532 Biotechnol. Prog.

2007, 23, 532−539

Production of Hepatitis B Surface Antigen in Recombinant Plant Systems: An

Update
G. B. Sunil Kumar, T. R. Ganapathi, and V. A. Bapat*
Plant Cell Culture Technology Section, Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Center,
Trombay, Mumbai 400 085, India

There is a growing interest to develop oral vaccines for infectious diseases, as it is the most
convenient and effective way to attain mucosal immunity. Hepatitis B continues to be a major
infectious disease in many developing countries despite the availability of recombinant vaccine.
On a global scenario, Hepatitis B Virus infection is probably the single most prevalent cause of
persistent viraemia in humans. There are about 350 million chronic carriers of HBV, which is
about 5% of the total world population. It is estimated that 75-100 million of them will die of
liver cirrhosis and/or hepatocellular carcinoma. Progress in plant genetic engineering has enabled
the transfer of useful genes for desirable traits. The recent trend is to use this technique to
exploit plants as biofactories for the production of therapeutic proteins including vaccines. Rapid
progress has been made in this area to develop plant-based vaccines for hepatitis B. This review
describes the expression, characterization, and immunogenicity studies of hepatitis B vaccines
produced in recombinant plant systems and their implications for developing a plant-based
vaccine.

Contents On a global scenario, HBV infection is probably the single most

prevalent cause of persistent viraemia in humans. There are
Introduction 532
about 350 million chronic carriers of HBV, which is about 5%
Structural Components of HBV 532 of the total world population. It is estimated that 75-100 million
Hepatitis B Surface Antigen 533 of them will die of liver cirrhosis and/or hepatocellular
Recombinant Hepatitis B Vaccines 533 carcinoma. The carrier state development probability is more
Plant-Based Vaccines for Hepatitis B 533 in youth than in older age. Up to 90% of the babies born to
Host Systems 534 carrier mothers may become carriers and are at a high risk of
Tobacco Plants 534 developing chronic liver disease at a younger age. The infection
may be mild and self-limiting, or it can cause fatal fulminant
Tobacco Cell Cultures 534
or sub-fulminant hepatic failure. The more serious consequences
Potato 534 of a chronic carrier state are progression to chronic liver disease,
Potato Hairy Roots 534 namely, chronic hepatitis and cirrhosis of the liver. It is one of
Banana 534 the major killer diseases of mankind (2). The continued
Lupin and Lettuce 535 transmission of HBV is thus assured by the existence of this
Optimization of HBsAg Expression in 535 large reservoir of human carriers. The late sequel of HBV
Recombinant Plant Systems infection is hepatocellular carcinoma (3). About 1 million new
Selection Strategies Employed To Develop 535 cases of hepatoma occur annually. The host range of this virus
Recombinant Plant Systems and the is limited to humans and chimpanzees, and the virus cannot be
Regulatory Requirements propagated in tissue culture. The virus is maintained in the
Optimization of Process Parameters for in 536 population mainly via maternal transmission and is transmitted
Planta HBsAg Expression by exposure to contaminated needles or instruments, by blood
Extraction of HBsAg from Plant Materials 536 transfusion, or by sexual contact (mucosal route). This Review
describes the present status of hepatitis B vaccine production
Structural Characterization of Plant-Derived 536
in recombinant plant systems.
HBsAg
Immunogenicity Studies of Plant-Derived 537
Structural Components of HBV
HBsAg
Future Perspectives 537 HBV infection results in secretion of high titers of an antigen
called Australia antigen in the sera (4). This antigen is found
on three types of particles: (1) pleomorphic spheres of ap-
Introduction proximately 20 nm diameter, (2) filaments of variable length
Hepatitis B virus (HBV) belongs to a family of viruses called and diameter of 20 nm, and (3) spherical double shelled particles
hepadnaviridae and causes serum or transfusion hepatitis (1). of 42 nm that are referred to as Dane particles. The inner protein
Its characteristic nature is to produce excess viral proteins shell of the Dane particle is called the core particle, which
without genome, which are known as virus-like particles (VLPs). contains core antigen (HBcAg). The antigen present on the outer
10.1021/bp0602754 CCC: $37.00 © 2007 American Chemical Society and American Institute of Chemical Engineers
Published on Web 03/10/2007
Biotechnol. Prog., 2007, Vol. 23, No. 3 533

Figure 1. Structural organization of the HBV ORF-S that encodes

the three forms of surface antigen proteins.

shell of the Dane particle is referred to as hepatitis B surface

antigen (HBsAg). The core antigen surrounds the viral genome,
a small DNA molecule of unusual structure. Another antigen,
which is a derivative of the core antigen and known as e antigen
(HBeAg) is found in various forms (5). Among the three
antigens, HBsAg was found to be protective antigen suitable
for vaccine development.

