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ORIGINAL RESEARCH PAPER
A classical swine fever virus E2 fusion protein produced
in plants elicits a neutralizing humoral immune response
in mice and pigs
Youngmin Park .Sangmin Lee .Hyangju Kang .Minhee Park .
Kyungmin Min .Nam Hyung Kim .Sungmin Gu .Jong Kook Kim .
Dong-Jun An .SeEun Choe .Eun-Ju Sohn
Received: 13 January 2020 / Revised: 1 April 2020 / Accepted: 14 April 2020 / Published online: 22 April 2020
ÓThe Author(s) 2020
Abstract Classical swine fever (CSF) is one of the
most important viral diseases of swine worldwide.
Although live or attenuated virus vaccines have been
used to control CSFV, it is difficult to distinguish
vaccinated pigs from infected pigs; this leads to
restrictions on import and export. Subunit vaccines
based on the CSFV E2 glycoprotein have been
developed using baculovirus or insect cell systems,
but some weaknesses remain. Here, we describe
production of an E2 recombinant protein using a
Nicotiana benthamiana plant expression system. To
do this, we took advantage of the ability of the swine
Fc domain to increase solubility and stability of the
fusion protein and to strengthen immune responses in
target animals. N. benthamiana expressed high
amounts of pFc2-fused E2 proteins, which were
isolated and purified by affinity chromatography to
yield a high pure recombinant protein in a cost-
effective manner. Native-polyacrylamide gel elec-
trophoresis and size exclusion chromatography con-
firmed that the pmE2:pFc2 fusion exists as a multimer
rather than as a dimer. Injection of recombinant pmE2
protein into mice or piglets generated anti-pmE2
antibodies with efficient neutralizing activity against
CSFV. These results suggest that a purified recombi-
nant E2 protein produced in N. benthamiana generates
high titers of neutralizing antibodies in vivo; as such,
the protein could be developed as a subunit vaccine
against CSFV.
Keywords Nicotiana benthamiana Classical swine
fever virus DIVA concept Subunit vaccine
Molecular farming Fc-fusion protein
Abbreviations
CSFV Classical swine fever virus
DIVA Differentiating infected from vaccinated
animals
ER Endoplasmic reticulum
CBD Cellulose-binding domain
BSA Bovine serum albumin
MLV Modified live vaccines
PCR Polymerase chain reaction
Electronic supplementary material The online version of
this article (https://doi.org/10.1007/s10529-020-02892-3) con-
tains supplementary material, which is available to authorized
users.
Y. Park S. Lee H. Kang M. Park K. Min
N. H. Kim S. Gu J. K. Kim E.-J. Sohn (&)
BioApplications Inc., Pohang Techno Park Complex, 394
Jigok-ro Nam-gu, Pohang, Korea
e-mail: ejsohn@postech.ac.kr
D.-J. An S. Choe
Virus Disease Division, Animal and Plant Quarantine
Agency, Gimcheon 39660, Gyeongbuk, Korea
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https://doi.org/10.1007/s10529-020-02892-3(0123456789().,-volV)(0123456789().,-volV)
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
Introduction
Classical swine fever (CSF), a highly contagious and
serious disease of pigs (Edwards et al. 2000), is caused
by the CSF virus (CSFV), which is a positive-strand
RNA virus belonging to the Pestivirus genus within
the Flaviviridae family (Moennig 2000). The disease
is endemic in Asia, Eastern Europe, and the Americas,
as well as in some African countries (Greiser-Wilke
et al. 2000; Tu et al. 2001; Deng et al. 2005; Cha et al.
2007; Postel et al. 2013). In Korea, live vaccines have
been used traditionally to control swine fever where all
pigs must be vaccinated early in life (Lim et al. 2016).
Although the vaccine works and is inexpensive, it
lacks stability, and it is hard to distinguish between
pigs infected with swine fever and pigs that have been
vaccinated (the DIVA concept; differentiating
infected from vaccinated animals), which limits the
pig trade. Because of this, DIVA vaccines have
emerged as important lines of study and many groups
have focused on generating marker vaccines.
CSFV has a genome of about 12.3 kb, which
encodes a 3898 amino acid polypeptide from which
four structural proteins (C, Erns, E1, and E2) and eight
nonstructural proteins (Npro, p7, NS2, NS3, NS4A,
NS4B, NS5A, and NS5B) are generated (Meyers et al.
1996). Of the four CSFV structural proteins, the E2
glycoprotein is the main target of neutralizing anti-
bodies generated during CSFV infection and is
considered a major antigenic protein in the vaccine
market (Ahrens et al. 2000; Bouma et al. 1999; Dewulf
et al. 2000). CSFV subunit vaccines have been
developed using recombinant E2 proteins expressed
in insect cells (Ahrens et al. 2000; Bouma et al.
1999,2000) and, BAYOVACÒCSF Marker (Adva-
sureÒ, Pfizer, UK) and PorcilisÒPesti (IDEXX CSF
marker, IDEXX Europe B.V., the Netherlands) are
commercially available in Europe market. Although
they were shown to provide protection against CSFV
infection, there were reports that they didn’t efficiently
provide early protection and transplacental transmis-
sion (Depner et al. 2001; van Oirschot 2003). In
addition to insect cells, other systems using yeast and
mammalian cells are also used to produce recombinant
E2 proteins (Lin et al. 2012; Hua et al. 2014).
However, large-scale manufacturing of these expres-
sion system remain expensive. The genotypes of
CSFV can be classified into three major groups with
several subgroups (Paton et al. 2000; Postel et al.
2013) and it has reported that antibodies specific to one
genotype E2 might not provide protection to other
CSFV genotypes (Luo et al. 2013). Therefore, in order
to effectively protect against the prevalent CSFV in
south Korea, it is necessary to develop a vaccine using
the E2 protein for this genotype.
