Vaccine 33 (2015) 117–125

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The large-scale production of an artiﬁcial inﬂuenza virus-like particle
vaccine in silkworm pupae
Kuniaki Nerome a,∗ , Shigeo Sugita b , Kazumichi Kuroda c , Toshiharu Hirose a ,Sayaka Matsuda a , Kei Majima d , Kazunori Kawasaki e , Toshikatsu Shibata f ,Okti Nadia Poetri g , Retno D. Soejoedono g , Ni L.P. Ika Mayasari g ,Srihadi Agungpriyono g , Reiko Nerome aa

The Institute of Biological Resources, 893-2, Nakayama, Nago, Okinawa 905-0004, Japan
Equine Research Institute, Japan Racing Association, 321-4, Tokami-cho, Utsunomiya, Tochigi 320-0856, Japanc
Division of Microbiology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabashi-ku, Tokyo 173-8610, Japand
Baculotechnologies Co., Ltd., 2217-16, Hayashi-cho, Takamatsu, Kagawa 761-0301, Japane
National Institute of Advanced Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japanf
Division of Gastroenterology and Hepatology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabshi-ku, Tokyo 173-8610, Japang
Faculty of Veterinary Medicine, Bogor Agricultural University IPB, JL Agatis Kampus IPB Darmaga, Bogor 16680, Indonesiab

a r t i c l e

i n f oArticle history:
Received 15 July 2014
Received in revised form 9 October 2014
Accepted 6 November 2014
Available online 18 November 2014
Keywords:
Vaccine
H5N1 inﬂuenza virus
Baculovirus
Virus-like particle

a b s t r a c t
We successfully established a mass production system for an inﬂuenza virus-like particle (VLP) vaccine
using a synthetic H5 hemagglutinin (HA) gene codon-optimized for the silkworm. A recombinant baculovirus containing the synthetic gene was inoculated into silkworm pupae. Four days after inoculation,
the hemagglutination titer in homogenates from infected pupae reached a mean value of 0.8 million
hemagglutination units (HAU), approximately 2,000 ␮g HA protein per pupa, more than 50-fold higher
than that produced with an embryonated chicken egg. VLPs ranging from 30 nm to 300 nm in diameterand covered with a large number of spikes were detected in the homogenates. The spikes were approximately 14 nm long, similar to an authentic inﬂuenza HA spike. Detailed electron micrographs indicatedthat the VLP spike density was similar to that of authentic inﬂuenza virus particles. The results clearlyshow that the expression of a single HA gene can efﬁciently produce VLPs in silkworm pupae. Whenchickens were immunized with the pupae homogenate, the hemagglutination inhibition titer in theirsera reached values of 2,048–8,192 after approximately 1 month. This is the ﬁrst report demonstratingthat a large amount of VLP vaccine could be produced by single synthetic HA gene in silkworm pupae.
Our system might be useful for future vaccine development against other viral diseases.
© 2014 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, occasional outbreaks of highly pathogenic avian
inﬂuenza (HPAI) viruses caused by the H5 and H7 subtypes haveoccurred in various regions of the world and they have killed atremendous number of chickens [1–3]. The 1997 H5N1 outbreakin Hong Kong was the ﬁrst case in which not only chickens but
also 18 humans were infected, 6 of whom died [3–5]. Since then,outbreaks of the virus have occurred worldwide, and the threat of
H5N1 infection remains.

∗ Corresponding author. Tel.: +81 980 54 3376; fax: +81 980 54 3457.
E-mail address: rnerome ibr@train.ocn.ne.jp (K. Nerome).
http://dx.doi.org/10.1016/j.vaccine.2014.11.009
0264-410X/© 2014 Elsevier Ltd. All rights reserved.

