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Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential therapeutics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-induced antiviral activity and its susceptibility to viral antagonism remain incompletely understood. iBET treatment transiently inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. Our functional assays confirmed JQ-1-mediated downregulation of ACE2 expression and multi-omics analysis uncovered induction of an antiviral NRF-2-mediated cytoprotective response as an additional antiviral component of JQ-1 treatment. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variants. JQ-1 antiviral activity was transient in human bronchial airway epithelial cells (hBAECs) treated prior to infection and absent when administered therapeutically. We propose that JQ-1 exerts pleiotropic effects that collectively induce a transient antiviral state that is ultimately nullified by an established SARS-CoV-2 infection, raising questions on their clinical suitability in the context of COVID-19.
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During the SARS-CoV-2 pandemic, multiple variants with differing amounts of escape from pre-existing immunity have emerged, causing concerns about continued protection. Here, we use antigenic cartography to quantify and visualize the antigenic relationships among 16 SARS-CoV-2 variants titrated against serum samples taken post-vaccination and post-infection with seven different variants. We find major antigenic differences caused by substitutions at spike positions 417, 452, 484, and possibly 501. B.1.1.529 (Omicron BA.1) showed the highest escape from all sera tested. Visualization of serological responses as antibody landscapes shows how reactivity clusters in different regions of antigenic space. We find changes in immunodominance of different spike regions depending on the variant an individual was exposed to, with implications for variant risk assessment and vaccine strain selection. One sentence summaryAntigenic Cartography of SARS-CoV-2 variants reveals amino acid substitutions governing immune escape and immunodominance patterns.
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PurposeSix-19% of critically ill COVID-19 patients display circulating auto-antibodies against type I interferons (IFN-AABs). Here, we establish a clinically applicable strategy for early identification of IFN-AAB-positive patients for potential subsequent clinical interventions. MethodsWe analysed sera of 430 COVID-19 patients with severe and critical disease from four hospitals for presence of IFN-AABs by ELISA. Binding specificity and neutralizing activity were evaluated via competition assay and virus-infection-based neutralization assay. We defined clinical parameters associated with IFN-AAB positivity. In a subgroup of critically ill patients, we analyzed effects of therapeutic plasma exchange (TPE) on the levels of IFN-AABs, SARS-CoV-2 antibodies and clinical outcome. ResultsThe prevalence of neutralizing AABs to IFN- and IFN-{omega} in COVID-19 patients was 4.2% (18/430), while being undetectable in an uninfected control cohort. Neutralizing IFN-AABs were detectable exclusively in critically affected, predominantly male (83%) patients (7.6% IFN- and 4.6% IFN-{omega} in 207 patients with critical COVID-19). IFN-AABs were present early post-symptom onset and at the peak of disease. Fever and oxygen requirement at hospital admission co-presented with neutralizing IFN-AAB positivity. IFN-AABs were associated with higher mortality (92.3% versus 19.1 % in patients without IFN-AABs). TPE reduced levels of IFN-AABs in three of five patients and may increase survival of IFN-AAB-positive patients compared to those not undergoing TPE. ConclusionIFN-AABs may serve as early biomarker for development of severe COVID-19. We propose to implement routine screening of hospitalized COVID-19 patients according to our algorithm for rapid identification of patients with IFN-AABs who most likely benefit from specific therapies.
