Received:
28 December 2016
Revised:3 March 2017
Accepted:
28 March 2017
Heliyon 3 (2017) e00286

Development of a Japanese
encephalitis virus-like particle
vaccine in silkworms usingcodon-optimised prM andenvelope genes
Sayaka Matsuda a, Reiko Nerome a , Kenichi Maegawa a , Akira Kotaki a,
Shigeo Sugita b , Kazunori Kawasaki c, Kazumichi Kuroda d , Ryoji Yamaguchi e ,Tomohiko Takasaki f , Kuniaki Nerome a, *a

The Institute of Biological Resources, 893-2, Nakayama, Nago-shi, Okinawa 905-0004, Japanb

Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke-shi, Tochigi 329-0412, Japanc

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

Division of Microbiology, Nihon University School of Medicine, 30-1, Oyaguchi-kamicho, Itabashi-ku, Tokyo

173-8610, Japane

Laboratory of Veterinary Pathology, Department of Veterinary, Faculty of Agriculture, University of Miyazaki,

1-1 Gakuenkibanadai-Nishi, Miyazaki 889-2192, Japanf

Department of Virology 1, National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku-ku, Tokyo 162-8640,

Japan* Corresponding author.
E-mail address: rnerome_ibr@train.ocn.ne.jp (K. Nerome).

Abstract
We have successfully prepared a Japanese encephalitis virus (JEV) − Nakayama
virus like particle (NVLP) vaccine using synthetic codon-optimized prM and E
genes. The expression of the recombinant JEV Nakayama-BmNPV (JEV-NNPV)
virus was determined in infected silkworm Bm-N cells by fluorescence andWestern blot analysis. The recombinant was inoculated into silkworm pupae andthe yield of Nakayama VLP (NVLP) reached a peak in the homogenates after 3
days. Additionally, in the peptide analysis of infected pupae homogenate, itappeared approximately 300–500 μg E protein/pupa were produced. When purified
the above eluates on the discontinuous sucrose density gradient centrifugation,

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NVLP showed a strong hemagglutination (HA) activity by using chicken red blood
cell in phosphate-buffered saline (PBS) free from Mg++ and Ca++ ions. The
immune antisera against NVLP strain could efficiently neutralize the plaqueformation of Nakayama, Beijing-1 and Muar strains, showing tendency of muchhigher reaction with heterologous Muar strain than homologous Nakayama strain.
Our findings suggest that the JEV-NVLP may be useful for JEV epidemic control
in many endemic areas of Asian countries as a widely effective and less expensive
JE vaccine.
Keywords: Immunology, Vaccines, Infectious disease

1. Introduction
The first confirmed epidemic of Japanese encephalitis disease was reported inJapan and Korea in 1924, damaging that 6000 or more patients infected and 60% ofthem died at the outbreak [1]. In 1935 and 1949, the causative viruses were isolated
from patients in Tokyo and Beijing, and they were serologically, virologicallycharacterised and designated the Nakayama and Beijing-1 strains, respectively.
The ecological background of JEV and its transmission through mosquitoes has
been investigated in detail [2, 3]; however, JEV epidemics caused the death ofapproximately another 5,000 individuals before the mid-1960s [4]. Owing toecological maintenance of the swine industry [2], the use of mouse brain-derived
JE vaccine was enforced by the Japanese government in 1954 [1], the incidence ofJapanese encephalitis has decreased dramatically. However, Japanese encephalitis
is still a major health threat, with numbers of patients showing symptoms in recent
years [5, 6, 7], and JE vaccines have been distributed throughout many parts of theworld to combat this disease [8, 9]. Notably, despite the use of effective vaccines,
JEV has actively and silently circulated in the mosquito-swine ecological system.
Moreover, the incidence of Japanese encephalitis has increased in many parts ofAsian and other Pacific countries [10, 11].
In recent years, the quality of the JE vaccine has been greatly improved byintroducing the different biotechnologies including Vero cell derived vaccine
production, transfected cell lines, etc. [8, 12, 13, 14, 15, 16, 17]. JE vaccines
produced in Vero cells have been used as safe and efficacious vaccines [15].
Similarly, adjuvant Vero cell-derived vaccines have been reported to elicit potent
antibody responses in mice and horses [16]. Additionally, potent antibody
responses and enhanced expression of JEV-E protein have been reported in a
Drosophila cell line [17]. Another type of JEV-VLP vaccine has also been
produced in cloned BHK cells following genetic engineering, and the resulting
cells showed increased JEV-E protein production [18]. Moreover, several JEVVLP vaccines have been produced using the baculovirus system [19, 20, 21].

