sgRNAs in the context of VOC Alpha infection, immune gene expression in infected cells did not differ between VOC Alpha and B.1. However, inferior spreading and entry efficiencies of VOC Alpha corresponded to lower abundance of proteolytically cleaved spike products presumably linked to the T716I mutation. In addition, we identified a bronchial cell line, NCI-H1299, which supported 24-fold increased growth of VOC Alpha and is to our knowledge the only cell line to recapitulate the fitness advantage of VOC Alpha compared to B.1. Interestingly, also VOC Delta showed a strong (595-fold) fitness advantage over B.1 in these cells. Comparative analysis of chimeric viruses expressing VOC Alpha spike in the backbone of B.1, and vice versa, showed that the specific replication phenotype of VOC Alpha in NCI-H1299 cells is largely determined by its spike protein. Despite undetectable ACE2 protein expression in NCI-H1299 cells, CRISPR/Cas9 knock-out and antibody-mediated blocking experiments revealed that multicycle spread of B.1 and VOC Alpha required ACE2 expression. Interestingly, entry of VOC Alpha, as opposed to B.1 virions, was largely unaffected by treatment with exogenous trypsin or saliva prior to infection, suggesting enhanced resistance of VOC Alpha spike to premature proteolytic cleavage in the extracellular environment of the human respiratory tract. This property may result in delayed degradation of VOC Alpha particle infectivity in conditions typical of mucosal fluids of the upper respiratory tract that may be recapitulated in NCI-H1299 cells closer than in highly ACE2-expressing cell lines and models. Our study highlights the importance of cell model evaluation and comparison for in-depth characterization of virus variant-specific phenotypes and uncovers a fine-tuned interrelationship between VOC Alpha- and host cell-specific determinants that may underlie the increased and prolonged virus shedding detected in patients infected with VOC Alpha.

Viral infections in hospitalized children in Germany during the COVID-19 pandemic: Association with non-pharmaceutical interventions.

Background: Non-pharmaceutical interventions (NPI) during the COVID-19 pandemic aimed at prevention of SARS-CoV-2 transmission also influenced transmission of viruses other than SARS-CoV-2. The aim of this study was to describe and compare the burden of common viral respiratory and gastrointestinal infections in children admitted to Berlin University Children's Hospital (BCH) before and during the COVID-19 pandemic at different levels of public NPI measures. Methods: In this retrospective study, we analyzed the frequency of detection of common human respiratory and gastrointestinal viruses from January 2016 through January 2022 in all patients admitted to BCH. We compared virus detection before and during the COVID-19 pandemic at different levels of public NPI measures. Results: The frequency of detection of seasonal enveloped and non-enveloped viruses [Boca-, Corona-, Influenza-, Metapneumo-, Parainfluenza-, Rota-, and Respiratory Syncytial Viruses (RSV)] was diminished during the COVID-19 pandemic, whereas detection rates of non-seasonal viruses (Rhino-/Entero-, and Adenoviruses) were stable during the pandemic. After withdrawal of major NPI measures, we observed an out of season surge of the detection rates of Boca-, Corona-, Parainfluenzaviruses, and RSV. In contrast, no increased detection frequency was observed for Influenza-, Metapneumo-, and Rotaviruses as of January 2022. Conclusion: Corona-, Boca-, Parainfluenzaviruses, and RSV returned as frequently detected pathogens after withdrawal of major NPI measures. The out of season rise might be attributed to an "immune-debt" due to missing contact to viral antigens resulting in waning of population immunity during the COVID-19 pandemic.

ELF5 is a potential respiratory epithelial cell-specific risk gene for severe COVID-19.

