Plant expression, lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation

Current immunisation programmes against hepatitis B virus (HBV) increasingly often involve novel tri-component vaccines containing—together with the small (S-HBsAg)—also medium and large surface antigens of HBV (M- and L-HBsAg). Plants producing all HBsAg proteins can be a source of components for a potential oral ‘triple’ anti-HBV vaccine. The objective of the presented research was to study the potential of M/L-HBsAg expression in leaf tissue and conditions of its processing for a prototype oral vaccine. Tobacco and lettuce carrying M- or L-HBsAg genes and resistant to the herbicide glufosinate were engineered and integration of the transgenes was verified by PCR and Southern hybridizations. M- and L-HBsAg expression was confirmed by Western blot and assayed by ELISA at the level of micrograms per g of fresh weight. The antigens displayed a common S domain and characteristic domains preS2 and preS1 and were assembled into virus-like particles (VLPs). Leaf tissues containing M- and L-HBsAg were lyophilised to produce a starting material of an orally administered vaccine formula. The antigens were distinctly sensitive to freeze-drying conditions and storage temperature, in the aspect of stability of S and preS domains and formation of multimeric particles. Efficiency of lyophilisation and storage depended also on the initial antigen content in plant tissue, yet M-HBsAg appeared to be approximately 1.5–2 times more stable than L-HBsAg. The results of the study provide indications concerning the preparation of two other constituents, next to S-HBsAg, for a plant-derived prototype oral tri-component vaccine against hepatitis B

Low-dose oral immunization with lyophilized tissue of herbicide-resistant lettuce expressing hepatitis B surface antigen for prototype plant-derived vaccine tablet formulation

Efficient immunization against hepatitis B virus (HBV) and other pathogens with plant-based oral vaccines requires appropriate plant expressors and the optimization of vaccine compositions and administration protocols. Previous immunization studies were mainly based on a combination of the injection of a small surface antigen of HBV (S-HBsAg) and the feeding with raw tissue containing the antigen, supplemented with an adjuvant, and coming from plants conferring resistance to kanamycin. The objective of this study was to develop a prototype oral vaccine formula suitable for human immunization. Herbicide-resistant lettuce was engineered, stably expressing through progeny generation micrograms of S-HBsAg per g of fresh weight and formed into virus-like particles (VLPs). Lyophilized tissue containing a relatively low, 100-ng VLP-assembled antigen dose, administered only orally to mice with a long, 60-day interval between prime and boost immunizations and without exogenous adjuvant, elicited mucosal and systemic humoral anti-HBs responses at the nominally protective level. Lyophilized tissue was converted into tablets, which preserved S-HBsAg content for at least one year of room temperature storage. The results of the study provide indications on immunization methodology using a durable, efficacious, and convenient plant-derived prototype oral vaccine against hepatitis B