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However, despite the development of the above-mentioned vaccines, novel
vaccines that are inexpensive, safe, and efficacious are still needed in developing
countries. On the other hand, the molecular mechanisms of viral morphogenesis
and the role of protective JEV E antigen have been well characterized [22]. In the
11-kb, single-stranded, positive-sense RNA JEV genome, a single, long open
reading frame (ORF) synthesises a single polyprotein, which is then cleaved into
11 structural (C, pr M, E) and nonstructural (NS1, NS2A, NS2B, NS3, NS4A,
NS4B, and NS5) proteins.
In this study, we describe the biological, morphological, and immunologicalcharacteristics of a VLP vaccine containing the E protein of JEV Nakayama strain
based on its important role as a protective antigen [22].

2. Materials and methods
2.1. Cells and viruses
Silkworm Bm-N cells were maintained in TC-100 medium and minimal essential
medium containing 10% foetal calf bovine serum. For construction and expressionof target vaccine DNA, the P6E strain of the baculovirus Bombyx mori nuclear
polyhedrosis virus-BmNPV [21] was used to generate the recombinantbaculovirus-JEV Nakayama virus. Baculovirus was multiplied in Bm-N cells.
Vero cells (cat. no. JCRB 9013, lot no. 11292002; Japanese Collection of Research
Bioresources [JCRB] cell bank) were also used for propagation of the Nakayama,
Beijing-1, and Muar JEV strains, which were obtained from the National Instituteof Infectious Diseases (Tokyo, Japan). The strains were propagated in Vero cells in
MEM containing 10% calf foetal serum.

2.2. Construction and generation of recombinant JEV-NPV
The methods for construction and preparation of JEV-Nakayama-BmNPV (JEVNNPV) were described previously [23]. To enhance expression in silkworms, prM/
E genes were codon-optimized for silkworms as described previously. Synthesis of
the designed prM/E DNA was carried out by GENEWIZ (Saitama, Japan). The
resulting DNA was inserted into the pBM-8 plasmid as described previously [24]using the In-Fusion technique (Clontech, Mountain View, CA, USA) to generate
pBM-8-JEV-NNPV. Subsequently, pBM-8-JEV-NNPV and linearized BmNPVNNPV DNA were cotransfected into serum-free cultured Bm-N cells. After 5 h ofincubation, the medium was changed to foetal calf serum-enriched TC-100
medium, and the cells were incubated for an additional 5–7 days. The resulting
medium was obtained for subsequent virological examination. The harvested
medium was centrifuged at 3,000 rpm for 5 min to separate the sedimented celldebris. The resultant supernatant was used as recombinant virus (JEV-NNPV).

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2.3. Fluorescence imaging and western blot analysis
Bm-N cells were infected with recombinant JEV-NNPV baculovirus harbouring
the codon-optimised JEV-E gene and maintained in MEM containing 10% calf
serum at 25 °C in a 5% CO2 incubator for 1–3 days. After confirmation of theinfection based on cytopathic effects, Bm-N cells on the cover slip glass were
washed in phosphate-buffered saline (PBS), fixed, and permeabilised. The cells
were then treated with mouse or rabbit antiserum against JEV, followed bytreatment with fluorescein isothiocyanate (FITC)-conjugated anti-mouse or rabbit
IgG antibodies (West Grove, PA, USA). For western blot analysis, goat polyclonalanti-JEV rabbit serum was obtained from NIID (Tokyo, Japan), and FITCconjugated rabbit anti-goat IgG-AP antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) were used. The final signal was detected using ECL plus reagents(GE Healthcare, Port Washington, NY, USA).

2.4. 50% Plaque Reduction Neutralization Test (PRNT50)
Neutralizing antibodies to JEV were measured by PRNT50 in Vero cells. Each JEVstrain was mixed by equal volume with four-fold serial dilutions (1:40 to 1:10,240)
of sera from rabbit or mice immunized with VLP antigens, and then incubated at
35 °C for 90 min. Vero cell monolayers were inoculated with these mixtures in 6well plates and incubated at 35 °C for 90 min. Subsequently, overlay medium
containing 1% methylcellulose was added and the cells were incubated at 35 °C for
4–6 days. The cells were fixed using a 3.7% formalin/PBS solution and stainedusing methylene blue. The PRNT50 titre, expressed as the logarithm (log 10) of thereciprocal serum dilution, was calculated based on plaque numbers compared withthose in the control wells, containing only a virus-diluent mixture (non-serum
control).

