title: Plant expression, lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation.
abstract: 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.
authors.affiliation: Institute of Plant Genetics, Polish Academy of Sciences, Strzeszyńska 34, 60-479 Poznan, Poland. tpni@igr.poznan.pl
authors.forename: Tomasz
authors.initials: T
authors.lastname: Pniewski
authors.forename: Józef
authors.initials: J
authors.lastname: Kapusta
authors.forename: Piotr
authors.initials: P
authors.lastname: Bociąg
authors.forename: Anna
authors.initials: A
authors.lastname: Kostrzak
authors.forename: Olga
authors.initials: O
authors.lastname: Fedorowicz-Strońska
authors.forename: Marcin
authors.initials: M
authors.lastname: Czyż
authors.forename: Michał
authors.initials: M
authors.lastname: Gdula
authors.forename: Paweł
authors.initials: P
authors.lastname: Krajewski
authors.forename: Bogdan
authors.initials: B
authors.lastname: Wolko
authors.forename: Andrzej
authors.initials: A
authors.lastname: Płucienniczak
mesh_terms: D005612:Freeze Drying; D006509:Hepatitis B / Q000517:prevention & control*; D006514:Hepatitis B Surface Antigens / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017325:Hepatitis B Vaccines / Q000008:administration & dosage*; D006515:Hepatitis B virus / Q000276:immunology*; D018545:Lactuca / Q000235:genetics / Q000378:metabolism*; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D028321:Plant Preparations; D030821:Plants, Genetically Modified; D055550:Protein Stability; D013696:Temperature; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D006514:Hepatitis B Surface Antigens; D017325:Hepatitis B Vaccines; D028321:Plant Preparations