Vaccine 35 (2017) 738–746

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Quantitative analysis of the yield of avian H7 influenza virus
haemagglutinin protein produced in silkworm pupae with the use of thecodon-optimized DNA: A possible oral vaccine
Kuniaki Nerome a,⇑, Sayaka Matsuda a, Kenichi Maegawa a, Shigeo Sugita b, Kazumichi Kuroda c,Kazunori Kawasaki d, 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
National Institute of Advanced Science and Technology (AIST), 1-8-31, Midorigaoka, Ikeda, Osaka 563-8577, Japanba r t i c l e

i n f oArticle history:
Received 5 July 2016
Received in revised form 25 November 2016
Accepted 22 December 2016
Available online 5 January 2017
Keywords:
Avian influenza virus
Vaccine development
Recombinant antigen
Virus-like particles
Oral vaccinea b s t r a c t
In this study, we aimed to quantitatively compare the increased production of three H7 influenza viruslike particle (VLP) haemagglutinin (HA) with the use of a codon-optimized single HA gene in silkworm
pupae. Recombinant baculovirus (Korea H7-BmNPV) could produce 0.40 million HA units per pupa, corresponding to 1832 lg protein. The yield of the HA produced in larva was estimated to be approximately
0.31 million HA units per larva, and there were no significant differences between the three HA proteins.
We could establish efficient recovery system of HA production in larvae and pupae with the use of threecycles sonication methods. Next, we compared yields of HA proteins from three different H7 and two H5
recombinant baculoviruses based on the amount of mRNA synthesized in BmN cells, suggesting thatmRNA synthesis may be also a useful indicator for the production of HA. Based on HA titres from fourrecombinants, the yield of HA had a great influence on the codon-optimized effect and the characteristicsof the viral HA gene. The recombinant containing codon optimized HA DNA of A/tufted duck/
Fukushima/16/2011 (H5N1) did produce more than one million HA units, although another recombinant
including of the wild H5N1 strain failed to show HA activity. Electron microscopy revealed the presenceof large VLP and small HA particle in the heavy and light fractions. The purified VLPs reacted with theauthentic anti-H7 antibodies and the antibodies prepared after immunization with the VLP H7 antigen.
Also H5 and H7 VLPs could produce HI antibody in chickens and mice with oral immunization. The antibodies elicited with oral immunization were confirmed in fluorescent antibody analysis and western
blotting in Korea H5-BmNPV and H7 HA-BmNPV recombinant infected BmN cells. Taken together, thesefindings provided important insights into future oral vaccine development.
Ó 2017 Elsevier Ltd. All rights reserved.

1. Introduction
Since the first isolation of avian H5N1 influenza virus from a 5year-old boy in Hong Kong in 1997 [1–3], subsequent circulation of
the causative highly pathogenic H5 avian influenza (HPAI) virus inhumans has posed a serious threat to public health [4]. Accordingly, scientists have recently developed an H5 vaccine [5,6], andmany important advancements are being made in the development of vaccines targeting H5N1 virus. For example, in our laboratory, we successfully established large-scale production of the H5⇑ Corresponding author.
E-mail address: rnerome_ibr@train.ocn.ne.jp (K. Nerome).
http://dx.doi.org/10.1016/j.vaccine.2016.12.058
0264-410X/Ó 2017 Elsevier Ltd. All rights reserved.

VLP haemagglutinin vaccine in the silkworm using optimized synthetic chimeric HA DNA [7].
The H7N9 virus recently appeared in March 2013 in southern
China as a second possible pandemic strain in humans [8–12]. During this outbreak, 440 human cases were reported, leading to 122
fatalities. Importantly, epidemiologists and epizootiologists are
particularly interested in studying infection with avirulent viruses,
unlike in cases of H5N1 HIPV infection in humans and chickens[4,13]. Accordingly, we have been studying avirulent avian
H7N10 viruses isolated from wild ducks in Korea in 2010 [14], withthe goal of developing an H7 influenza virus haemagglutinin (HA)
vaccine in silkworms using HA DNA as a model. Avirulent viral
DNA may be particularly useful because of its evolutionarily posi-