Hepatitis B Surface Antigen

The S region of HBV genome, which codes for the HBsAg,
further consists of two regions, preS (pre S1 and pre S2) and S,
with an initiation codon in each region. As a result, HBsAg
exists in three forms, namely, small (SHBs), middle (MHBs),
and large (LHBs). Each of the three proteins encoded by preS/S
arise from separate translation initiation at each of the three
in-frame start codons. The smallest SHBs, encoded by the S
region, exists in glycosylated (gp 27) and unglycosylated (p24)
forms. The middle MHBs encoded by the pre S2/S domain
consists of a hydrophilic N-terminal extension of 55 aminoacids
arising from translation of the pre S2 region. It exists as mono-
(gp33) and diglycosylated (gp36) forms. Translation at the pre
S1 region results in the production of LHBs with an additional
108 or 109 aminoacids from the pre S1 region (depending on
subtype). LHBs exists as glycosylated (gp42) and unglycosylated
(p39) forms (6). SHBs is the major component in all three
particles. The structural organization of the ORF S that encodes
the three forms of surface antigen is depicted in Figure 1.
SHBs carries at least one neutralizing epitope. Two other
determinants have been described for HBsAg (6). One deter-
minant has either d or y specificity and the other has either w
or r specificity. All combinations of determinants are found,
resulting in four subtypes of HBV: adw, adr, ayw, and ayr (7).
HBV strains are further classified in to nine serotypes, ayw 1,
2,3,4, ayr, adw 2,4, adrq- and adrq+, on the basis of their
antigenic determinants and sub-determinants of their HBsAg
(8, 9). The occurrence of escape mutants in many regions of
the world with different SHBs subtypes suggests that subtype-
specific immunity may not play an important role if SHBs is
the only vaccine component. Intramuscular injection of serum-
or yeast-derived recombinant HBsAg in healthy individuals
results in effective immunization and protection from viral
infection (10). The technical advisory group of the World Health
Organization (WHO) has recommended the addition of hepatitis
B vaccine in the Expanded Programme of Immunization (EPI)
in all countries with moderate to high endemicity of infection
(11, 12).
Recombinant Hepatitis B Vaccines
The recombinant subunit vaccines derived from yeast and
mammalian cells (Chinese hamster ovary cells) are effective at
eliciting high titers of protective anti-HBs antibodies in vac-
cinees and are licensed for human use. However, processes
based on recombinant yeast generally have been more produc-
tive. As a result, most of the second-generation hepatitis B
vaccine supplied worldwide is derived from recombinant strains
of yeast (Saccharomyces cereVisiae, Pichia pastoris).
Figure 2. Different expression systems used to express hepatitis B
surface antigen (HBsAg): (A) banana fruits, (B) tobacco plants, (C)
soybean callus, (D) potato hairy roots, (E) potato microtubers, (F)
tomato fruits.
Plant-Based Vaccines for Hepatitis B
Recombinant vaccine derived from yeast is expensive, and
this restricts its use in the mass immunization programs of
developing countries. Plant-based production of vaccine for
hepatitis B may be an economically feasible alternative. It offers
advantages such as production using existing agricultural
practice rather than fermentation or bioreactor systems. The
technology for harvesting and processing of plant material on
a large scale is already available, and the purification require-
ment can be eliminated when the plant tissue containing the
recombinant antigen is used as a food (edible vaccines) (13).
As HBV is transmitted through contaminated needles and by
sexual contact, effective vaccination should induce both systemic
and mucosal immune responses. Vaccines delivered orally
confer mucosal immunity. Conventional vaccines involving
parenteral immunization may not induce secretory antibodies
