I reviewed several manuscripts, books, grants and project proposals. This is one of the paper I reviewed recently published in Plant Biotechnology Journal

1. A transgenic plant cell-suspension system for expression
of epitopes on chimeric Bamboo mosaic virus particles
Thangarasu Muthamilselvan1
, Chin-Wei Lee1
, Yu-Hsin Cho1
, Feng-Chao Wu1
, Chung-Chi Hu1
, Yu-Chuan Liang2
,
Na-Sheng Lin1,3
and Yau-Heiu Hsu1,
*
1
Graduate Institute of Biotechnology, National Chung Hsing University, Taichung, Taiwan
2
Agricultural Biotechnology Research Center, Academia Sinica, Nankang, Taipei, Taiwan
3
Institute of Plant and Microbial Biology, Academia Sinica, Nankang, Taipei, Taiwan
Received 25 November 2014;
revised 5 March 2015;
accepted 12 March 2015.
*Correspondence (Tel +886 4 22856468;
fax +886 4 22856468;
email yhhsu@nchu.edu.tw)
Keywords: Bamboo mosaic virus,
post-transcriptional gene silencing,
chimeric virus particles, cell-suspension
culture, vaccine, BaMV viral vector.
Summary
We describe a novel strategy to produce vaccine antigens using a plant cell-suspension culture
system in lieu of the conventional bacterial or animal cell-culture systems. We generated
transgenic cell-suspension cultures from Nicotiana benthamiana leaves carrying wild-type or
chimeric Bamboo mosaic virus (BaMV) expression constructs encoding the viral protein 1 (VP1)
epitope of foot-and-mouth disease virus (FMDV). Antigens accumulated to high levels in BdT38
and BdT19 transgenic cell lines co-expressing silencing suppressor protein P38 or P19. BaMV
chimeric virus particles (CVPs) were subsequently purified from the respective cell lines (1.5 and
2.1 mg CVPs/20 g fresh weight of suspended biomass, respectively), and the resulting CVPs
displayed VP1 epitope on the surfaces. Guinea pigs vaccinated with purified CVPs produced
humoral antibodies. This study represents an important advance in the large-scale production of
immunopeptide vaccines in a cost-effective manner using a plant cell-suspension culture system.
Introduction
The advent of high-throughput genetic screening and epitope
mapping of infectious pathogens has opened up new ways of
identifying short antigenic peptides, which are potentially immu-
noprotective (Seib et al., 2012; Sette and Peters, 2007). Such high-
fidelity antigenic peptides are fused to the viral coat protein (CP)
and thus displayed on the surface of assembled chimeric virus
particles (CVPs). Presentation of these peptides (antigens) enables
the development of vaccines (Ca~nizares et al., 2005; Hefferon,
2014; Usha et al., 1993). Such epitopes have been successfully
expressed on the CP surface of human and other animal viruses
in vitro and used to elicit immune response in vaccinated animals
(Ca~nizares et al., 2005; Hefferon, 2014). So far, all previous
investigations reported for the production of CVPs using plant viral
vectors have been in the whole-plant systems (Hefferon, 2014).
Using these systems, a copious amount of CVPs can be achieved
due to the transient nature (Gleba et al., 2004, 2007; Hefferon,
2014; Shih and Doran, 2009), but requires the time and manpower
for inoculation. In addition, whole-plant systems suffer difficulties
in preventing field contamination and maintaining consistent
growth environment (Ca~nizares et al., 2005; Werner et al., 2011).
To overcome the pitfalls of the whole-plant system and the
methodological problems growing cells in vitro, plant cell-suspen-
sion culture system provides an alternative approach for conve-
niently producing recombinant proteins on a large scale (Xu et al.,
2011). The efficacy of plant viral-based vectors has been previously
demonstrated by expressing recombinant proteins in a cell-
suspension culture (Dohi et al., 2006; Hefferon and Fan, 2004;
Huang et al., 2009, 2010; Larsen and Curtis, 2012; Zhang and
Mason, 2006). Cell-suspension culture has several advantages over
whole-plant systems, such as high reproducibility and simple
maintenance of aseptic conditions (Hellwig et al., 2004; Mustafa
et al., 2011). Furthermore, it is amenable to current good
manufacturing practice (cGMP) and assures high-quality target
protein (Hellwig et al., 2004; Huang and McDonald, 2009).
However, plant viral vectors have not been used to produce CVPs
as potential vaccine candidates in cell-suspension cultures. Our
current work is designed to explore and contribute to this area.
We have previously demonstrated that Bamboo mosaic virus
(BaMV)-based expression viral constructs produced CVPs in
whole-plant systems, elicited protective antibodies in swine or
chicken vaccinated with CVPs that displayed the epitopes of foot-
and-mouth disease virus (FMDV) viral protein 1 (VP1) or infectious
bursal disease virus VP2 (Chen et al., 2012; Yang et al., 2007). In
this study, we have combined transgenic technology with an
antigen-presentation strategy, to develop a novel transgenic cell-
suspension culture system, which continually produces self-
replicating chimeric BaMV RNAs and CPs that self-assemble into
CVPs displaying target epitopes. Specifically, we established
transgenic cell-suspension cultures from callus derived from
transgenic plant lines expressing different BaMV expression
cassettes encoding FMDV VP1 epitope. To prevent the post-
transcriptional gene silencing (PTGS) triggered by the high
expression of replicable RNAs (Angell and Baulcombe, 1997),
we included a suppressor of gene silencing either P38 (Qu et al.,
2003) or P19 (Voinnet, 2002) by replacing the viral gene region
encoding the triple-gene-block proteins (TGBp1-3), which are not
required in the cell-suspension culture system. Using this new
approach, we were able to generate transgenic cell lines with
high yields of CVPs. We further validated the utility of this
approach, using the assembled CVPs to vaccinate guinea pigs; the
immunized animals produced humoral antibodies and showed
high sensitivity to FMDV-rVP1.
ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd 231
Plant Biotechnology Journal (2016) 14, pp. 231–239 doi: 10.1111/pbi.12377

2. Results
Construction of chimeric BaMV expression cassettes
The constructs in Figure 1 were assembled and transformed into
the Nicotiana benthamiana plant genome via Agrobacterium-
mediated transformation (Horsch et al., 1985). The pBaMV-S
construct contains the complete BaMV cDNA and was further
modified to pBVP1 (Yang et al., 2007). Other constructs, namely
pBdT38-VP1 and pBdT19-VP1, were built using pBVP1 as a
template, by replacing the triple-gene-block sequence (encoding
TGBp1-3) with that of P38 or P19 silencing suppressor. These
plant virus expression cassettes were placed under the control of
a dual constitutive 35S promoter of Cauliflower mosaic virus and
a nopaline synthase (nos) terminator.
The primary advantage of combining transgenic plant and viral
vector technologies is that stably integrated RNA viral vectors can
replicate autonomously. Continual transcription of viral RNA and
assembly of complete virions suggest that the host cells should
hypothetically be able to produce high levels of CVPs. Selfed-
progeny transgenic plant lines (F2) were selected for homozygos-
ity, and high-level antigen accumulation was confirmed by ELISA
and immunoblotting (manuscript in preparation). The transgenic
lines selected through this study are shown in Table 1.
Establishment of transgenic cell-suspension culture
The major problems associated with transgenic plant technologies
(combining nuclear and plant viral vectors) are the environmental
impact, consistency of production and compliance with cGMP
requirements. To overcome these difficulties, we established
transgenic cell-suspension cultures from calli derived from trans-
genic N. benthamiana leaves expressing wild-type or chimeric
BaMV replicons (Figure S1a,b). A nontransgenic cell line was used
as a control. Sigmoidal growth curves were observed for both
non-transgenic- and transgenic-suspended biomasses (Fig-
ure S1c), indicating that unimpeded replication of chimeric virus
did not have detrimental effects on the cell growth. Cell growth
remained in the lag phase for 3 days after subculture, followed by
an exponential (log) phase; growth peaked at day 21. Biomasses
significantly increased between days 8 and 15 after the
subculture. Cells reached stationary phase after day 22.
Transgenic cell-suspension cultures expressing chimeric
BaMV coat protein
We next examined whether our plant cell-suspension cultures
express wild-type or chimeric VP1 epitope-fused BaMV CP
(designated BVP1) by Western blot analysis of the total protein
from suspended biomass with specific antibodies against BaMV
CP and FMDV VP1. Coomassie Brilliant Blue staining revealed a
distinct band around 31 kDa in the samples from cell lines BdT38
and BdT19, but not from B2B27 or BVP1-16-7 (Figure 2a).
Similar results were observed in Western blot analysis with a
specific BaMV CP antibody (Figure 2a). A high level of BaMV CP
was detected in the cell lines BdT38 and BdT19, but not in the
BVP1-16-7 or B2B27 transgenic cell lines. The difference in
molecular weight between the wild-type and chimeric BaMV CP
indicated stable fusion of VP1epitope with the BaMV CP
(Figure 2a). To confirm that the FMDV VP1 epitope was fused
to BaMV CP, we probed the same protein extracts with anti-
FMDV VP1 antibody. BVP1 was expressed in all transgenic cell
lines except B2B27, as expected (Figure 2a). Probing with anti-
BaMV CP antibody revealed degradation of BVP1 in all the
samples, while probing with anti-FMDV VP1 antibody uncovered
significant amounts of the fused FMDV VP1 epitope on BaMV
CPs (Figure 2a).
Detection of silencing suppressor protein
To confirm the involvement of P19 and P38 in the dramatically
enhanced expression levels of BVP1, we performed Western blot
analysis of the total proteins using specific antibodies against P38
or P19. The result revealed that P38 and P19 proteins were
detected in suspension cells of BdT38 or BdT19, respectively, but
not in BVP1-16-7 or B2B27 (Figure 2a), further suggesting that
the significant difference in BVP1 accumulation levels might be
due to the suppression of PTGS system in cell-suspension culture.
Quantification of BVP1
Next, we estimated that the BVP1 constituted about 0.25% of
total soluble protein (TSP) in the BVP1-16-7 transgenic cell line at
day 21 postsubculture; the level of BVP1 in transgenic cell lines
BdT38 and BdT19 was 4.7% and 5% of TSP, respectively
(Figure 2b). The BVP1 expression in BdT38 or BdT19 transgenic
cell lines was 19-to 20-fold higher than that in the BVP1-16-7 cell
line.