Because the World Health Organization has highlighted the riskof a human pandemic resulting from the HPAI virus [6], avian
inﬂuenza vaccines for chickens and humans have been developedor are currently under development [7,8].
Although studies to create more effective and safer vaccines are
ongoing worldwide [7–11], the development of a vaccine against
HPAI H5 viruses is associated with intrinsic problems such as the
low immunogenicity of the virus [12] and the biohazard risk ofusing an infectious HPAI virus for the vaccine production. To overcome these hurdles, we synthesized an H5HA gene that lacks 4basic amino acids associated with the pathogenicity of HPAI viruses
located between the HA1 and HA2 subunits. The synthesized gene
was codon-optimized for the silkworm. We then produced a recombinant baculovirus containing the synthetic HA gene and used it to
inoculate silkworm pupae for mass production of the HA protein.

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Using this method, we produced large amounts of the HA proteinpresent in inﬂuenza virus-like particles (VLPs).
2. Materials and methods
2.1. Evolutionary analysis
The sequence data used for the analysis are shown in Supplemental Tables 1–5. The synonymous substitution distances were
estimated using the Nei and Gojobori method [13]. Phylogenetic
trees were constructed using the neighbor-joining method [13,14].
2.2. Cells and viruses
Silkworm Bm-N cells and MDCK cells were maintained in TC100 medium and minimal essential medium containing 10% fetal
bovine serum, respectively. The P6E strain of the Bombyx morinuclear polyhedrosis virus (BmNPV)[15] was used to generate the
H5HA-BmNPV recombinant virus. Baculoviruses were propagatedin Bm-N cells. The avian inﬂuenza viruses used for the hemagglutination inhibition (HI) test (shown in Fig. 4D) were propagated in
10-day-old embryonated chicken eggs (37 ◦ C, 2 days).
2.3. Generation of H5HA-BmNPV
Synthesis of the H5HA gene was carried out by Dragon Genomics(Shiga, Japan). The synthesized gene was inserted into the pBM-8
plasmid [15] using the In-Fusion technique (Clontech, Mountain
View, CA, USA) to produce pBM-8-H5HA. Next, pBM-8-H5HA andlinearized BmNPV genomic DNA were co-transfected into Bm-N
cells, which were then cultured in TC-100 medium without serum.
After 5 h, the medium was changed to TC-100 containing 10% fetal
bovine serum and the cells were further cultured for 5–7 days.
The medium was then harvested and centrifuged at 3000 rpm for
4 min to remove cell debris. The resulting supernatant was used asrecombinant virus H5HA-BmNPV.
2.4. Antibodies and Western blot analysis
The anti-FLAG mouse monoclonal antibody M2 and the antiDYKDDDDK-tag mouse monoclonal antibody were purchased from
Sigma (St. Louis, MO, USA) and Wako (Osaka, Japan), respectively. The ﬂuorescein isothiocyanate-conjugated goat anti-mouse
IgG antibody and the peroxidase-conjugated goat anti-mouse IgG
antibody were purchased from Jackson ImmunoResearch Laboratories (West Grove, PA, USA). For the Western blot analysis, theperoxidase-conjugated IgG antibody was visualized using an ECL
detection system (GE Healthcare, Port Washington, NY, USA), following the manufacturer’s instructions.
2.5. Hemadsorption test
Bm-N cells infected with viruses were washed 3 times withphosphate-buffered saline (PBS) and then overlaid with 0.5% (v/v)
chicken erythrocytes in PBS. After 30 min, the cells were washedthoroughly with PBS and observed under a microscope.
2.6. Hemagglutination test and hemagglutination inhibition tests
Hemagglutination (HA) and HI tests were performed as
described previously [16].
2.7. Production of HA protein in pupae
Silkworm pupae were injected with 100 ␮L of H5HA-BmNPVin TC-100 (Fig. 4A). Four days later, the infected pupae were

