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The COVID-19 pandemic has greatly impacted the global economy and health care systems, illustrating the urgent need for timely and inexpensive responses to a pandemic threat in the form of vaccines and antigen tests. The causative agent of COVID-19 is SARS-CoV-2. The spike protein on the virus surface interacts with the human angiotensin-converting enzyme (ACE2) via the so-called receptor binding domain (RBD), facilitating virus entry. The RBD thus represents a prime target for vaccines, therapeutic antibodies, and antigen test systems. Currently, antigen testing is mostly conducted by qualitative flow chromatography or via quantitative ELISA-type assays. The latter mostly utilize materials like protein-adhesive polymers and gold or latex particles. Here we present an alternative ELISA approach using inexpensive materials and permitting quick detection based on components produced in the microbial model Ustilago maydis. In this fungus, heterologous proteins like biopharmaceuticals can be exported by fusion to unconventionally secreted chitinase Cts1. As a unique feature, the carrier chitinase binds to chitin allowing its additional use as a purification or immobilization tag. In this study, we produced different mono- and bivalent SARS-CoV-2 nanobodies directed against the viral RBD as Cts1 fusions and screened their RBD binding affinity in vitro and in vivo. Functional nanobody-Cts1 fusions were immobilized on chitin forming an RBD tethering surface. This provides a solid base for future development of an inexpensive antigen test utilizing unconventionally secreted nanobodies as RBD trap and a matching ubiquitous and biogenic surface for immobilization.
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We provide follow-up data on the humoral immune response after COVID-19 vaccinations of a cohort aged below 60 and over 80 years. While anti-SARS-CoV-2 spike-specific IgG and neutralization capacity waned rapidly after initial vaccination, additional boosters highly benefitted humoral immune responses including neutralization of Omikron variants in the elderly cohort.
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BackgroundModification of vaccination strategies is needed to improve the immune response to SARS-CoV-2 vaccination in kidney transplant recipients (KTRs). MethodsThis multicenter observational study aimed to determine antibody kinetics among 60 seropositive KTRs and analyzed the effects of the third vaccination against SARS-CoV-2 in 174 previously seronegative KTRs. We investigated whether mycophenolate mofetil (MMF) dose reduction by 25-50% prior the third vaccination influences vaccination success. Results18 of 60 (30%) seropositive KTRs became seronegative in the serological assay within six months. Loss of antibodies was predicted by low initial antibody levels ([≤]206.8 BAU/ml), older age, and impaired graft function. A third vaccination in previously seronegative KTRs induced seroconversion in 56 of 174 (32.1%) KTRs with median antibody levels 119 (76-353) BAU/ml and median neutralizing capacity titer of 1:10 (0- 1:40). Multivariate logistic regression revealed that initial antibody levels (OR 1.39, 95% CI 1.09-1.76), graft function (OR 0.05, 95% CI 0.01-0.39), time after transplantation (OR 1.04, 95% CI 1.02-1.07) and MMF trough levels (OR 0.43, 95% CI 0.21-0.88) correlated with seroconversion, p<0.05. After controlling for these confounders, the effect of MMF dose reduction was calculated using propensity score matching. KTRs in the MMF reduction group had significantly lower MMF serum concentrations prior to the third vaccination and were more likely to develop antibody levels [≥]35.2 BAU/ml than their matched KTRs (p=0.02). ConclusionsTemporary reduction in MMF dose might be a promising approach to improve the immune response in KTRs.
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Integration of genomic surveillance with contact tracing provides a powerful tool for the reconstruction of person-to-person pathogen transmission chains. We report two large clusters of SARS-CoV-2 cases ("Delta" clade, 110 cases combined) detected in July 2021 by Integrated Genomic Surveillance in Dusseldorf. Structured interviews and deep contact tracing demonstrated an association to a single SARS-CoV-2 infected return traveller (Cluster 1) and to return travel from Catalonia and other European countries (Cluster 2), highlighting the importance of containing travel-imported SARS-CoV-2 infections.