There are many bioreactors that produce pharma-
ceutical proteins. Among other things, plant-based
production of recombinant proteins is an attractive
platform because plants are easy to grow, produce
large amounts of protein, and do not require expensive
facilities (Nandi et al. 2016; Sabalza et al. 2014;
Lomonossoff and D’Aoust 2016; Rybicki 2017). The
E2 protein produced by Arabidopsis thaliana reacts
with antibodies against the native E2 protein. In
addition, Arabidopsis thaliana-derived E2 antibodies
in a mouse can recognize the native E2 protein
suggesting that structure of plant-produced E2 is
comparable to native E2 protein (Sohn et al. 2018). In
addition, transient expression of recombinant E2
protein from the leaves of Nicotiana benthamiana or
from the leaves of transgenic N. benthamiana lines
generates antibodies that can neutralize CSFV in pigs
and protect them against subsequent challenge with
CSFV (Laughlin et al. 2019; Park et al. 2019).
Immunoglobin (Ig)G, one of the most abundant
proteins in plasma, has a long half-life. The tail (Fc
region) of these immunoglobulin molecules modulates
immune cell activity by interacting with cell surface
receptors and with proteins belonging to the comple-
ment system (Nimmerjahn and Ravetch 2008). Fusion
of the Fc domain with a specific antigenic protein
ensures an immune response to that antigen. The main
reason for fusing proteins to the Fc domain is to
enhance solubility and the half-life of an antigenic
protein (Kontermann 2011; Levin et al. 2015). In
addition, the Fc region enables easy and cost-effective
purification of recombinant protein by protein A
chromatography (Carter 2011; Ghose et al. 2006;
Huang 2009).
Here, we asked whether a fusion of the CSFV E2
protein and the Fc domain of porcine IgG could be
produced in N. benthamiana at low cost. The resulting
recombinant protein formed an oligomer rather than a
dimer, and injection into mice and pigs generated
neutralizing antibodies specific for CSFV.
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1248 Biotechnol Lett (2020) 42:1247–1261
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Materials and methods
Plant growth condition
Plants were grown under a 16:8 h light:dark cycle in a
growth room maintained at 25 ±2°C and 50 ±5%
relative humidity.
Plasmid construction
We used the pCAMBIA1300 MELCHE2 construct
(Park et al. 2019) that contains cellulose binding
domain (CBD)-fused E2 as a backbone. The CSFV E2
coding sequence was derived from GenBank (Acces-
sion number YP_009508222, amino acid positions
691-1030) and a codon-optimized synthetic gene was
purchased (Bioneer Inc. Daejeon, Korea). The
pCAMBIA1300 MELCHE2 construct was digested
with endonucleases XmaI and SacI to remove the
TEV:CBD:HDEL domain. The pFc2 coding sequence
from GenBank (BAM66310.1) was codon-optimized
for expression in N. benthamiana. Then, it was
amplified by polymerase chain reaction (PCR) using
the following primers: XmaI/pFc2-F, which contains a
XmaI restriction site and 21 nt of the pFc2 coding
sequence, and the HDEL/pFc2-R reverse primer,
which contains 12 nt of ER retention signal (His-
Asp-Glu-Leu) and 21 nt of the pFc2 coding sequence.
Since the HDEL sequence contains a SacI restriction
site, the PCR product was digested with XmaI and
SacI, and ligated into pCAMBIA1300 MELCH to
generate pCAMBIA1300::pmE2:pFc2:HDEL.
Transient expression of chimeric E2 protein
The pCAMBIA1300::pmE2:pFc2:HDEL construct
was introduced into A. tumefaciens strain LBA4404
by electroporation. Separate cultures of Agrobac-
terium harbouring pCAMBIA1300::pmE2:pFc2:H-
DEL and Agrobacterium harbouring p38, silencing
repressor, were grown overnight in YEB liquid
medium. Agrobacterium cells were collected by
centrifugation at 35009gfor 20 min and resuspended
in infiltration buffer (10 mM MES, 10 mM MgSO
4
,
100 lM acetosyringone, pH 5.6) to reach OD
600
of 1.0
and mixed each in a 1:1 ratio (v/v). Leaf tissues of 5 to
7-week-old N. benthamiana plants were co-infiltrated
with the Agrobacterium-mixture of suspension cells.
Plants were returned to the greenhouse and grown for a
further 4 DPI (day post infiltration). For expression
analysis of the fusion protein, fresh leaf tissues were
ground under liquid nitrogen to a fine powder in
protein extraction buffer (50 mM Tris-HCl, pH 7.5,
150 mM NaCl, 0.1% [v/v] Triton X-100). Total
soluble proteins (TSP) were extracted from the ground
tissue samples. After filtering with Miracloth (EMD
Millipore Corp., Billerica MA, USA; Cat. No:475855-
1R), lysates were clarified by centrifugation
(13,0009g) for 20 min and Soluble and insoluble
fractions were collected.
Generation of transgenic plants
The pCAMBIA1300::pmE2:pFc2:HDEL construct
was introduced into A. tumefaciens strain LBA4404
by electroporation. For plant tissue culture, N. ben-
thamiana leaves were fragmented into 0.5 cm 90.5
cm pieces, and incubated for 10 min with A. tumefa-
ciens transformed with pCMABIA1300::E2:pFc2 in
Murashige and Skoog (MS) liquid medium containing
2.0 mg/l a-naphthaleneacetic acid (NAA) and 0.5 mg/l
6-benzylaminopurin (6-BAP). Next, the culture med-
ium was removed, and the leaf fragments were placed
upside down (i.e., basal side up) on solid MS medium
(which had the same composition as the liquid
medium) and incubated for 3 days. After washing
with MS liquid medium, the leaves were placed upside
down on MS solid medium containing 1.0 mg/l NAA,
0.5 mg/l 6-BAP, 200 mg/l kanamycin, and 250 mg/l
cefotaxime, and incubated in the dark for 7–10 days.
