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

HBV: Genomic Structure, HBVsAg Isolation and innovative Virotherapy Initiation in the Middle East

“…STD-HBV could be a good tool for vaccination using HBVsAg in those countries. The indings of scientists [7][8][9], on small amount of expressed protein for the HBV-target genes (HBVsAg, L-and M-HBVsAg, HBVcAg) makes us to speculate another system of plant viral vector construct of chimeric virus.…”

“…Huang et al have got 0.8 mg/g of expressed protein when he used HBVcAg. Pniewski et al [8,9], have got few μg/g of the protein using HBVsAg and L-and M-HBVsAg.…”

Is An Oral Plant-based Vaccine against Hepatitis B Virus Possible?

“…Western blot analyses showed that S-HBsAg in plant cell was of proper size of 24 kDa and non-glycosylated, similarly to the yeast-derived antigen [41,46,50,51,59,71]. Yet, glycosylation of S-HBsAg as well as M and L antigens in plants was also reported [54,56,67,70], although putatively the antigens were modified according to the plant glycosylation pattern. However, the HBs antigens of plant origin most probably were properly folded [57] as for the most cases they were detected using specific antibodies or diagnostic kits designed to native HBsAg , (see Table 1).…”

Advances in hepatitis immunization (A, B, E)

“…Plants (tobacco and lettuce) producing all HBsAg proteins (S-HBsAg, M-HBsAg and L-HBsAg) can be a source of components for a potential oral 'triple' anti-HBV vaccine [29].…”