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IntroductionProphylactic vaccination against SARS-CoV-2 is one of the most important measures to contain the COVID-19 pandemic. Recently, break-through infections following vaccination against this virus have been reported. Here, we describe the humoral immune response of break-through infections in fully vaccinated individuals of old age from an outbreak in a nursery home. MethodsIn cooperation with the local health authority, blood samples from fully vaccinated and infected as well as fully vaccinated and uninfected residents of the nursery home were collected four weeks after the onset of the outbreak. The humoral immune response was determined in a neutralisation assay with replication-competent virus isolates and by a quantitative ELISA. ResultsIn this outbreak a total of 23 residents and four health care workers were tested positive for SARS-CoV-2. Four residents were unvaccinated, including one with a severe course of disease who later deceased. Despite their old age, all vaccinated residents showed no or only mild disease. Comparison of the humoral immune response revealed significantly higher antibody levels in fully vaccinated infected individuals compared to fully vaccinated uninfected individuals (p<0.001). Notably, although only a minority of the vaccinated uninfected group showed neutralisation capacity against SARS-CoV-2, all vaccinated and infected individuals showed high-titer neutralisation of SARS-CoV-2 including the alpha and beta variant. DiscussionLarge SARS-CoV-2 outbreaks can occur in fully vaccinated populations, but seem to associate with mild disease. SARS-CoV-2 infection in fully vaccinated individuals is a strong booster of the humoral immune response providing enhanced neutralisation capacity against immune evasion variants.
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BackgroundThe SARS-CoV-2 pandemic has led to the development of various vaccines. Real-life data on immune responses elicited in the most vulnerable group of vaccinees over 80 years old is still underrepresented despite the prioritization of the elderly in vaccination campaigns. MethodsWe conducted a cohort study with two age groups, young vaccinees below the age of 60 and elderly vaccinees over the age of 80, to compare their antibody responses to the first and second dose of the BNT162b2 COVID-19 vaccination. ResultsWhile the majority of participants in both groups produced specific IgG antibody titers against SARS-CoV-2 spike protein, titers were significantly lower in elderly participants. Although the increment of antibody levels after the second immunization was higher in elderly participants, the absolute mean titer of this group remained lower than the <60 group. After the second vaccination, 31.3 % of the elderly had no detectable neutralizing antibodies in contrast to the younger group, in which only 2.2% had no detectable neutralizing antibodies. ConclusionOur data suggests that lower frequencies of neutralizing antibodies after BNT162b2 vaccination in the elderly population may require earlier revaccination to ensure strong immunity and protection against infection.
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BackgroundThe role of pre-school children as a source and distributor of SARS-CoV-2 infections is still unclear. Daycare facilities that care particularly for young children with limited hygiene measures may contribute to the infection dynamics during the pandemic. The aim of this study was to implement and evaluate a voluntary SARS-CoV-2 screening program in daycare facilities. MethodsThe study was conducted over a period of 4 weeks, from June 10th to July 7th 2020. The aim was to screen a representative group of 5000 individuals (children and staff at a ratio 3:1) attending daycare facilities in Dusseldorf, North Rhine-Westphalia. Tests were performed twice per week with oral rinsing water as sample material for the detection of SARS-CoV-2-RNA by molecular pool testing. ResultsA total number of 5210 participants (75.9% children and 24.1% staff) from 115 day care centers participated in the study. Of a total of 34,068 returned samples (81.7%) during the study period, only one SARS-CoV-2 infection of a child was detected in the study cohort with one likely secondary infection within the daycare facility. Of note, during the study phase, no increase of SARS- CoV-2 infections was observed in daycare center compared to the overall incidence in Dusseldorf. ConclusionsA voluntary screening program for SARS-CoV-2 infections could successfully be implemented in daycare facilities. Although the low overall incidence during the study period precludes firm conclusions, there was no evidence for increased transmission in children attending daycare facilities compared to the general population of Dusseldorf. SummarySARS-CoV-2 screening programs in daycare facilities may help to detect asymptomatic infections at an early stage and thereby support containment. Here, a large screening study was evaluated suggesting similar infection rates in daycare facilities compared to the general population.
