(2021) 52:102
Masuda et al. Vet Res
https://doi.org/10.1186/s13567-021-00971-5

RESEARCH ARTICLE

Open Access

Stable trimer formation of spike proteinfrom porcine epidemic diarrhea virus improves
the efficiency of secretory production
in silkworms and induces neutralizing
antibodies in mice
Akitsu Masuda1, Jae Man Lee2, Takeshi Miyata3, Takeru Ebihara1, Kohei Kakino1, Masato Hino4, Ryosuke Fujita4,
Hiroaki Mon1 and Takahiro Kusakabe1*

Abstract
Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen of watery diarrhea that causes serious economic loss to the swine industry worldwide. Especially because of the high mortality rate in neonatal piglets, avaccine with less production cost and high protective effect against PEDV is desired. The intrinsically assembled
homotrimer of spike (S) protein on the PEDV viral membrane contributing to the host cell entry is a target of vaccine
development. In this study, we designed trimerized PEDV S protein for efficient production in the silkworm-baculovirus expression vector system (silkworm-BEVS) and evaluated its immunogenicity in the mouse. The genetic fusionof the trimeric motif improved the expression of S protein in silkworm-BEVS. A small-scale screening of silkworm
strains to further improve the S protein productivity finally achieved the yield of about 2 mg from the 10 mL larval
serum. Mouse immunization study demonstrated that the trimerized S protein could elicit strong humoral immunity,
including the S protein-specific IgG in the serum. These sera contained neutralizing antibodies that can protect Verocells from PEDV infection. These results demonstrated that silkworm-BEVS provides a platform for the production oftrimeric S proteins, which are promising subunit vaccines against coronaviruses such as PEDV.
Keywords: Porcine epidemic diarrhea virus, spike protein, trimeric motif, silkworm-baculovirus expression vector
system, subunit vaccine
Introduction
Since its first observation in Europe in the 1970s, porcine
epidemic diarrhea virus (PEDV) has spread worldwide
due to its highly contagious nature and has been causing significant economic losses [1, 2] by causing watery*Correspondence: kusakabe@agr.kyushu-u.ac.jp
1
Laboratory of Insect Genome Science, Kyushu University Graduate
School of Bioresource and Bioenvironmental Sciences, Motooka 744,
Nishi‑ku, Fukuoka 819‑0395, Japan
Full list of author information is available at the end of the article

diarrhea, vomiting, dehydration, and a high mortality
rate in neonatal pigs [3]. PEDV has a single positivestranded RNA genome and belongs to the genus Alphacoronavirus in the family Coronaviridae. Coronaviruseshave spike protein (S), membrane protein (M), envelope
protein (E), and nucleocapsid protein (N) as four major
structural proteins in the budded enveloped viruses [3].
Among these, the highly glycosylated S protein, consisting of S1 and S2 domains, forms a homotrimer and plays
an important role in facilitating host cell attachment andentry [4]. Coronaviruses use their S1 domain to bind to© The Author(s) 2021. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing,
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Masuda et al. Vet Res(2021) 52:102

various receptors, such as porcine aminopeptidase N:
APN (Transmissible gastroenteritis virus), angiotensinconverting enzyme 2: ACE2 (Human coronavirus NL63),
carcinoembryonic antigen-cell adhesion molecule:
CEACAM1, or dipeptidyl peptidase: DDP4 (MERSCoV), transferrin receptor 1 (PEDV), on host cells andtheir S2 domain to fuse with the cell membrane [5–9].
Therefore, there are many reports that the neutralizing
epitopes are in the S protein [10–13]. Notably, it has beenreported that several neutralizing epitopes are concentrated in a region called the CO-26 K equivalent epitope(COE), which contains amino acid residues 499–638[10, 14] as a priority candidate region. In addition, theN-terminal domain of the S protein, which is unique tothe genus Alphacoronavirus, has been reported to play
an important role in PEDV infection in the intestine, andthis second candidate region is thought to have the ability to bind to a type of sialic acids unique to the intestine[15]. Furthermore, as a third candidate region, several
linear neutralizing epitopes have been reported in the
N-terminal region of the S2 domain [16].
To develop a safe and effective vaccine, a rational strategy is to use recombinant proteins targeting the S protein of PEDV. An immunological study has shown thatthe administration of recombinant S1 protein producedby cultured porcine cells to pregnant sows can protect
newborns from PEDV infection through passive immunity [17]. Similarly, enveloped virus-like particles with S,
M, and E proteins produced in cultured insect cells wereable to induce neutralizing antibodies equivalent to thoseof inactivated PEDV vaccines, although their production
efficiency was lower [18]. It is generally reported that
protein complexes such as VLPs, which have complex
and regular structures, can induce very strong immunity.
If the native structure of the coronavirus S protein, whichforms a trimer, could be stabilized, it would have stronger
immunogenicity than the monomeric S1 protein, andstructural stabilization would enable mass production. In
this study, we used a baculovirus expression system using
individual silkworms, which is useful as a mass expression system for such a complex recombinant protein. In
addition, by trimer formation, the recombinant S protein is expected to have a structure similar to that on the
PEDV envelope, and a selective neutralizing antibody will
be inducted.
For the above reasons, we attempted to construct a stable expression system for the PEDV S protein by introducing exogenous trimerization motifs. Most trimericmotifs are coiled-coils that bundle several α-helices andare also present in the S2 region of coronaviruses. However, the trimeric motifs formed by the ectodomains of S
proteins detached from the membrane are unstable, andthus exogenous trimeric motifs have been employed inPage 2 of 14