Despite two years of intense global research activity, host genetic factors that predispose to a poorer prognosis of COVID-19 infection remain poorly understood. Here, we prioritise eight robust (e.g., ELF5) or suggestive but unreported (e.g., RAB2A) candidate protein mediators of COVID-19 outcomes by integrating results from the COVID-19 Host Genetics Initiative with population-based plasma proteomics using statistical colocalisation. The transcription factor ELF5 (ELF5) shows robust and directionally consistent associations across different outcome definitions, including a >4-fold higher risk (odds ratio: 4.88; 95%-CI: 2.47-9.63; p-value < 5.0 × 10-6) for severe COVID-19 per 1 s.d. higher genetically predicted plasma ELF5. We show that ELF5 is specifically expressed in epithelial cells of the respiratory system, such as secretory and alveolar type 2 cells, using single-cell RNA sequencing and immunohistochemistry. These cells are also likely targets of SARS-CoV-2 by colocalisation with key host factors, including ACE2 and TMPRSS2. In summary, large-scale human genetic studies together with gene expression at single-cell resolution highlight ELF5 as a risk gene for severe COVID-19, supporting a role of epithelial cells of the respiratory system in the adverse host response to SARS-CoV-2.

Psychosocial Wellbeing of Schoolchildren during the COVID-19 Pandemic in Berlin, Germany, June 2020 to March 2021.

The COVID-19 pandemic and related restrictions have affected the wellbeing of schoolchildren worldwide, but the extent and duration of specific problems are still not completely understood. We aimed to describe students' psychosocial and behavioral parameters and associated factors during the COVID-19 pandemic in Berlin, Germany. Our longitudinal study included 384 students from 24 randomly selected Berlin primary and secondary schools, assessing psychosocial wellbeing at four time points between June 2020 and March 2021. We analyzed temporal changes in the proportions of anxiety, fear of infection, reduced health-related quality of life (HRQoL), physical activity and social contacts, as well as sociodemographic and economic factors associated with anxiety, fear of infection and HRQoL. During the observation period, the presence of anxiety symptoms increased from 26.2% (96/367) to 34.6% (62/179), and fear of infection from 28.6% (108/377) to 40.6% (73/180). The proportion of children with limited social contacts (<1/week) increased from 16.4% (61/373) to 23.5% (42/179). Low physical activity (<3 times sports/week) was consistent over time. Low HRQoL was observed among 44% (77/174) of children. Factors associated with anxiety were female sex, increasing age, secondary school attendance, lower household income, and the presence of adults with anxiety symptoms in the student´s household. Fear of infection and low HRQoL were associated with anxiety. A substantial proportion of schoolchildren experienced unfavorable psychosocial conditions during the COVID-19 pandemic in 2020/2021. Students from households with limited social and financial resilience require special attention.

Impact of lockdown during the COVID-19 pandemic on health status in patients with cystic fibrosis: a mono-centre observational study.

FEV1 % predicted decreased substantially in paediatric patients with #cysticfibrosis during the first lockdown of the ongoing #SARSCoV2 pandemic in Germany. More information on consequences of repetitive shutdowns in people with cystic fibrosis is needed. https://bit.ly/3fZwuIb.

Impact of lockdown during the COVID-19 pandemic on health status in patients with cystic fibrosis- a mono-center observational study

Patients with cystic fibrosis (CF) suffer from impaired mucociliary clearance making them more susceptible to a spectrum of inhaled pathogens including bacteria, fungi and viruses [1, 2]. Therefore, patients with CF were considered at high risk for serious illness following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leading to “shielding or cocooning policies” [3]. Usually, patients with CF are cared for in specialised CF clinics with regular check-up visits. It was shown that regular care by multidisciplinary CF teams with highly trained staff lead to improvement in clinical outcomes in patient with CF [4]. At our centre, patients are usually seen at least quarterly. Between March and May 2020, at the peak of the first wave of SARS-CoV-2 infection in Germany, national authorities imposed an 8-week lockdown period on the entire population. During this period, hospitals and health care facilities reduced elective health care services including outpatient clinics to a minimum to focus their resources on the care of patients with coronavirus disease 2019 (COVID-19). Accordingly, all scheduled visits to our CF outpatient clinic were postponed and patients were advised to stay in home isolation. Additionally, due to lockdown measures, the patients’ possibilities for performing supportive therapies such as chest physiotherapy or physical exercise were limited. Hospital admission in case of severe clinical deterioration, e.g. pulmonary exacerbation, was available. Replacement of scheduled visits by e.g. video consultation or care by non-physician members of the multidisciplinary team was not yet implemented at this first lock-down period.