at mucosal surfaces. Mucosal immunization can be an effective
means of inducing not only secretory IgA but also systemic
antibody and cell-mediated immune responses. Mucosally
administered vaccines have potential to enhance stability and
increase shelf life, avoid cold storage (when delivered in edible
plant tissues/seeds), and eliminate the use of needles and the
need for trained personnel to deliver vaccines (14). Different
plant-based host systems were studied for their advantages and
disadvantages to produce hepatitis B vaccine (HBsAg) (15).
Some of the recombinant plant systems employed for HBsAg
production are shown in Figure 2.
534
Table 1. Host Systems Used for the Expression of Hepatitis B
Vaccine
host systerm protein expression levels ref
tobacco S 66 ng/mg TSP 17, 18
potato S 16 µg/g FW 22
PreS2 and S 37
potato hairy roots S 97.1 ng/g FW 23
lettuce and lupin S 150 ng/g FW 26
carrot S 25 ng/g FW 38
macroalga S 2.497 µg/mg TSP 39
tobacco cell cultures S 2 µg/g FW 19
226 ng/mg TSP 21
banana S 1 ng/g FW (fruits) 25
38 ng/g FW (leaves)
cherry tomatillo S 330 ng/g FW (leaves) 40
10 ng/g FW (fruits)
Host Systems
Vegetable and fruit crops would be ideal host systems for
the production of oral hepatitis B vaccine. However, non-food/
feed crops such as tobacco and in vitro systems such as plant
cell cultures and hairy roots have other advantages, which are
discussed below, and were also employed for hepatitis B vaccine
production. These systems are listed in Table 1.
Tobacco Plants. Tobacco offers several advantages for
HBsAg production as optimized tissue culture protocols and
genetic transformation methods are well established. Tobacco
is a prolific seed producer (3000 seeds/capsule), and availability
of a wide range of germplasm enables customization of genetic
backgrounds to remove metabolites such as nicotine that may
affect the end use. It is a non-food/non-feed crop, restricting
the transgenic plants’ entrance to the food chain. Biomass
production and protein extraction procedures have been ad-
equately standardized. High density (100,000 plants/ha) field
planting and multiple harvests from regrowth following the first
cutting produce fresh weight in excess of 50,000 kg/ha/season.
Soluble protein levels in tobacco range between 2.5% and 2.8%
of total biomass and concentrations as high as 9.2% were
possible when stems are excluded. Yield of extractable protein
varied between 155 and 228 kg/ha (16).
Mason et al. (17) reported the expression of HBsAg in
transgenic tobacco plants for the first time. The plants were
genetically engineered to produce HBsAg using the “s” gene
of HBV. The maximum expression levels of 66 ng/mg total
soluble protein (TSP) in leaves was obtained as measured by
ELISA using a monoclonal antibody directed against human
serum derived HBsAg. The amount of HBsAg in the extracts
of transformed leaves correlated with mRNA abundance,
suggesting that there were no major inherent limitations of
transcription or translation of this gene in tobacco. Further Sunil
Kumar et al. (18) demonstrated the accumulation of HBsAg in
tobacco seeds. Seed-based expression may enable long-term
storage of the vaccines at room temperature without any
significant degradation of the recombinant protein. A maximum
expression of 19.4 ng/g fresh weight (FW) of leaves and 4 ng/g
FW in seeds were obtained in this study. The HBsAg expressed
in tobacco needs to be purified prior to the formulation of the
vaccine, which adds to the costs of production. These factors
have to be considered for developing tobacco as a vaccine
production system.
Tobacco Cell Cultures. Tobacco cell cultures were explored
as production systems for recombinant proteins. The Nicotiana
tabaccum 1 (NT-1) cell line of tobacco is the most extensively
characterized and used plant cell line. Plant cell cultures allow
the production of recombinant proteins under controlled condi-
tions, enabling the implementation of good manufacturing