Next, leaves were exposed to light, leading to growth
of shoots and roots. The plants were transferred to soil
and expression of pmE2:pFc2 was analyzed by
western blot analysis of leaf extracts prepared by
homogenizing leaves in extraction buffer, followed by
centrifugation to yield a supernatant. After harvesting
seeds, T3 generation transgenic plants were selected in
the presence of hygromycin at a segregation ratio of
3:1.
Protein purification and western blot analysis
Briefly, 0.5 kg (fresh weight) leaves from transgenic
plants were harvested and homogenized in a blender
(32,000 rpm) in the presence of 1 L protein extraction
buffer (50 mM sodium phosphate buffer, pH 8.0, 300
mM NaCl, 100 mM glycine, 0.5% Triton X-100). To
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Biotechnol Lett (2020) 42:1247–1261 1249
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remove debris, extracts were centrifuged for 40 min at
20,0009gand supernatants were filtered through
Miracloth. Extracts were incubated for 1 h with 20
mL Protein A Agarose Resin (Amicogen, Jinju,
Republic of Korea; Cat. No: 1010200). Next, the
extract and resins were loaded onto a column at a flow
rate of 20 mL/min and washed three times with 200
mL washing buffer (50 mM sodium phosphate buffer,
pH 8.0, 300 mM NaCl). Recombinant E2 proteins
were eluted using elution buffer (100 mM sodium
citrate, pH 3.0, 300 mM NaCl), followed by addition
of 3 M Tris-Cl to obtain a pH of 7.2. Each fraction was
collected and subjected to western blot analysis.
Briefly, proteins were run on 10% SDS-PAGE gels,
transferred to PVDF (Polyvinylidene difluoride) mem-
branes (Merck Milipore Ltd., Tullagreen Carrigt-
wohill; Cat. No:IPVH00010), and incubated with an
antibody specific for HRP-conjugated swine IgG
(Bethyl Laboratoris, Montgomery, USA; Cat. No:
A100-250P) or CSFV (Median, Chuncheon, Republic
of Korea; Cat. No.9011) coupled with anti-mouse IgG
HRP-conjugated (Bethyl, Montgomery, USA).
Immunoblotting bands were visualized using SUPEX
Solution kit (Neutronex, Goryeong, Republic of
Korea; Cat. No. NXECL-2011) as a substrate and
images were obtained with a Chemiluminescence
Imaging system (Vilber, FRANCE). Bands were
visualized with Coomassie Brilliant Blue R-250
(Biosolution, Suwon, South Korea; Cat. No: BC006).
For concentration of purified protein, eluted frac-
tion was subjected to centrifuge at 30009gusing
Ultrafiltration unit (Sartorius, United Kingdom, Cat.
No. VS6021).
Size exclusion chromatography and native-PAGE
Size exclusion chromatography was performed using
the A
¨KTA Prime chromatography system and a
HiLoadTM 16/60 Superdex 200 pg (GE Healthcare,
Madison, WI, United States) column. The column was
washed and equilibrated with 120 ml buffer (50 mM
Tris-Cl, pH 7.2, 300 mM NaCl, 0.5 mM EDTA) prior
to loading of pmE2 proteins at a flow rate of 1.5 ml/
min. Absorbance at 280 nm was monitored, and
fractions were collected and subjected to 8% poly-
acrylamide gel electrophoresis (PAGE) without
sodium dodecyl sulfate (SDS) under non-reducing
conditions. Blue-Dextran (2000 kD), Alcohol dehy-
drogenase (150 kD), Bovine Serum Albumin (66 kD),
Carbonic Anhydrase (29 kD), and Cytochrom C (12.4
kD) for Size exclusion chromatography and Thy-
roglobulin (669 kD), Ferritin (440 kD), Catalase (232
kD), Lactate dehydrogenase (140 kD), and BSA (67
kD) for PAGE were used as protein molecular-weight
standards. The gels were stained with Coomassie
Brilliant Blue R-250.
Immunization of mice with pmE2:pFc2
and ELISA
Mice (C57BL/6J, male, 6 weeks old) were used for
immunization. All animals were handled in accor-
dance with the guidelines and protocols approved by
the Pohang University of Science and Technology
(POSTECH) Animal Care and Use Committee. Mice
were kept isolated for 1 week prior to the experiments
to ensure that they were healthy. For single dose
immunization, mice received a subcutaneous injection
of 1 lg pmE2:pFc2 fusion protein in Freund’s
complete adjuvant (Sigma, Cat. No. F5881) (Yoshikai
et al. 1990). Bloods were collected from Retro-Orbital
plexus weekly and tested in an ELISA. For double
immunizations, mice received 1 lg pmE2:pFc2 fusion
protein in Freund’s complete adjuvant, followed 2
weeks later by a second dose in Freund’s incomplete
adjuvant (Sigma, Cat. No. F5506). Blood samples
were collected prior to the primary injection and then
weekly (weeks 3 to 8) after the first injection.
Blood samples were centrifuged for 15 min at
30009gto obtain sera and the resulting sera were
further analyzed by a commercially available ELISA
kit (A VDProÒCSFV AB ELISA kit; Median,
Chuncheon, Republic of Korea, Cat. No. ES-CSF-
01) to detect anti-pmE2:pFc2 antibodies. Briefly, the
96-well microplate coated with CSFV E2 antigen and
all kit reagents were placed on a bench and allowed to
reach room temperature for at least 30 min before use.