(a)
(b)
(c)
(d)
(e)
Figure 1 Schematic representations of wild-type and recombinant
Bamboo mosaic virus (BaMV)-based expression cassettes. (a) The plasmid
harbouring the full-length infectious cDNA of BaMV under the control of a
dual 35S promoter and nopaline synthase (nos) terminator was designated
pBaMV-S. The BaMV open reading frames encode RNA-dependent RNA
polymerase (RdRp, 155 kDa); movement proteins triple-gene block
(TGBp1-3, TGBp1, 28 kDa), TGBp2 (13 kDa) and TGBp3 (6 kDa); coat
protein (CP). Open reading frames P19 or P38 encode the respective
silencing suppressor protein. (b) The pBS-d35CP vector was generated
from pBaMV-S by replacing the N-terminus of CP (35 amino acids) with a
multiple cloning site (Yang et al., 2007). (c) The pBVP1 vector is a
recombinant plasmid in which the N-terminus of CP (35 amino acids) is
replaced by the foot-and-mouth disease (FMD) viral protein 1 (VP1)
epitope (37 amino acids, T128
-N164
, Yang et al., 2007). (d) and (e) pBdT38
VP1 and pBd19 VP1 are recombinant plasmids derived from pBVP1 by
replacing the coding sequences for TGBp1-3 with those for the silencing
suppressor P38 or P19, respectively.
ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 14, 231–239
Thangarasu Muthamilselvan et al.232

3. Detection of chimeric BaMV genomic RNA
To further confirm whether the silencing suppressor protein P38 or
P19 augments chimeric virus replication through suppressing the
PTGS, we examined the accumulation of chimeric BaMV RNAs in
BdT38 and BdT19 and compared with the BVP1-16-7 and B2B27
transgenic cell lines by northern blot hybridization. Total RNA from
the suspended biomass of nontransgenic and transgenic lines was
hybridized with 32
P-labelled probes specific to the sense-strand of
the 30
UTR of BaMV CP gene. The wild-type or chimeric BaMV
genomic RNAs were barely detected in B2B27 or BVP1-16-7 cell
suspensions, but were significantly increased in BdT38 or BdT19
cell-suspension cultures (Fig. 2c). These results indicate that the
high accumulation of BaMV replicons in transgenic BdT38 or BdT19
cell suspensions was due to suppression of PTGS by P38 or P19,
respectively. Therefore, the substantial increase in replicating
chimeric BaMV RNAs in transgenic cell lines expressing P19 or
P38 enhanced the production of BVP1; the cells lacking such
silencing suppressors exhibited a decreased accumulation of viral
genomic RNAs and low yields of BVP1. Overall, our results suggest
that the silencing suppressors P38 and P19 play a significant role in
the accumulation of BVP1 in BdT38 or BdT19 cell lines.
Stability of chimeric BaMV genome in transgenic cell
lines
To examine the stability of different chimeric BaMV RNA in cell
suspension culture, we extracted the total RNA from suspended
biomass at day 21 postsubculture and produced the cDNAs by
reverse transcription PCR (RT-PCR). The coding region for N-
terminus of FMDV-VP1-BaMV CP was amplified using specific
primers in PCR. The amplified fragments were separated on a 1%
agarose gel, which showed that the amplified PCR products were
approximately ~1 kb in size as expected (Figure 3a). Further, these
PCR products were sequenced, and the results (Figure 3b) showed
that different chimeric BaMV genomes have retained the expected
nucleotide sequences at N-terminal region of the chimeric protein.
These results suggested that the different chimeric BaMV RNA were
stably replicating in the cell-suspension culture.
Characterization of CVPs purified from suspended
biomass
Transmission electron microscopy was used to investigate
whether the wild-type or CVPs undergo self-assembly in the
transgenic suspension cells. The formation of rod-shaped BaMV-
like structures was observed by negative staining (Figure 4).
Probing with antibodies specific to BaMV CP or FMDV VP1
followed by labelling with immunogold-conjugated secondary
antibodies revealed BaMV CP or FMDV VP1 on the surface of
CVPs. The anti-FMDV VP1 antibody did not react with the wild-
type BaMV. Similarly, the pre-immune serum showed no gold
decorations on the CVPs or wild-type BaMV (Figure 4).
To confirm the purity of CVPs, the proteins were analysed by
SDS-PAGE. The chimeric proteins showed significantly slower
migration as compared to that of the wild-type BaMV CP (Figure 5),
indicating the stable fusion of FMDV-VP1 peptide with BaMV CP.
The yields of CVPs in the BdT38 andBdT19 transgenic cell lines were
1.2–1.5 mg CVPs/20 g and 1.8–2.1 mg/20 g fresh weight of
suspended biomass, respectively; these estimates were performed
as previously described (Lin and Chen, 1991). The yields of B2B27
and BVP1-16-7 transgenic cell-suspension lines could not be
estimated due to low level accumulation of wild-type BaMV or
CVPs. These data demonstrate the stable assembly of CVPs from
chimeric BaMV CP with VP1 epitope fused to the N-terminus.
Elicitation of specific antibodies in BVP1-immunized
guinea pigs
To evaluate the efficacy of CVPs to induce antibodies, a group of
three guinea pigs were subcutaneously injected with CVPs
Table 1 Designations of Nicotiana benthamiana transgenic plant
lines stably expressing wild-type or chimeric BaMV replicons
Vector Transgenic line Designation Description
1. pBaMV-S B2B27 B2B27 Wild-type BaMV
2. pBVP1 BVP1-16-7 BVP1-16-7 Chimeric BaMV
3. pBdT38-VP1 BdT38-VP1-19-9 BdT38 Chimeric BaMV with P38
4. pBdT19-VP1 BdT19-VP1-26-7 BdT19 Chimeric BaMV with P19
(a)
(b)
(c)
Figure 2 Accumulation of wild-type or chimeric BaMV protein in
transgenic cell-suspension cultures. (a) Total protein was extracted from
nontransgenic or transgenic cell-suspension cultures at day 21
postsubculture and separated by 10% SDS-PAGE; gels were stained with
Coomassie blue or transferred to PVDF membranes and probed with
rabbit sera against BaMV CP (anti-CP) or VP1 (anti-VP1), or antibodies
against P38 (anti-P38) or P19 (anti-P19). CBS, Coomassie Brilliant Blue
staining; M, standard molecular marker or prestained marker or purified
BVP1; Nt, nontransgenic; *, degraded BVP1. (b) ELISA against BVP1, using
rabbit anti-FMDV VP1 sera. Data are means Æ SD from three replicates.
