Archives of Virology
https://doi.org/10.1007/s00705-018-3941-4

ORIGINAL ARTICLE

Overexpression of a virus‑like particle influenza vaccine in Eri
silkworm pupae, using Autographa californica nuclear polyhedrosis
virus and host‑range expansion
Kenichi Maegawa1 · Toshikatsu Shibata2 · Ryoji Yamaguchi3 · Kotomi Hiroike3 · Uda Zahli Izzati3 ·Kazumichi Kuroda4 · Shigeo Sugita5 · Kazunori Kawasaki6 · Reiko Nerome1 · Kuniaki Nerome1
Received: 4 April 2018 / Accepted: 28 June 2018
© Springer-Verlag GmbH Austria, part of Springer Nature 2018

Abstract
Ecological investigations of silkworms have revealed that Eri silkworms (Samia cynthia ricini) possess useful morphologicaland ecological characteristics for virus-like particle (VLP) production, namely non-seasonal breeding, longer lengths, andheavier weights than Bombyx mori silkworms. Furthermore, when vector DNA from Bombyx mori nuclear polyhedrosis
virus (BmNPV), which is unable to replicate in Sf9 cells from Eri silkworms, was replaced with the Autographa californica
nuclear polyhedrosis virus (AcNPV) vector, three improved AcNPV influenza virus recombinants capable of replicationin Sf9 cells were obtained. Although VLP antigens produced previously in silkworms were not evaluated individually, thepresent recombinant Fukushima (FkH5) and Anhui (AnH7) VLP antigens were detected in tissue fluids and fat bodies of
Eri silkworms. Here, we aimed to determine the function of the AcNPV vector and P143 gene by expressing recombinantsin Sf9 cells and eri silkworm pupae. The FkH5 recombinant produced high yields of haemagglutinin (HA)-positive VLPs,
showing a mean HA titre of 1.2 million. Similarly, high production of H7 HA VLPs was observed in the fat bodies of erisilkworm pupae. Antigenic analysis and electron microscopy examination of Eri-silkworm-produced H5 HA VLPs showedcharacteristic antigenicity and morphology similar to those of the influenza virus. Although FkH5 recombinants possessing
the AcNPV vector did not replicate in Bm-N cells, the introduction of the helicase p143 gene from BmNPV resulted in theirproduction in Bm-N and Sf9 cells.

Introduction
Handling Editor: Ayato Takada.
* Kuniaki Nerome
rnerome_ibr@train.ocn.ne.jp
1

The Institute of Biological Resources, 893‑2, Nakayama,
Nago, Okinawa 905‑0004, Japan

2

Division of Gastroenterology and Hepatology, Nihon
University School of Medicine, 30‑1, Oyaguchi‑kamicho,
Itabashi‑ku, Tokyo 173‑8610, Japan

3

Laboratory of Veterinary Pathology, Department
of Veterinary, Faculty of Agriculture, University of Miyazaki,
1‑1 Gakuenkibanadai‑Nishi, Miyazaki 889‑2192, Japan

4

Division of Microbiology, Nihon University Schoolof Medicine, 30‑1, Oyaguchi‑kamicho, Itabashi‑ku,
Tokyo 173‑8610, Japan

5

Equine Research Institute, Japan Racing Association, 1400‑4,
Shiba, Shimotsuke, Tochigi 329‑0412, Japan

6

National Institute of Advanced Science and Technology(AIST), 1‑8‑31, Midorigaoka, Ikeda, Osaka 563‑8577, Japan

Development of influenza vaccines first began after the
pandemic of 1918–1919 and the isolation of the causative
agent, influenza A virus [1, 2]. The severe outbreak, designated the Spanish influenza epidemic, caused 20–40 million deaths worldwide and drove rapid development of theinfluenza vaccine [3–5]. Then, the first licenses for civilian
vaccines were authorised, and full-scale production of influenza vaccines was performed using embryonated chicken
eggs, resulting in 70–80% effectiveness [6, 7]. Despite these
efforts, and although egg-grown influenza vaccines are traditionally used in many parts of the world, new antigenic
variants have recently been observed following passage ofmammalian-derived vaccine viruses in embryonated chicken
eggs [8]. To overcome this problem, mammalian-cellderived influenza vaccines produced in Madin–Darby canine
kidney (MDCK) cells have been used in the United States ofAmerica (USA) and Europe [9]. Additionally, recent circulation of the potentially pandemic H5 and H7 avian influenza