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ObjectiveTo analyze humoral and cellular immune responses to SARS-CoV-2 vaccinations and infections in anti-CD20 treated patients with multiple sclerosis (pwMS). Methods181 pwMS on anti-CD20 therapy and 41 pwMS who began anti-CD20 therapy were included in a prospective, observational, single-center cohort study between March 2020 and August 2021. 51 pwMS under anti-CD20 treatment, 14 anti-CD20 therapy-naive pwMS and 19 healthy controls (HC) were vaccinated twice against SARS-CoV-2. We measured SARS-CoV-2 spike protein (full-length, S1 domain and receptor binding domain) immunoglobulin (Ig)G and S1 IgA and virus neutralizing capacity and avidity of SARS-CoV-2 antibodies. SARS-CoV-2 specific T cells were determined by interferon-{gamma} release assays. ResultsFollowing two SARS-CoV-2 vaccinations, levels of IgG and IgA antibodies to SARS-CoV-2 spike protein as well as neutralizing capacity and avidity of SARS-CoV-2 IgG were lower in anti-CD20 treated pwMS than in anti-CD20 therapy-naive pwMS and in HC (p<0.003 for all pairwise comparisons). However, in all anti-CD20 treated pwMS vaccinated twice (n=26) or infected with SARS-CoV-2 (n=2), in whom SARS-CoV-2 specific T cells could be measured, SARS-CoV-2 specific T cells were detectable, at levels similar to those of twice-vaccinated anti-CD20 therapy-naive pwMS (n=7) and HC (n=19). SARS-CoV-2 S1 IgG levels (r=0.42, p=0.002), antibody avidity (r=0.7, p<0.001) and neutralizing capacity (r=0.44, p=0.03) increased with time between anti-CD20 infusion and second vaccination. Based on detection of SARS-CoV-2 antibodies, SARS-CoV-2 infections occurred in 4/175 (2.3%) anti-CD20 treated pwMS, all of whom recovered fully. InterpretationThese findings should inform treatment decisions and SARS-CoV-2 vaccination management in pwMS.
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Virus mutations have the potential to impact the accuracy of diagnostic tests. The SARS-CoV-2 B.1.1.7 lineage is defined by a large number of mutations in the spike gene and four in the nucleocapsid (N) gene. Most commercially available SARS-CoV-2 antigen-detecting rapid tests (Ag-RDTs) target the viral N-protein, encoded by the N-gene. We conducted a manufacturer-independent, prospective diagnostic accuracy study of three SARS-CoV-2 Ag-RDTs that are currently under review by the WHO Emergency Use Listing Procedure (Espline -Fujirebio Inc.; Sure Status -Premier Medical Corporation Private Limited; Mologic -Mologic Ltd.) and report here on an additional sub-analysis regarding the B.1.1.7 lineage. During the study, in Berlin and Heidelberg, Germany, from 20 January to 15 April 2021, B.1.1.7 rapidly became the dominant SARS-CoV-2 lineage at the testing sites and was detected in 220 (62%) of SARS-CoV-2 RT-PCR positive patients. All three Ag-RDTs yielded comparable sensitivities irrespective of an infection with the B.1.1.7 lineage or not. There is only limited data on how N-gene mutations in variants of concern may impact Ag-RDTs. Currently, no major changes to test performance are anticipated. However, test developers and health authorities should assess and monitor the impact of emerging variants on the accuracy of Ag-RDTs.
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BackgroundRapid antigen-detecting tests (Ag-RDTs) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transform pandemic control. Thus far, sensitivity ([≤]85%) of lateral-flow assays has limited scale-up. Conceivably, microfluidic immunofluorescence Ag-RDTs could increase sensitivity for SARS-CoV-2 detection. Materials and MethodsThis multi-centre diagnostic accuracy study investigated performance of the microfluidic immunofluorescence LumiraDx assay, enrolling symptomatic and asymptomatic participants with suspected SARS-CoV-2 infection. Participants collected a supervised nasal mid-turbinate (NMT) self-swab for Ag-RDT testing, in addition to a professionally-collected nasopharyngeal (NP) swab for routine testing with reverse transcriptase polymerase chain reaction (RT-PCR). Results were compared to calculate sensitivity and specificity. Sub-analyses investigated the results by viral load, symptom presence and duration. An analytical study assessed exclusivity and limit-of-detection (LOD). In addition, we evaluated ease-of-use. ResultsStudy conduct was between November 2nd 2020 and January 21st 2021. 761 participants were enrolled, with 486 participants reporting symptoms on testing day. 120 out of 146 RT-PCR positive cases were detected positive by LumiraDx, resulting in a sensitivity of 82.2% (95% CI: 75.2%-87.5%). Specificity was 99.3% (CI: 98.3-99.7%). Sensitivity was increased in individuals with viral load [≥] 7 log10 SARS-CoV2 RNA copies/ml (93.8%; CI: 86.2%-97.3%). Testing against common respiratory commensals and pathogens showed no cross-reactivity and LOD was estimated to be 2-56 PFU/mL. The ease-of-use-assessment was favourable for lower throughput settings. ConclusionThe LumiraDx assay showed excellent analytical sensitivity, exclusivity and clinical specificity with good clinical sensitivity using supervised NMT self-sampling.
