Stable expression of Human Papillomavirus (HPV) Type 18 L1 antigen in Tomato

Abstract

Cervical cancer, attributed to human papillomavirus (HPV) infection, ranks fourth among women-related cancers globally. Predominantly prevalent in low to middle income countries, cervical cancer poses a significant health challenge due to limited access to vaccination. Existing vaccines are costly, necessitate cold chain maintenance, and demand professional administration. Consequently, the imperative for a cost-effective and robust cervical cancer vaccine is evident. The present study focuses on utilizing HPV-18 from the Human Papillomavirus as a prospective vaccine candidate. Employing plants as an expression system for subunit vaccines is advantageous, and this research aims to establish an efficient and stable Agrobacterium-mediated transformation protocol for Solanum lycopersicum L. cv. Rio Grande to express the HPV-18 L1 antigen. The investigation encompassed optimizing conditions for seed sterilization, germination, and regeneration of tomato explants. Promising outcomes were achieved with seeds germinated on half MS media following sterilization with 0.1% mercuric chloride. Zeatin at a concentration of 2mg/L demonstrated favorable regeneration efficiency. Nodal explants of Solanum lycopersicum L. exhibited optimal growth on 75mg/L Kanamycin, the preferred concentration for transformed explant selection. Successful transformation with HPV 18 antigen was accomplished, with an infection time of 8 minutes and a co-cultivation period of 2 days yielding the highest transformation efficiency of 60%. PCR validation using gene-specific primers confirmed the success of the transformation. Transgenic tomato explants were subjected to quantitative real-time PCR (qRT-PCR) analysis, comparing transgene expression to the β-actin gene. Additionally, protein expression was verified through Dot blot, Western blot, and ELISA. In conclusion, the accomplished stable transformation of Solanum lycopersicum L. with HPV-18 holds promise for the development of an economically viable subunit vaccine against cervical cancer. Keywords: Solanum lycopersicum L., HPV-18 L1, Cervical Cancer, LTB-L1, Agrobacterium-mediated transformation, qRT-PCR, Western blotting, ELISA