Inducible Expression of Human Papillomavirus-16 L1 and LTB-L1 Fusion Protein in Plastomes of Nicotiana tabacum

Abstract

Cervical cancer is caused by human papillomavirus (HPV) and ranked at the fourth position among women-related cancers, worldwide. It is the most prevalent Sexually Transmitted Disease (STD) that originates from the cervix with abnormal cell growth invading other body parts. The three approved and commercially available vaccines against cervical cancer are expensive, require maintenance of cold chain, and professional expertise are required for vaccine administration. Women in low to middle-income countries (LMICs) are the major victims of cervical cancer as the lowest opportunities to get vaccinated against cervical cancer are available in these countries. Therefore, the production of a cost-effective and stable vaccine against cervical cancer is the need of the hour. Previously, attempts were made to constitutively express HPV type 16 L1 antigen (with capsomeric assembly) alone and in fusion with biological adjuvant LTB as LTB-L1 in tobacco plastomes. However, their constitutive expression led to severe pleiotropic effects such as male sterility, chlorosis, and growth retardation in the transplastomic plants. Thus, the present project was designed to inducibly express L1 and LTB-L1 genes in plastomes of Nicotiana tabacum. The expression of transgenes was under the control of the lambda bacteriophage-derived T7 promoter. The final expression vectors for both transgenes were obtained by Gateway® cloning. The transformation was done using biolistic gene gun method. Transformation and achievement of homoplasmy were confirmed by polymerase chain reaction (PCR) and Southern blot analysis, respectively. For the induction of transgene expression, 5% ethanol was sprayed on plants, and the sprayed plants were kept under constant observation for all morphological changes. Fertility of transplastomic plants was checked by the studying ultra-micro structure of pollen grains collected from transplastomic lines under scanning electron microscopy (SEM). Transplastomic plants with inducible expression of transgenes were perfectly normal, and fertile as compared to the plants with constitutive expression of transgenes. The expression of proteins was detected with the help of immunoblot analysis, where HPV-16 L1 specific antibody MD2H11 was used as the primary antibody. Expression was recorded as 2.5% and 2% for HPV-16 L1 and LTB-L1 of total soluble protein (TSP), respectively. Antigen capture ELISA confirmed the retention of conformational epitopes in plant-expressed L1 antigens. Plants expressed HPV-16 L1 and the LTB- Abstract Inducible Expression of Human Papillomavirus-16 L1 and LTB-L1 Fusion Protein in Plastomes of Nicotiana tabacum xi L1 fusion proteins were purified with the help of heparin-based affinity chromatography. Transplastomic plants were further studied to investigate the possible cause of pleiotropy using protein-protein interaction studies via yeast two-hybrid system. The results showed that LTB-L1 directly and significantly interacted with imidazole glycerol-phosphate dehydratase, that is involved in histidine metabolism, leading to defects in chlorophyll synthesis. Similarly, ECERIFERUM3-like protein, involved in long-chain fatty acid metabolism, was also observed to be affected by LTB-L1, resulting in the defective pollen-stigma interaction, leading to male sterility in the transplastomic plants. The purified L1 and LTB-L1 proteins were checked for their immunogenic potential in mice along with the commercially available vaccine Gardasil-9 against cervical cancer. HPV-16 L1 directly fused with LTB adjuvant (as adjuvant-antigen couple) showed highest degree of immunogenicity among all tested groups. In conclusion, the present study establishes the plant-safe expression of L1 and LTB L1 antigens in plastomes of Nicotiana tabacum using ethanol inducible system. The higher immunogenicity of LTB-L1 in mice in comparison to commercially available vaccine against cervical cancer can lay a strong basis for a plant-based cost-effective capsomere-based vaccine against cervical cancer.