2.5. Extraction of JEV-NVLP from silkworm pupa homogenates
The recombinant baculovirus (JEV-NNPV) consisting of codon-optimized DNA of
JEV Nakayama strain was inoculated into silkworm pupa. After three days, 0.01%
formalin and phenylthiourea were added, and the inactivated pupae were thenhomogenised with three cycles of sonication. The homogenates were centrifuged at
16,000 rpm for 30 min. Estimation of E protein was carried out by slab gelelectrophoresis (e-PAGEL; Atto). The resulting supernatant was further centrifuged for 4 h [24] and subjected to the electrophoresis.

2.6. Purification of VLP antigens
The silkworm pupae homogenates were first centrifuged at 16,000 rpm for 30 min
using SW28 rotor with a Beckman centrifuge, and partially purified bycentrifugation at 32,000 rpm for 16 h on a 10–50% (w/w) discontinuous sucrose

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gradient in SW 50.1 rotor. The resultant sucrose gradient specimen which produced
20 fractions was subjected to the sucrose concentration, protein concentration and
HA activity analyses.

2.7. Hemagglutination test
In this study, we found a phenomenon that silkworm derived NVLP could
apparently agglutinate chicken RBC in PBS free from Mg++ and Ca++ ions (PBS(−)) and we carried out the HA test by using a chicken RBC which prepared bycommon PBS (−) (pH 7.4). NVLP samples were diluted 2-fold serially with 50 μL
PBS (−) in 96 microtitre plates, and 50 μL of 0.5% (v/v) chicken RBC in PBS (−)
was added and mixed well. After 30 min at room’s temperature, the HA plates
were read as previously described [24].

2.8. Immunization with the JEV-NVLP vaccine
Mouse and rabbit experiments were approved and carried out in accordance with
the Fundamental Rules for Animal Experiments and the Guidelines for Animal
Experiments Performed at The Institute of Biological Resources published by the
Animal Welfare and Animal Care Committee including Animal Ethics Committeeof the Institute of Biological Resources, Okinawa, Japan. The partially purified
JEV-NVLP was used for immunisation in rabbits and mice. JEV-E protein contents
were adjusted to 20 μg per dose into a mouse (inbred ddY, female, 4 weeks old;
Japan SLC) and 40 μg per dose in a rabbit (Std: NZW, female, 10 weeks old, Japan
SLC), as adjuvant or incomplete adjuvant vaccines. In the first immunization (firstdose), NVLPs were mixed with complete adjuvant. In the second immunisation(second dose), incomplete adjuvant vaccine was used after a 2-week interval. A
third immunisation (third dose) without adjuvants was given after 4 weeks, andthen, blood samples were collected after 2 weeks.

3. Results
3.1. Construction and expression of the target DNA coding
structural gene
The target DNA for vaccine production was designed to include the complete pr M,
and target E genes. When we designed the chimeric DNA, assumed the pr M
protein may play an important role in morphogenesis and that introduction ofsilkworm-derived codons may enhance the expression of the E protein. Based onthese assumptions, 2001 silkworm codon-optimised E genes were designed. Of
these, 1557 (77.8%) sequences were derived from the authentic E gene of human
JEV-Nakayama strain and the remaining 444 (22.2%) sequences were designed toinclude original silkworm codons which were frequently used in silkworm (Fig. 1).

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Fig. 1. Design of target chimeric DNA consisting of the authentic JEV prM and E gene and silkwormderived codons.

As shown in Fig. 2A, recombinant JEV-NNPV expressed E protein, demonstrating
E protein-specific fluorescence of JEV monoclonal antibody (a) and JEV rabbit
antiserum (c). Negative control did not show any fluorescence (b, d). Apparently,
the expression of JEV Nakayama E protein in the cell lysates was determined bywestern blot analysis and it appeared to increase with time (Fig. 2B(a)). The reason
that E protein was undetected in the culture fluids was more likely due to lack of asignal peptide in the front of prM protein (Fig. 2B(b)).