(c) Northern blot analysis of wild-type or chimeric BaMV RNA in transgenic
suspended biomass (at day 21 postsubculture). BaMV genomic RNA
(6.4 kb), subgenomic RNA (2.0 kb, 1.0 kb) were detected with a BaMV-
specific probe. The film was exposed for 1 h (middle panel) and 2 h (upper
panel), respectively; the bottom panel indicates the rRNA loading control
stained with EtBr. Nt, nontransgenic.
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Plant cell-suspension system for vaccine production 233

4. purified from the cell lines (BdT38 or BdT19), respectively.
Similarly, a group of two guinea pigs were subcutaneously
injected with CVPs or wild-type BaMV purified from the leaves
and were used as positive and negative controls, respectively.
Sera were subsequently collected from the vaccinated guinea
pigs, and the antibody titres were determined by its sensitivity
towards FMDV recombinant VP1 antigen (FMDV-rVP1) using
ELISA. Antisera collected from guinea pigs vaccinated with CVPs
purified from BdT38 exhibited high sensitivity (OD450 > 0.7) to
FMDV recombinant VP1 (FMDV-rVP1) protein (Figure 6a) at
1 : 16 000 dilution (final bleed), while the sera collected from
those vaccinated by CVPs purified from BdT19 showed lower titre
at about 1 : 8000 (OD450 = 0.6). CVPs from BdT38 cell line show
the equivalent sensitivity to guinea pigs vaccinated with CVPs
purified from leaves. In contrast, guinea pigs vaccinated with
BaMV-S showed a weak sensitivity against rVP1 (Figure 6a).
Western blot analysis further confirmed that injection of CVPs
resulted in the production of specific antisera against BVP1 and
FMDV-rVP1 (Figure 6b). Therefore, purified CVPs can trigger the
production of specific antibodies in guinea pigs.
Discussion
Here, we describe the development of a novel system to obtain
antigenic small peptides using cell-suspension cultures. In recent
years, enormous progress has been made in the field of
biotechnology and vaccine production, most significantly in the
utilization of whole plants or their derivatives as inoculators and
hosts to generate antigenic molecules (Chargelegue et al., 2001;
Davies, 2010; Rybicki, 2009; Tiwari et al., 2009). For example, a
high-yield viral vaccine antigen generated in transgenic tomato
fruit was reported to accumulate to 8% of TSP (Zhang et al.,
2006). However, the distribution of this antigen in edible regions
varied considerably in vaccine dose (Molina et al., 2005). A
separate study reported the use of chloroplast system to express a
vaccine antigen with a yield of approximately 31% of TSP (Daniell
et al., 2009); however, this system is limited to certain applica-
tions due to the lack of post-translational modifications. Alter-
nately, RNA viruses may be of use in producing candidate epitope
vaccines, as viral vector systems have the advantage of relatively
simple purification processes (Mallory et al., 2002; Yusibov and
Rabindran, 2008; Yusibov et al., 2006). However, viral vector
systems involve transient expression strategies that require
additional inoculation to maintain production of CVPs in the
whole plant (Ca~nizares et al., 2005). Furthermore, the cultivation
and harvesting of plant biomass is a tedious and relatively
expensive process, which is accompanied by other limitations,
including environmental concerns when compare with the cell-
suspension culture (Hellwig et al., 2004).
To overcome the aforementioned constraints, we developed an
innovative transgenic cell-suspension culture-based system to
produce self-assembling CVPs. The major advantage of our
system is that the proreplicon (from which replicable RNA is
transcribed) is stably integrated, thereby providing a permanent
genetic resource for CVP production. Additional benefits of this
system include its technical ease, obviated need for manual
inoculation or Agrobacterium transfection, production of homog-
enous biomass and its intrinsic safety and the efficiency of
post-translation modification (Hellwig et al., 2004; Xu et al.,
2011); however, the VP1 epitope used in this study does not have
glycosylation site as predicted through online glycosylation
prediction tool (NetNGlyc 1.0 Server, Center for Biotechnological
Sequence Analysis, Technical University of Denmark, Lyngby,
Denmark). In our system, transgenic plant lines with different viral
expression cassettes are used to establish cell-suspension cultures.
To counter the reduction in CVP yield from gene-silencing
mechanisms, we incorporated two suppressors of RNA silencing,
(a)
(b)
Figure 3 Analysis of genetic stability of chimeric BaMV RNA in transgenic
cell lines by RT-PCR and sequencing. (a) The amplified PCR fragments were
separated on a 1% agarose gel. M, marker 100 kb. (b) Confirmation by
nucleotide sequencing. Partial nucleotide and translated amino acid
sequences of the 50
region of amplified fragments corresponding to the
chimeric protein from transgenic cell lines (BVP1-16-7, BdT38 and BdT19)
were shown. Letters in blue represent the coding region of VP1128-164
;
bold letters indicate the start codon; the down-arrow signs indicate the
beginning and end of the VP1 epitope coding sequence. The nucleotide
positions relative to the chimeric BaMV genome are indicated on top of
the sequence.