the expression of S proteins of various coronavirus genera [19–21]. The previous studies reported that the fusionof T4 fibritin of bacteriophage T4 and yeast transcription
factor GCN4 to S proteins enhanced its trimerizationand stabilization [19, 22].
In this study, to develop a subunit vaccine, we designed
several constructs suitable for the expression of PEDV S
protein using the silkworm-baculovirus expression vector system (silkworm-BEVS). The silkworm-BEVS utilizing recombinant Bombyx mori nucleopolyhedrovirus(BmNPV) to express a protein of interest (POI) is a useful platform for manufacturing biopharmaceuticals [23].
Under the control of powerful polyhedrin promoter, avariety of POIs expressed from recombinant BmNPVs
accumulate in silkworm larvae and pupae in large quantities [24–27].
In the silkworm-BEVS, the introduction of the trimerization domain was very effective for stable mass secretion expression, and the vaccine candidate molecules
produced by this system were able to induce strong
humoral immunity and neutralizing antibodies against
PEDV in mice.

Materials and methods
Silkworm cells and strain

The NIAS-Bm-Oyanagi2 cell (BmO2: kindly provided
from Dr Imanishi) was maintained in IPL41 insect
medium (Sigma, St. Louis, MO, USA) with 10% fetal
bovine serum (Gibco, Grand Island, NY, USA) at 27 °C.
The silkworm strains used in this study were provided by
the Institute of Genetic Resources at Kyushu University,and the silkworm larvae were reared on mulberry leaves
at 24–29 °C.
Transmembrane prediction of PEDV S protein

The transmembrane domain of S protein from the PEDV
83P-5 strain (GenBank accession number, AB548621.1)
was predicted using TMHMM v.2.0 software [28] or
SOSUI software [29].
Plasmid construction

The full-length spike gene from the PEDV 83P-5
strain was chemically synthesized [30]. The DNA
fragment encoding amino acid 1–1332 of the S protein was amplified by PCR and subcloned into a
modified pENTR11 vector containing a lobster L21
sequence (translation enhancer) at the N-terminal
and affinity tags at the C-terminal in the originalpENTR11 vector [24]. All primers and the PCR products for the constructs produced in this study arelisted in Table 1. The resulting construct was namedMasuda et al. Vet Res(2021) 52:102

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Table 1 Primer sequences and the names of PCR products and generated vectors used in this study
Primer name

Sequence (5ʹ to 3ʹ)PCR product name

Vector nameS1-1332-noATG-F

ACG​CCT​T TA​ATT​TAC​T TC​TGG​T TG​T TC​T TA​CPEDV/S(1–1332)-XhoI

pENTR11L21-PEDV/S(1–1332) + TEVdH8S

PEDV/S(1–1332)-Tags

pENTR11L21-PEDV/S(1–1332) + Tags

PEDV/S(1–1320)-Tags

pENTR11L21-PEDV/S(1–1320) + Tags

S1-1332-stopXhoI-F GGG​GCT​CGA​GAT​AAA​AAT​AAT​CAA​CCA​AAC​CCAC​
S1-1332-F

AAG​CAC​AAA​GGA​AGC​TAG​ACC​CAG​C TT​TCT​TGT​ACA​AAG​

S1-1332-R

CAT​GCT​GCC​CCC​TCC​ATG​ATG​ATG​GTG​ATG​ATG​C TT​T TCG​

S1-1320-F

TCG​AGT​GCA​GGC​GAA​AAC​C TG​TAC​T TCC​

S1-1320-R

GAT​ATA​TGT​C TC​AAC​TCG​GTT​GAG​CCA​C TC​AAGG​PEDVS-TEV-F

TCG​AGT​GCA​GGC​GAA​AAC​C TG​TAC​T TCC​S+CMP-R

GAT​ATA​TGT​C TC​AAC​TCG​GTT​GAG​CCAC​

CMP-F

CCG​AGT​TGA​GAC​ATA​TAT​CGA​AGA​AGA​CCC​GTG​TGA​GTG​
TAA​AAGC​

CMP-R

GGT​T TT​CGC​C TG​CAC​TCG​AGA​TAA​T TT​TAT​TCT​CCA​GCG​
CTT​CAA​TGC​G

pENTR11L21-PEDV/S(1–1332) + TEVdH8S.
pENTR11L21-PEDV/S(1–1332) + Tags(TEVdH8SKtag)
in which Ktag (MKHKGS) is inserted into thepENTR11L21-PEDV/S(1–1332) + TEVdH8S
wasprepared by inverse PCR. Also, pENTR11L21PEDV/S(1–1320) + Tags was prepared by inverse
PCR with the pENTR11L21-PEDV/S(1–1332) + Tags
as the template. In this study, trimerization motif
from chicken cartilage matrix protein (CMP) was
inserted into the C-terminal of the S ectodomain.
The DNA fragment for the CMP trimerization motif
was amplified by PCR using the chemically synthesized DNA coding the motif as a template [31].
The vector backbone was amplified from the pENTR11L21-PEDV/S(1–1332) + Tags by PCR and named
PEDV/S(1–1320)-Tags-gib. These PCR products wereassembled using the Gibson assembly method to generatepENTR11L21-PEDV/S(1–1320) + CMP + Tags
vectors.
Preparation of recombinant baculovirus

