Harnessing plant chloroplasts for oral delivery of a multi-epitope HPV vaccine: toward cost-effective systemic and mucosal immunization.

Human papillomavirus (HPV) is a major causative agent of cervical and other mucosal cancers. However, the distribution and accessibility of current prophylactic vaccines remain limited, especially in low- and middle-income countries (LMICs), due to high production costs, cold-chain dependency, and limited induction of mucosal immune responses. To addressing these challenges, we designed a rationally constructed multi-epitope HPV vaccine (HPV_MEV) incorporating conserved cytotoxic T lymphocyte (CTL), helper T lymphocyte (HTL), and B-cell epitopes from diverse high- and low-risk HPV genotypes. The construct includes the Toll-like receptor 4 (TLR4) agonist RS09 to enhance innate immune activation and cholera toxin B subunit (CTB) as a mucosal adjuvant to facilitate uptake and presentation at mucosal surfaces. The codon-optimized gene was stably integrated into the chloroplast genome of Nicotiana tabacum via biolistic transformation. Molecular analyses confirmed site-specific integration, homoplasmy, and high-level antigen accumulation (~3.6 mg/g fresh weight; ~20.8% of total soluble protein). Immunogenicity was evaluated in BALB/c mice following intraperitoneal administration of purified antigen or oral gavage of lyophilized transplastomic leaf tissue. Oral administration induced antigen-specific systemic IgG and mucosal IgA responses, with higher levels of vaginal IgA observed compared with parenteral delivery, reflecting enhanced mucosal antibody induction. The chloroplast-produced HPV_MEV exhibited immunogenicity comparable to its E. coli-expressed counterpart, supporting its structural and functional integrity. Overall, this study demonstrates the feasibility of plastid biotechnology as a platform for producing a thermostable, orally deliverable HPV vaccine candidate and provides preliminary immunogenicity data supporting its application as a promising vaccine candidate for accessible vaccination strategies against HPV and other mucosally transmitted pathogens in resource-limited settings.