Next, serum samples (diluted 1:10,000) were added to
the plates in duplicate. Positive and negative controls
were diluted 20-fold. Samples (100 lL) were added to
the plate and incubated at 37 °C for 1 h. Next, 100 lL
horseradish peroxidase (HRP)-conjugated anti-mouse
IgG (1:5000 dilution) was added to each well for 1 h at
37 °C. After rinsing with wash buffer, 100 lL ABTS
substrate was added to each well, followed by
incubation for 10 min at room temperature. The
reaction was stopped by addition of 100 lL Stop
Solution. Absorbance at 405 nm was read in an ELISA
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1250 Biotechnol Lett (2020) 42:1247–1261
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Reader (Thermo Fisher Scientific, MULTISKAN FC,
Cat. no. N07710). Sample to positive ratio (S/P value)
was calculated by (OD
sample
-OD
negative control mean
)/
OD
positive control mean
. Test samples with an S/P
ratio C0.14 are positive; those with an S/P ratio \
0.14 are negative. The experiments were repeated
three times and presented a representative result.
Vaccination of pigs with pmE2:pFc2
All experiments involving pigs complied with the
current laws of South Korea. Animal care and
treatment were conducted in accordance with guide-
lines established by the Animal and Plant Quarantine
Agency Animal Care and Use Committee (QIA-
ACUC). The study was approved by QIA-ACUC
(permit number 2017-369).
Six piglets (aged 40 days) were allotted randomly to
a control group (n = 2) or a vaccinated group (n = 4).
The vaccine was prepared by simple hand mixing of
150 lg pmE2:pFc2 with an oil-in-water emulsion
adjuvant (ISA 15A VG; SEPPIC MONTANIDE
TM
,
Paris, France). The final volume was 1 ml, which was
injected intramuscularly. Piglets from the control
group were injected with the same volume of PBS.
Pigs received a booster vaccination 20 days after the
primary vaccination (the same vaccine formulation
was used). Blood samples were collected on Day 0,
immediately before the second vaccination, and at 20,
60, 90, and 110 days after the second vaccination;
neutralizing antibody responses were then examined.
Serum anti-CSFV neutralizing antibody assay
Sera were tested for the presence of anti-CSFV
neutralizing antibodies using a neutralizing peroxi-
dase linked assay in accordance with the standards set
out in the manual of the OIE (OIE 2013). In brief, sera
were serially diluted to twofold (1:2 to 1:2048) in
serum-free MEM media containing 1 lg/ml trypsin
and 50 ll of aliquots were added in a 96-well plate. An
equal volume of 200 TCID
50
/mL of CSFV LOM strain
was added to each well and incubated for 1 h at 37 °C
in 5% CO
2
. 100 ll of CPK (cloned porcine kidney)
cells (1 910
6
/ml) was added to each well and
incubated for 3 days at 37 °Cin5%CO
2
. The cells
were fixed with 100 ll of pre-chilled 80% acetone for
7 min at -20 °C. After drying a plate at 37 °C,
commercial anti-LOM antibody (Median Diagnostics;
Cat. No. 9011) (100 ll of 200-fold dilution) was added
and incubated for 1 h at 37 °C. After rinsing out the
plate with PBS three times, biotinylated goat anti-
mouse IgG antibody (Vector Lab; Cat. No. BA-9200)
(100 ll of 200-fold dilution) was added and incubated
for 1 h at 37 °C. After washing three times with PBS,
VECTASTAIN ABC-HRP Kit (Vector Lab; Cat. No.
PK-4000) was added according to the manufacturer’s
instructions and incubated for 1 h at 37 °C. After
washing three times with PBS, ImmPACT DAB
Peroxidase (HRP) Substrate (Vector Lab; Cat. No.
SK-4100) was added according to the manufacturer’s
instructions. Neutralizing antibody titers in serum
samples were expressed as the reciprocal of the
highest dilution that yielded 50% neutralization.
Results
Generation of the pmE2:pFc2 fusion construct
and protein expression in N. benthamiana
To investigate whether the recombinant pmE2:pFc2
(porcine Fc fragment) fusion was effective as a
vaccine, we first constructed a DNA construct for
expression in plants. The E2 protein was expressed at
high levels after targeting to the endoplasmic reticu-
lum (ER). This was achieved by adding an upstream
BiP leader sequence and a downstream HDEL ER
retention signal. In addition, we inserted 17 nucleo-
tides into the 50UTR region and an Arabidopsis HSP
terminator into the 30UTR region to increase expres-
sion (Kim et al. 2014; Nagaya et al. 2010). Previously,
we tested three different Fc domains (pFc1, pFc2, and
pFc3) from Sus scrofa and found out that pFc2 showed
strongest expression in N. benthamiana (data not
shown). Therefore, we fused the pFc2 domain down-
stream of the pmE2 coding sequence to generate the
pmE2:pFc2 fusion construct (Fig. 1a). The
pFc2:pmE2 fusion protein generated by transient
expression in N. benthamiana leaves using Agrobac-
terium tumefaciens was detected in the soluble frac-
tion, indicating that pFc2 increases the solubility of the
antigenic protein (Fig. 1b, c). When we looked more
closely, we noted a strong band at around 70 kD. The
expected size of the E2 recombinant protein is 64 kD;
therefore, the strong band appeared to be larger than
expected. We assume that this is due to N-glycosyla-
tion since E2 protein has putative 7 N-glycosylation
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Biotechnol Lett (2020) 42:1247–1261 1251
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sites (Laughlin et al. 2019). Consistent with this,
previous studies reported that CBD-fused E2 recom-
binant proteins have several N-glycosylation sites and
are larger than expected when expressed in plants
(Park et al. 2019; Sohn et al. 2018). In addition, the
pFc2 domain contains one N-glycosylation site
(kDa)
35
63
75
100
48
M T S P WT
A
B
UTR::L pFc2 HDELpmE2 (GP55) Hsp-T
35S
- 1300::pmE2:pFc2
M
pmE2:CBD
pmE2:pFc2
D
100
(kDa)
35
63
75
48
100
(kDa)
35
63
75
M T S P WT
Anti-CSFV CBB staining
Anti-CSFV CBB staining Anti-Pig CBB staining
C
Fig. 1 Construction of 1300::pmE2:pFc2:HDEL and expres-
sion in N. benthamiana.aSchematic showing the
1300::pmE2:pFc2:HDEL construct. 35S, double cauliflower
mosaic virus 35S promoter; UTR::L, 50untranslated region and
BiP endoplasmic reticulum (ER)-leader sequence; pmE2,
transmembrane domain-deleted classical swine fever virus
envelope glycoprotein E2; pFc2, IgG heavy chain constant
region from Sus scrofa; HDEL, ER retention signal; Hsp-T,
Arabidopsis HSP terminator. band cWestern blot analysis of
expression and solubility of the recombinant protein. Total
protein was prepared from leaves and separated to soluble and
insoluble fractions by centrifugation at 20,0009gfor 15 min.