Figure 4 Transmission electron microscopy of VP1 epitopes on the
surface of CVPs. Purified CVPs were coated on a grid and treated with
rabbit pre-immune sera or antibodies specific to BaMV CP or FMDV VP1,
and then reacted with gold-labelled (12 nm) goat anti-rabbit IgG
conjugates. Scale bars = 100 nm.
ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 14, 231–239
Thangarasu Muthamilselvan et al.234

5. P38 and P19; transgenes encoding these suppressors increased
CVP yield significantly. Suppression of gene-silencing enabled
consistent and stable expression of chimeric viral RNAs and CVPs.
It is also important to note that high yield was observed not only
in the long-term cultures, but also in the subsequent subcultures
of transgenic cell suspensions.
Constitutive expression of replicable viral vectors can have toxic
effects on the host cells, because of autonomous amplification
and accumulation of viruses. However, no detrimental effects
were observed on the growth of our cell-suspension culture
(Figure S1c). Nevertheless, we observed a strong reduction in viral
RNA replication in B2B27 and BVP1-16-7 transgenic lines; this
reduction could be overcome by co-expression of the amplicon
with a silencing suppressor protein (Mallory et al., 2002). In
general, the TGBp1 of potexviruses serves as silencing suppressor
protein. However, we observed that the TGBp1 of BaMV is not an
efficient silencing suppressor (Figure 2a). Therefore, the trans-
genic lines B2B27 and BVP-16-7 produced lower accumulation of
virions when compared with those in cell lines BdT38 and BdT19.
It has been previously reported that the silencing suppressor
proteins can enhance the recombinant protein expression in cell-
suspension cultures (Boivin et al., 2010; Larsen and Curtis, 2012).
In these earlier studies, the majority of the silencing suppressors
were transiently expressed from plasmids cotransfected into the
suspension cells. However, the efficiency of cotransfection is
lower than transfecting a single plasmid. For example, only 4%–
5% of protoplasts prepared from leaf tissue infected with two
Tobacco mosaic virus (TMV)-based viral vectors expressed both
the reporter genes (Giritch et al., 2006). In contrast, here we
inserted the silencing suppressor genes directly upstream of the
target gene, replacing the TGBp1-3 open reading frames, which
are not required by the chimeric virus in suspension cells. This
approach guaranteed the persistent co-expression of the silencing
suppressor and target protein in the same cell, resulting in a
marked increase in BVP1 accumulation in the transgenic cell lines
BdT38 and BdT19 (up to 4.7% or 5% of TSP, respectively).
Notably, the expression of silencing suppressor proteins is
associated with negative effects on the growth and development
of transgenic plants (Siddiqui et al., 2008). In our study, we
observed moderate growth defects in 94% of transgenic plants
expressing silencing suppressor protein P19 or P38, but the
remaining 6% transgenic plants appeared to be healthy and
yielded a significant amounts of BVP1 [Na-Sheng Lin (NSL), Chin-
Wei Lee (CWL), Ying-Wen Huang (YWH), Ming-Ru Liou (MRL),
and Yau-Heiu Hsu (YHH), unpublished data.] Consequently, we
used these transgenic lines to establish cell-suspension cultures
and measured the accumulation of BVP1. Our results revealed
that despite significant expression of suppressor protein P19 in
the transgenic cell line BdT19, it had no detrimental effect on the
cell growth, which was also reported in other study (Boivin et al.,
2010). A similar trend was observed in another cell line BdT38,
which expresses suppressor protein P38. Most importantly,
although these silencing suppressor proteins suppresses the PTGS
in a plant cell through different pathways (Incarbone and
Dunoyer, 2013), there are no significant differences in the yields
of BVP1 between BdT38 and BdT19 transgenic cell lines. This
result indicates that the suppressor protein P19 or P38 exhibited
similar effect on BVP1 expression. These results suggested the use
of the silencing suppressor protein as a versatile tool to improve
the production of foreign proteins in the transgenic cell lines.
Certain studies reported that insert size and isoelectric point
(pI) of the fusion protein are critical factors, which influence the
stability of chimeric viral particle (Bendahmane et al., 1999; Porta
et al., 2003; Uhde-Holzem et al., 2007). As a consequence, when
the epitopes were presented on TMV and Potato virus X, the
fusion protein amino acid sequences often showed point muta-
tions and deletions during the infection on the plants to
compensate the pI values (Bendahmane et al., 1999; Uhde-
Holzem et al., 2007). On the other hand, the sequencing of the
chimeric BaMV RNA from suspended biomass of transgenic cell
(a)
(b)
Figure 6 Titration of sera from guinea pigs vaccinated with CVPs. Guinea
pigs were vaccinated with 200 lg purified CVPs or BaMV-S. (a)
Determination of anti-sera titres against FMDV-rVP1 protein. Sera were
collected on day 35 after vaccination. (b) Representative Western blot
analysis of sera from immunized-guinea pigs, using FMDV-rVP1 or purified
CVPs as the targets. *, degraded BVP1.
Figure 5 Purification of CVPs. CVPs were purified from transgenic cell-
suspension cultures, separated by 10% SDS-PAGE and stained with
Coomassie Brilliant Blue. M, standard molecular marker; *, degraded
BVP1.
