Analysis of the SARS-CoV-2 spike protein revealed that blocked receptor-binding domain antigenicity decreases the production of neutralizing antibodies in vivo

The identification of SARS-CoV-2 spike protein distribution and function in target cells has raised concerns about its possible impacts on vaccine efficacy and pathogenic effect in host cells. Thus, a better understanding of such consequences is necessary. In this study, we studied the biological characteristics of six variants of SARS-CoV-2 in A549 and HEK293 cells using four different technologies. The results showed that compared to the other fragments, the full-length spike protein exhibited the highest expression on the cell surface and was detectable in the cell supernatant, cytoplasm, and nucleus. Except for the cell surface, the S1 subunit generally expressed higher than the full-length spike protein. RBD and S2 subunits were expressed in the cytoskeleton. The SS-RBD peptide, which consists of a 19-amino acid signal peptide sequence (SS)-linked RBD, exhibited the highest expression in the cell supernatant among all other studied peptides. The SS positively enhanced the expression, migration, and secretion of SS-RBD from the cytoskeleton to the supernatant. Importantly, the FACS assay results showed that neutralizing antibodies (NAbs) could recognize SS-RBD but not RBD in the transfected cells, suggesting that RBD was tightly bound by ACE2 in HEK293 cells. In contrast, the antigenicity of the RBD in the spike protein was revealed and efficiently monitored only by 6-His-tag mAbs. Thus, our findings demonstrated that ACE2 blocks crucial immunogenic epitopes of the RBD, and the full-length spike protein mainly induces non-neutralizing antibodies in vivo. Therefore, we suggest that reducing ACE2 binding affinity and exposing the immunogenicity of the RBD on the spike protein is imperative for improving vaccine efficacy and generating new SARS-CoV-2 mRNA vaccines. ### Competing Interest Statement The authors have declared no competing interest.