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K. Maegawa et al.

viruses has also promoted the development of human andavian influenza vaccines [10–14].
Previously, to improve immune responses to inactivatedinfluenza vaccines, we developed artificial ribosomal vaccines using muramyl-dipeptide; this vaccine is morphologically similar to current virus-like particle (VLP) vaccines
and induces higher levels of haemagglutination-inhibition(HI) antibodies and cellular immunity in mice and humans[15–17]. Researchers have investigated the efficacy of current VLP vaccines in different field settings [18–21]. We
have also developed influenza VLP vaccines, using singlecodon-optimized H5 and H7 haemagglutinin (HA) genes inBombyx mori silkworms, and have demonstrated extremely
high yields of these gene products [22, 23]. In a recent reporton the development of a Japanese encephalitis vaccine[24], the use of Bombyx mori nuclear polyhedrosis virus(BmNPV) showed enhanced expression levels in Bm-N cells[25]. For example, a BmNPV H5 Fukushima (Fk) recombinant produced 800,000 HA units per pupa when a singlecodon-optimized HA gene was used, and a BmNPV Anhui(An) H7 recombinant showed an HA titre of approximately
400,000 per pupa. In contrast, Pushko et al. [26] produced
moderate yields of H5, H7, and H9 VLP HA antigens in
Sf9 cells originating from Spodoptera frugiperda. Furthermore, high expression levels of the Escherichia coli lacZ
gene were confirmed in Sf-21 and Bm-N cells coinfectedwith Autographa californica nuclear polyhedrosis virus(AcNPV) and BmNPV [27]. Kumar et al. [28] successfully
produced large amounts of foot-and-mouth disease VLP protein in eri silkworms using an AcNPV vector. Interestingly,
hybrid baculoviruses containing sequences from B. mori and
A. californica NPVs can grow well in both Bm-N and Sf-21
cells, and Maeda et al. [29] and Kim et al. [27] demonstrated
expansion of the host range by constructing recombinants
between Bm-NPV and AcNPV. Based on these studies, the143-kDa protein P143 was found to be necessary for viral
DNA synthesis and therefore important for host range specificity [30, 31].
The aim of this study was to determine the function of
the above-described AcNPV vector and the P143 gene byexpressing the recombinants in Sf9 cells and Eri silkworm
pupae. We describe dramatic overexpression of an avian
influenza HA VLP vaccine (H5 subtype), as well as the morphological antigenic characteristics of the vaccine.

Materials and methods
Comparison of the ecological characteristics
of silkworms
In this study, 10 B. mori silkworms and 10 Eri silkworms
were collected at the fifth-instar phase of 5-day-old larvae to13measure their lengths and body weights. Similarly, 10 female
pupae of each species of silkworm were also collected forbody weight measurements. B. mori silkworms were purchased from Baculotechnologies Co., Ltd. (Kagawa, Japan),and Eri silkworms were provided by Okinawa UKAMI Sericulture Co. Ltd. (Okinawa, Japan).

Cells and viruses
Sf9 cells (originating from S. frugiperda) were purchasedfrom Sigma–Aldrich (Tokyo, Japan), and Bm-N cells (originating from B. mori) were provided by Baculotechnologies
Co. Ltd. Both cell lines were maintained in Grace’s insect
medium (Thermo Fisher Scientific) supplemented with 10%
foetal bovine serum. MDCK cells were maintained in TCMEM containing 10% foetal bovine serum and used to produce influenza virus.