Each entry clone for PEDV S protein and pDEST8 vector (Invitrogen, Carlsbad, CA, USA) were subjected
to gateway LR reaction according to the manufacturer’s protocol to generate the transfer plasmid for
production of recombinant baculovirus. According to
the protocols described previously, the transfer plasmid was introduced into E. coli DH10bac to generate
recombinant BmNPV T3 bacmid by Tn7-mediatedtransposition [32]. In this study, the modified BmNPV
T3 bacmid deleting six dispensable genes (chitinase A,
cathepsin, egt, p26, p10, and p74) was used from ourlaboratory stock to improve protein expression [26, 33–35]. The recombinant baculoviruses were generated byintroducing these bacmids into silkworm BmO2 cells

PEDV/S(1–1320)-Tags-gib pENTR11L21-PEDV/S(1–1320) + CMP + Tags
CMP

and amplified by serial infection. The viral titers were
determined by plaque assay.
Expression analysis of PEDV spike protein in cultured
insect cells and silkworm

Recombinant BmNPVs at an MOI of 1 were used for
infection to 1 × ­106 BmO2 cells. The cell pellet andmedium of infected cells were separated and collected
at 4 days post-infection (dpi) by centrifugation at 1000 g
for 10 min at 4 °C. After the centrifugation, the cell pellet
was lysed with 1 mL of 1 × T buffer (20 mM Tris–HCl,
0.5 M NaCl, pH 7.5) and sonicated for 5 min by TOMY
UD-100 (Tomy Seiko Tokyo, Japan) at 4 °C. After centrifugation at 9000 g for 10 min at 4 °C, the supernatant
was collected, and the precipitated was resuspended withthe same volume of 1 × Phosphate-buffered saline (PBS;
137 mM NaCl, 2.7 mM KCl, 10 mM ­Na2HPO4·12H2O,
1.8 mM ­KH2PO4, pH 7.4).
Silkworm larvae were injected with recombinant BmNPVs of 1 × ­104 plaque-forming unit per larva. The sera ofinfected larvae at 4 dpi were collected and centrifugedat 3200 g for 10 min at 4 °C. The supernatants were collected and stored at −80 °C until use.
Purification of the PEDV spike protein

As for the large-scale purification, 10 mL of the larval serum from the silkworm infected with BmNPVPEDV/S(1–1320) + CMP + Tags were thawed and mixed
with 40 mL of buffer A (20 mM Tris–HCl, 0.5 M NaCl,
pH 7.5). After sonication by TOMY UD-100 (TomySeiko) at 4 °C for 10 min, the sample was centrifuged at
12 110 g for 30 min at 4 °C. The supernatant was filteredusing a 0.45 µm filter (Millipore, Milford, USA).
Two-step affinity chromatography was performed
to purify the S protein. Briefly, the filtered sample was
loaded onto a 5 mL HisTrap excel column (Cytiva, Tokyo,

Masuda et al. Vet Res(2021) 52:102

Japan) equilibrated with buffer A containing 20 mM imidazole followed by the column wash with buffer A containing 30 mM imidazole (25 mL). The bound proteins
were eluted with buffer A containing 500 mM imidazole.
The 25 mL of the eluent were pooled in a 50 mL tube.
The elution containing PEDV S protein was ultrafiltratedusing Amicon ultra-15 100 K filters (Merck, USA) to substitute the buffer to 1 × PBS. A final 50 mL diluted solution was applied to 5 mL STREP-tactin columns (IBA
GmbH, Germany), followed by washing with 1 × PBS(25 mL) and elution with the buffer containing 2.5 mM
desthiobiotin (25 mL). The eluent was concentrated
by ultrafiltration using Amicon ultra-15 100 K filters(Merck) and dialyzed against 1 × PBS. The purified PEDVS protein was quantified by bradford assay using ImageJsoftware version 1.51 s using BSA as a standard.
SDS‑PAGE and Western blotting

The fivefold dilution of the larval serum and all otherspecimens were mixed with an equal volume of 2 × SDS
sample buffer (0.1 M Tris–HCl pH 6.8, 0.2 M dithiothreitol, 4% SDS, 20% glycerol, and 0.02% bromophenol blue)and denatured at 96 °C for 10 min. Subsequently, samples
were separated on 12% SDS-PAGE gels. After separation,gels were stained by Coomassie Brilliant Blue (CBB). In
the Western blotting analysis, proteins separated by SDSPAGE were transferred to PVDF membrane (Millipore).
Membranes were treated with blocking buffer (Tris-buffered saline containing 5% skim milk) and incubated with
HisProbe-HRP (Thermo Scientific, Waltham, USA) for
1 h at room temperature. The specific protein bands werevisualized using the Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). In the comparisonof the S protein expression among silkworm strains, theintensities of each specific band were measured using
ImageJ software version 1.51 s, and the relative intensities were calculated by setting the mean intensity of f38
strain to 1.
Blue‑native PAGE

Protein samples were mixed with a 4 × NativePAGE
sample buffer (Thermo Fisher Scientific, Waltham, MA,
USA) and were electrophoresed in 3 to 12% Bis–Tris
NativePAGE gel (Thermo Fisher Scientific) for 115 min at
150 V. As a cathode buffer, NativePAGE running buffer(Thermo Fisher Scientific) containing NativePAGE cathode buffer additive (Thermo Fisher Scientific) was used.
NativePAGE running buffer was used as an anode buffer.
NativeMark unstained protein standard (Thermo Fisher
Scientific) was used as a size standard. The gels after electrophoresis were stained using CBB.

