Efficient Assembly of Coxsackievirus A16 Virus‐Like Particles in Insect Cells via VP4‐Deficient P1 Expression

Coxsackievirus A16 (CVA16) is a major causative agent of hand, foot, and mouth disease (HFMD), yet efficient virus‐like particle (VLP)–based vaccine development remains challenging due to enteroviral structural complexity and the cytotoxicity of the 3CD protease required for polyprotein processing. In this study, we established an optimized production strategy for CVA16 VLPs using the baculovirus expression vector system (BEVS) by balancing structural protein expression and proteolytic cleavage. To mitigate 3CD‐mediated cytotoxicity, we applied an attenuation strategy using the modified p10 ‐BS5 promoter, which reduced transcriptional strength by ~90% and delayed protease expression, thereby extending the production window. We further examined the impact of P1 structural protein conformation and expression levels on VLP assembly. Unlike Enterovirus 71 (EV71), which favors high‐level expression of full‐length P1, CVA16 assembly was highly sensitive to expression dynamics. Optimal VLP formation was achieved using a standard expression vector (pAcPol) combined with a VP4‐deficient P1 construct (ΔVP4). Under optimized conditions (multiplicity of infection 1, 7 days postinfection), the rAcPol‐CV‐ΔVP4‐BS5 construct yielded the highest level of assembled particles. Transmission electron microscopy confirmed uniform, spherical VLPs (30–35 nm) resembling native CVA16 virions. These findings demonstrate that precise control of transcriptional stoichiometry and structural configuration is critical for high‐yield CVA16 VLP production, providing a robust platform for multivalent HFMD vaccine development.