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ObjectivesThe aim of this diagnostic accuracy study was direct comparison of two different nasal sampling methods for an antigen-based rapid diagnostic test (Ag-RDT) that detects severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, the accuracy and feasibility of self-sampling was evaluated. MethodsThis manufacturer-independent, prospective diagnostic accuracy study, compared professional anterior nasal (AN) and nasal mid-turbinate (NMT) sampling for a WHO-listed SARS-CoV-2 Ag-RDT. A second group of participants collected a NMT sample themselves and underwent a professional nasopharyngeal swab for comparison. The reference standard was real-time polymerase chain reaction (RT-PCR) using combined oro-/nasopharyngeal sampling. Individuals with high suspicion of SARS-CoV-2 infection were tested. Sensitivity, specificity, and percent agreement were calculated. Self-sampling was observed without intervention. Feasibility was evaluated by observer and participant questionnaires. ResultsAmong 132 symptomatic adults, both professional AN- and NMT-sampling yielded a sensitivity of 86.1% (31/36 RT-PCR positives detected; 95%CI: 71.3-93.9) and a specificity of 100.0% (95%CI: 95.7-100). The positive percent agreement (PPA) was 100% (95%CI: 89.0-100). Among 96 additional adults, self NMT- and professional NP-sampling yielded an identical sensitivity of 91.2% (31/34; 95%CI 77.0-97.0). Specificity was 98.4% (95%CI: 91.4-99.9) with NMT- and 100.0% (95%CI: 94.2-100) with NP-sampling. The PPA was 96.8% (95%CI: 83.8-99.8). Most participants (85.3%) considered self-sampling as easy to perform. ConclusionProfessional AN- and NMT-sampling are of equivalent accuracy for an Ag-RDT in ambulatory symptomatic adults. Participants were able to reliably perform the NMT-sampling themselves, following written and illustrated instructions. Nasal self-sampling will likely facilitate scaling of SARS-CoV-2 antigen testing.
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BackgroundAntigen-detecting rapid diagnostic tests (Ag-RDTs) have been widely recommended as a complement to RT-PCR. Considering the possibility of nasal self-sampling and the ease-of-use in performing the test, self-testing may be an option. Methods and FindingsWe performed a manufacturer-independent, prospective diagnostic accuracy study of nasal mid-turbinate self-sampling and self-testing when using a WHO-listed SARS-CoV-2 Ag-RDT. Symptomatic participants suspected to have COVID-19 received written and illustrated instructions. Procedures were observed without intervention. For comparison, Ag-RDTs with nasopharyngeal sampling were professionally performed. Estimates of agreement, sensitivity, and specificity relative to RT-PCR on a combined oro-/nasopharyngeal sample were calculated. Feasibility was evaluated by observer and participant questionnaires. Among 146 symptomatic adults, 40 (27.4%) were RT-PCR-positive for SARS-CoV-2. Sensitivity with self-testing was 82.5% (33/40 RT-PCR positives detected; 95% CI 68.1-91.3), and 85.0% (34/40; 95% CI 70.9-92.9) with professional testing. The positive percent agreement between self-testing and professional testing on Ag-RDT was 91.4% (95% CI 77.6-97.0), and negative percent agreement 99.1% (95% CI 95.0-100). At high viral load (>7.0 log10 SARS-CoV-2 RNA copies/ml), sensitivity was 96.6% (28/29; 95% CI 82.8-99.8) for both self- and professional testing. Deviations in sampling and testing (incomplete self-sampling or extraction procedure, or imprecise volume applied on the test device) were observed in 25 out of the 40 PCR-positives. Participants were rather young (mean age 35 years) and educated (59.6% with higher education degree). Most participants (80.9%) considered the Ag-RDT as rather easy to perform. ConclusionsAmbulatory participants suspected for SARS-CoV-2 infection were able to reliably perform the Ag-RDT and test themselves. Procedural errors might be reduced by refinement of the Ag-RDTs for self-testing, such as modified instructions for use or product design/procedures. Self-testing may result in more wide-spread and more frequent testing. Paired with the appropriate information and education of the general public about the benefits and risks, self-testing may therefore have significant impact on the pandemic.