Generation of recombinant viruses
The construction of recombinant expression plasmids using
the BmNPV vector was described previously [22, 23].
DNA sequences encoding FkH5 HA, AnH7 HA, and A/
duck/Korea/A76/2010 H7N7 (KoH7) HA DNA were synthesised by Dragon Genomics (Shiga, Japan) and inserted
into the pFastBacl vector using a Gibson Assembly Cloning
Kit (NEB). The resultant constructs were used to transform
DH10Bac cells (purchased from Thermo Fisher Scientific,
K.K., Japan). Subsequently, the recombinant AcNPV DNA
was purified and used to transfect Sf9 cells. One week after
transfection, supernatants from the infected cells were recovered to obtain the AcNPV recombinants. The helicase genefrom the BmNPV vector was also inserted into the region
between the V-cath and chitinase structural genes of the
AcNPV vector. Recombinant DNA encoding FkH5, KoH7,
or AnH7 HA proteins was extracted from the BmNPV vector
using a High Pure Viral Nucleic Acid Kit (Roche, Switzerland), and HA DNA was amplified by PCR. The resultant
PCR products were inserted downstream of the pFastbac1
polyhedrin promoter, and the resultant plasmids were used
to transform DH10Bac. Subsequently, the recombinant
AcNPV DNA was purified and used to transfect Sf9 cells.
One week later, culture supernatants containing the recombinant AcNPV were collected (Fig. 2a).

Preparation of viral antigens and their antibodies
Low-pathogenic avian influenza viruses were kindly provided by Dr. R. G. Webster, St. Jude Children’s Research
Hospital, Memphis, TN, USA (Import Permit No.: 29 douken
322, issued on 12 June 2017 by the Ministry of Agriculture, Forestry and Fisheries, Japan). These included RG-A/
Barn Swallow/Hong Kong/1161/2010-A/PR/8/34 H5N1 [R]VLP influenza vaccine from Eri silkworm pupae(6+2): (Swallow HK H5:SwHKH5), RG-A/Anhui/2013-A/
PR/8/34 H7N9 [R] (6+2): (Anhui H7:AnH7), and A/HongKong/308/2014 -A/PR/8/34 H9N2 [R] (6+2): (HKH9).
The SwHKH5, AnH7, and HKH9 viruses were produced in
10-day-old embryonated chicken eggs. After a 2-day incubation, the infected allantoic fluids were harvested and concentrated by centrifugation at 110,000×g for 2 h. Antisera wereprepared from mice after three intraperitoneal immunisations
conducted at 1-week intervals. The above-mentioned viruses
were used to characterise the antigenicity of H5 and H7 VLP
antigens.

infected pupae were homogenized in PBS containing 0.5%
sodium thiosulfate as an antioxidant. Homogenization was
performed using a UR-20P Handy Sonic Ultrasonic Disruptor (Tomy Digital Biology, Tokyo, Japan). Sonication was
performed on ice with six pulses, each lasting 2 min. Eachhomogenate was then centrifuged at 3,800×g for 30 min.
The supernatant was further centrifuged at 72,000×g for 2 h
on a 20% (w/w) sucrose gradient in a SW28 swing rotor(Beckman Coulter, Japan). The resulting pellet was resuspended in PBS for subsequent experiments.

Fluorescent antibody (FA) test

Histopathological examination of eri silkworm
pupae infected with AcNPV‑FkH5 and AcNPV‑AnH7
recombinants

The recombinant AcNPV-FkH5, AcNPV-AnH7, and AcNPVKoH7 viruses were inoculated into Bm-N or Sf9 cells on glass
coverslips. Three days later, the cells were washed with phosphate-buffered saline (PBS). The cells were then treated withmouse anti-influenza antibodies against H5 and H7 viruses
and subjected to subsequent procedures, as described previously [22, 23]. The samples were observed under a fluorescent
microscope (Olympus, Tokyo, Japan) at 400× magnification.