homogenized in PBS containing 0.01% formalin and phenylthiourea
as an antioxidant; homogenization was performed using a Handy
Sonic (Model Tomy UR-20P). Sonication was performed on ice byusing 6 pulses, each lasting 2 min. The resultant homogenate wasthen centrifuged at 6,000 rpm for 30 min. The supernatant was further centrifuged at 16,000 rpm for 30 min. The supernatant fromthe second centrifugation was used for subsequent experiments.
2.8. Sucrose density gradient centrifugation and electron
microscopy
The homogenate of H5HA-BmNPV-infected pupae was centrifuged through a 10–50% (w/w) sucrose density gradient at
25,000 rpm for 120 min using an SW28 swing rotor (Hitachi). The
gradient was fractionated and the fractions were examined for
HA activity and protein concentration. Six fractions (21–26) that
had high HA activity (greater than 8,192) were collected and centrifuged at 25,000 rpm for 4 hr. The pellet was examined under an
H-7600 electron microscope (Hitachi) after staining with 2% phosphotungstic acid.
3. Results
3.1. Selection of the target strain used for the anti-H5N1
inﬂuenza vaccine
We selected a vaccine strain based on an evolutionary analysisof H5HA genes. Through precise evolutionary analyses of genotype
Z viruses, which appeared in 2002 and became the predominant genotype associated with HPAI outbreak in domestic poultry[17], we found that viruses from subclade 2.3, which included
the A/tufted duck/Fukushima/16/2011 (H5N1: duck-Fukushima)strain, show discontinuous evolution (i.e., evolution that did notoccur at a single rate over time) at the slow rates of 0.002 substitutions/site and 0.008 substitutions/site per year (subclades 2.3 (i)and (ii) in Fig. 1B, respectively). Considering the slow evolutionary rate of wildfowl inﬂuenza viruses (0.005 substitutions/site per
year, as shown in Fig. 1A), we hypothesized that fast continuous
linear evolution would occur in domestic poultry (Fig. 1B red line)
and the slow rate of subclade 2.3 would occur in wildfowl. The
atypical evolution of subclade 2.3 may be explained if subclade 2.3
viruses are maintained in poultry (Fig. 1B Clade 2.3, red line) but
occasionally infect wildfowl, in which they would then evolve ata lower evolutionary rate. If the hypothesis were correct, subclade
2.3 viruses should be the major virus type disseminated; therefore,
we selected the duck-Fukushima strain as the target for vaccine
preparation.
3.2. Design of the synthetic H5HA gene and the generation of
H5HA-BmNPV
The synthetic gene for the production of the H5HA protein insilkworms was designed based on the nucleotide sequence of the
HA gene from the duck-Fukushima strain. Duck-Fukushima is avirulent strain that is highly pathogenic to chickens. The polybasic sequence of RERRRKRG, located at the end of the HA1 subunit,
is associated with the high pathogenicity of H5N1. Because thissequence is present, the HA0 precursor is cleaved to HA1 and HA2
in almost any cell in which the HA protein is produced. By cleavage,the HA protein obtains membrane fusion activity and is destabilized in acidic environments such as in intracellular transport
vesicles [18,19]. Therefore, the RERRRKRG sequence was replacedwith RDTRG to avoid this cleavage in insect cells (Fig. 2). By the
introduction of RDTRG sequences instead of RERRRKRG, virulent
DNA was converted to avirulent DNA.

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Fig. 1. Evolutionary rates of the hemagglutinin (HA) gene in H5N1 viruses. (A) Distances from the putative root for wildfowl viruses isolated between 1996 and 2004,
plotted against isolation year. The evolutionary rate was estimated as 0.0050 substitutions per site per year. The viruses and the distances used for the calculation are listed
in Supplemental Table 1. (B) Evolutionary rates of viruses from different clades. The distances that contribute to the graphs are shown in Supplemental Tables 2–5. The
evolutionary trajectory of viruses belonging to subclade 2.3 is atypical compared with that of viruses in other clades. While the latter have evolved linearly (i.e., a constantrate of evolution over time, as shown red line), subclade 2.3 viruses have evolved discontinuously (green and blue lines). The duck-Fukushima strain, which belongs to
subclade 2.3 (red symbol) was selected as the target for vaccine preparation. (For interpretation of the references to color in this ﬁgure legend, the reader is referred to the
web version of this article.)

