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

Abstract

Summary 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.

Production of the SARS-CoV-2 Spike protein and its Receptor Binding Domain in plant cell suspension cultures

“…Samples were added to the sample buffer without the presence of reducing agents and were not boiled, to preserve disulfide bonds. We firstly detected the trimer in the cell extract, similarly to what has been reported in N. benthamiana leaves (Jung et al, 2022). WB analysis (Figure 7A) detected two bands in the HEK-S sample, corresponding to the glycosylated monomer, around 180 kDa, and a higher molecular weight band that may correspond to the trimer.…”

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

“…The SARS-CoV-2 S protein produced in N. benthamiana has a molecular weight of about 140 kDa in reducing conditions [17]. Post-translational modifications of this recombinant protein were investigated by mass spectrometry analysis of peptides generated by endoprotease digestions.…”

“…VLPs containing the S protein from SARS-CoV-2 isolate Wuhan-Hu-1 (NC_045512.2) were expressed in N. benthamiana and purified as previously described [17]. As determined by ELISA measurement of the amount of S protein in the VLP sample, 50 µg of SARS-CoV-2 S protein were separated by electrophoresis on an 8% (w/v) SDS-PAGE gel.…”

“…For the current study, we have chosen to use a version of the S protein that contains the native amino acid sequence, including the leader peptide evaluated [17]. This material is intended as a non-infectious surrogate for the native virus in studies on various aspects of the viral replication cycle, such as cell attachment.…”

High-Yield Production of Chimeric Hepatitis E Virus-Like Particles Bearing the M2e Influenza Epitope and Receptor Binding Domain of SARS-CoV-2 in Plants Using Viral Vectors

“…Kentucky BioProcessing is developing a recombinant subunit RBD-based vaccine in plants and has already started phase 1–2 clinical trials (NCT04473690). In other studies, it was shown that simultaneous transient expression of envelop (E), membrane (M), and S proteins in Nicotiana benthamiana resulted in VLP formation [ 47 , 48 ]; as well as the co-expression of M, E and nucleocapsid (P) [ 49 ].…”