Haemagglutination (HA) and haemagglutination
inhibition (HI) tests
Test samples were serially diluted twofold in 50 µL of PBS
in 96-well U-bottom plastic plates, and 50 µL of 0.5% (v/v)
chicken erythrocytes in PBS was placed in all wells andmixed well. After a 30-min incubation at room temperature(23-25 °C), the HA titres were estimated based on the lastcomplete HA [22, 23]. For HI testing, similar twofold dilution series of antiserum were prepared in 25 µL of PBS, andan equal volume of four units of test HA antigen was added
to each well and mixed. After the addition of 50 µL of 0.5%
chicken erythrocytes and thorough mixing, the plates wereincubated at room temperature for 30 min, and their HI profiles were determined as the highest inhibition titres.

Haemadsorption (HAD) tests
The culture medium was removed from infected cells, andthe cells were washed once with PBS. Subsequently, a 0.5%
chicken erythrocyte suspension was were added to the cells.
After a 30-min incubation at room temperature, the test cells
were washed in PBS and observed under a stereoscopic
microscope (Olympus, Tokyo, Japan) at 400× magnification.

Production and purification of the HA VLP antigen
in eri silkworm pupae
Two recombinants, AcNPV-AnH7 and AcNPV-FkH5, were
inoculated into Eri silkworm pupae. Nine days later, thePupa samples were collected, fixed in methanol-Carnoy’s
solution, processed for paraffin embedding, sectioned at
3 µm, and stained with haematoxylin and eosin (HE). Immunohistochemistry was performed with DYKDDDDK-tagged
antibodies (Wako, Osaka, Japan) and mouse antibodies
against reference strains of SwHKH5 and AnH7 influenza
viruses.

Electron microscopy
The purified homogenate of AcNPV-FkH5-infected Eri
silkworm pupae was centrifuged through a 10–50% (w/w)
sucrose density gradient at 141,000×g for 120 min in an
SW28 swing rotor. The gradient was fractionated, and the
HA activity and protein concentration of each fraction were
determined. Six fractions showing high HA activity (greaterthan 8,192) were collected, and sucrose was removed fromthe samples using column filtration (PD-10 Desalting Columns; GE Healthcare Bio-Sciences AB, Sweden). The filtrates were examined under an H-7600 electron microscope(Hitachi, Japan) after staining with 2% phosphotungstic acid.

Results
Comparison of the physical characteristics of Eri
and B. mori silkworms
As shown in Fig. 1a, the face morphology, larva shape (withmany spines and black spots), pupa size, and wing colour(black wings with white stripes) of Eri silkworms are quite
different from those of B. mori silkworms (Fig. 1b). Eri
silkworm larvae were longer and approximately two times
heavier than B. mori silkworms (Fig. 1c). These differences
may have influenced the yield of functional VLP proteinsof the viruses.

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K. Maegawa et al.

Fig. 1  Ecological comparisons of B. mori and Eri silkworms. (a,b) Head appearance (left) and comparison of the sizes and weightsof larvae and pupae (centre and right). Comparison of wing shapes

and the colours of eri silkworm moths and B. mori silkworms (lower
right). (c) Summary of data comparing the physical attributes of B.
mori and Eri silkworms

Comparison of the replication of BmNPV and AcNPV
recombinants

Histopathological examinationof AcNPV‑recombinant‑infected eri silkworm pupae

First, three recombinant viruses were examined in growth
tests in Bm-N and Sf9 cells. The results of FA tests showedthat the recombinant AcNPV-FkH5, AcNPV-KoH7, and
AcNPV-AnH7 viruses failed to express HA genes in Bm-N
cells (Fig. 2b). In contrast, the same H5 and H7 HA genes
encoded within the AcNPV vector showed clear FA reactions in Sf9 cells (Fig. 2b). Similarly, all three recombinants adsorbed to Sf9 cells infected with the recombinant AcNPV-FkH5 and AcNPV-AnH7 viruses. These
results were confirmed by performing HAD tests (Fig. 2b).
Consistent with the FA and HAD results, we found that
the AcNPV-FkH5, AcNPV-KoH7, and AcNPV-AnH7
recombinant viruses produced HA antigen, with titres of1024–2048 (Table 1).