Untimely TGFß responses in COVID-19 limit antiviral functions of NK cells.

SARS-CoV-2 is a single-stranded RNA virus that causes COVID-19. Given its acute and often self-limiting course, it is likely that components of the innate immune system play a central part in controlling virus replication and determining clinical outcome. Natural killer (NK) cells are innate lymphocytes with notable activity against a broad range of viruses, including RNA viruses1,2. NK cell function may be altered during COVID-19 despite increased representation of NK cells with an activated and adaptive phenotype3,4. Here we show that a decline in viral load in COVID-19 correlates with NK cell status and that NK cells can control SARS-CoV-2 replication by recognizing infected target cells. In severe COVID-19, NK cells show defects in virus control, cytokine production and cell-mediated cytotoxicity despite high expression of cytotoxic effector molecules. Single-cell RNA sequencing of NK cells over the time course of the COVID-19 disease spectrum reveals a distinct gene expression signature. Transcriptional networks of interferon-driven NK cell activation are superimposed by a dominant transforming growth factor-ß (TGFß) response signature, with reduced expression of genes related to cell-cell adhesion, granule exocytosis and cell-mediated cytotoxicity. In severe COVID-19, serum levels of TGFß peak during the first two weeks of infection, and serum obtained from these patients severely inhibits NK cell function in a TGFß-dependent manner. Our data reveal that an untimely production of TGFß is a hallmark of severe COVID-19 and may inhibit NK cell function and early control of the virus.

A Retrospective Outbreak Investigation of a COVID-19 Case Cluster in a Berlin Kindergarten, November 2020.

While SARS-CoV-2 infection activity in German kindergartens during the first year of the pandemic appeared to be overall low, outbreaks did occur. We retrospectively investigated an outbreak in November and December 2020 in a Berlin kindergarten participating in the Berlin Corona School and Kindergarten Study (BECOSS). Interviews were conducted with affected families regarding symptomatology, contact persons and possible sources of infection, as well as relevant information on the conditions on-site and infection prevention measures. A chronology of the outbreak was elaborated, and based on data on contacts and symptoms, we mapped the most likely chains of infection. Overall, 24 individuals, including ten educators, seven children, and seven household members, were infected with SARS-CoV-2 in a four-week time interval. Courses of infection ranged from asymptomatic to severe, with children less affected by symptoms. Viral spread within the facility seemed to occur mainly through kindergarten staff, while children primarily transmitted infections within their families. Interviewees reported that hygiene measures were not always adhered to inside the facility. To prevent outbreaks in kindergartens, especially in the light of current and newly emerging viral variants of concern, strict compliance to hygiene rules, staff vaccinations against SARS-CoV-2, and immediate reaction to suspected cases by quarantining and frequent testing seem reasonable measures.

Analysis of Severe Acute Respiratory Syndrome 2 Replication in Explant Cultures of the Human Upper Respiratory Tract Reveals Broad Tissue Tropism of Wild-Type and B.1.1.7 Variant Viruses.

BACKGROUND: The upper respiratory tract (URT) is the primary entry site for severe acute respiratory syndrome 2 (SARS-CoV-2) and other respiratory viruses, but its involvement in viral amplification and pathogenesis remains incompletely understood. METHODS: In this study, we investigated primary nasal epithelial cultures, as well as vital explanted tissues, to scrutinize the tropism of wild-type SARS-CoV-2 and the recently emerged B.1.1.7 variant. RESULTS: Our analyses revealed a widespread replication competence of SARS-CoV-2 in polarized nasal epithelium as well as in the examined URT and salivary gland tissues, which was also shared by the B.1.1.7 virus. CONCLUSIONS: In our analyses, we highlighted the active role of these anatomic sites in coronavirus disease 2019.

SARS-CoV-2 infection and transmission in school settings during the second COVID-19 wave: a cross-sectional study, Berlin, Germany, November 2020.