A FLAG tag (DYKDDDDK) was also added to the C-terminus
of HA2 to label the modiﬁed HA protein (see Fig. 2). Because
codon usage in silkworms is distinct from that in vertebrates,
the nucleotide sequence of the H5HA gene was codon-optimized
for silkworms. Because of these modiﬁcations to the original nucleotide sequence of the duck-Fukushima HA gene, thesequence homology between the authentic HA gene and the synthesized gene was 77.5% at the nucleotide level and 98.4% at
the amino acid level. The synthesized gene was inserted intoa baculovirus transfer vector, pBM-8, to obtain pBM-8-H5HA.
Then, the recombinant baculovirus H5HA-BmNPV was generated via co-transfection of pBM-8-H5HA into Bm-N cells together

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Fig. 2. Comparison of the nucleotide sequences for the authentic HA gene from the duck-Fukushima strain (light green) and the synthetic HA gene (orange). The codon
usage of the H5HA gene was optimized for the silkworm, B. mori; the amino acid sequence connecting the H1 and H2 subunits (linked to the high pathogenicity of the
duck-Fukushima strain) was changed from RERRRKRG to RDTRG, and a FLAG tag (DYKDDDDK) was added to the C-terminus of the H2 peptide. (For interpretation of thereferences to color in this ﬁgure legend, the reader is referred to the web version of this article.)with the linearized genomic DNA from the wild-type baculovirus
BmNPV.

3.3. Expression of the synthetic H5HA gene in cultured cells
To examine the expression of the synthetic H5HA gene, we
inoculated H5HA-BmNPV and wild-type BmNPV into Bm-N cells
and performed a hemadsorption test on the following day. As
shown in Fig. 3A, chicken red blood cells adhered to the recombinant virus-infected Bm-N cells (indicated by black arrows in
Fig. 3A, H5HA-BmNPV) but not to mock-infected and wild-typevirus-infected cells (BmNPV, Mock in Fig. 3A). Occlusion bodies
were observed in the nuclei of BmNPV-infected cells (indicatedby arrowheads in Fig. 3A, BmNPV), which were indicators of viral
infection. In contrast, mock-infected Bm-N cells showed neither
hemadsorption nor occlusion bodies.
In addition, an immunoﬂuorescence test using serum from mice
immunized with the homogenate of H5HA-BmNPV-infected pupae
showed ring-shaped ﬂuorescence in H5HA-BmNPV-infected Bm-N
cells, but not in mock-infected and wild-type BmNPV-infected cells(Fig. 3B).
Western blot analysis of H5HA-BmNPV-infected Bm-N cells
using an anti-FLAG antibody conﬁrmed the expression of proteinfrom the synthetic H5HA gene; a band of molecular weight 72.4 kD
was clearly visible in lysates from H5HA-BmNPV-infected cells
but not in those from wild-type BmNPV-infected cells (Fig. 3C).
The expression of the H5HA protein in HeLa cells transfectedwith a pCAGGS expression vector [20] containing the synthetic
H5HA gene was also conﬁrmed by Western blot analysis (seeFig. 3C). The molecular weight of the H5HA protein in HeLa
cells (78.3 kD) was greater than that in Bm-N cells. This difference may have been due to the structure of the carbohydrate
chain attached to the HA protein, as the carbohydrate chain produced by insect cells is shorter than that produced by mammaliancells.