Each fraction was subjected to western blotting with an anti-
CSFV antibody and an anti-Pig antibody. The membrane was
stained with Coomassie Brilliant Blue. Mprotein markers
(molecular weights in kD are shown on the left), Tplant total
extract, Ssoluble fraction, Ppellet fraction, WT wild-type
plants. dComparison of expression levels of CBD- or pFc2-
fused pmE2. Total extracts were subjected to western blot
analysis using anti-CSFV antibody. The arrows indicate
recombinant pmE2 proteins
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suggesting that the larger size is caused by N-glyco-
sylation of both pmE2 and pFc2. To test expressional
superiority of the pFc2 fusion, we compared expres-
sion levels of pmE2:pFc2 with cellulose-binding
domain (CBD):pmE2 by western blotting (Fig. 1d).
CBD is derived from Clostridium thermocellum and
binds to cellulose; therefore, it can be used as a tag for
protein purification (Park et al. 2019). As a result, the
amount of pmE2:pFc2 was much higher than that of
CBD-fused pmE2 when total extracts were prepared
from identical fresh weight of leaves. There results
suggested that pFc2 fusion increases the expression
level of pmE2 protein as well as solubility.
Generation of pmE2:pFc2 in transgenic N.
benthamiana
Next, we tried to generate pmE2:pFc2 in transgenic
plants to ensure more consistent expression. Similar to
transient expression, we performed tissue culture
using A. tumepaciens after introduction of pCAMBIA
1300::E2:pFc2. We obtained dozens of T0 lines.
Western blot analysis revealed that several lines
expressed high amounts of recombinant protein;
therefore, we selected homogenous lines from T2
plants. To select the most strongly expressed line
among four T2 generation plants, we performed
further western blot analysis and finally obtained total
extracts from two or three lines (Fig. 2a). Expression
of recombinant protein was strongest in transgenic line
42. When we compared this transgenic plant with a
wild-type plant, we found no significant difference in
biomass. Therefore, we used this line to generate
recombinant protein (Fig. 2b).
Purification of recombinant pmE2:pFc2
To purify pmE2:pFc2 recombinant protein, total
protein extracts were prepared from the leaves of the
transgenic plant using protein extraction buffer. The
pmE2:pFc2 fusion was purified by affinity chromatog-
raphy on protein A beads. All fractions including total,
flow-through contained unbound E2, wash-off and
elution were monitored by western blot analysis
(Fig. 3a). Compared with the total protein extract,
the flow-through fractions contained around 10%
unbound E2:pFc2. In addition, the first-wash fraction
exhibited only faint bands, while the second- and
third-wash fractions contained very little E2, indicat-
ing tight binding of E2:pFc2 to protein A beads.
Almost all the E2 protein was present in the eluted
fraction; very little remained on the beads after
elution, indicating that the elution buffer stripped the
beads effectively.
Producing a vaccine from a recombinant protein
requires a concentration step to generate a high
concentration of antigenic protein in a small volume.
Therefore, purified proteins must be concentrated, a
process that can result in loss of protein due to
aggregation. When we concentrated the eluted fraction
by centrifugal filtration, it was enriched without
aggregation. Next, the proteins were loaded onto
SDS-PAGE gels along with known concentrations of
bovine serum albumin (BSA) (Fig. 3b). We estimated
that the amount of pmE2 protein was about 1.135 lg/
ll, and that 302 mg pmE2 protein could be produced
from 1 kg plant leaves.
Characterization of pmE2:pFc2
Previous reports suggest that the CSFV E2 protein
forms a dimer (Lin et al. 2009; Hua et al. 2014; Risatti
et al. 2007; Thiel et al. 1991). However, we showed
previously that the E2 protein fused to a CBD was
expressed in plants as an oligomer (Park et al. 2019).
Moreover, pFc2 forms dimers via the tail part of the
antibody (Janeway et al. 2001). Thus, we performed
SDS-PAGE under reducing and non-reducing condi-
tions to investigate whether purified pmE2 protein
forms dimers or oligomers when expressed in plants.
We observed a band that was much larger than that
expected for the protein only under non-reducing
conditions (Fig. 4a). This indicates that the recombi-
nant protein was multimeric. To confirm this, we
subjected the purified protein to size exclusion chro-
matography (Fig. 4b). We observed a small peak at 54
ml and a major peak at 60 ml; no peak was observed in
the fraction corresponding to a monomer (78 ml). The
major peak was slightly larger than the peak corre-
sponding to 150 kDa (64.18 ml). To confirm that these
peaks were pmE2, each fraction from each peak was
analyzed by native-PAGE; bands were observed at
sizes corresponding to a tetramer or a pentamer
(Fig. 4c). By contrast, no protein was in the fraction
corresponding to the peak at 54 ml (data not shown).