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Plant cell-suspension system for vaccine production 235

6. lines has revealed no change in the amino acid sequence of the
fusion protein (Figure 3b). In addition, suspension cells are
continuously dividing in the culture medium, and a cell
generation time is estimated to be about 44 h/L (Hellwig et al.,
2004). Therefore, in our system, suspension cells can undergo at
least 5–6 generations (log phase) in a subculture (suspended
biomass harvested at day 21 postsubculture). Simultaneously,
stably integrated proreplicon can continuously produce new
copies of RNA transcript and export to cytoplasm and start
translation and self-replication in the newly divided cells. The
observation that the integrated viral constructs has retained the
original sequences over 21 days suggested the stability of
replicons in the transgenic cell-suspension cultures. In our
previous report, BaMV-based viral constructs were stable and
resulted in successful assembly of CVPs in whole-plant systems up
to three serial passages (Chen et al., 2012).
Here, we observed consistent production of BVP1 and efficient
assembly of CVPs in the suspension cells of transgenic lines BdT38
or BdT19 through subsequent weekly subculturing up to
12 weeks. However, the yields of BVP1 in inoculated leaves were
0.2–0.5 mg/g of leaf, which were similar to wild-type BaMV-S
(Yang et al., 2007), whereas the yields of CVPs in plant cell
suspension cultures were 1.5 or 2.1 mg/20 g fresh weight of
suspended biomass from transgenic cell lines BdT38 and BdT19.
On the other hand, we suspect that physical shearing could be
one of the reason for the degradation of BVP1 during the
purification process or the fused epitope may cause the fusion
protein structurally labile compared to native CP due to cultural
conditions such as shaking or unknown factors (Doran, 2006), as
we have included protease inhibitor cocktails in the extraction
buffer. However, currently, we do not have experimental data to
prove our hypothesis for the degradation of BVP1 in cell-
suspension culture. Therefore, future studies on optimizing the
purification conditions and processes are required.
Another significant advantage of BaMV-based CVPs is that
they are composed of about 1300 identical CP subunits [as has
been reported for a filamentous potexvirus (Lico et al., 2006)].
Furthermore, CVPs can display FMDV VP1 epitopes on the surface
of each CP. Such properties facilitate the production of high titres
of humoral antibodies in guinea pigs. In addition, BaMV virions
might serve as adjuvants to enhance the antigenicity of the
vaccine candidates, as reported for Papaya mosaic virus (PapMV,
Savard et al., 2011, 2012). In this study, the sera of immunized-
guinea pigs were found to specifically recognize BVP1 and FMDV-
rVP1 protein.
This is the first report to demonstrate the production of fully
assembled wild-type or CVPs from suspended biomass. We
describe an innovative system for co-expressing foreign antigenic
peptides with silencing suppressors in transgenic cell-suspension
cultures, which enables consistent viral expression and simple
purification, thereby providing a cost-effective and efficacious
means of producing vaccine candidates. Thus, our results reveal
the potential of using transgenic cell-suspension cultures to
produce vaccine candidates based on CVPs. Further optimization
of this system may provide lucrative and viable option for large-
scale industrial grade production.
Experimental procedures
Construction of recombinant expression cassettes
The plasmids pBaMV-S and pBVP1 (Yang et al., 2007) were used
as starting materials for the construction of other constructs. The
pBS-d35CP vector was derived from pBaMV-S by deletion of the
coding sequence for the 35 N-terminal amino acids of the CP.
The sequence coding for the FMDV VP1 epitope (37 amino acids,
corresponding to T128
-N164
of FMDV VP1 serotype O/Taiwan/97)
was used to replace the truncated CP N-terminus in pBS-d35CP,
thereby generating the BVP1 construct. Constructs pBdT38-VP1
or pBdP19-VP1 were derived from BVP1, by replacing the TGBp1-
3 (1079 bp) region of BaMV with the coding sequences for the
silencing suppressor protein P38 (1056 bp, GenBank accession
no. HQ589261) or P19 (519 bp, GenBank accession no.
AJ288926), respectively. The templates for P38 (Turnip crinkle
virus) and P19 (Tobacco bushy stunt virus) constructs were
obtained (Chapman et al., 2004). The coding sequences for P38
or P19 were amplified by PCR with primers containing DraIII
restriction sites. The BVP1 vector was digested with the DraIII
restriction enzyme to remove the TGBp1-3 region from this
vector. The P38 or P19 PCR products were also subjected to DraIII
restriction digestion and then cloned into the BVP1 vector;
successful cloning was confirmed by nucleotide sequencing. The
expression cassettes were digested with the respective enzymes
and subcloned into the pKn binary vector (Khang et al., 2005);
the resulting plasmids were transformed into Agrobacterium
strain pGV3850 (Zambryski et al., 1983) via electroporation (Bio-
Rad Gene Pulser II, Bio-Rad, Hercules, California) and used in the
generation of transgenic plants (Horsch et al., 1985) as listed in
Table 1.
Plant material and callus induction
Wild-type (nontransgenic) and transgenic N. benthamiana plant
lines (Table 1) were grown in a glasshouse at 25 °C under a 16-h/
8-h light/dark photoperiod. Transgenic plant leaves were har-
vested at day 50 postgermination, washed under running tap
water and then surface sterilized using 10% bleaching solution.