To investigate the distribution of VLP HA antigens in
Eri silkworm pupae, AcNPV-FkH5 and AcNPV-AnH7
recombinants were inoculated into Eri silkworm pupae,
and whole thin sections were examined 9 days later using
an anti-DYKDDDDK-tag monoclonal antibody and mouse
antibodies against SwHKH5 and AnH7 influenza viruses.
Numerous vacuoles were detected in the fat body tissues ofpupae infected with FkH5 and AnH7 recombinant viruses.
As shown in Fig. 3a and b, fat body cells were well stainedwith HE reagents. Further, the vacuoles showed prominent
staining with the anti-DYKDDDDK-tag monoclonal antibody (Fig. 3c and d).

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VLP influenza vaccine from Eri silkworm pupae
Fig. 2  Construction and confirmation of replacement expression plasmids. (a) Separation
model of the H5 and H7 HAtarget genes from the BmNPV
vector (left). Insertion model ofthe AcNPV H5 and H7 target
genes into the ­pFastBacTM1
plasmid (right). (b) Expressionof the FkH5, KoH7, and AnH7
recombinant viruses inserted
in the AcNPV vector. FA tests
in Bm-N and Sf9 cells (leftand centre). HAD test with theabove recombinants in Sf9 cells(right)Table 1  HA activity of AcNPV
antigens produced in Bm-N andSf9 cells infected with H5 andH7 AcNPV recombinant viruses

Recombinant

AcNPV-FkH5
AcNPV-KoH7
AcNPV-AnH7

Cell line
Bm-N

Sf9

<4
<4
<4

2,048
1,024
1,024

Production of an influenza virus HA VLP antigen
in eri silkworms and examination by electron
microscopy
The AcNPV-FkH5 and AcNPV-AnH7 recombinant viruses
were then inoculated into Eri silkworm pupae. AcNPVFkH5 recombinant-inoculated Eri silkworm pupae wereseparated into two groups containing 22 and 15 pupae. Since
extremely high HA titres were measured in each homogenate, a representative HA titration microplate (Fig. 3e) isshown to illustrate this. Importantly, HA VLP antigens
appeared to be produced in both the fat body and body fluids (Fig. 4a and b). This may be the first evidence of the
production of VLP antigens in both tissues in Eri silkworm.
H5 VLP antigens produced in the fat body tissues of Eri silkworm pupae showed extremely high HA titres (16.7 and 8.3
million in the first (1) (n = 22) and second (2) (n = 15) group(Fig. 3e and Fig. 4a). The mean HA titre was 12.5 million(Fig. 4a). Interestingly, the HA VLP yield of FkH5 VLPs per
pupa was 39.2 million. However, the AcNPV-AnH7 recombinant still produced 0.58 million HA VLP antigens, similar
to previous reports (Fig. 4a). For reference, the titres of H5

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K. Maegawa et al.
Fig. 3  (a, b) HE staining of fat
body tissues in Eri silkworm
pupae infected with H5 HA(a) and H7 HA (b). Scale bar(a, b): 50 µm. (c, d) Immunohistochemical staining withan anti-DYKDDDDK-tag
monoclonal antibody showing
widespread cytoplasmic staining in silkworm pupae infectedwith H5 HA (c) and H7 HA(d). Scale bar: 100 µm (c) and50 µm (d). (e) An HA-titration
plate used to estimate the HA
activity of fat body homogenates inoculated with FkH5 HA
recombinants and body fluids ofpupae infected with the recombinants. Numbers on the top andbottom of the plate indicate the
HA titre of the well, and whitelines show the endpoint of HA
titration

VLP and H7 VLP produced in B. mori silkworm infectedwith FkH5 BmNPV were 0.8 and 0.4 million [22, 23].