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tion and ease of handling [14]. Indeed, an avian H7 VLP vaccine wasalready developed using the Anhui/1/2013(H7N9) strain, and theimmune responses induced by this vaccine were clearly confirmed
in ferrets, supporting the potential efficacy of the vaccine [15].
Similarly, the above H7 vaccine prepared in insect cells has been
evaluated in humans and has been shown to induce high seroconversion [16].
Recent epidemiological studies have revealed that human H7N9
isolates are distributed throughout different parts of China [17].
Therefore, in this study, we prepared recombinant baculovirus
containing the HA gene of A/Anhui/1/2013 and A/Shanghai/1/2013
viruses and performed a comparative analysis of the yield of HA
protein in order to improve vaccine development.
2. Materials and methods
2.1. Design and construction of three chimeric HA DNAs and thegeneration of three H7HA- BmNPVs
Codon-optimized chimeric DNA was designed on the basis of
the HA gene of A/duck/Korea/A76/2010 (H7N7) virus by introduction of frequently used silkworm codons (Fig. 1). A total of 384 silkworm codons (22.9%) were designed to include 1295 (77.1%) viralcodons. As a result, 1680 codon-optimized DNAs were then clonedin Escherichia coli for preparation of recombinant baculoviruses.
Furthermore, Flag-tagged codons corresponding to eight aminoacids (DYKDDDDK) were added to the end of the HA2 molecule.
According to these procedures, chimeric DNAs containing HA genesof A/Shanghai/1/2013 (H7N9) and A/Anhui/1/2013 (Anhui H7)739

viruses were also constructed. The synthetic H7HA gene was prepared by Dragon Genomics (Shiga, Japan), and the resulting gene
was inserted into the pBM-8 plasmid [18] using the In-Fusion technique (Clontech, Mountain View, CA, USA) to produce pBM-8H7HA, as described previously [7].
2.2. Quantification of mRNA
RNA was extracted using an RNeasy Mini Kit (Qiagen, Valencia,
CA, USA) according to the manufacturer’s instructions. After RNA
extraction, RNA was treated with DNase 1 (Qiagen). RNA(1000 ng) was then reverse transcribed using SuperScript III
reverse transcriptase (Life Technologies Japan Ltd. Tokyo, Japan)
and random-hexamer primers (Life Technologies Japan Ltd.)according to the manufacturer’s instructions. The cDNA concentrations were measured using SYBR Premix Ex-Taq II (Takara, Japan)
according to the manufacturer’s instructions. Polymerase chain
reaction (PCR) was performed using the following primer sets:
influenza virus HA for Fukushima, TCAAACGAACAAGGTTCAGGT(forward) and TTGTTTGTCACACCGTCGAT (reverse); for Korea,
CACAATCGACCTGGCTGACT (forward) and CGAAGCAACCTGTACCGTCT (reverse); for Anhui and Shanghai, TCACCTGGTGCTAGACCTCA (forward) and GAGCGATGAAAGCACCGTTG (reverse);
and baculovirus GP64, CTGTAACGGCGGCTTGTT (forward) and
GTCGCTTTCCAGCTGTACG (reverse). The amplification conditions
were as follows: one cycle at 95 °C for 30 s and then 40 cycles at
95 °C for 10 s and 60 °C for 30 s. Copy numbers were estimatedfrom the standard curve obtained using serial 10-fold dilutions of
the PCR product as the template.

Fig. 1. Functional design of the target chimera DNA for the production of Korea H7 haemagglutinin protein by a combination of the authentic Korea H7 haemagglutinin gene
(Wild n.t.) and silkworm-derived codons (Opt. n.t.). Because the authentic gene did not contain basic virulent sequences, the last portion of the HA1 gene was not modified
with the introduction of the further codons. However, the Flag tag (GACTACAAAGACGACGACGACAAATAA) was added to the C-terminus of the H2 peptide. For interpretationof the references to codons in this legend, the reader is referred to the web version of this article. Additionally, codons that are frequently used in the silkworm are shown to
the left of the nucleotide and amino acid sequences.

