Abstract
First production of virus-like particles as a vaccine candidate in a non-vascular plant. Virus-like particles (VLPs) are self-assembling nanoparticles composed of viral structural proteins which mimic native virions but lack viral DNA and infectivity. VLPs are a resourceful class of biopharmaceuticals applied as subunit vaccines or as delivery vehicles for drugs and nucleic acids. Similar to viruses, VLPs are diverse in structure, composition, and assembly, requiring a tailored production platform aligned with the intended application. The moss plant Physcomitrella (Physcomitrium patens) is an emerging expression system offering humanized N-glycosylation, scalability, and adaptability to existing industry settings. Here, we used Physcomitrella to produce human papillomavirus (HPV) 16 VLPs. HPV VLPs are composed of the major structural protein L1 and are used as vaccines against HPV infections which are the main causal agent of cervical and other anogenital cancers. We characterized Physcomitrella chloroplast transit peptides, which we used for targeting of moss-produced L1 to chloroplasts, leading to higher recombinant protein yield compared to nuclear or cytoplasmic localization. We confirmed subcellular localization with confocal laser scanning microscopy and found L1 to accumulate within the chloroplast stroma. Production in 5-L photobioreactors yielded over 0.3 mg L1 per gram fresh weight. We established a purification protocol for moss-produced L1 using a combination of ammonium sulphate precipitation and cation exchange chromatography. Purified samples were subjected to a controlled dis- and reassembly, yielding fully assembled HPV-16 L1 VLPs. This is the first report of production, purification, and assembly of VLPs in a non-vascular plant.