3.4. H5HA protein production in H5HA-BmNPV-infected pupae
To examine the potency of silkworm pupae as a protein production system, we injected H5HA-BmNPV into silkworm pupae. HA
protein production was then monitored by testing homogenatesof H5HA-BmNPV-injected pupae up to 4 days after injection. We
found that HA titers increased linearly over the 4-day observation
period, ﬁnally reaching approximately 1 million (Fig. 4A). Given
this result, we thereafter harvested infected pupae at day 4. A
homogenate of 220 pupae in approximately 200 mL (0.9 mL/pupa)
PBS had an HA titer of 1,048,300. The amount of HA protein in
an individual pupa (0.9 mL of homogenate with an HA titer of
1,048,300 is equal to 953,000 HAU) was more than 50 times higher
than that obtained with an embryonated chicken egg, which generally produces approximately 10 mL allantoic ﬂuid with an HA titerof 1,024–2,048, that is, 10,240–20,480 HAU/egg. Our rough estimation of the amount of HA protein produced by one silkworm
pupa was 2,000 ␮g/pupa, which was based on the protein contentsof the fractions with high HA titers obtained from sucrose density
gradient centrifugation (see Fig. 5A).
To conﬁrm the high level of HA protein production in pupae, we
repeated the experiment twice using different numbers of pupae
each time. When 47 and 158 pupae were homogenized in 50 mL and
150 mL PBS, respectively, 1115,000 HAU/pupa were obtained fromthe former and 497,000 HAU/pupa from the latter. The average HA
protein production in the 3 experiments was 855,000 HAU/pupa(Fig. 4B). Even though the HA titers obtained from 3 experiments
varied slightly, the amount of HA protein produced in a pupa werealways much more than that produced in a chicken egg.
3.5. Immunogenicity of HA protein produced in pupae
To conﬁrm the immunogenicity of the HA protein produced insilkworm pupae, we diluted the pupae homogenate in PBS to adjust
the HA titer to 16,000. Then, 0.5 mL of the diluted homogenate
was injected intradermally into 1-month-old female chickens, andK. Nerome et al. / Vaccine 33 (2015) 117–125

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Fig. 3. Expression of the H5HA gene in H5HA-BmNPV-infected Bm-N cells. (A) Bm-N cells infected with H5HA-BmNPV and wild-type BmNPV were overlaid with chicken
erythrocytes and incubated for 30 min at room temperature. Next, the cells were washed thoroughly with PBS and observed using phase-contrast microscopy. Mock-infected
cells were also examined. Adsorption of chicken erythrocytes to H5HA-BmNPV infected cells was detected (black arrows). Occlusion bodies (arrow heads) were clearlyvisible in the nuclei of BmNPV-infected cells. Un-adsorbed erythrocytes (white arrows) were observed in H5HA-BmNPV, wild-type BmNPV, and mock-infected cells. Neither
hemadsorption nor occlusion bodies were observed in mock-infected cell. (B) H5HA-BmNPV and mock-infected Bm-N cells were ﬁxed and permeabilized. The cells were ﬁrst
incubated with serum from mice immunized with the homogenate of H5HA-BmNPV-infected pupae and then incubated with an FITC-conjugated anti-mouse IgG antibody.
The stained cells were then observed by ﬂuorescence microscopy. BmNPV-infected cells were also observed. (C) Bm-N cells were infected with H5HA-BmNPV or BmNPV.
HeLa cells were transfected with pCAGGS containing the synthetic H5HA gene (pCAGGS-H5HA) or empty pCAGGS. The infected and transfected cells were then analyzed
with Western blotting using an anti-FLAG tag antibody and a peroxidase-conjugated anti-mouse IgG antibody that was detected with an ECL detection system.

