A novel approach to HPV16 VLP construction and production with improved accessibility and particle sorting potential.

Human papillomavirus (HPV), a member of the Papillomaviridae family, infects a broad range of animal hosts and is responsible for the majority of cervical cancers and a substantial proportion of oropharyngeal cancers. Current treatment strategies for HPV-associated cervical disease depend largely on disease stage, with preinvasive and early invasive lesions commonly managed through surgical excision or radiation. Additionally, more advanced cases often require chemotherapy, immunotherapy, or combination regimens. Prophylactic vaccination remains the most effective preventive approach, and currently available vaccines, including Gardasil, Gardasil-9, and Cervarix, rely on L1-based virus-like particles (VLPs) to induce protective immunity. At the same time, increasing interest has emerged in the use of nanomaterials and viral capsids as platforms for targeted drug and gene delivery, alongside the development of higher-throughput methods for nanoparticle and viral characterization. Modified HPV capsids have shown promise for cell-specific targeting and more efficient cargo delivery with reduced off-target cytotoxicity. In this study, the HPV16 structural proteins L1 and L2 were fused to the N- and C-terminal halves of the Venus fluorophore, respectively, using a specially designed linker intended to preserve capsid assembly while reducing VLP aggregation associated with current production methods. The resulting bimolecular fluorescence complementation (BiFC)-based VLPs demonstrated reduced aggregation and improved total particle yield. In addition, the particles generated a detectable YFP signal, supporting fluorescence-based monitoring of VLP assembly and providing a potential basis for high-throughput particle characterization and sorting. This intrinsic fluorescent signal may also reduce reliance on separately packaged reporter constructs for downstream analysis.