Biotechnol. Prog., 2007, Vol. 23, No. 3
practices. The most important advantage of cell cultures is the
ease of recombinant protein isolation and purification, especially
when it is secreted into the spent medium. HBsAg was expressed
both as intracellular and secreted forms in the NT-1 cell line of
tobacco. The expression levels of the antigen at 2 µg/g FW of
cells and 31µg/L of spent medium were reported (19, 20).
N-Terminal fusion of plant signal peptide to HBsAg resulted
in enhanced accumulation of the fusion protein in the NT-1 cell
line of tobacco (226 ng/mg TSP) and stimulated higher levels
of serum IgG response in mice (21). This report demonstrated
that HBsAg tolerates the polypeptide fusion at the amino
terminus and improved the antigen expression levels and
immune responses when injected into mice. These results
suggest that HBsAg could be expressed in plant cell cultures
in the required form and elicited antigen-specific immune
response in mice. However, there is a need to enhance and
optimize the expression levels and maintain the stability of plant
cell cultures for continued and constant expression levels in
long-term cultures.
Potato. Potato is one of the important non-cereal food crops,
providing half of the world’s annual production of all roots and
tubers. It offers several advantages for the production of HBsAg,
which include efficiency of genetic transformation by Agro-
bacterium tumefaciens with relatively short transformation and
generation times, clonal propagation, availability of tissue-
specific promoters, and capability of microtuber production and
storage potential. Richter et al. (22) reported highest expression
levels of 16 µg HBsAg per gram potato tubers. Although potato
has several advantages for vaccine production, the raw potatoes
are not palatable to humans and cooking may degrade the
antigen. It is more suitable for evaluating the oral vaccine
efficacy.
Potato Hairy Roots. Hairy roots have been used in a wide
range of research applications such as plant improvement, plant
secondary metabolism studies, plant interaction with the envi-
ronment, and for the production of recombinant proteins. The
genetic stability, biosynthetic potential, growth in hormone-free
media, and short doubling time of hairy roots make them an
attractive system for the production of biopharmaceuticals under
controlled conditions. The expression levels of the recombinant
proteins in the transgenic plants could be influenced by
environmental factors. The implementation of good manufactur-
ing practice (GMP) requires their cultivation under controlled
conditions. This could be best achieved by using plant cell
cultures or hairy roots as expression systems. Recently, our
group reported the initiation, growth optimization and generation
of potato hairy roots for the production of HBsAg using two
different expression cassettes (23). Higher expression levels were
noted in hairy root cultures (97.1 ng/g FW) when compared to
transgenic plants (19.11ng/g FW) from which they were derived.
However, expression levels need to be further enhanced by using
strong root specific or inducible promoters to make it cost-
effective. Rhizosecretion is an attractive means for HBsAg
production that can be explored with the hairy roots.
Banana. Banana is considered as an ideal host for the
production of oral vaccines for hepatitis B. It offers advantages
such as digestibility and palatability by infants and availability
throughout the year in the tropics and subtropics where
economical vaccines for hepatitis B are required to immunize
large segments of the population (24). The majority of edible
bananas are vegetatively propagated through suckers and do not
set seeds. Hence it is a suitable candidate for gene containment
with no segregation of the transgene. Recently, we have reported
the expression of HBsAg in banana fruits (25). Expression level
Biotechnol. Prog., 2007, Vol. 23, No. 3 535