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2.3. Virus antigen, antibodies, and western blot analysis

2.8. Sucrose density gradient centrifugation

The mouse monoclonal anti-FLAG 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 Jackson ImmunoResearch Laboratories (WestGrove, PA, USA). Western blot analysis was carried out as described
previously [7]. Inactivated A/Barn Swallow/Hong Kong/1161/2010(H5N1) and A/Anhui/1/2013 (H7N9) and their ferret antisera werekindly provided by Dr. R. G. Webster and Dr. R. Webby of St. JudeChildren’s Research Hospital (Memphis, USA).

VLP crude samples were prepared from concentrated pupae
homogenates after centrifugation at 27,000 rpm for 4 h on a 20%(w/w) sucrose cushion in an SW28 swing rotor. Samples were thenpurified on a sucrose density gradient as described previously [7],and centrifugation was carried out for 4 h. For electron microscopic
examination, sucrose was removed from the samples using column
filtration (PD-10 Desalting Columns; GE Healthcare Bio-sciences
AB, Sweden).

2.4. Production of VLP-HA protein

3.1. Design of chimeric H7 HA DNA and the generation of the Korea H7
BmNPV recombinant strain

A 0.2-mL aliquot of recombinant Korea H7-BmNPV virus was
inoculated into silkworm pupae. Four 4 days later, all pupae thatexhibited a colour change from brown to black were harvested.
The skin was removed, and naked pupae were homogenized in
PBS containing 0.01% formalin and phenylthiourea as an antioxidant; homogenization was performed on ice using six pulses inthree cycles at 2-min intervals with a Handy Sonic (Model Tomy
UR-20P). The resulting homogenates were centrifuged at 4200g
for 30 min. The supernatant was further centrifuged at 30,000g
for 30 min. This crude samples were sedimented in the presenceof 20% sucrose (w/w) by centrifugation at 110,000g for 4 h. The
supernatants were purified as described previously [7], but withcentrifugation for 4 h.

2.5. Immunological investigation of the VLP vaccine
Four-week-old female ddY mice were separated into threegroups (n = 5 mice per group). The first group was immunizedorally without adjuvant at week 0 using H7 VLP containing
16,384 HA titres and then 1 month (4 weeks) later. The second
group was immunized orally with two doses at weeks 0 and 1.
The third group of mice were immunized orally three times at 1week intervals with the same amount (16,384 HA titres) of H7
VLP antigen. Blood samples were collected from all mice at
4 weeks. Additionally, immune responses in mice immunizedintraperitoneally with two doses of H7 VLP were determined.
Additionally, six-month-old chickens were also immunized
three times orally with 16,384 HA titres of H7 VLP. The H5Fukushima VLP vaccine was simultaneously administered orally
at 16,384 HA titres, and the immune responses of the chickens
were compared with those of the mice described above. Antiserum
to Korea H7-VLP was also prepared in chickens via intramuscularimmunization three times.

2.6. Haemagglutination and haemagglutination inhibition (HI) tests
Test samples were diluted with 50 lL phosphate-buffered saline (PBS) in 96-well microtitre plates, and haemagglutination andHI tests were carried out as described previously [7].

2.7. Haemadsorption tests
The culture medium from infected cells was removed andwashed once with PBS. Next, a 0.5% chicken erythrocyte mixture
was added to the infected cells. After a 30-min incubation, the test
cells were washed through with PBS and observed under stereoscopic microscope (Olympus. Tokyo, Japan).