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BackgroundSince the beginning of the coronavirus disease 2019 (COVID-19) pandemic, there has been increasing demand to identify predictors of severe clinical course in patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Human leukocyte antigen alleles (HLA) have been suggested as potential genetic host factors. We sought to evaluate this hypothesis by conducting an international multicenter study using HLA sequencing with subsequent independent validation. MethodsWe analyzed a total of 332 samples. First, we enrolled 233 patients in Germany, Spain, and Switzerland for HLA and whole exome sequencing. Furthermore, we validated our results in a public data set (United States, n=99). Patients older than 18 years presenting with COVID-19 were included, representing the full spectrum of the disease. HLA candidate alleles were identified in the derivation cohort (n=92) and tested in two independent validation cohorts (n=240). ResultsWe identified HLA-C* 04:01 as a novel genetic predictor for severe clinical course in COVID-19. Carriers of HLA-C* 04:01 had twice the risk of intubation when infected with SARS-CoV-2 (hazard ratio 2.1, adjusted p-value=0.0036). Importantly, these findings were successfully replicated in an independent data set. Furthermore, our findings are biologically plausible, as HLA-C* 04:01 has fewer predicted bindings sites with relevant SARS-CoV-2 peptides as compared to other HLA alleles. Exome sequencing confirmed findings from HLA analysis. ConclusionsHLA-C* 04:01 carriage is associated with a twofold increased risk of intubation in patients infected with SARS-CoV-2. Testing for HLA-C* 04:01 could have clinical implications to identify high-risk patients and individualize management.
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BackgroundAdequate patient allocation is pivotal for optimal resource management in strained healthcare systems, and requires detailed knowledge of clinical and virological disease trajectories. MethodsA cohort of 168 hospitalized adult COVID-19 patients enrolled in a prospective observational study at a large European tertiary care center was analyzed. ResultsForty-four percent (71/161) of patients required invasive mechanical ventilation (IMV). Shorter duration of symptoms before admission (aOR 1.22 per day less, 95%CI 1.10-1.37, p<0.01), age 60-69 as compared to 18-59 years (aOR 4.33, 95%CI 1.07-20.10, p=0.04), and history of hypertension (aOR 5.55, 95%CI 2.00-16.82, p<0.01) were associated with need for IMV. Patients on IMV had higher maximal concentrations, slower decline rates, and longer shedding of SARS-CoV-2 than non-IMV patients (33 days, IQR 26-46.75, vs 18 days, IQR 16-46.75, respectively, p<0.01). Median duration of hospitalization was 9 days (IQR 6-15.5) for non-IMV and 49.5 days (IQR 36.8-82.5) for IMV-patients. ConclusionOur results indicate a short duration of symptoms before admission as a risk factor for severe disease and different viral load kinetics in severely affected patients.