of 1 ng/g FW in fruits and 38 ng/g FW in leaves were noted.

Although the expression levels obtained in this study are less,
it has demonstrated the feasibility of HBsAg expression in the
fruits in a required form. It is necessary to increase the level of
HBsAg expression in banana fruits by adopting various mo-
lecular strategies.
Lupin and Lettuce. Lupin produces high protein seeds like
other legumes. It is grown both as a forage and grain legume.
Lupin seed can be fed directly to poultry and livestock whole,
cracked, or ground. These characteristics allow lupin to be used
as a host system for the production of oral vaccines. Lettuce is
another host system often grown as a leafy vegetable. It is
typically eaten cold and raw in salads, burgers, and other dishes.
Kapusta et al. (26) reported HBsAg expression at levels of 150
ng/g FW in lupin callus and 5.5 ng/g FW in lettuce leaves.
Expression of HBsAg in lupin seeds would be advantageous
for storage at room temperatures. However, storage and
distribution of lettuce for longer periods would be a challenge
to be addressed.

Optimization of HBsAg Expression in Recombinant

Plant Systems
The optimization of expression levels is required to develop
plant-based hepatitis B vaccine as a commercially viable
production system. These include cellular and molecular ap-
proaches. Richter et al. (22) constructed a series of HBsAg
expression vectors all of which included 35S promoter and
examined the role of 5′ untranslated regions (5′ UTRs) from
tobacco etch virus and tobacco mosaic virus, the signal peptide
from soybean vegetative storage protein (VSP), a plastid transit
peptide, and three different 3′ flanking sequences to optimize
the HBsAg expression in potato. The constructs with either VSP
3′ UTR or potato pin2 gene resulted in higher levels of HBsAg
mRNA compared with those with NOS 3′ elements. VSP 3′
UTR gave the highest levels of expression in potato leaves.
These results indicate that post-transcriptional effect contributes
strongly to the enhanced expression. However, potato lines with
higher expression levels grew poorly in the greenhouse or had
poor tuber yield, indicating that HBsAg at high levels may be
phytotoxic. There was no striking difference between the
constructs of different 5′ elements. The incorporation of VSP
signal peptide at the N-terminus of HBsAg resulted in higher
expression levels and also the C-terminal ER retention signal
allowed better accumulation of HBsAg in plant cells. The use
of plastid transit peptide did not produce measurable HBsAg.
Some of the representative expression cassettes used for HBsAg
expression in plant systems are schematically represented in
Figure 3.
The immunogenicity and assembly of HBsAg is dependent
on disulfide bonding and extensive cross-linking, and mono-
clonal antibodies recognize the properly folded HBsAg particle
only. We have investigated this by incorporating a C-terminal
ER retention signal (SEKDEL) to test whether the proportion
of monoclonal antibody binding to HBsAg could be increased.
Our results indicated that incorporating the ER retention signal
improved the proportion of monoclonal antibody reactive
HBsAg (49.3-67.8%) in transgenic tobacco and banana plants
(18, 25). The endoplasmic reticulum harbors protein-folding
machinery including protein disulfide isomerase (27), which
might have contributed to the proper folding of HBsAg. Sojikul
et al. (21) obtained more stable and immunogenic HBsAg when
it was expressed as a fusion protein with a plant signal peptide.
The use of ethylene forming enzyme promoter (EFE) of the
banana (28) allowed post-harvest enhanced expression of
Figure 3. Expression cassettes used for HBsAg production in
recombinant plant systems. D 35 S: CaMv promoter with dual
enhancer, TEV, HBS: the “s” gene of hepatitis B virus. NOS: 3′ region
from A. tumefaciens nopaline synthase gene terminator. VSP R S: 3′
region from soybean vegetative storage protein gene with a signal
peptide. VSP R L: 3′ region from soybean vegetative storage protein
gene with a putative vacuolar targeting signal. VSP 3′: 3′ region from
soybean vegetative storage protein gene. PIN2 3′: 3′ region from potato
proteinase inhibitor II gene. TEV: tobacco etch virus 5′ UTR. TPSS:
transit peptide from the small subunit of Rubisco. SEKDEL: hexapep-
tide ER retention signal. UBQ 3: UBQ3 promoter from Arabidopsis
thaliana. EFE: ethylene forming enzyme promoter from banana.
HBsAg in wounded leaves of transgenic tobacco and banana
leaves. This was further enhanced by treating wounded leaves
with plant growth regulators (25). The expression levels in cell
cultures/hairy roots could be further enhanced by manipulating
various process conditions.
Selection Strategies Employed To Develop
Recombinant Plant Systems and the Regulatory
Requirements
The npt II gene that confers resistance to aminoglycoside
antibiotics such as kanamycin and geneticin was extensively
used to select transgenic plant systems expressing HBsAg. The
use of antibiotic resistance genes as markers for the selection