Morphological and antigenic analysis of influenza
HA VLP antigens produced in Eri silkworms
The structure of the authentic influenza virus (Fig. 4c) andH5 HA VLP antigen obtained from Eri silkworms (Fig. 4d)
were fundamentally similar, based on the clear projections arising from the surface of authentic influenza virus
and VLPs. For example, approximately 50–200 nm of thesurface of several H5 VLPs was covered with 14-nm-long13

spikes (Fig. 4d). Although individual projections were notclearly recognized, projections detected on the surface of
native virions were more densely arranged than those of H5
VLP. In contrast, the individual H5 HA projections wereclearly separated from each other. Although different sizesof VLP were observed in the VLP samples (data not shown),
overall, all of the HA projections on the VLPs were similarin length. The results of the antigenic analysis of the FkH5
HA antigen are shown in Fig. 5a. Although antigenic analysis of silkworm-derived AcNPV H5 VLP and BmNPV H5
VLP was performed with antisera prepared in mice against
the reference strains of H5 and H7 influenza viruses, AcNPVVLP influenza vaccine from Eri silkworm pupae

Fig. 4  Production of two VLP antigens (FkH5 VLP and AnH7 VLP)
in Eri silkworm pupae and electron microscopic examination. (a)Comparison of the HA VLP yield produced in Eri silkworm pupae.
The yields of FkH5 VLPs produced in the fat bodies of Eri silkworm pupae are shown. The AcNPV-FkH5 recombinant was inoculated into 37 pupae. Nine days after infection, pupae were separated(first group, (1), n = 25; second group, (2), n = 15). M, mean HA titreof group 1 and group 2 pupae homogenates. (b) HA titre of group
1 and group 2 body fluids. (c, d) Electron micrographs of H5 influenza virus and FkH5 VLP antigens. (c) Purified SwHKH5 influenza
virus. (d) Purified FkH5 VLP antigens produced in the fat bodies of
Eri silkworm pupae

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K. Maegawa et al.

H5 VLP and BmNPV H5 VLP antigens appeared to react
specifically with the antisera to the H5 reference strain andfailed to react with the anti-H7 antibody. Similarly, haemagglutination of AcNPV H7 antigen was inhibited with H7
antibody, but not with H5 antiserum. Additionally, antiserum
against reference H7 virus did inhibit the haemagglutination of AcNPV H7 VLP antigen, showing a high HI titre of
4,096. It was also interesting that both AcNPV FkH5 and
AcNPV AnH7 VLP fat antigens produced in tissue fluids
reacted specifically with H5 and H7 antibodies prepared inmice.

Host‑range expansion by introducing the helicase
gene from BmNPV into the AcNPV recombinantFig. 5  Antigenic analysis of the influenza VLP antigen produced in
Eri silkworms and construction of the AcNPV expression plasmidwith introduction of the helicase gene. (a) Antigenic analysis of the
VLPs. (b) The presence and insertion locus of the helicase gene in the

AcNPV vector (c). (d) Expansion of the helicase gene area from the
AcNPV plasmid. (e) The helicase gene already present in the AcNPV
vector, resulting in the AcNPV H5 and H7 recombinants having two
helicase genes

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Functionally important sites are distributed throughout the AcNPV genome [36], and we targeted the site
between the V-cath and chitinase gene regions for insertion of foreign genes (Fig. 5b). We inserted the helicase gene from BmNPV into that region (Fig. 5c), andas shown in Fig. 5b–e, the improved AcNPV-FkH5
and AcNPV-AnH7 recombinants contained two different helicase genes in each vector. As a result, the two

VLP influenza vaccine from Eri silkworm pupae

helicase-containing recombinants (AcNPV-FkH5+heli
and AcNPV-ANH7+heli) expressed their structural genes
in both Bm-N and Sf9 cells, as indicated by positive
HAD (Fig. 6a) and FA (Fig. 6b) reactions. Additionally,
AcNPV-FkH5+heli and AcNPV-AnH7+heli recombinants
appeared to produce HA VLP antigens in Bm-N and Sf9
cells (Fig. 6c). When these recombinants were inoculated
into B. mori pupae, they both produced high HA titres
even in B. mori silkworm systems (Fig. 6d). These HA

titres increased linearly over time. The mean HA activity titres of FkH5 and AnH7 VLP were approximately
300,000 and 240,000, respectively (Fig. 6d). BmNPVFkH5 and BmNPV-AnH7 replicated well in B. mori silkworms (Fig. 6d). However, the AnH7 HA titre was lower
than those of FkH5 in B. mori silkworms, which weresimilar to those produced using the BmNPV recombinant
in the B. mori silkworm system (Fig. 6d). Thus, AcNPV
recombinants containing two helicase genes could replicate well in both silkworm cells and silkworm hosts.