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BackgroundNasopharyngeal (NP) swab samples for antigen-detecting rapid diagnostic tests (Ag-RDTs) require qualified healthcare professionals and are frequently perceived as uncomfortable by patients. MethodsWe performed a manufacturer-independent, prospective diagnostic accuracy study, comparing professional-collected nasal mid-turbinate (NMT) to nasopharyngeal swab, using the test kits of a WHO-listed SARS-CoV-2 Ag-RDT (STANDARD Q COVID-19 Ag Test, SD Biosensor), which is also being distributed by Roche. Individuals with high suspicion for COVID-19 infection were tested. The reference standard was RT-PCR using a combined oro-/nasopharyngeal swab sample. Percent positive and negative agreement, as well as sensitivity and specificity were calculated. ResultsAmong the 179 participants, 41 (22.9%) tested positive for SARS-CoV-2 by RT-PCR. The positive percent agreement of the two different sampling techniques for the Ag-RDT was 93.5% (CI 79.3-98.2). The negative percent agreement was 95.9% (CI 91.4-98.1). The Ag-RDT with NMT-sampling showed a sensitivity of 80.5% (33/41 PCR positives detected; CI 66.0-89.8) and specificity of 98.6% (CI 94.9-99.6) compared to RT-PCR. The sensitivity with NP-sampling was 73.2% (30/41 PCR positives detected; CI 58.1-84.3) and specificity was 99.3% (CI 96.0-100). In patients with high viral load (>7.0 log10 SARS-CoV-2 RNA copies/swab), the sensitivity of the Ag-RDT with NMT-sampling was 100% and 94.7% with NP-sampling. ConclusionThis study demonstrates that sensitivity of a WHO-listed SARS-CoV-2 Ag-RDT using a professional nasal-sampling kit is at least equal to that of the NP-sampling kit, although confidence intervals overlap. Of note, differences in the IFUs of the test procedures could have contributed to different sensitivities. NMT-sampling can be performed with less training, reduces patient discomfort, and it enables scaling of antigen testing strategies. Additional studies of patient self-sampling should be considered to further facilitate the scaling-up of Ag-RDT testing.
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As children are under-represented in current studies aiming to analyse transmission of SARS-coronavirus 2 (SARS-CoV-2), their contribution to transmission is unclear. Viral load, as measured by RT-PCR, can inform considerations regarding transmission, especially if existing knowledge of viral load in other respiratory diseases is taken into account. RT-PCR threshold cycle data from 3303 patients who tested positive for SARS-CoV-2 (out of 77,996 persons tested in total, drawn from across Germany) were analysed to examine the relationship between patient age and estimated viral load. Two PCR systems were used. In data from the PCR system predominantly used for community and cluster screening during the early phase of the epidemic (Roche LightCycler 480 II), when such screening was frequent practice, viral loads do not differ significantly in three comparisons between young and old age groups (differences in log10 viral loads between young and old estimated from raw viral load data and a Bayesian mixture model of gamma distributions collectively range between -0.11 and -0.43). Data from a second type of PCR system (Roche cobas 6800/8800), introduced into diagnostic testing on March 16, 2020 and used during the time when household and other contact testing was reduced, show a credible but small difference in the three comparisons between young and old age groups (differences, measured as above, collectively range between -0.43 and -0.83). This small difference may be due to differential patterns of PCR instrument utilization rather than to an actual difference in viral load. Considering household transmission data on influenza, which has a similar viral load kinetic to SARS-CoV-2, the viral load differences between age groups observed in this study are likely to be of limited relevance. Combined data from both PCR instruments show that viral loads of at least 250,000 copies, a threshold we previously established for the isolation of infectious virus in cell culture at more than 5% probability, were present across the study period in 29.0% of kindergarten-aged patients 0-6 years old (n=38), 37.3% of those aged 0-19 (n=150), and in 51.4% of those aged 20 and above (n=3153). The differences in these fractions may also be due to differences in test utilization. We conclude that a considerable percentage of infected people in all age groups, including those who are pre- or mild-symptomatic, carry viral loads likely to represent infectivity. Based on these results and uncertainty about the remaining incidence, we recommend caution and careful monitoring during gradual lifting of non-pharmaceutical interventions. In particular, there is little evidence from the present study to support suggestions that children may not be as infectious as adults.