High‐yield rapid production of hepatitis B surface antigen in plant leaf by a viral expression system

Abstract

SummaryRecombinant hepatitis B surface antigen (HBsAg) constitutes currently used vaccines against hepatitis B virus, and has been successfully employed as a carrier for foreign epitopes.With the aim of developing an inexpensive, easily administered vaccine source for global immunization, several groups have expressed HBsAg in plant systems. Transgenic plant-derived HBsAg assembles into virus-like particles (VLPs) and is immunogenic in both mice and humans. However, HBsAg expression is relatively low in transgenic plant systems.The time-consuming and labour-intensive process of generating transgenic plants also significantly limits high-throughput analyses of various HBsAg fusion antigens. In this paper, the high-yield rapid production of HBsAg in plant leaf using a novel viral transient expression system is described. Nicotiana benthamiana leaves infiltrated with the MagnICON viral vectors produced HBsAg at high levels, averaging 295 μ g/g leaf fresh weight at 10 days post-infection, as measured by a polyclonal enzyme-linked immunosorbent assay.Transiently expressed HBsAg accumulated as the full-length product, formed disulphidelinked dimers, displayed the conformational 'a' antigenic determinant and assembled into VLPs. Immunization of mice with partially purified HBsAg elicited HBsAg-specific antibodies.Furthermore, it was found that transient production of HBsAg using vacuum infiltration of whole plants, rather than syringe infiltration of leaves, was readily scalable, and greatly improved the accumulation of correctly folded HBsAg that displays the protective 'a' determinant.

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

“…Plant-expressed S-HBsAg had buoyant density comparable to the antigen from human plasma or recombined in yeast [35,45,49,52,54,56,58]. 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.…”

Plant-based vaccines against human hepatitis B virus

“…In this case, the transient expression system using plant virus-based vectors have the advantage of rapid high-level expression of the recombinant protein as compared with stable transgenic plants. Recent work has demonstrated that tobacco leaves infiltrated with MagnlCON viral vectors produced HBsAg at a high level (~300 µg/g fresh leaf weight) on day 10 after infection [40], which exceeds the best results achieved for transgenic plants by several times (TABle 1). It was demonstrated that transiently expressed HBsAg was correctly assembled and was immunogenic in intraperitoneally injected mice.…”

“…When using plants as alternative expression systems for accumulation of antigens, the work is also directed towards an increase in the yield of recombinant protein in plant tissues [40,47,49]. In this case, the transient expression system using plant virus-based vectors have the advantage of rapid high-level expression of the recombinant protein as compared with stable transgenic plants.…”

“…To increase the efficiency of edible vaccines, it is necessary to solve the problem of how to elevate the production of the target antigen in the developed transgenic plants [40,41].…”

An Oral Vaccine for Hepatitis B: Challenges, Setbacks, and Breakthroughs

“…An incomplete backcross and hybrid breeding program has already produced material that expresses upward of 500 μg/g (unpublished data) and therefore shows exceptional promise as a candidate oral vaccine. Nicotiana benthamiana, using the transient MagnICON viral vector system, has averaged 295 μg/g of fresh weight (Huang et al 2008), but this system is designed for purification and injection of the antigen, rather than oral delivery. Nicotiana species typically produce noxious secondary compounds that are not compatible with an oral delivery system and therefore are not good candidates for oral vaccines in an unpurified form.…”