This result is consistent with data derived from CBD-
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Biotechnol Lett (2020) 42:1247–1261 1253
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
fused pmE2. Therefore, we believe that these charac-
teristics are due to pmE2 itself, rather than to the tags,
when expressed in plants.
Immunogenicity of pmE2:pFc2 in mice
Prior to testing the vaccine in pigs, we used
pmE2:pFc2 to immunize mice to assess whether
recombinant pmE2 protein produced from N. ben-
thamiana acts as a vaccine. Mice (C57BL/6J, male, 6
weeks old) received a subcutaneous injection of 1 lg
pmE2:pFc2 in complete Freund’s adjuvant or the same
volume of PBS (as a control). After one-dose injec-
tion, serum samples were collected on a weekly basis
and antibody levels were tested in an ELISA (Fig. 5).
Anti-CSFV antibody levels in all mice increased
gradually, peaking at 4–5 weeks. These levels were
maintained or decreased slightly until week 8 indicat-
ing that the chimeric protein produced from plant acts
as an antigen. In addition, we used mouse serum (week
8) to test for neutralizing antibodies (Table 1) in order
to investigate whether the antibody against recombi-
nant pmE2 can provide protection against CSFV. Most
sera had neutralizing antibody titers of 2
7
–2
9
, although
slight variations were observed. This level of neutral-
izing antibodies suggests that the fusion protein has
great potential as a vaccine.
Verification of CSFV antibody-mediated
neutralization in serum from pigs immunized
with pFc2:pmE2
Finally, piglets were injected with 150 lg pmE2:pFc2
in ISA 15A VG, and neutralizing antibody responses
were verified by testing serum in a virus neutralization
Transgenic plant Wild-type plant
AB
18 42 81 99 (T0)
M 1 7 10 5 7 11 12 6 10 12 (T2)
(kDa)
28
35
63
75
48
Anti-Pig
CBB staining
Fig. 2 Selection of 1300::pmE2:pFc2:HDEL-expressing trans-
genic plants. aWestern blot analysis of 1300::pmE2:pFc2:H-
DEL expression in transgenic plants (T2 generation). The same
amount of total protein extract was prepared from two or three
independent lines of four T2 generation transgenic plants and
subjected to immunoblotting with an anti-Pig antibody. The blot
was visualized by staining with Coomassie Brilliant Blue in
order to verify the clarity of total protein extraction processes.
M, protein markers; upper numbers indicate transgenic lines at
T0; bottom numbers indicate transgenic lines at T2. bPheno-
typical comparison of wild-type and transgenic plants. Plants
were grown side by side from the same germination stage and
the morphology was compared at 37 days-of-age
123
1254 Biotechnol Lett (2020) 42:1247–1261
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
test (Table 2). Four of six pigs (aged 40 days) were
vaccinated with 150 lg pmE2:pFc2 and then boosted
20 days later with the same vaccine formulation. The
other two pigs were injected with the same volume of
PBS (control). Serum was obtained from each pig at
20, 60, 90, and 110 days post-second vaccination and
neutralizing antibody titers were measured. Although
some neutralizing antibodies were detected as early as
48
63
75
35
100
M T FT W1 W2 W3 E EB
100
35
25
45
63
75
(kDa)
BSA ( )
pmE2:
pFc2 210.5
BSA( )
OD 595nm
10.068
20.148
40.264
pmE2
2 0.156
•1.135
y = 0.0643x + 0.01
R² = 0.9929
0
0.05
0.1
0.15
0.2
0.25
0.3
012345
OD 595nm
BSA(
A
Anti-Pig CBB staining
BC
Fig. 3 Purification and quantitative analysis of the pmE2 fusion
protein. aPurification of the pmE2:pFc2 recombinant protein by
protein A chromatography. Total extracts were prepared from
transgenic plant leaves by incubation in extraction buffer,
followed by centrifugation. Samples were loaded onto a column
containing protein A resin. After binding, resin-bound recom-
binant proteins were washed three times and then eluted in
elution buffer. Each fraction was separated by SDS-PAGE and
subjected to western blot analysis with an antibody specific for
anti-pig IgG (whole). Ttotal fraction, FT flow-through, W1-3
wash-off fractions, Eelution fraction, EB post-elution resin
content. bQuantification of purified pmE2:pFc2. Purified
protein from awas concentrated by centrifugal filtration, and
subjected to perform Bradford assay. Linear regression was
generated by using 1, 2 and 4 lg of bovine serum albumin (BSA)
and 2 lg of pmE2:pFc2 was used for quantification. cQuantifi-
cation of pmE2:pF2 by SDS-PAGE. 2 lg of pmE2:pFc2 and
simultaneously, 0.5, 1, and 2 lg bovine serum albumin (BSA)
was run for comparison. The gel was visualized by Coomassie
Brilliant Blue staining and band intensities were compared
123
Biotechnol Lett (2020) 42:1247–1261 1255
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
20 days after the primary vaccination, a marked
increase in titer was observed at 20 days post-booster
vaccination. The high neutralizing antibody titers were
maintained at values [6 (log2) for 170 days, although
they fell gradually after reaching a peak at aged 80
days. Thus, pmE2:pFc2 generated neutralizing anti-
bodies specific for CSFV in the natural host.
Discussion
Most CSFV vaccines in use today in CSF-endemic
areas, including Asia, are modified live vaccines
(MLV); although MLVs provide good protection
against CSFV infection, it is difficult to distinguish
between infected and vaccinated pigs (Blome et al.
2006; Deng et al. 2005; van Oirschot 2003). Therefore,
it is difficult to prove that the area is free from this
disease, which constrains import and export of pigs.