subsequent injections were performed on days 16 and 33 postimmunization (Fig. 4C). Their sera were subjected to HI tests ondays 17, 31, 45, and 55 post-immunization. On each day, sera from
2 to 4 different chickens were prepared. As a result, an average HI
titer of 128 was observed at 17 days after the ﬁrst immunization,and high (4,096 and 3,327) titers were attained after 31 and 45 days.
Furthermore, an HI titer of 1,448 was still observed even on 55 days
post immunization. All chickens immunized with the VLP vaccine
derived from silkworms did not show any side effects, including
reduction in body weight. In contrast, unimmunized control chickens did not show any HI titers (data not shown).
We determined the HI titer of the chicken serum against H5inﬂuenza viruses including epizootic Indonesian viruses (Fig. 4D).
We found that the serum reacted with all of the viruses examined.
In addition, the serum inhibited plaque formation by the authentic
A/duck/Singapore-Q/F119-3/97 (H5N3) virus (Fig. 4E).
3.6. Analysis of homogenates from virally infected pupae using
sucrose density gradient centrifugation
To characterize the structure with hemagglutination activity, we fractionated the homogenate of H5HA-BmNPV-infectedpupae and that of BmNPV-infected pupae using sucrose density
gradient centrifugation and examined the HA titers and protein

concentrations of the fractions. The analysis of H5HA-BmNPVinfected pupae showed that the protein concentration peaked in
fractions 1, 25, and 40, which corresponded to 47.8%, 26.5%, and 10%
sucrose, respectively, with the highest peak observed in fraction 40(Fig. 5A). The HA titer showed 2 peaks at fractions 1 and 25 with thehighest peak in fraction 25. On the contrary, none of the fractions ofwild-type BmNPV-infected pupae had detectable HA titers and only
1 peak of the highest protein concentration was detected at fraction
36, corresponding to 10.2% sucrose (Fig. 5B). Based on these results,
we assumed that fraction 25 of H5HA-BmNPV-infected pupae contained the H5HA protein. To conﬁrm this assumption, Western blot
analysis of fraction 25 was performed using anti-FLAG antibody.
The analysis clearly showed a band of 72.4 kD, whereas the analysis
of fraction 40 showed no clear band (Fig. 5C). Given these observations, we concluded that recombinant HA protein was concentrated
in fraction 25 and its neighboring fractions (21–26) in the analysisof H5HA-BmNPV-infected pupae.
It is noteworthy that the density of fraction 25 was slightly
less than that of the fraction in which intact inﬂuenza virions
were detected. This was shown in the analysis of intact virions on
the same sucrose density gradient where the peak HA titer was
detected in the fraction corresponding to 38.5% sucrose (indicatedby an arrow in Fig. 5A). The difference of density between VLP
and intact virus may be attributable to the difference of protein

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Fig. 4. Production of H5HA protein in silkworm pupae. (A) H5HA-BmNPV was injected into silkworm pupae. After the number of days indicated, the pupae were homogenized
in PBS via sonication. The homogenate was centrifuged at 5,000 rpm for 30 min and then at 15,000 rpm for 30 min using an SW28 rotor. The HA titers of the supernatant werethen determined. (B) A comparison of HA protein production levels from 3 experiments. For these experiments, H5HA-BmNPV-infected pupae were homogenized at day 4
post-injection. (C) The homogenate prepared from H5HA-BmNPV-infected pupae was diluted in PBS to an HA titer of 16,000. The diluted homogenate (0.5 mL) was injectedsubcutaneously into eight 1-month-old female chickens. The same amount was subsequently injected on days 16 and 33 after the ﬁrst immunization. Blood was harvested ondays 0, 17, 31, 45, and 55. Sera were prepared from the blood samples and used to determine the HI antibody titer against pupae homogenate. (D) The HI titer of chicken serum
against epizootic H5 inﬂuenza viruses. The HI titer of the pooled serum of chickens immunized with H5HA-BmNPV-infected pupae homogenate on day 45 post-immunization
was determined against various H5 inﬂuenza viruses. The HI titer against H5HA-BmNPV-infected pupae homogenate was also assayed. (E) A/duck/Singapore-Q/F119-3/97(100 plaque forming units [PFUs]) was mixed with 2-fold serially diluted serum from chickens immunized with the homogenate of H5HA-BmNPV-infected pupae and thenincubated for 30 min at room temperature. Then, the PFUs of each virus treated with serially diluted serum were determined. % inhibition = 100 × (100 − PFUs of treatedvirus)/100.

