Systematic characterization of SARS-CoV-2 spike protein subunit trafficking and secretion reveals enhanced strategies...

Despite the widespread deployment of current COVID-19 vaccines, significant gaps remain in understanding the complete biological behavior of the SARS-CoV-2 spike (S) protein. Through systematic characterization of mammalian expression systems, this study shows that the full-length S protein exhibits a complex intracellular distribution, predominantly localizing not only to the cell membrane but also to the cytoplasm, nucleus, and extracellular compartments. Comparative analyses revealed distinct subunit-specific trafficking patterns. The S1 subunit showed increased intracellular accumulation and secretion compared to the full-length S protein, although with reduced surface expression. Conversely, the receptor-binding domain (RBD) and S2 domains were mainly associated with cytoskeletal (CS) structures. Notably, the signal sequence-enhanced RBD (SS-RBD) construct engineered in this study demonstrated dramatically enhanced extracellular accumulation, approximately 100-fold higher than the full-length S protein and 10-fold greater than S1, as measured by proximity extension assay (PEA). Signal peptide modification effectively redirected RBD from CS retention to efficient secretion, significantly improving detection sensitivity. PEA outperformed conventional methods such as flow cytometry (FACS), Western blotting (WB), and immunofluorescence, offering sensitivities several orders of magnitude higher. Consequently, these findings provide: (1) a structural framework for rational antigen design by distinguishing essential versus dispensable domains; (2) experimental support for SS-RBD as a promising vaccine candidate due to its high secretion efficiency and preservation of neutralizing epitopes; and (3) a robust platform using PEA for high-sensitivity antigen characterization. This study enhances the fundamental understanding of spike protein biology and offers actionable insights for developing next-generation vaccines targeting SARS-CoV-2 and related coronaviruses.