of transgenic crops has resulted in the public concern that these
genes may be transferred to pathogenic bacteria. Despite the
availability of risk assessment reports, the public concerns about
their safety have resulted in significant delays in the release of
transgenic varieties. During gene stacking for multiple trait
incorporation, the selectable marker genes will also get amplified
and result in homology dependent gene silencing. To overcome
these problems, the positive selection systems may enable
reduced negative effects and enhance the efficiency of trans-
formation. However, this technology may not overcome all of
the problems. Therefore, it is desirable to remove selectable
markers from the transgenic plants. To achieve this, different
methods were attempted: (1) Co-transformation of plant cells
536
using two different T-DNAs with a selectable marker and gene
of interest on different T-DNA; if the two T-DNAs integrated
into unlinked loci, the selectable marker can be eliminated by
crossing. (2) Site-specific recombination system that utilizes the
Cre/lox system of bacteriophage P1, which consists of the
recombinase (Cre) and its recognition site (loxP). Cre, which
causes recombination, results in the excision of a DNA segment
between two directly adjacent loxP sites; the selectable marker
gene placed in between two loxP sites will be excised from the
genome and the marker free transgenic plants can be obtained
by crossing. (3) Recently, two types of multi-auto-transformation
(MAT) vectors (ipt-type and rol-type) have been developed to
remove the selectable marker genes from transgenic plants (29).
The implementation of one of the above strategies in the
development of transgenic systems may address the concerns
of the antibiotic marker genes.
Optimization of Process Parameters for in Planta
HBsAg Expression
Process parameters were studied for HBsAg expression in
soybean and tobacco cell cultures. Expression of HBsAg in
soybean cell suspension cultures resulted in 10 times higher
levels of antigen accumulation than that obtained in tobacco
cell cultures. The expression levels of HBsAg remained ap-
proximately constant during the exponential phase of growth
and triggered an accumulation in the stationary phase. However,
the level of assembly (monoclonal antibody reactive HBsAg)
was substantially better in early exponential phase than in
stationary phase cells of soybean, but there was a less marked
dependence on culture age in tobacco cell cultures (30). The
HBsAg should be characterized for its assembly and immuno-
genicity by extracting and purifying this antigen from recom-
binant plant systems.
Extraction of HBsAg from Plant Materials
Protocols for the extraction of recombinant HBsAg from
transgenic plant materials were studied. The effects of reducing
agent 2-mercaptoethanol (BME), protease inhibitors, extraction
buffers, temperature, storage at 4 °C, detergent, antioxidant
concentrations, and stabilizers (skimmed milk powder) were
analyzed to optimize the extraction protocols to recover and
obtain the required form of HBsAg from recombinant potato
tubers (31, 32).
The optimum temperature was found to be 50 °C for the
extraction of HBsAg. Increasing the temperature of the sample
for about 1 min increased the measurable antigenic activity.
Three-fold enhancement of antigenic activity was noted when
assayed with monoclonal antibody based ELISA (mAb ELISA),
although no enhancement was obtained with the polyclonal
antibody based ELISA (pAb ELISA). Temperature might have
an effect on the conformation of the epitopes to which the
monoclonal antibodies bind or alter the fluidity of the surface
lipids. BME also increased the proportion of monoclonal
antibody reactive HBsAg by 4-fold with no increase using pAb
ELISA. This result also demonstrated that BME may affect the
structure or presentation of the “a” epitope. A proteinase
inhibitor, leupeptin, also increased the antigenic activity. This
could be due to the protection of the antigen from proteolytic
degradation by leupeptin from sulfhydryl proteases. Despite the
fact that both BME and temperature could increase the antigenic
activity of HBsAg individually, their combined interaction was
antagonistic.
Two different extraction buffers were tried in the extraction
procedure to aid the release of HBsAg particles that are encased
Biotechnol. Prog., 2007, Vol. 23, No. 3
within the membrane of the endoplasmic reticulum. One was
based on phosphate buffer (pH 7.4) and the other was sodium
carbonate buffer (pH 11). The low salt, high pH conditions of
the carbonate buffer converts vesicles into sheets releasing ER
lumen contents, while leaving integral membrane proteins
associated with the ER intact (33). When carbonate buffer was
used, the detectable polyclonal antibody reactive HBsAg (pAb-
HBsAg) increased by 50% indicating the improved HBsAg
release. However, only a 5-fold increase was obtained with the
addition of detergents to either buffer. The levels of the
antioxidant sodium ascorbate in the extraction buffer influenced
the monoclonal antibody reactive HBsAg (mAb-HBsAg). A 14-