AB
C
M
Reducing & Boiling
Reducing & Non-Boiling
Non-Reducing
Fig. 4 Characterization of pmE2:pFc2 recombinant Status of
pmE2:pFc2 under different conditions. Purified protein was
treated with b-mercaptoethanol and/or boiling, and then
separated by SDS-PAGE. The gel was visualized by Coomassie
Brilliant Blue staining. bSize exclusion chromatography of
pmE2:pFc2. Purified recombinant proteins were injected onto a
size exclusion column and separated (bottom panel). Blue-
Dextran (2000 kD), Alcohol dehydrogenase (150 kD), Bovine
Serum Albumin (66 kD), Carbonic Anhydrase (29 kD), and
Cytochrom C (12.4 kD) were used as protein molecular-weight
standards (upper panel). cNative-PAGE analysis of recombi-
nant proteins eluted from the column. The fraction correspond-
ing to the major peak from bwas separated on 8% Native-PAGE
gels and visualized by CBB staining
123
1256 Biotechnol Lett (2020) 42:1247–1261
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
To solve these problems, subunit marker vaccines are
being developed. Many studies are examining the
efficacy of vaccines based on the CSFV E2 glycopro-
tein; indeed, some products have come to market:
BAYOVACÒCSF Marker (AdvasureÒ, Pfizer, UK)
and PorcilisÒPesti (IDEXX CSF marker, IDEXX
Europe B.V., the Netherlands). The two viral glyco-
proteins E2 and E
rns
are necessary for viral attachment
to host cells; of these, E2 is the major envelop
glycoprotein that induces production of anti-CSFV
neutralizing antibodies (Ko
¨nig et al. 1995; Weiland
et al. 1990). Previously, we demonstrated that an E2
glycoprotein fused with a CBD was immunogenic in
pigs and confirmed that it provided efficient protection
against CSFV challenge (Park et al. 2019). Here, we
intended to use the recombinant protein Fc domain
from pig IgG as an alternative to the CBD and evaluate
its efficacy in animals. An ideal vaccine should not
only be efficient and safe, but also affordable for end-
users. Simple and successful expression of large
amounts of recombinant protein with high immuno-
genicity is the key to cost-effectiveness. This is why
we chose the porcine IgG Fc domain as the fusion
partner. The Fc domain improved solubility and
increased expression of E2 when expressed in plants.
In addition, the Fc domain makes isolation of recom-
binant proteins by protein A affinity chromatography
both easy and cheap. Although protein A resin is not
cheap, appropriate use and management through a
cleaning-in-place protocol means that hundreds of
purification cycles can be performed without disas-
sembly of the equipment, resulting in significant
0
1
2
3
4
Absorbance at 405nm
pmE2:pFc2 mouse 1
pmE2:pFc2 mouse 2
pmE2:pFc2 mouse 3
pmE2:pFc2 mouse 4
pmE2:pFc2 mouse 5
PBS con. mouse 1
PBS con. mouse 2
012345678
Week post vaccination
Fig. 5 Immunogenicity of
pmE2:pFc2 in mice.
PmE2:pFc2 fusion protein
(1 lg) combined with
Freund’s complete adjuvant
was injected into mice. Sera
were collected every week
post-vaccination and
subjected to ELISA. The
negative control comprised
the volume of PBS. Signal
intensity was measured at
A405
Table 1 Single- and
double-dose vaccination
and classical swine fever
virus (CSFV)
neutralizingantibody titers
from mouse serum
VNA virus neutralizing
antibody, CSFV
neutralizing antibody titers
were measured as described
in the Materials and
methods section
Vaccination Antigen Mouse ID# VNA titer
Single-dose vaccination pmE2:pFc2 1 128
2 256
332
4 256
5 512
Double-dose vaccination pmE2:pFc2 1 256
2 256
364
4 128
564
Back titers 177
Negative control \4
Positive control 2048
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Biotechnol Lett (2020) 42:1247–1261 1257
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
economic benefits (Gronberg et al. 2011; Zhang et al.
2017). The most important advantages of the Fc
domain are that it increases the half-life of an antigenic
peptide in plasma and interacts with Fc receptors on
immune cells, thereby acting as a molecular adjuvant.
Taking advantage of these benefits, we produced 302
mg recombinant pmE2 protein from 1 kg tobacco
leaves. This is around tenfold higher than that reported
previously using CBD:pmE2 (30.3 mg/kg) (Park et al.
2019). Although the benefits of low manufacturing
costs have been mentioned in producing recombinant
proteins from plants, there has been much debate due
to the lack of research. There are a few techno-
economic analyses for producing recombinant pro-
teins in plants (Buyel anf Fisher 2012; Tuse et al.
2014; Walwyn et al. 2015; Nandi et al. 2016). These
simulation reports suggest that cost-effective produc-
tion with plant expression system can be achieved over
alternative platforms although it depends on the
products. In addition, high expression of recombinant
proteins contributes to lower capital requirements and
cost of final products. Therefore, the subunit vaccine
based on pFc2-fused pmE2 chimeric protein can help
to provide protection against CSFV at a lower price.
Previous reports show that E2 protein exists as a
dimer, as does the Fc domain. However, we found that
pFc2:pmE2 was expressed as a multimer. Consistent
with this, when CBD-fused E2 was expressed in
plants, the product was also multimeric (Park et al.
2019; Sohn et al. 2018). According to size-exclusion
chromatography, the protein seemed to form trimer
considering that the peak was slightly larger than the
peak corresponding to 150 kD. However, native-
PAGE displayed the band corresponding to tetra or
pentamer size. Because the migration of the protein on
native-PAGE is affected by diverse factors such as
size, folding and isoelectric point, it may not perfectly
match with molecular markers. Therefore, more
research is needed to establish the structure of
pmE2:pFc2, and to examine how it affects the
immunogenicity of recombinant proteins and their
potential use as vaccines.
