Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

Plant-based expression and characterization of SARS-CoV-2 virus-like particles presenting a native spike protein.

We have investigated the use of transient expression to produce virus-like particles (VLPs) of severe acute respiratory syndrome coronavirus 2, the causative agent of COVID-19, in Nicotiana benthamiana. Expression of a native form of the spike (S) protein, either alone or in combination with the envelope (E) and membrane (M) proteins, all of which were directed to the plant membranes via their native sequences, was assessed. The full-length S protein, together with degradation products, could be detected in total protein extracts from infiltrated leaves in both cases. Particles with a characteristic 'crown-shaped' or 'spiky' structure could be purified by density gradient centrifugation. Enzyme-linked immunosorbent assays using anti-S antibodies showed that threefold higher levels of VLPs containing the full-length S protein were obtained by infiltration with S alone, compared to co-infiltration of S with M and E. The S protein within the VLPs could be cleaved by furin in vitro and the particles showed reactivity with serum from recovering COVID-19 patients, but not with human serum taken before the pandemic. These studies show that the native S protein expressed in plants has biological properties similar to those of the parent virus. We show that the approach undertaken is suitable for the production of VLPs from emerging strains and we anticipate that the material will be suitable for functional studies of the S protein, including the assessment of the effects of specific mutations. As the plant-made material is noninfectious, it does not have to be handled under conditions of high containment.

Mobile computed tomography for the efficient allocation of medical resources in patients with COVID-19 pneumonia: A case report.

RATIONALE: The highly contagious Coronavirus 2019 (COVID-19) infection raise social and economic burden. Medical staff and resources are being diverted for the care of patients with COVID-19. There are problems for healthcare systems, including burnout syndrome for the medical staff and exhaustion of medical resources. PATIENT CONCERNS: The patient was a 65-year-old woman presenting with fever, cough, and dyspnea due to COVID-19 pneumonia. She received antiviral agents, broad-spectrum antibiotics, and conservative treatment. Although her clinical condition improved, there was no significant improvement in portable chest X-ray results. DIAGNOSES: Due to concerns over the propagation of infection when transferring to patients for scanning and the need for excessive medical personnel to move patients, we moved a mobile chest computed tomography (CT) machine to an isolation ward for CT scanning. INTERVENTIONS: We report our experience using mobile chest CT to effectively allocate medical resources and assess treatment response in patient with COVID-19 pneumonia. OUTCOMES: Follow-up mobile CT scans disclosed progressive resolution of the multifocal ground-glass opacities and mixed consolidations distributed peripheral to subpleural spaces. During the mobile chest CT scan, there were no adverse or unforeseen events. Three medical personnel were required to performed mobile chest CT, including a clinician, a nurse, and a radiologist. LESSONS: As a result of using mobile chest CT on COVID-19 patients, the number of medical personnel required for CT scanning decreased by about 83%, rapid, and safe compared with a patient who performed conventional CT.