and 4.6-fold increase was observed in crude extracts of
transgenic tobacco and soybean suspension cells, respectively,
with the addition of sodium ascorbate. However, sodium
ascorbate had no effect on mAb-HBsAg in potato tuber crude
extracts.
The HBsAg was unstable in crude extracts with a complete
loss of pAb-HBsAg after 15-20 days storage at 4 °C. This loss
was exacerbated by the presence of elevated levels of sodium
ascorbate. Lowering the detergent to cell concentration has
improved stability, with pAb-HBsAg levels stable for 30-40
days with storage at 4 °C. Both pAb and mAb-HBsAg were
maintained at initial levels for at least 60 days with the addition
of skimmed milk powder, whereas in the extracts lacking
skimmed milk powder pAb-HBsAg was lost within 20 days
and mAb-HBsAg within 3 days in soybean cell extracts. Similar
results were obtained for tobacco cell lysates where stability of
the pAb-HBsAg was maintained at initial levels for 90 days.
During the course of storage at 4 °C, the mAb-HBsAg levels
were increased in crude lysates of both soybean and tobacco
cells. This increase was influenced by the presence of sodium
ascorbate. Although the initial mAb-HBsAg increased with
increasing levels of antioxidants, it was retarded by the reducing
environments later (31, 32). The formation of multimeric particle
is essential for the immunogenicity of HBsAg. Thus it is
necessary to characterize it, for structure and assembly. A flow
chart of the key steps involved in HBsAg expression in
recombinant plant systems for developing oral vaccines are
shown in Figure 4.
Structural Characterization of Plant-Derived HBsAg
The plant-derived HBsAg was characterized for its structure
by buoyant density studies and transmission electron micros-
copy. In the first report transgenic tobacco derived HBsAg was
studied for its buoyant density properties (17). It was observed
that the tobacco-derived HBsAg had buoyant density similar
to that of human serum derived HBsAg. Tobacco cell line as
well as transgenic banana derived HBsAg was also assessed
for its buoyant density properties, and it was found that HBsAg
derived from these plant systems also had a buoyant density
comparable to that of human serum derived HBsAg (19, 25).
The affinity purified HBsAg from tobacco plants had a size of
22 nm, comparable to VLPs found in carriers of HBV as
observed by transmission electron microscopy (17). Potato-
derived HBsAg accumulated as 17 nm particle structures in
leaves (34) and as tubular structures within dilated ER mem-
branes in tuber tissues (35). This is analogous to those observed
in a mammalian cell line (Chinese hamster ovary cells)
expressing HBsAg, but plant-derived HBsAg had a complex
size distribution. These reports suggest that HBsAg assembles
into a required particulate form, which is essential for its
immunogenicity.
Biotechnol. Prog., 2007, Vol. 23, No. 3
Figure 4. Flow chart for the production of plant derived vaccine (oral vaccine ) for hepatitis B.
Immunogenicity Studies of Plant-Derived HBsAg
The plant-derived HBsAg was assessed for its immunoge-
nicity in both animals and humans. The first study to demon-
strate the immunogenic potential of transgenic plant derived
HBsAg was carried out by Thanavala et al. (36). HBsAg purified
from transgenic tobacco leaves could induce an immune
response that was qualitatively similar to the responses obtained
by immunizing mice with yeast-derived HBsAg. Anti-HBsAg
response in mice immunized with the tobacco-derived HBsAg
included HBsAg-specific antibodies of all IgG subclasses and
also IgM. On the contrary, mice immunized with yeast-derived
HBsAg produced predominantly IgG1 and substantial amounts
of IgG2b and lower levels of IgG2a and IgM antibodies.
Additionally, T cells obtained from mice primed with the
tobacco-derived HBsAg could be stimulated in vitro by the
yeast-derived HBsAg. The primed T-cells proliferated in vitro
after stimulation with a monoclonal anti-idiotype antibody and
anti-idiotype derived peptide, demonstrating that both B and T
cell epitopes of HBsAg are preserved when the antigen was
expressed in transgenic tobacco plants.
Kong et al. (34) reported oral immunization of mice with
transgenic potato tubers expressing HBsAg along with a mucosal
adjuvant, cholera toxin. Mice fed with HBsAg-transgenic
potatoes produced HBsAg-specific serum antibodies that ex-
ceeded the protective levels and upon parenteral boosting
generated a strong long-lasting secondary antibody response.
Further mice immunized (injection) with HBsAg fused to a plant
signal peptide at the N-terminus had stronger antibody responses
and a greater number of responders than mice that received
537
unmodified HBsAg. The induction of balanced Th 1 and Th 2
responses would be preferred to obtain an effective humoral
and cell mediated immune responses. Both yeast-derived HBsAg
and plant signal peptide HBsAg fusion protein induced relatively
balanced immune responses, whereas the unmodified HBsAg
induced a more Th 2 biased response (21). Recently, Thanavala