In this study, we used pmE2:pFc2 expressing
transgenic plants instead of transient expression
system. Transient expression generally has been
shown to give high amount of target protein than
stably expressing transgenic plants (Yamamoto et al.
2018) and we also obtained same results with this fact
(data is not shown in here). Thus, assuming that
transient expression uses the same biomass, more
protein can be produced in a short period and many
plant-based pharmaceutical companies are already
using the platform. However, there are several reason
why we used transgenic plants to produce the recom-
binant protein. In case of transient expression system,
the expression level of target genes is not uniformed.
On the other hand, transgenic plant provide stable ex-
pression of recombinant genes once the homozygous
transgenic lines are generated. This leads to large-
scale production easier than transient system. In
Table 2 CSFV-neutralizing antibody responses in pmE2:pFc2 vaccinated piglets
Age 40-day 60-day 80-day 110-day 150-day 170-day
Piglet
ID#
1st
injection
2nd
injection
20 days post-
boosting
60 days post-
boosting
90 days post-
boosting
110 days post-
boosting
PBS control 1 0 0 0 0 0 0
2000000
Ave. VNA titers
(log
2
)
0 0 0.0 0.0 0.0 0.0
pmE2:pFc2 35 0 3 10 8 6 6
36 0 8 11 7 7 6
38 0 6 10 7 6 5
39 0 5 12 \887
Ave. VNA titers
(log
2
)
0 5.5 10.75 7.5 6.75 6
VNA virus neutralizing antibody, CSFV neutralizing antibody titers were measured as described in the Materials and methods section
123
1258 Biotechnol Lett (2020) 42:1247–1261
Content courtesy of Springer Nature, terms of use apply. Rights reserved.
economic point of view, upstream processes in
transient expression system require additional equip-
ment and material for A.tumefaciens-mediated infil-
tration while they can be eliminated in transgenic
approach, which enables to produce recombinant
proteins more inexpensively (Garabagi et al. 2012).
Moreover, transient expression system requires addi-
tional step to avoid concerns relating to endotoxins
that is caused by the use of A.tumefaciens for vacuum
infiltration. For these reasons, we think it is more
appropriate to use transgenic plants in situations where
vaccines must be produced steadily, whereas transient
expression system is suitable for urgent situations.
Next, we examined neutralizing antibody responses
to CSFV virus in mice and pigs. We found that
vaccination of mice and pigs with pmE2:pFc2 gener-
ated anti-CSFV neutralizing antibodies. The pig
experiments showed a slight increase in the titer of
neutralizing antibodies at 20 days post-primary vac-
cination, and a dramatic increase in titer after the
second vaccination. Neutralizing antibody titers were
maintained at 6 (log2) until 170 days, although they
did fall slightly thereafter. In addition, neutralizing
antibodies were readily detectable in mouse serum
even at 8 weeks post-vaccination. It is desirable to
minimize the number of vaccination to reduce the
burden on end users. We confirmed a single vaccina-
tion generated antibodies at levels similar to a double
vaccination indicating that one-dose injection with 1
lg of pmE2:pFc2 is enough to induce immune
responses in mice. Taken together, the results suggest
that pmE2:pFc2 induces an efficient neutralized
antibody response to CSFV. A limitation is that we
vaccinated animals with a single dose (150 lg) and
used only one type of adjuvant; further studies should
vary both parameters to achieve better results. In
addition, further studies should address several impor-
tant issues. For instance, we need to clarify whether
vaccination of pigs affects viral shedding and the viral
load. Also, we need to examine effects on vertical and
horizontal transmission. In other words, it is important
to examine whether vaccinated pregnant sows can
prevent transplacental transmission of CSFV to the
fetus.
We confirmed the presence of anti-pFc2 antibodies
in immunized pigs (data is not shown). Antibodies
typically contains complex bi-antennary glycans
whereas the heterogenous pFc2 may have high
mannose type of glycans due to ER accumulation.
N-glycans at CH2 domain influence the folding of the
Fc part (Mimura et al. 2001). Therefore, it is possible
that pFc2 can be recognized as antigen. Nevertheless,
the vaccinated pigs hardly displayed adverse effects
during the study. Highly sophisticated approach using
glyco-engineering is required for minimizing autoim-
mune reactions and improved vaccine development
based on Fc fragment.
In conclusion, we expressed pmE2:pFc2 in trans-
genic N. benthamiana plants, and isolated and purified
the recombinant fusion protein at high yield and low
cost. In addition, we confirmed that vaccination of
mice and pigs with the fusion protein generated a
neutralizing antibody response against CSFV. Taken
together, this recombinant protein could be developed
as a subunit vaccine against CSFV in a cost-effective
manner.
Author contributions EJS designed and organized the overall
study. YP and SL wrote the manuscript. HK performed cloning
and generated constructs. MP selected and generated transgenic
plants. SG, JK, D-JA and SC, organized and carried out piglet-
related work and antibody analysis. KM carried out protein
purification and western blot analysis. NK performed ELISA
assay and mouse vaccination.
Supporting information Supplemental Table 1—The list of
nucleotides sequences used in this study.
Conflict of interest The authors declare that they have no
conflict of interest.
Open Access This article is licensed under a Creative
Commons Attribution 4.0 International License, which
permits use, sharing, adaptation, distribution and reproduction
in any medium or format, as long as you give appropriate credit
to the original author(s) and the source, provide a link to the
Creative Commons licence, and indicate if changes were made.
The images or other third party material in this article are
included in the article’s Creative Commons licence, unless
indicated otherwise in a credit line to the material. If material is
not included in the article’s Creative Commons licence and your
intended use is not permitted by statutory regulation or exceeds
the permitted use, you will need to obtain permission directly
from the copyright holder. To view a copy of this licence, visit
http://creativecommons.org/licenses/by/4.0/.
123
Biotechnol Lett (2020) 42:1247–1261 1259
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