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Viral genome sequencing can address key questions about SARS-CoV-2 evolution and viral transmission. Here, we present an integrated system of genomic surveillance in the German city of Dusseldorf, combining a) viral surveillance sequencing, b) genetically based identification of infection clusters in the population, c) analysis of hospital outbreaks, d) integration of public health authority contact tracing data, and e) a user-friendly dashboard application as a central data analysis platform. The generated surveillance sequencing data (n = 320 SARS-CoV-2 genomes) showed that the development of the local viral population structure from August to December 2020 was consistent with European trends, with the notable absence of SARS-CoV-2 variants 20I/501Y.V1/B.1.1.7 and B.1.351 until the end of the local sampling period. Against a background of local surveillance and other publicly available SARS-CoV-2 data, four putative SARS-CoV-2 outbreaks at Dusseldorf University Hospital between October and December 2020 (n = 44 viral genomes) were investigated and confirmed as clonal, contributing to the development of improved infection control and prevention measures. An analysis of the generated surveillance sequencing data with respect to infection clusters in the population based on a greedy clustering algorithm identified five candidate clusters, all of which were subsequently confirmed by the integration of public health authority contact tracing data and shown to be represent transmission settings of particular relevance (schools, care homes). A joint analysis of outbreak and surveillance data identified a potential transmission of an outbreak strain from the local population into the hospital and back; and an in-depth analysis of one population infection cluster combining genetic with contact tracing data enabled the identification of a previously unrecognized population transmission chain involving a martial arts gym. Based on these results and a real-time sequencing experiment in which we demonstrated the feasibility of achieving sample-to-turnaround times of <30 hours with the Oxford Nanopore technology, we discuss the potential benefits of routine ultra-fast sequencing of all detected infections for contact tracing, infection cluster detection, and, ultimately, improved management of the SARS-CoV-2 pandemic.
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We present a small molecule chemotype, identified by an orthogonal drug screen, exhibiting nanomolar activity against members of all the six viral families causing most human respiratory viral disease, with a demonstrated barrier to resistance development. Antiviral activity is shown in mammalian cells, including human primary bronchial epithelial cells cultured to an air-liquid interface and infected with SARS-CoV-2. In animals, efficacy of early compounds in the lead series is shown by survival (for a coronavirus) and viral load (for a paramyxovirus). The drug target is shown to include a subset of the protein 14-3-3 within a transient host multi-protein complex containing components implicated in viral lifecycles and in innate immunity. This multi-protein complex is modified upon viral infection and largely restored by drug treatment. Our findings suggest a new clinical therapeutic strategy for early treatment upon upper respiratory viral infection to prevent progression to lower respiratory tract or systemic disease. One Sentence SummaryA host-targeted drug to treat all respiratory viruses without viral resistance development.
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We analysed SARS-CoV-2 specific antibody responses in 42 social and working contacts of a super-spreader from the Heinsberg area in Germany. Consistent with a high-prevalence setting 26 individuals had SARS-CoV-2 antibodies determined by in-house neutralisation testing. These results were compared with four commercial assays, suggesting limited sensitivity of the assays in such a high-prevalence setting. Although SARS-CoV-2 nucleocapsid-restricted tests showed a better sensitivity, spike-based assays had a stronger correlation with neutralisation capacity.
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COVID-19 pandemic caused by SARS-CoV-2 infection is a public health emergency. COVID-19 typically exhibits respiratory illness. Unexpectedly, emerging clinical reports indicate that neurological symptoms continue to rise, suggesting detrimental effects of SARS-CoV-2 on the central nervous system (CNS). Here, we show that a Dusseldorf isolate of SARS-CoV-2 enters 3D human brain organoids within two days of exposure. Using COVID-19 convalescent serum, we identified that SARS-CoV-2 preferably targets soma of cortical neurons but not neural stem cells, the target cell type of ZIKA virus. Imaging cortical neurons of organoids reveal that SARS-CoV-2 exposure is associated with missorted Tau from axons to soma, hyperphosphorylation, and apparent neuronal death. Surprisingly, SARS-CoV-2 co-localizes specifically with Tau phosphorylated at Threonine-231 in the soma, indicative of early neurodegeneration-like effects. Our studies, therefore, provide initial insights into the impact of SARS-CoV-2 as a neurotropic virus and emphasize that brain organoids could model CNS pathologies of COVID-19. One sentence summaryCOVID-19 modeling in human brain organoids
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We whole-genome sequenced 55 SARS-CoV-2 isolates from Western Germany and investigated the genetic structure of SARS-CoV-2 outbreaks in the Heinsberg district and Dusseldorf. While the genetic structure of the Heinsberg outbreak indicates a clonal origin, reflective of superspreading dynamics during the carnival season, distinct viral strains are circulating in Dusseldorf, reflecting the citys international links. Limited detection of Heinsberg strains in the Dusseldorf area despite geographical proximity may reflect efficient containment and contact tracing efforts.
