Development of HiBiT-tagged mumps virus-like particles assembled from authentic viral structural proteins for neutralizing testing.

Despite widespread vaccination programs, mumps outbreaks persist even in highly vaccinated populations, raising concerns about current vaccine effectiveness against circulating strains. Existing serological methods exhibit limitations in practical implementation and antigenic specificity. To address these challenges, we developed a rapid neutralization assay using HiBiT-tagged mumps virus-like particles (hiMuV-VLPs) composed entirely of authentic viral structural proteins. Transmission electron microscopy confirmed these hiMuV-VLPs morphologically resembled native mumps virions. The VLPs demonstrated efficient cellular entry, quantifiable via luminescent signals generated by HiBiT-LgBiT complementation. Validation against conventional plaque reduction neutralization tests (PRNT) using human sera revealed the biological relevance and practicability of our assay. A key innovation was the successful incorporation of hemagglutinin-neuraminidase (HN) proteins from multiple mumps virus genotypes into the hiMuV-VLP platform, enabling assessment of strain-specific neutralizing antibody responses. This system represents a valuable tool for large-scale seroepidemiological surveillance, evaluation of vaccine-induced immunity against heterologous strains, and prediction of population susceptibility to emerging mumps virus variants.