quantity between them. For example, the former contained only
HA, but the latter was comprised of all viral proteins.
To characterize the structure with hemagglutination activity
in fractions with high HA titers, we combined fractions 21–26(see Fig. 5A) and analyzed them with negative-staining electron
microscopy. The analysis revealed VLPs, as shown in Fig. 6A, C, and
D. The particles had diameters of 30–300 nm and were covered witha large number of spikes. These spikes were approximately 14-nm
long, similar to the previously reported lengths of inﬂuenza HA
spikes [21]. The analysis of the fractions prepared from wild-type
BmNPV-infected pupae revealed the presence of relatively small
vesicles, but spikes similar to those on the surface of the VLPs were
not observed (Fig. 6B).
The shape of the spikes was more clearly observed on the surfaces of VLP subpopulations with relatively low spike densities (seeFig. 6C). Triangular structures were discerned on the surfaces of themembranes (indicated by an arrow in Fig. 6D), consistent with thetriple globular heads of HA trimers. In addition, rod-like shapes
projecting from the VLP surfaces (two-headed arrows in Fig. 6A
and arrowheads in Fig. 6D) were consistent with the side view of

HA molecules. The subpopulations of VLPs (see Fig. 6C) that wereapproximately 100 nm in width resembled typical inﬂuenza viralparticles of the H5N1 virus (A/HK/156/97) (see Fig. 6E). These structural characteristics observed in VLPs might be partly related to thecondition of sonication process.
Although several previous reports have described the production of VLPs containing HA proteins using insect culture cells inwhich M1 and other genes were simultaneously introduced in addition to the HA gene [22–27], this is the ﬁrst report demonstratingthat the expression of the HA gene alone in silkworm pupae is
sufﬁcient to produce VLPs.
4. Discussion
For improvement of the immunogenicity of inﬂuenza subunit
vaccines, the formation of VLPs by using a recombinant baculovirus
expression system has been successfully performed in cultured
insect cells [22–27]. In these previous studies, HA genes and otherinﬂuenza genes (such as the M1 and NA genes) were co-expressed.
By contrast, the system used in the present study, in which

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Fig. 5. Analysis of the homogenate of H5HA-BmNPV-infected pupae by using sucrose density gradient centrifugation. At day 4 post-infection, H5HA-BmNPV-infected (A) andwild-type baculovirus BmNPV-infected (B) pupae were homogenized in PBS via sonication. The homogenate was centrifuged using an SW28 rotor at 5,000 rpm for 30 min
and then at 15,000 rpm for 30 min. The resulting supernatant was then applied to a 10–50% (w/w) sucrose density gradient and centrifuged at 25,000 rpm for 120 min. The
gradient was fractionated from bottom to top, and the HA titer and protein concentration of each fraction were analyzed. The sucrose concentration of each fraction was also
determined using a saccharometer. The fraction corresponding to the sucrose concentration where an HA titer peak was observed by the analysis of authentic inﬂuenza virus
is indicated by an arrow. (C) Fractions 25 and 40 of H5HA-BmNPV-infected samples were analyzed with Western blotting using an anti-FLAG antibody. The homogenate of
infected pupae was also analyzed.

silkworm pupae were used instead of cultured cells, produced
a great deal of VLPs via the expression of a single synthetic HA
gene. We assume that the high level of H5HA protein production
observed in pupae is responsible for the efﬁcient VLP formation
using our system. Our preliminary study suggested an effect ofcodon optimization on the production yield of HA protein (at least
the H5 and H7 subtypes) in the silkworm.
With respect to the purity of the HA protein in VLPs, 0.01 ng
protein of the puriﬁed VLPs corresponded to 1 HAU based on the
HA titer and protein content in fractions 21–26 in a sucrose density
gradient analysis (Fig. 5 A). This estimated value indicated the highpurity of VLPs prepared from silkworms.
Jin et al. produced a recombinant baculovirus displaying the
H5HA protein (gp64HA fusion protein) on its surface using silkworm pupae, and 3% of the baculovirus protein was the fusion

