Hepatitis B surface antigen (HBsAg) expression in plant cell culture: Kinetics of antigen accumulation in batch culture and its intracellular form

Abstract

The production of edible vaccines in transgenic plants and plant cell culture may be improved through a better understanding of antigen processing and assembly. The hepatitis B surface antigen (HBsAg) was chosen for study because it undergoes substantial and complex post-translational modifications, which are necessary for its immunogenicity. This antigen was expressed in soybean (Glycine max L. Merr. cv Williams 82) and tobacco NT1 (Nicotiana tabacum L.) cell suspension cultures, and HBsAg production in batch culture was characterized. The plant-derived antigen consisted predominantly of disulfide cross-linked HBsAg protein (p24(s)) dimers, which were all membrane associated. Similar to yeast, the plant-expressed HBsAg was retained intracellularly. The maximal HBsAg titers were obtained with soybean suspension cultures (20-22 mg/L) with titers in tobacco cultures being approximately 10-fold lower. For soybean cells, electron microscopy and immunolocalization demonstrated that all the HBsAg was localized to the endoplasmic reticulum (ER) and provoked dilation and proliferation of the ER network. Sucrose gradient analysis of crude extracts showed that HBsAg had a complex size distribution uncharacteristic of the antigen's normal structure of uniform 22-nm virus-like particles. The extent of authentic epitope formation was assessed by comparing total p24(s) synthesized to that reactive by polyclonal and monoclonal immunoassays. Depending on culture age, between 40% and 100% of total p24(s) was polyclonal antibody reactive whereas between 6% and 37% was recognized by a commercial monoclonal antibody assay. Possible strategies to increase HBsAg production and improve post-translational processing are discussed.

Virus-like particles production in green plants

“…Dimers are then transported to the post-ER, pre-Golgi compartment where oligomerization and VLP formation occur prior to secretion [56]. For plant systems, HBsAg dimer formation was demonstrated by Western blot of protein extracts from cultured plant cells [53,57] and from infiltrated leaves [58], indicating that plants can provide a suitable environment for correct processing of HBsAg. The ability of plant-produced HBsAg to assemble VLPs was determined by sucrose gradients and electron microscopy.…”

“…Production of HBsAg in plant cell culture systems has also been attempted. The yield of HBsAg approached 22 mg/L in soybean culture while it was one tenth of that in tobacco culture [53]. NT1 cell lines were used to express S gene either with or without a C-terminal ER retention signal [54].…”

Two-phase fed-batch modification for 48 hour peak expression of hepatitis B surface antigen in Pichia pastoris shake flask system

“…In tobacco cell suspension cultures, HBsAg production achieved 2 µg g -1 of HBsAg fresh weight [29]. Cell suspension cultures of soybean were reported to produce a maximum of 20-22 mg L -1 of HBsAg [30]. Constitutive expression of HBsAg in recombinant Saccharomyces cerevisiae generated approximately 10 mg L -1 by fed-batch fermentation [18].…”

Production of vaccines for treatment of infectious diseases by transgenic plants

“…Besides using different plants, Shekhawat et al (2010) (Smith et al, 2002) this study proved promising for further investigations of stable HBsAg expression in suspension cells of sandalwood.…”

“…Since the first reported plant-made HB Ag in A. tumefaciens strain LBA4404-transformated tobacco by Mason et al (1992) there has been a huge research on expressing major HBV viral proteins. Recombinant HBsAg has been expressed in lupine and lettuce (Kapusta et al, 1999), carrot suspension cells (Zhao et al, 2002), soybean (Smith et al, 2002), peanut (Chen et al, 2002), tomato (Ma et al, 2002;Carolina and Francisco, 2004;Wang and Li, 2008;Srinivas et al, 2008), cherry tomatillo (Gao et al, 2003;Guan et al, 2010), potato (Shulga et al, 2004), banana (Sunil-Kumar et al, 2005), apple core and leaves and tomato (Lou et al, 2005), NT-1 cells of tobacco (Sunil-Kumar et al, 2007) and lettuce (Kostrzak et al, 2011). Chen et al (2002) reported a high level expression of HBsAg in peanut, obtained by Agrobacterium-mediated transformation, with the yield of 2.42 µg HBsAg/g of fresh weight.…”