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

Biomass allocation to anchoring structures in the aquatic macrophytes from the subcontinental and Atlantic climates in Europe

Biomass allocation to anchoring structures in 10 species of aquatic macrophytes in two types of the cool and humid temperate climate in Europe, subcontionental (NW Poland) and Atlantic (W France), was examined. The study focused on the weight of anchoring organs in Chara delicatula Ag., Ch. fragilis Desv., Juncus bulbosus L., Littorella uniflora (L.) Asch., Luronium natans (L.) Raf., Potamogeton pectinatus L., P. perfoliatus L., P. natans L. and Ranunculus fluitans (Lam.) Wimm., as well as submerged structures in Hydrocharis morsus-ranae L. The plants were collected from 10 geographically distant lakes and 2 rivers with a current velocity of 0.3-0.5 m s-1. It was assumed that biomass allocation to anchoring structures in rooting macrophytes depends on the time that mechanical forces, which can remove the plants from the occupied area, exert their effect. It was found that, in the Atlantic and subcontinental climates, the ratio between the biomass of underground (or submerged) and above-ground structures (U/A index), calculated for the ramets of Chara fragilis, Hydrocharis morsus-ranae and Ranunculus fluitans, has similar values (p>0.05). Nevertheless, among seven species of Cormophyta, which were anchored in the lake or pond littoral, six (Juncus bulbosus, Littorella uniflora, Luronium natans, Potamogeton pectinatus, P. perfoliatus and P. natans) had higher values of the U/A index in the Atlantic climate than in the subcontinental type (p < 0.05). This can be explained by the lack of ice cover and, consequently, a longer time of wave action than in the subcontinental climate

Parenteral–Oral Immunization with Plant-Derived HBcAg as a Potential Therapeutic Vaccine against Chronic Hepatitis B

Chronic hepatitis B (CHB) is the cause of severe liver damage, cirrhosis, and hepatocellular carcinoma for over 240 million people worldwide. Nowadays, several types of treatment are being investigated, including immunotherapy using hepatitis B core antigen (HBcAg) assembled into highly immunogenic capsid-like particles (CLPs). Immunogenicity of plant-produced and purified HBcAg, administered parenterally or intranasally, was previously reported. In this study, a novel parenteral–oral vaccination scheme is proposed using plant-derived HBcAg preparations. The antigen for injection was obtained via transient expression in Nicotiana benthamiana. HBcAg-producing transgenic lettuce was lyophilized and used as an orally delivered booster. The intracellular location of plant-produced HBcAg CLPs implies additional protection in the digestive tract during oral immunization. BALB/c mice were intramuscularly primed with 10 µg of the purified antigen and orally boosted twice with 5 or 200 ng of HBcAg. A long-lasting and significant systemic response after boosting with 200 ng HBcAg was induced, with anti-HBc titer of 25,000. Concomitantly, an insignificant mucosal response was observed, with an S-IgA titer of only 500. The profile of IgG isotypes indicates a predominant Th1 type of immune response, supplemented by Th2, after injection–oral vaccination. The results demonstrate that a low dose of parenteral–oral immunization with plant-derived HBcAg can elicit a specific and efficient response. This study presents a potential new pathway of CHB treatment