In planta deglycosylation improves the SARS-CoV-2 neutralization activity of recombinant ACE2-Fc.

SARS-CoV-2 infects human cells via binding of the viral spike glycoprotein to its main cellular receptor, angiotensin-converting enzyme 2 (ACE2). The spike protein-ACE2 receptor interaction is therefore a major target for the development of therapeutic or prophylactic drugs to combat coronavirus infections. Various engineered soluble ACE2 variants (decoys) have been designed and shown to exhibit virus neutralization capacity in cell-based assays and in vivo models. Human ACE2 is heavily glycosylated and some of its glycans impair binding to the SARS-CoV-2 spike protein. Therefore, glycan-engineered recombinant soluble ACE2 variants might display enhanced virus-neutralization potencies. Here, we transiently co-expressed the extracellular domain of ACE2 fused to human Fc (ACE2-Fc) with a bacterial endoglycosidase in Nicotiana benthamiana to produce ACE2-Fc decorated with N-glycans consisting of single GlcNAc residues. The endoglycosidase was targeted to the Golgi apparatus with the intention to avoid any interference of glycan removal with concomitant ACE2-Fc protein folding and quality control in the endoplasmic reticulum. The in vivo deglycosylated ACE2-Fc carrying single GlcNAc residues displayed increased affinity to the receptor-binding domain (RBD) of SARS-CoV-2 as well as improved virus neutralization activity and thus is a promising drug candidate to block coronavirus infection.

Resilience and Protection of Health Care and Research Laboratory Workers During the SARS-CoV-2 Pandemic: Analysis and Case Study From an Austrian High Security Laboratory

The SARS-CoV-2 pandemic has highlighted the interdependency of healthcare systems and research organizations on manufacturers and suppliers of personnel protective equipment (PPE) and the need for well-trained personnel who can react quickly to changing working conditions. Reports on challenges faced by research laboratory workers (RLWs) are rare in contrast to the lived experience of hospital health care workers. We report on experiences gained by RLWs (e.g., molecular scientists, pathologists, autopsy assistants) who significantly contributed to combating the pandemic under particularly challenging conditions due to increased workload, sickness and interrupted PPE supply chains. RLWs perform a broad spectrum of work with SARS-CoV-2 such as autopsies, establishment of virus cultures and infection models, development and verification of diagnostics, performance of virus inactivation assays to investigate various antiviral agents including vaccines and evaluation of decontamination technologies in high containment biological laboratories (HCBL). Performance of autopsies and laboratory work increased substantially during the pandemic and thus led to highly demanding working conditions with working shifts of more than eight hours working in PPE that stressed individual limits and also the ergonomic and safety limits of PPE. We provide detailed insights into the challenges of the stressful daily laboratory routine since the pandemic began, lessons learned, and suggest solutions for better safety based on a case study of a newly established HCBL (i.e., BSL-3 laboratory) designed for autopsies and research laboratory work. Reduced personal risk, increased resilience, and stress resistance can be achieved by improved PPE components, better training, redundant safety measures, inculcating a culture of safety, and excellent teamwork

Pre-analytical sample stabilization by different sampling devices for PCR-based COVID-19 diagnostics.

The outbreak of the SARS-CoV-2 pandemic created an unprecedented requirement for diagnostic testing, challenging not only healthcare workers and laboratories, but also providers. Quantitative RT-PCR of various specimen types is considered the diagnostic gold standard for the detection of SARS-CoV-2, both in terms of sensitivity and specificity. The pre-analytical handling of patient specimens is a critical factor to ensure reliable and valid test results. Therefore, the effect of storage duration and temperature on SARS-CoV-2 RNA copy number stability was examined in various commercially available specimen collection, transport and storage devices for naso/oropharyngeal swabs and saliva. The swab specimen transport and storage devices tested showed no significant alteration of viral RNA copy numbers when stored at room temperature, except for one system when stored for up to 96 h. However, at 37 °C a significant reduction of detectable RNA was found in 3 out of 4 of the swab solutions tested. It was also found that detectability of viral RNA remained unchanged in all 7 saliva devices as well as in unstabilized saliva when stored for 96 h at room temperature, but one device showed marked RNA copy number loss at 37 °C. All tested saliva collection devices inhibited SARS-CoV-2 infectivity immediately, whereas SARS-CoV-2 remained infectious in the swab transport systems examined, which are designed to be used for viral or bacterial growth in cell culture systems.

Identification of contagious SARS-CoV-2 infected individuals by Roche's Rapid Antigen Test.

OBJECTIVES: Rapid antigen tests (RAT) can provide valuable information on the presence or absence SARS-CoV-2 within 15 min without the need of a laboratory. The analytical and diagnostic characteristics of available RATs has led to the question whether they can safely distinguish between infectious and non-infectious patients in an acute care setting. METHODS: Three nasopharyngeal swabs for the analysis by RAT, reverse transcriptase real time polymerase chain reaction (RT-qPCR), and a cell culture based infection assay were collected from 67 patients that presented to the emergency department of the University Hospital of Graz (Austria). The first swab was used for on-site RAT testing in the emergency department using the Roche SARS-CoV-2 RAT. The second swab was sent to the central laboratory of the hospital for RT-qPCR with two independent methods (Cepheid Xpert® Xpress SARS-CoV-2 assay and Roche Cobas SARS-CoV-2 Test) and repeat RAT testing using the same commercial test. With the third swab a cell culture-based infection assay was performed. RESULTS: The RATs performed from independent samples showed substantial agreement (Cohen's-kappa: 0.73, p<0.001). All patients with a positive RAT had positive RT-qPCR with cycle threshold (ct) values <25. Fifteen out of 55 RAT-negative samples were RT-qPCR positive with ct values between 25 and 40. The inoculation of cell cultures with RT-qPCR negative swabs and RT-qPCR positive swabs with ct values >25 did not induce cytopathic effects that were related to SARS-CoV-2. The infection assays from four RAT-negative patients showed cytopathic effects that were induced by other pathogens. CONCLUSIONS: The SARS-CoV-2 RAT from Roche Diagnostics is a valuable tool for managing symptomatic patients. RAT-negative patients may be regarded as non-contagious.

Structure-guided glyco-engineering of ACE2 for improved potency as soluble SARS-CoV-2 decoy receptor.

Infection and viral entry of SARS-CoV-2 crucially depends on the binding of its Spike protein to angiotensin converting enzyme 2 (ACE2) presented on host cells. Glycosylation of both proteins is critical for this interaction. Recombinant soluble human ACE2 can neutralize SARS-CoV-2 and is currently undergoing clinical tests for the treatment of COVID-19. We used 3D structural models and molecular dynamics simulations to define the ACE2 N-glycans that critically influence Spike-ACE2 complex formation. Engineering of ACE2 N-glycosylation by site-directed mutagenesis or glycosidase treatment resulted in enhanced binding affinities and improved virus neutralization without notable deleterious effects on the structural stability and catalytic activity of the protein. Importantly, simultaneous removal of all accessible N-glycans from recombinant soluble human ACE2 yields a superior SARS-CoV-2 decoy receptor with promise as effective treatment for COVID-19 patients.