The leaves were subsequently washed until the soap residue was
completely removed. A scalpel was used to cut small discs out of
leaves, and the discs were placed on solid MS medium plates
(Duchefa Biochemie, Haarlem, the Netherlands) supplemented
with 3% sucrose, 1 mg/L naphthalene acetic acid, 0.5 mg/L
kinetin, 100 mg/L myo-inositol and 200 mg/L casein hydrolyte. A
whitish callus developed in 5–6 weeks, and calli were subcultured
on the same plates for subsequent passages.
Plant cell-suspension culture
To prepare initial inoculum, approximately 2 g friable callus
biomass was transferred to 25 mL MS liquid media (the compo-
sition described in ‘Plant materials and callus induction’) in a 125-
mL Erlenmeyer flasks. Liquid media (25 mL) was added once a
week, and the cells were transferred to larger Erlenmeyer flasks to
increase biomass level and continue to maintain the cells in log
phase. The initial inoculum was cultured in a 500-mL Erlenmeyer
flask with a final volume of 150 mL. The cells were maintained on
a shaker at 120 rpm under a 16-h/8-h light/dark photoperiod of
at 45 L mol photons per m2
per second and 25 Æ 1 °C. The initial
inoculum was cultured for 7 days and served as seeds for further
experiments.
Growth determination
An equal amount of 7-day-old suspended biomass was trans-
ferred to 25 mL fresh hormonal media in a 125-mL flasks. The
suspension culture was incubated under a 16-h/8-h light/dark
photoperiod at 25 °C in a shaker at 120 rpm. Suspension cultures
were harvested at 3, 4, 7, 10, 14, 17, 21, 24, 27 and 30 days
ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 14, 231–239
Thangarasu Muthamilselvan et al.236

7. postsubculture for the determination of the growth rate and
expression of chimeric BaMV CP in the biomass. Fresh biomass
was analysed by filtering the solution from each flask with
Miracloth filter paper (Calbiochem, San Diego, CA) and washing
the collected biomass with sterile water to remove sugar and
other residual materials.
Immunoblotting assay
Total proteins were extracted from suspended biomass using
protein extraction buffer (50 mM Tris–HCl, pH 8, 10 mM KCl,
10 mM MgCl2, 1 mM EDTA, 20% glycerol, 2% SDS and 10% b-
mercaptoethanol) and loaded onto 10% polyacrylamide gel
containing 1% SDS. Following electrophoresis, proteins were
electroblotted onto Immobilon-p membranes (Perkin Elmer,
Waltham, MA), which were then incubated with rabbit primary
antibodies against BaMV CP, FMDV VP1, P38, or P19, or CVP-
immunized guinea pig sera, at 37 °C for 1 h; antibodies were
prepared as described previously (Yang et al., 2007). Membranes
were subsequently incubated with phosphatase-conjugated goat
(Jackson Immuno Research, West Grove, PA) or guinea pig
secondary antibodies (Jackson Immuno Research) at 37 °C for
1 h, and visualized by NBT/BCIP colour development (Thermo
Scientific, Waltham, MA).
Northern blot analysis
Total RNA from 200 mg nontransgenic or transgenic N. benth-
amiana suspended biomass was extracted by hot phenol extrac-
tion and LiCl precipitation (Pawlowski et al., 1994). Total RNA
was glyoxylated and analysed by northern blot using probes
specific to positive-strand BaMV RNA, as previously described (Lin
et al., 2010).
Stability of chimeric genomic BaMV RNA analysis by RT-
PCR
Total RNA extracted from nontransgenic and transgenic cell lines
(Table 1) suspended biomass at day 21 postsubculture. Subse-
quently, cDNA was prepared as described (Cheng et al., 2010), and
the FMDV-VP1-BaMV-CP coding region was amplified using
following a set of primers (Ba5353R-F, 50
-CACCATGTGAAATA
ATAATAAA CG-30
and Ba6366-R, 50
-TGGAAAAAACTGTAGA
AACCAAAAGG-30
). The amplified fragments were separated on
a 1% of agarose gel, and a corresponding band was excised and
purified. Subsequently, these purified products were sequenced on
the ABI Prism 3730 DNA Analyzer (Applied Bio systems, Waltham,
MA) with reagents kit, ABI Prism BigDyeTM Terminator v. 3.1.
ELISA
Total soluble protein was extracted using a protein extraction
buffer (50 mM Tris–HCl, pH 8.0, 1 mM EDTA, and 1 mM b-
mercaptoethanol). The 96-well microtitre plates were coated with
total proteins (5 lg/mL of 0.1 M carbonate/bicarbonate buffer,
pH 9.6, 100 lL/well) from non-transgenic- or transgenic-sus-
pended biomass or purified FMDV-rVP1 protein expressed by
Escherichia coli [Wang et al., 2003, (for the positive control and
standard curve)], and then incubated at 37 °C for 1 h. Plates
were washed three times with PBST (PBS containing 0.05%
Tween 20), and 100 lL blocking buffer [PBS containing 0.5%
bovine serum albumin (BSA)] was subsequently added to each
well. The plates were incubated at 37 °C for 1 h, washed and
incubated with rabbit polyclonal anti-FMDV VP1 antibody
(100 lL/well, diluted 1 : 5000 in blocking buffer) at 37 °C for
1 h. After a washing, alkaline phosphatase-conjugated goat
anti-rabbit IgG (Jackson Immuno Research, 100 lL/well 1 : 5000
PBS containing 0.5% BSA) was added, and the plates were
incubated at 37 °C for 1 h. After washes, the plates were
incubated with the p-nitrophenyl phosphate solution (Sigma, St.
