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Lecture 40&41
Edible Vaccines
Specific vaccines have been produced in plants as a result of transient or stable
expression of foreign genes. It has recently been shown that genes encoding antigens of
bacterial and viral pathogens can be expressed in plants in a form in which they retain
native immunogenic properties.
There are basically 2 strategies for edible vaccine production. These include:
1. Expression of foreign antigens in plant via stable transformation
2. Delivery of vaccine epitopes via plants virus
(Mason and Arntzen, 1995)
Figure 4
Strategies for the production of candidate vaccine antigens in plant tissues
(Mason and Arntzen, 1995)
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The first report of the concept of using a plant expression system for the
production of an edible vaccine appeared in a patent application published under the
international patent co-operatin treaty. It described a means to express a surface protein
(spa A) from Streptococus mutans in tobacco plants to a level of 0.02% of total leaf
protein, the gene had been stably inserted by Agrobacterium mediated transformation.
Data were presented on the oral immuno genicity of spa A produced in E.coli, which
stimulated the production of S-IgA in saliva (Bergmann and Waldman, 1998).
This paved the way of the concept of using plants as bioreactors for obtaining
edible vaccines against bacterial and viral diseases.
The use of strong and organ specific promoters help for targeting the proteins in
the specific organs. This helps in the stability of proteins (antigens). These antigens
stimulate serum as well as mucosal immune system to produce antibody against the
orally injected antigens. Thus an antigen produced in edible part act as vaccines.
Till now 5 antigens have been experimented in plants:

Rabies virus G protein in Tomato

Norwalk virus (cold virus) capsid protein in tobacco and potato

Hepatitis B surface antigens in tobacco and potato

E.Coli heat labile entertoxin B subunit (LT-B) in tobacco

Carbohydrateslera toxin B (CT-B) in potato
1. Hepatitis “B” surface antigens in Tobacco
Hepatitis “B” virus is one of the major causes of chronic viremia in humens.
Arntzen and his team (Mason et al., 1992), demonstrated that tobacco leaves can express
recombinant hepatitis surface B antigen (HBsAg) to a level of 0.01% of the soluble
protein content. The surface antigen (HBsAg) is produced in copious amounts in liver
cells of infected individuals (Richter et al., 2000). Some of this envelope protein is
secreted from liver cells can be recovered in serum in form of virus like particdes (VLPs).
The VLPs, consisting of HBsAg and membrane lipids were found to be highly
immunogenic and would elicit antibodies specific for authentic HBV. The gene encoding
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HBsAg was expressed in tobacco plants, targeting the plastid (chloroplast). Expression
of HBsAg in transformation tobacco leaves yield VLPs avg. 22mm in size. When used
for parenteral immunization of mice, the tobacco VLPs provoked BandT cell immune
response, comparable to yeast – derived vaccine.
However, because of possible
degradation of proteins in the gut, presentation of vaccine antigens by an oral route
requires much higher levels of immunogen than parenteral delivery so by increasing the
levels of HBsAg in plants tissues, it would enhance the utility of plants as source of
recombinant antigen.
Figure 4
Structure of expression cassettes
Structures of expression cassettes
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Figure 5
RNA Analyzed by Northern Blot Hybridised with HBs Ag coding sequence
E.coli heat labile enterotoxin (B subunit )in tobacco
The heat labile enterotoxin (LT) from E.coli is a multimeric protein consisting of
a 27KDA A subunit (LT-A) and a pentamer of 11.6Kda B sub unit (LT-B). LT-B
specifically binds to GM1, gangliosides on epithelial cells allowing entry of LT-A into
cells. The gene encoding LT-B was cloned into a plant expression vector behind CaMV
35S promoter and 5’ untranslated region of tobacco etch virus (TEV). The 3’ sequence
of soybean VSPB gene was used as a terminator. A second plasmid with short coding
sequence leading to the endoplasmic retention was ligated to the 3’ end of LT-B coding
sequence.
Both the plasmids were transformed into tobacco and potato plants by
Agrobacterium transformation (Haq et al., 1995). The antigen amount was found to be
0.0014% of total soluble proteins in tobacco leaves and 0.011% of microtuber proteins in
potato tubers. On feeding tubers to mices, both humoral and mucosal immune responses
were generated.
Rabies virus “G” protein in tomato
The gene for rabies virus glycoprotein including the signal peptide was linked to
CaMV 35 S promoter and transformation by Agrobaterium into tomato plants. Leaves,
fruits from transgenic tomato plants expressed the recombinant glycoprotein having a
molecular weight of 62 Kda. The protein was detected by Western blotting and found to
be localised in Golgibodies, vesicles, plasmalemma and parenchymatous cell walls (Mc
Garvey et al., 1995)
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HIV Virus
The glycoprotein (gp41) of HIV type, (HIV-1, IIIb) containing 22 aminoacids was
introduced between aminoacids 22 and 23 of small capsid subunit of CPMV (Porta et al.,
1994). 3 different HIV-1 strains were found to be neutralized in vitro and invivo by
antibodies recovered from mice previously injected with gp41 modified CPMV (McLain
et al., 1995).
Norwalk Virus (Cold Virus) capsid protein in tobacco and potato
Norwalk virus is the causative agent of acute epidemic gastroenteritis.
The
Norwalk virus capsid protein (NVCP) gene was fused to CaMV 35S promoter or potato
tuber specific patatin promoter using TEV translation enhancer (Mason et al., 1996).
Transformation was carried out using Agrobaterium tumefaciens. Upto 0.23% of soluble
protein in tobacco leaves and 0.37% of total soluble protein in potato tubers were
recovered as rNVCP.
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