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Size‑exclusion chromatography

Purified S protein was subjected to size-exclusion
chromatography using a superose 6 increase 10/300
GL column (Cytiva) in a 1 × PBS buffer. Thyroglobulin(669 kDa), Ferritin (440 kDa), and Aldolase (158 kDa)
were used as molecular weight markers.
Dynamic light scattering (DLS)

The size distribution of PEDV S proteins was analyzed
by dynamic light scattering using an ELSZ-2000ZS(Otsuka electronics CO., Ltd, Osaka, Japan). To assess
the thermal stability, 0.6 mg/mL of the S protein wereincubated at 4, 30, 40, 50, 60, 70, 80, 90 °C for 5 min
and measured the size changes. The measurement was
performed at room temperature. Before the heating,the sample was centrifuged at 15 000 rpm for 5 min toremove aggregates which interfere with obtaining precise spectra in DLS.
Mouse immunization

A total of 6 female Balb/c mice (7-week-old) were inoculated with 30 μg of PEDV S protein with micro-emulsion adjuvant (Montanide IMS 1313, Seppic, France)and boosted at 14 and 28 days after prime vaccination.
At 0, 21, and 42 days after prime vaccination, the sera
were collected and used for antigen-specific IgG ELISA
and neutralization assay.
S protein‑specific IgG response evaluation
with Enzyme‑linked immunosorbent assay

The 96-well ELISA plates (Sumilon type S, Sumitomobakelight co., ltd, Japan) were coated with 50 µL of
5 µg/mL of the S proteins in bicarbonate buffer (pH9.6)
and incubated overnight at 4 °C. The plates were
washed with PBS containing 0.1% Tween-20 (PBST)
and PBS and blocked with a blocking buffer (PBS containing 1% BSA) at room temperature for 1 h. Afterwashing, 50 µL of each serum sample was added into
each well. The plates were washed, and 50 µL of 0.5%
BSA containing a peroxidase-conjugated goat antimouse IgG Fab antibody (Jackson immunoresearch,
Laboratories, Inc., West Grove, PA, USA) was added
into each well and incubated for 1 h. Finally, the plates
were washed with PBST and PBS, and the color reaction was developed with 1-Step Turbo TMB-ELISA
substrate solution (Thermo Scientific). The optical density at 450 nm (OD450nm) was read using a microplate
reader.
Serum virus neutralization assay

Eagle MEM containing 10% fetal bovine serum (biosera, USA) and 50 µg/mL Gentamicin (Merck) wasMasuda et al. Vet Res(2021) 52:102

prepared as a growth medium for the neutralization
assay. The Immune sera of each mouse were heated at
56 °C for 30 min. Then, 25 μL of serially diluted sera
were mixed with an equal volume of 4000 ­TCID50/mL PEDV (P-5 V strain) and incubated in the 96 wellplates at 37 °C for 1 h [36]. The plate was added 50 μL of
Vero cells at 1 × ­105 cells/mL, and the cells were incubated in the growth medium at 37 °C with 5% ­CO2 for
5 days. The presence of the cytopathic effect (CPE) was
checked, and the highest dilution factor at which CPE
was not observed was used as the neutralizing antibody
titer.

Results
Expression of PEDV S ectodomain in silkworm‑BEVS

In order to secrete PEDV S protein into silkworm serum,
the transmembrane domain (TM domain) predicted were
removed from the expression constructs. Two software
programs, SOSUI and TMHMM v.2.0, predicted different amino acid regions 1333–1355 (Figure 1A, red line)
and 1324–1346 (Figure 1A, blue line) as a TM domainof the S protein, respectively. Therefore, two constructs
were designed based on the prediction and generated therecombinant BmNPVs expressing the candidates for S
ectodomain, PEDV/S(1–1332) and PEDV/S(1–1320) as
described under the Materials and Methods (Figure 1B).
PEDV/S(1–1332) and PEDV/S(1–1320) correspond to S
proteins without TM domains predicted by SOSUI and
TMHMM v.2.0, respectively. The expression analysisof the ectodomain in BmO2 cells and silkworm larvae
revealed that both constructs were secreted inefficiently
into the culture media and larval sera, respectively (Figure 1C). The TM domain is well conserved among coronaviruses, and in PEDV, residue 1321 corresponds to thebeginning of the TM domain of other coronaviruses [37].
Therefore, it is likely that the S1-1320 construct, in which
residues 1321 and later were removed, was more efficiently secreted. Importantly, a part of PEDV/S(1–1332)
in the serum was cleaved probably by trypsin (Figure 1C).
This construct partially retains the TM domain, and its
presence on the membrane may make it more susceptible to cleavage by trypsin. In conclusion, 1–1320 residueof PEDV S protein was stably secreted into the silkworm
serum without cleavage, but the expression levels wereinsufficient for developing an inexpensive vaccine.
Construction and expression of PEDV S protein fusedwith CMP trimerization motif