et al. (37) conducted double-blind placebo-controlled human
clinical trials to evaluate the immunogenicity of HBsAg
produced and delivered in potatoes orally to previously vac-
cinated individuals. When fed with 100 g of tuber (8.5 µg
HBsAg/g tuber), volunteers exhibited an increase in serum anti-
HBsAg titers in 10 out of 16 individuals who ate three doses
of potatoes and 9 out of 17 volunteers who ate two doses of
transgenic potatoes, whereas no increase was noted in volunteers
who ate non-transgenic potatoes. These results were obtained
without the co-administration of mucosal adjuvants or the need
for buffering stomach pH. The above observations conclude that
HBsAg derived from recombinant plant systems is immunogenic
in both animals and humans and to the extent achieved by
immunization with yeast-derived HBsAg.
Future Perspectives
The plant-based production of vaccines provides new op-
portunities to develop oral vaccines for hepatitis B. It is one of
the preferred ways to deliver vaccines for easy administration
and to attain mucosal immunity. Different host systems were
employed to produce hepatitis B vaccine. Among the different
systems, banana is considered as an ideal host for the production
and delivery of oral vaccine. However, the reported levels of
538
expression are inadequate to test the efficacy of the banana-
based hepatitis B vaccine. Further, optimization of expression
levels by adopting various strategies may allow higher levels
of HBsAg expression in transgenic banana fruits. Considerable
work has been carried out with potato, wherein the expression
levels were optimized in tuber tissue, HBsAg was characterized
for its structure, and immunogenicity studies were carried out
using raw potato tubers in animals and humans. These results
indicated the success of plant-based vaccine for hepatitis B.
However, the use of uncooked potatoes decreases the accept-
ability of the vaccine by the people.
There is a need to produce this vaccine in edible fruits such
as bananas to make it more palatable and acceptable. Processing
of the edible plant material by freeze drying and developing
semiprocessed plant products may be required to deliver the
required dose of vaccine. The effects of the environment and
concerns about safety of transgenic plants in the farmer’s fields
and transfer or mixing of transgenic material with the food crops
are the issues need to be addressed. The yield and the stability
of the antigen expressed is influenced by several factors, and
stable and high yielding lines that perform well in laboratory
condition must produce similar results under field conditions.
Plant cell cultures and hairy roots may enable the production
of vaccines under cGMP and cGLP conditions, which facilitate
easier approvals of plant-based vaccines from the regulatory
agencies. One such example is the recent approval of plant cell
culture based vaccine for a viral disease of poultry granted to
Dow Agro Sciences by the USDA.
There is a necessity to produce mucosal adjuvants along with
vaccines in the edible plant tissues, especially for antigens such
as HBsAg, which has poor immunogenicity when administered
alone. The recent reports on the use of heat-labile enterotoxin
B subunit (LT-B) and saponins along with the plant-derived
vaccines indicated that these adjuvants can be used effectively
to enhance the immunogenicity. Non-food and non-feed crops
such as tobacco would be suitable host systems to avoid mixing
of transgenic material with the food chain. However, it requires
that vaccines produced in these systems should be purified to
remove harmful secondary metabolites before their delivery.
Seed-based expression would be ideal to store vaccines for
longer periods at room temperature and for transportation to
distant places.
The challenges needing to be addressed include enhancing
the expression levels of HBsAg in recombinant plant systems
and addressing the regulatory issues. Plant viral vectors and
chloroplast transformation have provided the highest expression
levels of recombinant proteins in plant systems. However, these
methods may suffer from certain limitations; for example,
protein folding and assembly required for immunogenicity and
the formation of virus-like particles as in case of HBsAg may
limit their use. Contained facilities such as greenhouses, positive
selection methods/removal of selectable marker genes, and the
use of inducible promoters may reduce the risk factors. Plant
tissue processing and formulation should be implemented by
adopting safety guidelines. There is a need to gain social
acceptance of plant-derived vaccines in many countries. The
optimization of HBsAg expression levels in edible plant parts,
developing plant tissue processing methods and formulation
protocols, coexpression or delivery of mucosal adjuvants, and
the incorporation of stabilizers may result in an effective plant-
based vaccine for hepatitis B in the near future.
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Received September 8, 2006. Accepted February 14, 2007.
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