The Cellular Characterization of SARS-CoV-2 Spike Protein in Virus-Infected Cells Using the Receptor Binding Domain Binding Specific Human Monoclonal Antibodies

Abstract

The SARS-CoV-2 spike (S) protein receptor binding domain (RBD) mediates the attachment of SARS-CoV-2 to the host cell. This interaction plays an essential role in initiating virus infection, and the S protein RBD is therefore a focus of therapeutic and vaccine interventions.

Evidence that the SARS-CoV-2 S protein undergoes a conformational change at the Golgi Complex that leads to the formation of virus neutralising antibody binding epitopes in the S1 protein subunit.

“…On this basis and by default, we therefore concluded that the PD4 and sc29 antibodies bind to the S protein at sites located within the S2 domain rather than the S1 domain. This is consistent with our previous study that demonstrated high binding affinity of the PD4 and sc29 antibodies to the S protein, but that they did not bind to the purified RBD and nor did they exhibit virus neutralising activity [17].…”

“…2D). Based on the staining intensity exhibited by the transfected cells we concluded that both sc23 and PD5 exhibited similar binding properties to the S1 domain, which is consistent with sc23 and PD5 exhibiting similar binding affinities for the S protein in our previous study [17]. However, in this current study we failed to detect sc23-staining in the RBD-expressing cells, suggesting that sc23 binds to other regions of the S1 protein subunit that are distinct from the RBD.…”

“…This suggested a small degree of overlap of the PD4 and PD5 staining in this region of the cell and further suggested that the S protein species recognised by PD5 and sc23 may also contain the PD4 and sc29 epitopes. This was consistent with our previous imaging analysis made in virus-infected cells when the different staining patterns by these different antibodies were compared [17]. In addition, in this previous study we described Singapore SARS-CoV-2 isolates that exhibited some minor differences in the amino acid sequence of the S protein when compared with that of the S protein sequence of the SARS-CoV-2/WIV04 strain.…”

“…The construction of the S protein expression vectors has been described previously [17]. Briefly, the S gene of SARS-CoV-2 (accession no.…”

“…These antibodies were functionally characterised based on their capacity to neutralise virus infection and to bind to the SARS-CoV-2 S protein RBD. In our earlier study we showed that in virus-infected cells the RBD-binders exhibited an antibody-specific staining pattern that localised within the Golgi complex of infected cells [17]. We had earlier hypothesised that during the export of the S protein through the secretory pathway these antibodies may recognise a conformational change in the S protein in which the RBD becomes surface accessible thus allowing antibody binding to the exposed epitopes.…”