From the above results, it is assumed that PEDV S proteins are difficult to form homotrimer spontaneously in
the insect cells. To improve secretion by forming stablehomotrimer of PEDV S protein, we fused chicken cartilage matrix protein (CMP) to the S protein, following a

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previous study in which CMP was used to stabilize thetrimeric antigen [38]. As described under the Materials and Methods, PEDV/S(1–1320) was C-terminally
fused with the trimeric motif of CMP and generated therecombinant BmNPV (Figures 2A and B). The aminoacid sequence of PEDV/S(1–1320) + CMP + Tags wasshown in the Additional file 1. The expression analysis byWestern blotting demonstrated that C-terminal fusionof CMP to the PEDV S ectodomain improved the secretion efficiency drastically compared to that without CMP(Figure 2C). In the CBB staining of polyacrylamide gel,the clear protein bands corresponded to PEDV S protein
fused with CMP from 0.5 µL of the silkworm sera was
detected, although at the same position, a non-specificband is also observed in the serum of silkworm expressing PEDV/S(1–1320) without CMP (Figure 2C).
The expression levels of the PEDV S protein differedamong silkworm strains

To further improve the production of PEDV S protein,small-scale screening of the S protein expression using
25 silkworm strains was performed. The recombinant
BmNPV expressing PEDV/S(1–1320) + CMP + Tags was
injected into each larva, and the sera of three individuals
infected were collected to analyze the S protein expression levels. Comparison of the specific bands by Western
blotting analysis using HisProbe-HRP revealed that n22
and xa50 strains showed relatively high S protein expression (Figures 3A and B). The n22 and xa50 strains preserved in Kyushu University are inbred lines, and they
are not suitable for mass-rearing, and the size of the last
instar larvae is relatively small. Since these shortcomings can be overcome by utilizing hybrid vigor throughF1 hybrid formation, we crossed n22 and xa50 strains(n22 × xa50 and xa50 × n22) and confirmed that theexpression level of S protein was not altered. As shown in
Figure 3C, the expression of S protein in serum of thesehybrids infected with the recombinant BmNPV was comparable to those of n22 and xa50 strains. Although there
was no synergistic effect in the amount of expression, thecrossbreeding of these strains was easier to raise, and thevolume of serum recovered from each larva was greatly
increased.
Characterization of the multimer formation of PEDV S
protein fused with CMPThe PEDV/S(1–1320) + CMP + Tags proteins were purified from the serum of silkworm (xa50 × n22) using
two-step affinity chromatography as described under
the Materials and Methods. As shown in Figure 4A, theS protein was appeared as a single band after the STREP
purification, and the final yield from 10 mL sera was
about 2 mg. To determine whether the S proteins form

Masuda et al. Vet Res(2021) 52:102

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Figure 1 Expression of the ectodomain of PEDV S protein in cultured silkworm cells and silkworm larva. A Transmembrane (TM) domain
prediction of PEDV S protein. The TM domains predicted using TMHMM v.2.0 software and SOSUI software are indicated by a blue and red
underline, respectively. B The constructions of baculovirus transfer vector. pPH: polyhedrin promoter; B1 and B2: recombination sites for Gateway
cloning; L21: translational enhancer; Tags: TEV protease cleavage site, 8 × histidine tag, STREP(II) tag, 6 × histidine tag, and Ktag (MKHKGS); SV40pA:
polyadenylation signal (SV40). C Expression of PEDV S(1–1320) and S(1–1332) protein in BmO2 cells (left panel) and silkworm larva (right panel).
Medium: culture medium; Soluble: soluble samples of cell lysates; Insoluble: insoluble samples of cell lysates. All samples from BmO2 cells (5μL) and silkworm sera (0.5 μL) were resolved on an 8% SDS-PAGE. The recombinant PEDV S protein was also detected by Western blotting using
HisProbe-HRP. Orange and blue arrows indicate the PEDV S protein and the cleaved products, respectively.

homotrimer, Blue-Native PAGE (BN-PAGE) and sizeexclusion chromatography analyses were carried out. In
the BN-PAGE analysis, a band was observed between 480
and 720 kDa of the molecular weight marker (Figure 4B).

The molecular weight of PEDV/S(1–1320) + CMP + Tags
protein monomer is calculated as 153 kDa, and thatof the trimer is 459 kDa. Because of the post-translational modifications such as glycosylation, theMasuda et al. Vet Res(2021) 52:102

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Figure 2 Genetic fusion of trimerization motifs to the S protein. A, B Scheme of PEDV S protein fused with the trimeric motif. The CMP
coiled-coil was appended at the C terminus of the S protein. C Expression analysis of the S protein fused with the trimeric motif. Medium: culture
medium; Soluble: soluble samples of cell lysates; Insoluble: insoluble samples of cell lysates. All samples from BmO2 cells (5 μL) and silkworm sera
(0.5 μL) were resolved on an 8% SDS-PAGE. The recombinant PEDV S protein was also detected by Western blotting using HisProbe-HRP. Orange
arrows indicate the PEDV S protein.

molecular weight of PEDV/S(1–1320) + CMP + Tags
protein monomer reached about 180 kDa from our
SDS-PAGE analysis, and based on this calculationthat of the trimer is expected to be about 540 kDa(Figure 2C). Consistent with this result, the peak of
PEDV/S(1–1320) + CMP + Tags protein analyzed by