BackgroundSchool attendance during the COVID-19 pandemic is intensely debated.AimIn November 2020, we assessed SARS-CoV-2 infections and seroreactivity in 24 randomly selected school classes and connected households in Berlin, Germany.MethodsWe collected oro-nasopharyngeal swabs and blood samples, examining SARS-CoV-2 infection and IgG antibodies by RT-PCR and ELISA. Household members self-swabbed. We assessed individual and institutional prevention measures. Classes with SARS-CoV-2 infection and connected households were retested after 1 week.ResultsWe examined 1,119 participants, including 177 primary and 175 secondary school students, 142 staff and 625 household members. SARS-CoV-2 infection occurred in eight classes, affecting each 1-2 individuals. Infection prevalence was 2.7% (95% confidence interval (CI): 1.2-5.0; 9/338), 1.4% (95% CI: 0.2-5.1; 2/140), and 2.3% (95% CI: 1.3-3.8; 14/611) among students, staff and household members. Six of nine infected students were asymptomatic at testing. We detected IgG antibodies in 2.0% (95%CI: 0.8-4.1; 7/347), 1.4% (95% CI: 0.2-5.0; 2/141) and 1.4% (95% CI: 0.6-2.7; 8/576). Prevalence increased with inconsistent facemask-use in school, walking to school, and case-contacts outside school. For three of nine households with infection(s), origin in school seemed possible. After 1 week, no school-related secondary infections appeared in affected classes; the attack rate in connected households was 1.1%.ConclusionSchool attendance under rigorously implemented preventive measures seems reasonable. Balancing risks and benefits of school closures need to consider possible spill-over infection into households. Deeper insight is required into the infection risks due to being a schoolchild vs attending school.

Maintenance of Elective Patient Care at Berlin University Children's Hospital During the COVID-19 Pandemic.

Background: In Germany, so far the COVID-19 pandemic evolved in two distinct waves, the first beginning in February and the second in July, 2020. The Berlin University Children's Hospital at Charité (BCH) had to ensure treatment for children not infected and infected with SARS-CoV-2. Prevention of nosocomial SARS-CoV-2 infection of patients and staff was a paramount goal. Pediatric hospitals worldwide discontinued elective treatments and established a centralized admission process. Methods: The response of BCH to the pandemic adapted to emerging evidence. This resulted in centralized admission via one ward exclusively dedicated to children with unclear SARS-CoV-2 status and discontinuation of elective treatment during the first wave, but maintenance of elective care and decentralized admissions during the second wave. We report numbers of patients treated and of nosocomial SARS-CoV-2 infections during the two waves of the pandemic. Results: During the first wave, weekly numbers of inpatient and outpatient cases declined by 37% (p < 0.001) and 29% (p = 0.003), respectively. During the second wave, however, inpatient case numbers were 7% higher (p = 0.06) and outpatient case numbers only 6% lower (p = 0.25), compared to the previous year. Only a minority of inpatients were tested positive for SARS-CoV-2 by RT-PCR (0.47% during the first, 0.63% during the second wave). No nosocomial infection of pediatric patients by SARS-CoV-2 occurred. Conclusion: In contrast to centralized admission via a ward exclusively dedicated to children with unclear SARS-CoV-2 status and discontinuation of elective treatments, maintenance of elective care and decentralized admission allowed the almost normal use of hospital resources, yet without increased risk of nosocomial infections with SARS-CoV-2. By this approach unwanted sequelae of withheld specialized pediatric non-emergency treatment to child and adolescent health may be avoided.

SARS-CoV-2 infections in kindergartens and associated households at the start of the second wave in Berlin, Germany-a cross-sectional study.

Actual surveys in kindergartens on SARS-CoV-2 infections are rare. At the beginning of the second pandemic wave, we screened 12 randomly selected kindergartens in Berlin, Germany. A total of 720 participants (pre-school children, staff and connected household members) were briefly examined and interviewed, and SARS-CoV-2 infections and anti-SARS-Cov-2 IgG antibodies were assessed. About a quarter of the participants showed common cold-resembling symptoms. However, no SARS-CoV-2 infection was detected, and only one childcare worker showed IgG seroreactivity. Against a backdrop of increased pandemic activity in the community, this cross-sectional study does not suggest that kindergartens are silent transmission reservoirs.

Cross-reactive CD4+ T cells enhance SARS-CoV-2 immune responses upon infection and vaccination.

