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, yet 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 immunity. To overcome these challenges, we designed a 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 using biolistic transformation. Molecular analyses confirmed site-specific integration, homoplasmy, and high-level expression of the recombinant antigen (~ 3.6 mg/g fresh weight; ~20.8% of total soluble protein). Immunogenicity was evaluated in BALB/c mice via intraperitoneal injection of purified antigen or oral gavage of lyophilized transplastomic leaf tissue. Oral administration elicited strong systemic IgG and mucosal IgA responses, with mucosal immunity surpassing that of the injected formulations. The chloroplast-produced HPV_MEV was comparable in immunogenicity to its E. coli-expressed counterpart, validating its structural and functional integrity. This study highlights the potential of plastid biotechnology for producing an effective, thermostable, needle-free oral HPV vaccine. By integrating rational antigen design with a scalable plant-based production and delivery platform, this approach offers a promising solution for accessible immunization against HPV and other mucosal pathogens in resource-limited settings.