Masuda et al. Vet Res(2021) 52:102

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Figure 3 Screening of the suitable silkworm strain for PEDV S protein expression. A The Western blotting analysis of 25 silkworm strains
infected with recombinant baculovirus encoding PEDV/S(1–1320) + CMP + Tags. The sera of 0.5 μL were subjected to 8% SDS-PAGE, and thespecific band was detected using HisProbe-HRP. Three individual samples were prepared from each strain, and f38 sera were used as a control.
B Comparison of relative band intensities among silkworm strains by Western blotting detecting PEDV/S(1–1320) + CMP + Tags protein. The
intensities of each band were measured, and the relative intensities were calculated using the average band intensity of f38 strain as 1. Bars
represent mean relative intensities ± SE (N = 3). C Expression analysis of the hybrid silkworm of n22 strain and xa50 strain. Three individual serum
samples of each n22 (lane1-3), xa50 (lane4-6), and n22 × xa50 (lane7-9), and xa50 × n22 (lane10-12) were resolved in an 8% SDS-PAGE with CBBstain (upper panel) and Western blotting using HisProbe-HRP (lower panel). Orange arrows indicate the PEDV S protein.

size-exclusion chromatography was eluted between 440
and 669 kDa (Figure 4C), indicating that the PEDV S
protein fused with CMP formed homotrimer. Finally,

the thermal stability of PEDV/S(1–1320) + CMP + Tags
homotrimer was analyzed by dynamic light scattering (DLS). PEDV/S(1–1320) + CMP + Tags had a long

Masuda et al. Vet Res(2021) 52:102

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Figure 4 Characterization of PEDV/S(1–1320) + CMP + Tags protein. A Purification of the S protein from the silkworm serum. Two-step affinity
chromatography using HisTrap excel column and STREP-tactin column was executed described in the Materials and Methods. Lanes 1 and 6,
molecular weight marker; lanes 2–5, nickel affinity chromatography; lanes 7–10, STREP-tactin affinity chromatography. All samples were resolved
in 8% SDS-PAGE and visualized by CBB staining. An orange arrow indicates the PEDV S protein. B Blue-Native PAGE analysis of the S protein. Afterelectrophoresis, the gel was further stained by CBB. An orange arrow indicates the PEDV S protein trimer. C Size-exclusion chromatography of PEDVS protein. The chromatogram of the S protein is shown as a blue curve. The molecular weight markers are indicated as dashed lines. D The sizechanges of the S protein against heating. The thermal stability profile was collected by DLS.

diameter of about 24 nm measured at 4 °C that is slightly
longer than that observed in the cryo-EM [22]. The S protein showed the same diameter at 4, 30, 40, and 50 °C, butbegan to change from about 24 nm to 33 nm at 60 °C, andbecame even larger at 70 °C to 90 °C (Figure 4D). These
results indicate that the PEDV/S(1–1320) + CMP + Tags
protein purified from silkworm serum forms a stable
trimer up to about 50 °C.

PEDV S protein elicited humoral immunity in mice

Purified PEDV/S(1–1320) + CMP + Tags protein mixed
with micro-emulsion adjuvant (Seppic, France) was inoculated three times into 6 female Balb/c mice at a doseof 30 µg, 14 days apart (Figure 5A). This administration
condition was previously used to induce efficient antibody production in mice with a silkworm-derived vaccine antigen [26]. We decided to use this animal modelMasuda et al. Vet Res(2021) 52:102

because it is optimal to verify the induction of neutralizing antibodies. Serum samples at days 0, 21, and 42 after
prime vaccination were collected and subjected to ELISA
analysis to determine the induction of PEDV S proteinspecific IgG antibodies. The ELISA analysis showed thatthe first boost vaccination, given 14 days after the prime
vaccination, was sufficient to saturate the antibody production level, and the second boost vaccination did notenhance the antibody response (Figure 5B). The ELISAOD values of the immunized mice sera did not drop evenat a high dilution factor (Figure 5C), and as shown in
Additional file 2, the OD value did not drop as much asthat of the negative control group, indicating that the S
protein-specific antibody titer was higher than the maximum dilution factor in the experiment. High S proteinspecific IgG antibody levels were induced even after two
doses of 1 μg or 10 μg S protein moiety, suggesting thata sufficient immune response can be obtained even if
the dose and number of doses are reduced (Additional
file 2). A neutralization assay revealed that the S protein induced neutralizing antibodies against PEDV in

Page 10 of 14

the mice antiserum (Table 2). Collectively, we confirmed
PEDV/S(1–1320) + CMP + Tags induced sufficient antibodies that can block PEDV infection.