protein [28]. Dong et al. obtained approximately 500 ␮g of puriﬁed
secretory H5HA protein (anchorless HA) from 30 silkworm larvae[29]. Similarly, Gomez-Casado et al. obtained 113 ␮g of H1HA protein from 1 g of fresh Trichoplusia ni larvae [30]. However, to thebest of our knowledge, our present report is the ﬁrst to describe
the production of VLPs in insects via the expression of a single HA
gene.
Recently, Giles et al. successfully developed an H5N1 VLP vaccine prepared from the supernatants of 293 T cells expressing NA,
M1, and COBRA (computationally optimized broadly reactive antigen) HA genes [31]. This vaccine could produce effective and broad
immune responses in nonhuman primates against H5N1 inﬂuenza
viruses belonging to different evolutionary clades. If the COBRA
HA could be produced in our system, the VLP HA vaccine may
be of additional interest due to higher immune responses and the124

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insects [32]. Recently, recombinant baculoviruses express enzymes
that inhibit fucose addition to N-glycans have been successfully
produced and are used to produce recombinant glycoproteins[33,34]. This approach has the potential to be integrated into oursystem.
In any case, our present study suggests the possibility of largescale HA-VLP vaccine production in the near future.
Acknowledgments
The authors thank Ms. Emiko Kobayashi for her assistancewith microscope observations. The pCAGGS expression vector was
kindly provided by Dr. Jun-ichi Miyazaki. This work was primarily supported by a grant from the Okinawa New Industry Creation
Project for ﬁscal years 2011 and 2012 by the Okinawa Industry Promotion Public Corporation trusted from the Okinawa prefectural
government.
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.vaccine.
2014.11.009.
References

Fig. 6. Representative transmission electron microscopy images of virus-like particles produced by H5HA-BmNP-infected silkworm pupae. Combined fractions 21–26
from Fig. 5A were centrifuged at 25,000 rpm for 4 h. The resulting pellet was thenresuspended in PBS and negatively stained with 2% phosphotungstic acid (pH 7) andobserved using a TEM (Hitachi H-7600) operated at 80 kV. (A) Virus-like particles(VLPs) were pleomorphic and between 30 nm and 300 nm wide. Spike structures(two-headed arrows) approximately 14 nm long projected radially on the contoursof the particles. The spike alignment was so dense that it appeared as an almost continuous layer. On the faces of the particles, a high density of small grains (arrows)
were observed and attributed to the crowded heads of spikes 2-dimensionallyarranged perpendicular to the plane of the micrograph. (B) Wild-type BmNPVinfected pupae were also analyzed using the same methods. In the preparation of
BmNPV-infected pupae, vesicle-like structures were observed, but no spike wasobserved on their surfaces. (C, D) Examination of single spikes on subpopulations of
VLP membranes, with occasional lower densities of spikes ascertained as rod-likeshapes (arrowheads in C) and triangular heads (denoted by an arrow in D), consistent with the morphological features of HA spikes. (E) Viral particle images ofauthentic inﬂuenza strain A/HK/156/1997 (H5N1). Bar = 50 nm.

potential for mass production at a low cost. A series of studies to
explore more appropriate antigens may contribute to future vaccine development.
In a previous study, we developed an inﬂuenza virosome vaccine with HANA using synthetic muramyl dipeptide [16]. Although
the immunogenicity of this combination was higher than that ofan ordinary embryonated egg-derived vaccine, inoculation withthe vaccine resulted in local manifestations such as pain and erythema at the inoculation site. Because the system described in thisstudy contains no artiﬁcial components, such side effects might be
reduced. In fact, immunization with the VLP vaccine prepared in
the present study did not induce clinical side effects and reduced
the body weight of chickens and mice.
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