3.2. Production and purification of VLPs in silkworm pupae
The recombinant JEV-NNPV was injected into silkworm pupae, and the expressionof E protein antigen was examined based on ELISA, western blot and slab gelelectrophoresis analyses. As shown Fig. 3A(a), ELISA titres peaked on day 3 after
infection. Also, marked expression of the E protein was observed on day 3 bywestern blot analysis of purified VLPs (Fig. 3A(b)). The content and position of E
protein included in a large number (30) of pupae homogenates were confirmed byslab gel electrophoresis shown in Fig. 3B(a) and the migration profiles of thepeptides from infected pupae homogenates (Fig. 3B(b)). The result was also
reflected in the proportion (%) data of Exp. 2 (Fig. 3C) and its molecular weight
was estimated 53 kDa similar to that of JEV control (Fig. 3A(b) and 3B(a)). To
enhance E protein yields, the JEV-NVLP recombinant was further inoculated into
30 silkworm pupae. After three cycles of pupae homogenates-sonication, 9,480 μgof E protein was recovered from the homogenates of 30 pupae (Fig. 3C). Fromtentative calculations, each silkworm pupa appeared to produce more than 300 μg

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Fig. 2. Confirmation of the expression of JEV-NVLP E protein by fluorescent antibody analysis. A, (a),anti-JEV monoclonal antibody treated and recombinant-infected (JEV-NNPV) BmN cells. (b),noninfected BmN cells (negative control). (c), recombinant infected and anti-JEV rabbit antiserum
treated cells. (d), normal rabbit serum treated cells (negative control). B, Determination of theexpression of JEV-Nakayama E protein by western blot analysis. (a), pupae cell lysate and (b),supernatant specimens. JEV-C and MM represent JEV-control and molecular weight marker,
respectively.

E protein. Furthermore, in the second experiment, 15,840 μg E protein wasrecovered from 30 pupae, resulting in production of 528 μg E protein/pupa.
To establish the first level purification method, the pupae homogenate obtained
from the centrifugation at 16,000 rpm for 30 min was partially purified using small
scale sucrose gradient. For example, the harvested homogenates, the resulting
eluates were further centrifuged on a 10–50% (w/w) linear sucrose gradient in
SW50.1 roter, producing 20 fractions. The fractions were characterised by the
sucrose concentration, protein concentration and HA activity tests as shown in
Fig. 4A. The centrifuged pupae-homogenates showed two protein peaks in
fractions 5 (first) and 12 (second) (Fig. 4A). The first peak located in the 40%
sucrose fraction supported the possible presence of the heavy VLP based on HA
activity (Fig. 4A) and the migration profile in the electrophoresis (Fig. 4B). The
second peak may have been derived from the host components because definite E
protein was not detected in the second peak (Fig. 4B).

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Fig. 3. A, Extraction of JEV-NVLP and the corresponding E protein from silkworm pupae
homogenates. (a), time course production of NVLPs in the pupae, (b), identification of thecorresponding E protein by western blot analysis. B, JEV E protein analysis of the homogenates. (a),the content and position were confirmed by slab gel electrophoresis of homogenates pupae. (b), themigration profiles of the peptides obtained from infected pupae homogenates. C, Extraction andestimation of JEV-NVLP from pupae homogenates.

3.3. Immunogenicity and immune responses of JEV-NVLP
We investigated the immunogenicity of the JEV-NVLP vaccine produced insilkworm pupae by measuring antibodies produced in rabbits. Plaques produced in
Nakayama strain-infected Vero cells were extremely small, as compared with
Beijing-1 and Muar strains (Fig. 5A).
Immune responses of JEV-NVLP was examined in rabbits immunized with threedoses of NVLP vaccine. Antiserum against JEV-NVLP showed significant
PRNT50 mean titres (log10), 3.9, 3.1 and 4.7 against three challenge viruses,
homologous Nakayama, heterologous Beijing-1 and Muar strains, respectively.
Notably, the antiserum to Nakayama VLPs had a much higher titre against8

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Fig. 4. Partial purification of NVLP specimens on 10–50% (w/w) linear sucrose gradient andfractionated into 20 fractions. A, Fractions were examined by the sucrose concentration, protein
concentration and HA activity tests. B, Protein migration profile analyzed in the electrophoresis.

[(Fig._5)TD$IG]

Fig. 5. Plaque neutralization tests with antibodies produced in rabbits immunized with NVLP antigens.
A, Plaque phenotypes of the JEV Nakayama, Beijing-1 and Muar strains in Vero cells. B, PRNT50 titresof NVLP-rabbit antibody against three challenge viruses. C, Antibodies production elicited byimmunization of one and three doses of JEV-NVLP antigen in mice.