Discussion
PEDV causes major economic losses to the swine industry due to its high infectivity and mortality in neonatal
piglets. Therefore, vaccination of pigs is a logical strategy for its prevention. In this study, we have designed
the trimerized PEDV S protein with high immunogenicity by genetically fusing CMP trimeric motif andmass-produced using silkworm-BEVS. Induction of stable artificial trimer formation by the addition of CMP
greatly improved the protein expression in silkworm larvae compared to the S ectodomain, which is difficult toself-assemble. In mice administered with the trimerized
PEDV S proteins, S protein-specific IgG and neutralizing antibodies were generated in their sera. These results
demonstrated that silkworm-BEVS is expected to be a

Figure 5 Immunogenic study of the S protein in mice. A Schematic representation of vaccination schedule in mice. Female mice (N = 6) were
vaccinated with 30 μg of purified PEDV/S(1–1320) + CMP + Tags in a micro-emulsion adjuvant (IMS1313). The mice were boosted at 14- and 28-days
post-vaccination (dpv), and serum samples were collected at 0, 21, and 42 dpv. B ELISA analysis to detect the increase of PEDV S protein-specificantibodies. All serum samples collected at each time point from each mouse were diluted at 1:50. The OD 450 nm values from each mouse serum
are indicated as circles, and the average values are indicated as a bar. C ELISA analysis of the serum dilutions at 42 dpv. All serum samples are diluted
from 1:50 to 1:404 800. The OD 450 nm values from each mouse are indicated.

Masuda et al. Vet Res(2021) 52:102

Page 11 of 14

Table 2 The titer of the neutralizing antibody against PEDV in the sera of vaccinated mice
Antigen

PEDV/S(1–1320) + CMP + Tags

Adjuvant

IMS1313

Dose

3

Number6

Titre of neutralizing antibody
No. 1

No. 2

No. 3

No. 4

No. 5

No. 6

Geometric mean titer

64

256

128

256

128

1024

203

Sera were collected from the mice vaccinated with PEDV/S(1–1320) + CMP + Tags at 42 days post-vaccination, and a serum neutralization assay was performed using
Vero cells to determine the titer of the neutralizing antibody against PEDV.

very promising production system for developing subunit
vaccines against coronaviruses, including PEDV.
Using our powerful silkworm-BEVS, we successfully
purified about 2 mg of the antigenic PEDV S protein
trimer containing both S1 and S2 domains from 10 mLof silkworm serum. This expression level is much higher
than that of only the S1 domain of PEDV expressed using
Spodoptera frugiperda-9 cells (3.9 mg/L), and is likely
to be produced inexpensively [39]. When the full-length
S protein including the transmembrane domain was
expressed by BEVS in a commercial silkworm F1 hybrid,
about 17 μg of protein per pupa could be expressed, butoral administration of this pupa failed to induce a significant PEDV-specific immune response in piglets [40].
On the other hand, the full-length S protein displayed on
the recombinant baculovirus elicited higher neutralizing
antibodies than the S1 protein, but mass production ofrecombinant viruses remains a technical problem [41].
In this study, based on the example of successful trimerization of gp140 of human immunodeficiency virus
1 and Fiber-knob domain of egg-drop syndrome virus[38, 42], CMP was fused as a trimerization motif to form
homotrimers that mimic the native structure and also
improve the expression of S protein in silkworm serum.
Intermolecular disulfide bonds in the N-terminal regionof CMP are thought to stabilize the oligomer and contribute to the immunogenicity of the antigen fused [31].
In other examples of the use of trimerization motifs,
T4 fibritin and its deletion derivative, foldon, have been
employed to trimerize the PEDV S protein for cryoEM analyses [22, 43]. When foldon was used for trimerization, a simple analysis by non-reducing SDS-PAGE
showed that a portion of the S protein remained in thetrimeric state even after heating at 95 °C for 5 min, suggesting that it contributes to the stability of the S protein[44]. In the analysis using DLS, the formation of large
aggregates of S protein fused with CMP was not observed
even after heating at 90 °C for 5 min. These results suggest that the CMP fusion increases the expression of
the S protein in the silkworm by providing high stability to the S protein and allowing proper folding. For therational design of coronavirus spike proteins, further
studies are needed to determine which trimerization

motifs have the best properties in terms of solubility, stability, and immunogenicity.
In the production of recombinant proteins using silkworm-BEVS, it has been reported that the expression
level of the protein varies greatly depending on the strain
used. The previous researches showed the differences inthe sensitivity to both Autographa californica nucleopolyhedrovirus (AcNPV) from 163 silkworm strains and
BmNPV from the 12 strains [27, 45]. Therefore, we conducted a small-scale screening using a group of strains
maintained at Kyushu University with a relatively goodtrack record in recombinant protein production andfound n22 and xa50 as suitable silkworm strains for producing PEDV spike proteins. The reasons for these differences in expression levels may be based on the diversityof immune responses and differences in viral susceptibility to BmNPV, but the differences in expression levels depending on the target protein species cannot be
explained. For example, the d17 strain showed highexpression of intracellularly accumulated proteins suchas luciferase and DsRed, but not of PEDV S protein in theserum of this strain [27]. The n22 strain identified in thisstudy also shows high expression of mouse interleukin-4,
a secreted protein similar to S protein [25]. As mentioned
above, n22 and xa50 are inbred strains and are not suitable for mass breeding. Therefore, the strains n22 andxa50 were crossed to increase the body size and disease
resistance, and in addition, we expected to increase theexpression of the S protein of PEDV, but the expression
level of the hybrids was almost the same as that of theparent strain. However, the increase in size and resistance to disease due to hybrid vigor was remarkable,
indicating that it would be helpful for mass rearing andefficient protein production.
In this study, the immunogenic analysis of the trimerized S protein showed that the protein elicited highlevels of the S protein-specific IgG and the neutralizing
antibodies against PEDV infection. In a previous study,
three doses of a crude plant extract containing 18.76 μgof trimerized CO-26K equivalent epitopes induced a geometric mean neutralizing antibody titer of 57.6 in mouseserum, which was comparable to that of a commercial
vaccine [14]. In addition, administration of the B-cell
epitope of PEDV presented on hepatitis B virus-likeMasuda et al. Vet Res(2021) 52:102

