Occlusion body-incorporated SARS-CoV-2 S-RBD via cypovirus polyhedrin-derived peptide: a novel insect cell-expressed antigen for high-accuracy COVID-19 diagnosis, and humoral immune response and hybrid immunity evaluation

Accurate serological assays are critical for monitoring antibody responses to SARS-CoV-2 and for assessing vaccine efficacy. In this study, we engineered a recombinant baculovirus to express the SARS-CoV-2 spike receptor-binding domain (RBD) fused to an N-terminal peptide of cypovirus polyhedrin (S-RBD-POLH), thereby enabling its potential encapsulation within occlusion bodies. The construct was expressed in insect cells and employed to establish an in-house enzyme-linked immunosorbent assay (ELISA-S) for COVID-19 diagnosis and evaluation of the humoral immune response to SARS-CoV-2. We compared the performance of ELISA-S with a commercial lateral flow immunoassay (LFIA), a chemiluminescence immunoassay (CLIA), and an in-house ELISA targeting Escherichia coli-produced nucleocapsid (N) protein (ELISA-N). Both in-house ELISAs had excellent diagnostic performance (overall accuracy = 0.90), with ELISA-S achieving 98.9% sensitivity and 100% specificity. Inter-assay concordance was high (kappa coefficient > 0.92), with near-perfect agreement between ELISA-S and LFIA (kappa = 0.99). Longitudinal analysis revealed increasing anti-RBD IgG titers over time, in contrast to declining anti-N IgG levels, consistent with expected post-infection kinetics. Moreover, ELISA-S reliably detected antibodies in individuals with hybrid immunity across different periods of the pandemic. These findings establish a scalable platform for antigen encapsulation and production in insect cells to enhance antibody detection. The ELISA-S system represents a robust and adaptable approach for evaluating humoral immunity following infection, vaccination, or hybrid immunity, and can be used for all variants.