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heterologous Muar strain (4.7) than that of homologous Nakayama strain (3.9)(Fig. 5B).
Next, we further investigated the immune responses of JEV-NVLP in mice
immunised with one and three doses of NVLP vaccine. As shown in Fig. 5C,
higher neutralising titre was observed for the JEV-Muar strain with three dose, likeas observed in rabbits.

4. Discussion
In this study, we prepared large amounts of JEV-NVLP vaccine in silkworm pupae
through successful construction of codon-optimised chimeric DNA and generationof recombinant JEV-NNPV. The NVLP product appeared to contain VLPs ofapproximately 30–200 nm in diameter covered with densely arranged shortprojections (data not shown).
The recombinant baculovirus JEV-NNPV produced large amounts of NVLP
antigen in silkworm pupae; based on this yield, a suitable amount of E protein was
also confirmed. For example, the yield of E protein extracted from pupae
homogenates reached approximately 300–500 μg/pupa, with the protein identified
as a 53-kDa peptide band. Notably, the production of JEV-E protein has recently
been reported using a Drosophila cell line [15], baculovirus-insect cells, andhuman cells [16], with suitable yields. For example, the production of JEV-E
antigen in recombinant insect cells reached about 20 μg/mL on day 6 after infection[17]. Additionally, Bu et al. [15] reported a production level of 15–20 μg/mL JEVglycoprotein as a JEV-VLP vaccine in a human cell line. When compared with theabove production, the yield (approximately 300–500 μg/pupa) of JEV-E protein in
the present silkworm pupae appeared to be higher. Moreover, high antibody titres
against JEV-NVLP were observed in rabbit and mouse.
For example, antibodies against JEV-NVLP produced in rabbits showed more than
3.9 and 3.1 PRNT50 titres (log10) for homologous Nakayama and heterologous
Beijing-1 strains, both GIII viruses. In contrast to this strain, the titre for theheterologous Muar (GV) strain was estimated to be 4.7.
JEV is classified into five genotypes (GI-GV) based on its genomic sequence [25],of them the GIII strain was most widely distributed and most frequently isolated in
JE endemic areas until the 1990s. Therefore, all licenced inactivated and liveattenuated JE vaccines are derived from GIII JE viruses. However, the major
genotype of JEV isolated in Japan changed from GIII to GI in the early 1990s [26]and in recent years, a similar genotype shift has been observed in south Korea,
northern Vietnam, China, Taiwan, Thailand [27, 28, 29], and India since 2009
[30].

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One GV JEV strain was first isolated from a patient with encephalitis in Malaysia
in 1952, and it remained the only one instance of this genotype for over 50 years[27]. Recently, the GV JEV genome has been identified in Cultex mosquitoes in
China in 2009 and in South Korea in 2010 and 2012 [31, 32]. A group in China
succeeded in isolating a new GV strain from C. tritaeniorhynchus [32]. Thesefindings lead to the possibility that other species of Cultex mosquitoes may be
involved in the natural transmission cycle of the GV strain of JEV. Thus, GV JEV
may be emerging in other endemic areas, and that is why it is necessary to be more
focused on investigating the dynamics of circulating JEV strains and developmentof its vaccine used in these areas.
In summary, based on the above virological background, we have attempted toprepare potent JE vaccines for a variety of epidemic viruses including fivegenotypes (GI-GV). Thus, the silkworm system may be useful for large-scale
preparation of effective and inexpensive JE vaccines for future emerging viruses.

Declarations
Author contribution statement
Sayaka Matsuda, Reiko Nerome, Kenichi Maegawa, Akira Kotaki, Shigeo Sugita,
Kazunori Kawasaki, Kazumichi Kuroda, Ryoji Yamaguchi, Tomohiko Takasaki:
Conceived and designed the experiments; Performed the experiments; Analyzed
and interpreted the data; Contributed reagents, materials, analysis tools or data.
Kuniaki Nerome: Wrote the paper.

Funding statement
This work was supported by a grant from the Okinawa Communicable Disease
Research Hub Formation Promotion Project by Okinawa Prefectural Government
Commissioned Projects for fiscal years 2015 and 2016.

Competing interest statement
The authors declare no conflict of interest.

Additional information
No additional information is available for this paper.

Acknowledgments
We are grateful to Ms. Emiko Kobayashi for her assistance with electron
microscopy.

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