The functional relevance of preexisting cross-immunity to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a subject of intense debate. Here, we show that human endemic coronavirus (HCoV)­reactive and SARS-CoV-2­cross-reactive CD4+ T cells are ubiquitous but decrease with age. We identified a universal immunodominant coronavirus-specific spike peptide (S816-830) and demonstrate that preexisting spike- and S816-830­reactive T cells were recruited into immune responses to SARS-CoV-2 infection and their frequency correlated with anti­SARS-CoV-2-S1-IgG antibodies. Spike­cross-reactive T cells were also activated after primary BNT162b2 COVID-19 messenger RNA vaccination and displayed kinetics similar to those of secondary immune responses. Our results highlight the functional contribution of preexisting spike­cross-reactive T cells in SARS-CoV-2 infection and vaccination. Cross-reactive immunity may account for the unexpectedly rapid induction of immunity after primary SARS-CoV-2 immunization and the high rate of asymptomatic or mild COVID-19 disease courses.

Self-collected oral, nasal and saliva samples yield sensitivity comparable to professionally collected oro-nasopharyngeal swabs in SARS-CoV-2 diagnosis among symptomatic outpatients.

INTRODUCTION: Containing COVID-19 requires broad-scale testing. However, sample collection requires qualified personnel and protective equipment and may cause transmission. We assessed the sensitivity of SARS-CoV-2-rtPCR applying three self-sampling techniques as compared to professionally collected oro-nasopharyngeal samples (cOP/NP). METHODS: From 62 COVID-19 outpatients, we obtained: (i) multi-swab, MS; (ii) saliva sponge combined with nasal vestibula, SN; (iii) gargled water, GW; (iv) professionally collected cOP/NP (standard). We compared ct-values for E-gene and ORF1ab and analysed variables reducing sensitivity of self-collecting procedures. RESULTS: The median ct-values for E-gene and ORF1ab obtained in cOP/NP samples were 20.7 and 20.2, in MS samples 22.6 and 21.8, in SN samples 23.3 and 22.3, and in GW samples 30.3 and 29.8, respectively. MS and SN samples showed sensitivities of 95.2% (95%CI, 86.5-99.0) and GW samples of 88.7% (78.1-95.3). Sensitivity was inversely correlated with ct-values, and became <90% for samples obtained more than 8 days after symptom onset. For MS and SN samples, false negativity was associated with language problems, sampling errors, and symptom duration. CONCLUSION: Conclusions from this study are limited to the sensitivity of self-sampling in mildly to moderately symptomatic patients. Still, self-collected oral/nasal/saliva samples can facilitate up-scaling of testing in early symptomatic COVID-19 patients if operational errors are minimized.

Mild COVID-19 despite autoantibodies against type I IFNs in autoimmune polyendocrine syndrome type 1.

Autoantibodies against IFN-α and IFN-ω (type I IFNs) were recently reported as causative for severe COVID-19 in the general population. Autoantibodies against IFN-α and IFN-ω are present in almost all patients with autoimmune polyendocrine syndrome type 1 (APS-1) caused by biallelic deleterious or heterozygous dominant mutations in AIRE. We therefore hypothesized that autoantibodies against type I IFNs also predispose patients with APS-1 to severe COVID-19. We prospectively studied 6 patients with APS-1 between April 1, 2020 and April 1, 2021. Biobanked pre-COVID-19 sera of APS-1 subjects were tested for neutralizing autoantibodies against IFN-α and IFN-ω. The ability of the patients' sera to block recombinant human IFN-α and IFN-ω was assessed by assays quantifying phosphorylation of signal transducer and activator of transcription 1 (STAT1) as well as infection-based IFN-neutralization assays. We describe 4 patients with APS-1 and preexisting high titers of neutralizing autoantibodies against IFN-α and IFN-ω who contracted SARS-CoV-2, yet developed only mild symptoms of COVID-19. None of the patients developed dyspnea, oxygen requirement, or high temperature. All infected patients with APS-1 were females and younger than 26 years of age. Clinical penetrance of neutralizing autoantibodies against type I IFNs for severe COVID-19 is not complete.