Fig. 6  Replication of AcNPV
recombinants containing two
helicase genes and the BmNPV
recombinant in two different
insect cells from B. mori and S.
frugiperda. Replication of two
recombinants with two helicase
genes in Bm-N (a) and Sf9 (b)cells. Replication was examinedby performing HAD (a) andFA (b) tests. (c) Results of a
growth activity assay in Bm-N
and Sf9 cells infected with
FkH5 and AnH7 recombinants,
with (FkH5-H and AnH7-H)
or without (FkH5 and AnH7)
an additional helicase gene.
(d) Replication kinetics of theindicated recombinants and theeffects of the helicase genes.
BmNPV-FkH5 and BmNPVAnH7 were also produced in B.
mori silkworms

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K. Maegawa et al.

Discussion
Here, we describe the dramatic overexpression of an avian
influenza (subtype H5) HA VLP vaccine and its morphological antigenic characteristics.
Baculoviruses have been used to produce various influenza VLP vaccines in response to the appearance of possible
pandemic H5 and H7 avian influenza viruses [10–14]. For
example, a single injection of the H7 avian VLP vaccine
produced in Sf9 cells increased protective immunity against
H7N9 influenza virus in a mouse model [32]. Smith et al.
[33] developed an H7 VLP vaccine using Sf9 cells, and its
immune responses in mice reached protective levels against
the homologous human Anhui 2013 and heterologous Jalisco
2012 chicken isolates. Moreover, a recent report showed thata suitable amount of H7 VLP vaccine was produced in Sf9
cells, with an HA titre of 2,048. Additionally, immunisationin ferrets using 15 µg of the H7N9 VLP vaccine reduced
replication of the challenge H7N9 virus to approximately­104–105 plaque-forming units (PFU)/mL [34], and H5 andH7 VLP vaccines prepared using the AcNPV vector protected chickens against infection with highly pathogenic
H5 and H7 viruses [35]. In previous studies, influenza vaccine development was evaluated using a combination of the
AcNPV vector and insect cells.
In addition to an increase in the HA VLP yield, we wereinterested in a report by Maeda et al. [29], who isolated
a hybrid baculovirus from Sf-21 cells co-infected with
BmNPV and AcNPV that could replicate in both Bm-N andSf9 cells. A similar report also described host-range expansion for the E. coli lacZ gene and showed increased expression by a recombinant baculovirus [27].
In summary, we demonstrated that the use of two helicase genes from BmNPV and AcNPV in the recombinant
AcNPV-FkH5 and AcNPV-AnH7 vectors promoted thereplication in both Bm-N and Sf9 cells. Our experimental results suggest that different combinations of structuralgenes from the influenza virus genome, codon optimisation,
usage of the AcNPV DNA vector, and usage of Eri silkworm
as the host may be helpful in increasing the production of
AcNPV-influenza VLP recombinants.
Acknowledgements This study was funded by the Okinawa Communicable Disease Research Hub Formation Promotion Project of Okinawa
Prefectural Government Commissioned Projects, for fiscal years 2016
and 2017.

Compliance with ethical standards
Conflict of interest The authors declare that they have no conflict ofinterest.
Research involving animal and human participants Mouse experiments
were approved and performed in accordance with the Fundamental

13

Rules for Animal Experiments and the Guidelines for Animal Experiments Performed at The Institute of Biological Resources publishedby the Animal Welfare and Animal Care Committee, including theAnimal Ethics Committee of the Institute of Biological Resources,
Okinawa, Japan.

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