Comparison of plant- and mammalian cell-produced human papillomavirus pseudovirions.

High-risk human papillomaviruses (HPVs) are the primary etiological agents of cervical, anal and oropharyngeal cancers. While existing vaccines are effective in preventing infection, their impact in low-and middle-income countries (LMICs) is limited by type coverage, high costs and uptake. To address this gap, there is a critical need for next-generation vaccines that are both regionally tailored and cost-effective, along with efficient and accessible tools for evaluating their efficacy. HPV pseudovirions (PsVs), which encapsidate a reporter plasmid, are widely used in pseudovirion-based neutralisation assays (PBNAs) and in vivo murine models to assess vaccine-induced immunity - and have potential for use as DNA vaccine delivery systems. Traditionally, PsVs are produced in mammalian cells, which remain the gold standard due to their high infectivity and structural fidelity. However, recent studies have demonstrated the feasibility of producing PsVs in plants, a platform that offers lower infrastructure and reagent costs, scalability, and biosafety advantages. Although plant-derived PsVs have shown promise in PBNAs, their performance in in vivo models had not been evaluated prior to this study. Here, we compared mammalian cell-derived PsVs encapsidating either Gaussia or firefly luciferase reporter plasmids and found that firefly luciferase provided more consistent and robust signals in both in vitro and in vivo assays. Building on this, we generated PsVs encapsidating the firefly luciferase gene using both mammalian and plant expression systems, and assessed their infectivity. While plant-derived PsVs were capable of infecting HeLa cells and mice in a cervicovaginal challenge model, mammalian-derived PsVs exhibited significantly higher infectivity overall. These findings represent the first demonstration of in vivo infectivity of plant-produced HPV PsVs and highlight their potential as a cost-effective alternative for immunogenicity testing and potentially as vaccines. Although further optimization is needed, particularly in capsid assembly and purification, plant-based PsV production holds promise for expanding access to HPV research tools and supporting vaccine development in resource-limited settings.