Louis, MO, USA, 100 lL/well 1 tablet in 5 mL of PBS) at 37 °C for
45 min for colour development. Plate absorbance at 405–
490 nm was determined using a microplate reader (Spectramax
M2; Molecular Devices,Sunnyvale, California), and protein was
quantified by comparison with known amounts of the bacterial
FMDV-rVP1–antibody complex. All measurements were per-
formed in triplicate.
Purification of BaMV CVP
Wild-type or BaMV CVP was purified as previously described (Lin
and Chen, 1991), with slight modifications. The suspended
biomass (20 g) from BdT38 and BdT19 cell lines was homoge-
nized using liquid nitrogen and 2 volume (w/v) extraction buffer
(0.5 M boric acid, pH 8.5, 1 mM EDTA, and 0.5% b-mercapto-
ethanol) at 4 °C, and the homogenate was then centrifuged at
12 000 g for 10 min. The supernatant was mixed with 1% (v/v)
of 4 M K2HPO4, and 2% (v/v) of 2 M CaCl2 was then added drop-
by-drop to the mixture at 4 °C for 10 min. This mixture was
centrifuged at 12 000 g for 10 min. The supernatant was then
mixed with 2% Triton X-100 and PEG 6000 for 30 min at 4 °C
and before being centrifuged at 12 000 g for 10 min. The pellet
was suspended in BE buffer (0.05 M borate, pH 8.0, 1 mM EDTA)
and centrifuged at 8000 rpm for 5 min. The supernatant was
transferred to a tube and centrifuged through 5 mL of a 20%
sucrose cushion at 136 000 g for 1 h (Optima L-90k ultra-
centrifuge; Beckman Coulter, Brea, California). The purified virus
particles were suspended in BE buffer (pH 8) and stored at
À20 °C. We determined the yields based on the ultraviolet
absorption method using an extinction coefficient of 3 as
described for BaMV virions (Lin and Chen, 1991).
Transmission electron microscopy of immunogold-
labelled complexes
Immunogold labelling was performed as described previously (Lin,
1984). CVPs were purified from suspended biomass at day 21
postsubculture, and grids were inverted on purified CVP droplets
for 5 min. The grids were then incubated with anti-BaMV-CP or
anti-FMDV VP1 (1 : 100) antibodies and decorated with gold-
labelled goat-anti-rabbit IgG complexes. Grids were stained with
2% uranyl acetate and examined under a Philips CM 100 electron
microscope Philips Electron Optics, Hillsboro, Oregon.
Vaccination of guinea pigs
Pathogen-free guinea pigs were obtained in Taiwan. Guinea pigs
(three each) were subcutaneously injected with 200 lg purified
CVPs (BdT38 or BdT19) emulsified with Montanide ISA 206
(Seppic, Puteaux Cedex, France) at a 1 : 1 ratio (v/v). Guinea pigs
(two) injected with CVPs assembled from transgenic line express-
ing BVP1 or wild-type BaMV-S particles purified from leaves were
used as positive and negative controls, respectively. All animals
were boosted with the same amount of antigen at 7-day
intervals. The first booster was subcutaneously injected and the
second and third boosters were intramuscularly injected. Sera
were collected from immunized animals at days 1, 21, 28 and 35.
ELISA titration of CVP-immunized guinea pig sera
ELISA was performed as described previously (Yang et al., 2007),
with minor modifications. The 96-well microtitre plates were
ª 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd, Plant Biotechnology Journal, 14, 231–239
Plant cell-suspension system for vaccine production 237

8. coated with FMDV recombinant VP1 protein (FMDV-rVP1, 1 lg/
well) in phosphate-buffered saline (PBS) and incubated at 4 °C
overnight. Plates were blocked with 1% bovine serum albumin in
PBS and washed three times with PBST (PBS containing 0.05%
Tween-20). Samples of diluted serum were added to wells
(100 lL/well) and incubated for 1 h at 37 °C. After three
washings, alkaline phosphatase-conjugated guinea pig secondary
antibodies (Jackson Immuno Research, 100 lL/well 1 : 5000 PBS
containing 1% BSA) were added for 1 h at 37 °C. The plates
were subsequently washed, and the p-nitrophenyl phosphate
solution (Sigma, 100 lL/well 1 tablet in 5 mL of PBS) was added
for the colour development. Finally, an equal volume of 1% SDS
was added to stop reaction. The absorbance was measured by an
ELISA reader (Spectramax M2; Molecular Devices) at 450 nm. The
working dilution gave an absorbance at upper part of the linear
region of the titration curve.
Statistical analysis
Data obtained from three replicate samples are expressed as
mean Æ SD. Statistical analysis was performed using ANOVA.
A P-value of <0.05 was considered significant.
Acknowledgements
We thank Dr. Muni Subramani (USA) for his critical review and
comments on our manuscript. This work was supported by the
Ministry of Science and Technology (MOST 98-2321-B-005-005-
MY3) and Council of Agriculture (97AS-1.2.1-ST-a2), Taiwan,
Republic of China.
Competing financial interests
A patent application has been filed based on our results reported
in this paper.
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Supporting information
Additional Supporting information may be found in the online
version of this article:
Figure S1 Induction of cell-suspension cultures from N. benth-
amiana nontransgenic and transgenic leaves, and the resulting
plant cell growth curves.
Data S1 Supporting experimental procedures for supplementary
materials.
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Plant cell-suspension system for vaccine production 239