The infectious hepatitis B virus represents 42 nm spherical double-shelled particles. However, analysis of blood from hepatitis B virus carriers revealed the presence of smaller 22 nm particles consisting of a viral envelope surface protein. These particles are highly immunogenic and have been used in the design of hepatitis B virus vaccine produced in yeast. Upon expression in yeast, these proteins form virus-like particles that are used for parenteral immunization. Therefore, the DNA fragment encoding hepatitis B virus surface antigen was introduced into Agrobacterium tumerifacience LBA4404 and used to obtain transgenic lupin (Lupinus luteus L.) and lettuce (Lactuca sativa L.) cv. Burpee Bibb expressing envelope surface protein. Mice that were fed the transgenic lupin tissue developed significant levels of hepatitis B virus-specific antibodies. Human volunteers, fed with transgenic lettuce plants expressing hepatitis B virus surface antigen, developed specific serum-IgG response to plant produced protein.

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.

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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.Electronic supplementary materialThe online version of this article (doi:10.1007/s00299-011-1223-7) contains supplementary material, which is available to authorized users.

The idea of an oral vaccine administered as a portion of plant tissue requires a high level of antigen production. An improved protocol for the induction of transgenic yellow lupin calli or tumours, reaching 44% of transformation rate, is presented here. It has been developed by using the nptII marker gene and the uidA reporter gene as well as various Agrobacterium strains and plant explants. This method of seedling and hypocotyl transformation was applied to raise calli or tumours producing a small surface antigen of Hepatitis B Virus (S-HBsAg). Lupin tissue lines were long-term cultured on selection media maintaining the growth rate and high expression level of the native form of S-HBs, up to 6 microg per g of fresh tissue.

Mucosal immunity elicited by plant-based and other orally administered vaccines can serve as the first line of defense against most pathogens infecting through mucosal surfaces, but it is also considered for systemic immunity against blood-borne diseases such as hepatitis B (HB). Previous oral immunization trials based on multiple administration of high doses of HBs antigen elicited an immune response; however, a reproducible and long-lasting immunization protocol was difficult to design. The objective of this study was to evaluate the effect of dose and timing of orally delivered alum-adsorbed antigen on the magnitude of the anti-HBs humoral response. Mice were immunized orally by gavage intubation or parenterally by intramuscular injection three times, once every 2 weeks, with doses of 5, 50, or 500 ng alum-adjuvanted HBsAg. A low dose (10 ng) of HBsAg was orally administered three times in different time intervals: 2, 4, 6, and 8 weeks. The three consecutive 5-ng oral doses of the antigen induced immune response at the protective level (>or=10 mIU/ml), significantly higher than the reaction elicited by three 50 or 500 ng doses. In contrast, intramuscular delivery of these doses did not differ significantly; however, they induced a five to six times higher immune response than oral immunization. The 8-week period between each of the three oral immunizations appeared to be favorable to the anti-HBs humoral responses compared with the shorter schedules. The results presented here clearly identify the importance of low doses of antigen administered orally in extended intervals for a significantly higher anti-HBs response. This finding provides some indications concerning the strategy of orally administered vaccines, including plant-based ones.

Key message Micropropagation protocol of transgenic lettuce bearing S-, M- and L-HBsAg was developed for increased production of uniformised material for oral vaccine preparation. AbstractEffective manufacturing of plant-based biopharmaceuticals, including oral vaccines, depends on sufficient content of a protein of interest in the initial material and its efficient conversion into an administrable formulation. However, stable production of plants with a uniformised antigen content is equally important for reproducible processing. This can be provided by micropropagation techniques. Here, we present a protocol for micropropagation of transgenic lettuce lines bearing HBV surface antigens: S-, M- and L-HBsAg. These were multiplied through axillary buds to avoid the risk of somaclonal variation. Micropropagation effectiveness reached 3.5–5.7 per passage, which implies potential production of up to 6600 plant clones within a maximum 5 months. Multiplication and rooting rates were statistically homogenous for most transgenic and control plants. For most lines, more than 90 % of clones obtained via in vitro micropropagation had HBsAg content as high as reference plants directly developed from seeds. Clones were also several times more uniform in HBsAg expression. Variation coefficients of HBsAg content did not exceed 10 % for approximately 40–85 % of clones, or reached a maximum 20 % for 90 % of all clones. Tissue culture did not affect total and leaf biomass yields. Seed production for clones was decreased insignificantly and did not impact progeny condition. Micropropagation facilitates a substantial increase in the production of lettuce plants with high and considerably equalised HBsAg contents. This, together with the previously reported optimisation of plant tissue processing and its long-term stability, constitutes a successive step in manufacturing of a standardised anti-HBV oral vaccine of reliable efficacy.

The process of plant regeneration via somatic embryogenesis of two gentianas, Gentiana tibetica and G. cruciata was described. For this purpose seedling explants were cultured on agar medium and later maintained in cell suspension. For callus initiation seedling explants like: cotyledons, hypocotyl and root were plated on a callus induction medium (CIM) composed of MS (1962), supplemented with 0.5 mg/l 2,4-D and 1.0 mg/l Kin. For the formation of cell suspension culture, embryogenic callus was transferred into liquid maintained medium (MM) composed of MS (1962), supplemented with 1.0 mg/l Dic + 0.1 mg/l NAA + 2.0 mg/l BAP + 80.0 mg/l SA. The conversion of somatic embryo into plantlets required a new medium (ECM) based on MS (1962) mineral salts, supplemented with 0.5 mg/l GA₃ + 1.0 mg/l Kin + 0.5 mg/l NAA. For cytomorphological studies of particular stages of embryogenesis, specimens were stained with dyes and reagents: 1. PAS reaction with leukofucsin, 2. Safranin + fast green, 3. Erlich's hematoxylin