particles at 20 μg dosage resulted in average neutralizing
antibody titers between 20 and 30 [46]. Using the sameneutralization method, the average titer of neutralizing
antibodies in pigs inoculated with PEDV was about 128
at a viral titer of 5.62 ­TCID50/mL, and in that study, neutralizing activity was considered positive when the neutralizing antibody titer was 8 or higher [47]. Although
there are some differences in these assay protocols, thegeometric mean neutralizing antibody titer of 203 in thisstudy suggests the potential of the S protein as a vaccine candidate. Because neonatal piglets with immature
immune systems are highly susceptible to PEDV, a passive immunity through colostrum and milk from vaccinated sows effectively prevents them from the infection[48]. Additional studies are needed to determine whether
the trimeric S protein produced in this study can induce
secretory IgA antibodies necessary for passive immunity
in the vaccinated sow herd.
In summary, this study describes the improved
expression of the PEDV spike protein in silkworm larvae by fusing the CMP trimerization motif and using
a suitable silkworm strain. The immunogenicity of
the S protein was confirmed by immunization experiments in mice, and sufficient neutralizing antibodies
were induced in the sera. These results indicate that thesilkworm is capable of producing relatively large oligomeric proteins, and the stabilized trimeric S protein
might be a promising subunit vaccine candidate against
PEDV.
Abbreviations
PEDV: porcine epidemic diarrhea virus; S: spike; silkworm-BEVS: silkworm-baculovirus expression vector system; M: membrane; E: envelope; N: nucleocapsid; ApN: porcine aminopeptidase N; ACE2: angiotensin-converting enzyme
2; CEACAM1: carcinoembryonic antigen-cell adhesion molecule; DDP4:
dipeptidyl peptidase; COE: CO-26K equivalent epitope; BmNPV: Bombyx morinucleopolyhedrovirus; POI: proteins of interest; BmO2: NIAS-Bm-Oyanagi2;
CMP: Cartilage matrix protein; dpi: days post-infection; PBS: phosphatebuffered saline; CBB: Coomassie Brilliant Blue; DLS: dynamic light scattering;OD: optical density; CPE: Cytopathic effect; TM: transmembrane; AcNPV:Autographa californica Nucleopolyhedrovirus; dpv: days post-vaccination.

Supplementary Information
The online version contains supplementary material available at https://​doi.​
org/​10.​1186/​s13567-​021-​00971-5.
Additional file 1. The amino acid sequence of
PEDV/S(1–1320) + CMP + Tags. The sequences corresponding to PEDV
spike protein (1–1320 amino acids) and the CMP were indicated as orange
and blue, respectively.
Additional file 2. ELISA analysis of serum dilutions at low doses
and frequency of administration. Female Balb/c mice (N = 6) were
inoculated with 1 or 10 μg of purified PEDV/S(1–1320) + CMP + Tags in a
micro-emulsion adjuvant (IMS1313) and boosted at 14 days after prime
vaccination. At 21 days after prime vaccination, the sera of each vaccinated group were pooled and used for antigen-specific IgG ELISA. As anegative control, the group vaccinated only micro-emulsion adjuvant wasPage 12 of 14

used. The data were represented as mean ± standard error of the mean(in triplicate).
Acknowledgements
We appreciate Dr Imanishi (National Institute of Agrobiological Sciences,
Japan) providing the NIAS-Bm-oyanagi2 (BmO2) cell line.
Authors’ contributions
AM designed and operated the experiment and wrote the manuscript. JML
supervised the experiment. TM performed the animal experiment and revised
the manuscript. TE, KK, and HM contributed to rearing silkworm and baculovirus infection. RF and MH analyzed the data and provided ideas. TK provided
ideas, conceived the experiment, and revised the manuscript. All authors readand approved the final manuscript.
Funding
This work was supported by JSPS KAKENHI Grant Number JP19J21617.
Availability of data and materials
The datasets supporting the conclusions of this article are included within thearticle and its additional files.

Declarations
Ethics approval and consent to participate
All animal experimental protocols were approved by the Institutional Animal
Care and Use Committee of the Kagoshima University, and the experiments
were conducted according to the Ethical Guidelines for Animal Experiments of
the Kagoshima University.
Competing interests
The authors declare that they have no competing interests.
Author details
1
Laboratory of Insect Genome Science, Kyushu University Graduate Schoolof Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi‑ku,
Fukuoka 819‑0395, Japan. 2 Laboratory of Creative Science for Insect Industries,
Kyushu University Graduate School of Bioresource and Bioenvironmental
Sciences, Motooka 744, Nishi‑ku, Fukuoka 819‑0395, Japan. 3 Departmentof Biochemistry and Biotechnology, Faculty of Agriculture, Kagoshima University, 1‑21‑24 Korimoto, Kagoshima 890‑0065, Japan. 4 Laboratory of Sanitary
Entomology, Kyushu University Graduate School of Bioresource and Bioenvironmental Sciences, Motooka 744, Nishi‑ku, Fukuoka 819‑0395, Japan.
Received: 23 April 2021 Accepted: 21 June 2021

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