In Silico Design and Subsequent Expression of Human Papillomavirus-16 and -18 L1 Vaccine Antigens in Broccoli

<b>Background</b>: Cervical carcinoma remains a widespread cancer worldwide, primarily caused by persistent infection with high-risk human papillomavirus (HPV). HPV types 16 and 18 account for approximately 70% of cervical cancer cases. Although prophylactic HPV vaccines are commercially available, their high cost and reliance on expensive expression platforms limit their accessibility in developing countries. <b>Objectives</b>: This study aimed to develop a cost-effective, plant-based HPV vaccine candidate by expressing capsomeric HPV-16 and HPV-18 L1 antigens in <i>Brassica oleracea</i> (broccoli). <b>Methods</b>: Modified L1 from HPV types 16 and 18 were designed to retain capsomeric assembly and fused with heat-labile enterotoxin B subunit (LTB). Immunoinformatics analyses were used to assess antigenicity, epitope distribution, and structural characteristics. Codon-optimized genes were cloned using Gateway^® technology and expressed in broccoli via <i>Agrobacterium</i>-mediated transformation. Transgenic plants were validated by PCR and qRT-PCR. Protein accumulation was quantified, and immunogenicity was evaluated in mice. <b>Results</b>: PCR and qRT-PCR confirmed the stable integration of two copies of the <i>LTB-L1</i> transgenes in broccoli plants. Western blotting detected L1 protein at ~56.5 kDa, indicating the cleavage of the LTB-L1 fusion protein. The correct folding of L1 capsomeres was verified by antigen-capture ELISA. The recombinant proteins accumulated to approximately 0.33% and 0.35% of total soluble protein for HPV-16 and HPV-18, respectively. The immunization of mice with transgenic L1 induced significant humoral immune responses, comparable to those elicited by purified VLPs. <b>Conclusions</b>: The results demonstrate broccoli as a promising platform for the expression of immunogenic HPV L1 capsomeres and highlight its potential for the development of affordable, plant-based HPV vaccines.