3. Results

First, we aimed to design chimeric HA DNAs from A/duck/
Korea/A76/2010 (H7N7), A/Anhui/1/2013 (H7N9), and A/Shanghai/1/2013 (H7N9) viruses. In addition to the above H7 HA genomes, as shown in Fig. 1, 20 codons (UUC, GAC, GUU, CAA, AAC,
AAA, UGC, UAA, CAC, UAC, GAA, AGA, CUG, UGG, UCA, AUG, CCU,
GUG, AUC, and GGC), which are frequently used in the physiological metabolism of silkworms, were selected from 60 codons andwere replaced with the wild-type codons of the target viral geneat 1295 positions. The synthesized DNA was then inserted into abaculovirus transfer vector, pBM-8, to obtain Korea H7-BmNPV.
The above three recombinant baculoviruses were then generated
via cotransfection of the three H7-BmNPVs together with the linearized DNA from the wild-type baculovirus BmNPV, as described
previously [7]. According to the same method, expression plasmid
containing of a wild strain (A/tufted duck/Fukushima/16/2011(H5N1)) was also constructed.

3.2. Expression of the synthetic Korea H7-BmNPV genes in cultured
cells
As shown in Fig. 2A, infection with wild-type BmNPV was confirmed in cytopathic effect and western blot analysis specific forbaculovirus. Additionally, the expression of Korea H7 recombinant
was determined in haemadsorption, FA, and western blot analyses.
Haemadsorption tests revealed the expression of H7 Anhui, H7
Shanghai, and H5 Fukushima recombinants. For comparison of
HA yields and mRNA copy numbers, the above three recombinants
were injected into BmN cells and pupae. The results showed that
HA units and RNA synthesis of codon-optimized H5 Fukushima
had the highest titres (Fig. 2B, C), followed by both of the Korea
H7 recombinant (Fig. 2B, C). It was of interest to know that recombinant including HA-DNA of H5 virus showed HA activity morethan one million, although a recombinant containing HA DNA ofwild stain did not produce any HA protein. In contrast, mRNAand HA yields of H7 Anhui and H7 Shanghai (HA yield data notshown) were extremely low. Thus, titres obtained from mRNAand HA titration were consistent, suggesting that mRNA synthesis
may be a useful marker for understanding the production level of
HA molecules.
Notably, when interpreting these results to determine theeffects of codon optimization, we must emphasize that the transfer
vector for the H5 Fukushima recombinant was different from thoseof the three other H7 recombinants. In any case, the yields of HA
protein for each recombinant were greatly influenced by the insertion of silkworm codons and by the genomic structures of viral HA.
Interestingly, the insertion of the recombinant gene in the baculovirus did not influence the synthesis of baculovirus Gp64 mRNA.

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Fig. 2. Examination of protein expression on the recombinant baculovirus (Korea H7-BmNPV) using western blotting of baculovirus (negative control), haemadsorption, andfluorescent antibody tests (A). The expression of Korea H7-BmNPV, Anhui H7-BmNPV, and Shanghai was also examined using western blotting (Korea H7-BmNPV),
haemadsorption and haemagglutinin tests. HA production of H7 recombinant, H5-wild strain and H5-codon optimized HA recombinant (B). (C) Comparative analysis ofmRNA yield based on codon optimization and differences in the specific genome structure of the virus. (D) Number of mRNA copies following codon optimization, and mRNA
expression of baculovirus-specific genes including HA DNA of H5 wild type Fukushima strain.

3.3. Production of the H7 VLP vaccine in silkworm pupae
Life stages of silkworms are shown in Fig. 3A and B. The Korea
H7 recombinant appeared to produce moderate levels of HA
units/larva (314,026), which was judged as +4. After infection with
the Korea H7 recombinant, the pupae changed colours from brown

to black. Moreover, despite infection with the recombinant virus,
some pupae grew up to the cocoon and moth stages. In order toconfirm the increased yield of H7 Korea VLP, recombinant Korea
H7-BmNPV virus was infected in 40 pupae. After infection, the skincolours of the pupae gradually changed, acquiring a deep black colour after day 4. These results suggested that the peak HA titre of

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Fig. 3. Larva, pupae, moths, and cocoons of silkworms were used for the production of haemagglutinin (A). (B). Comparative analysis of the yield of haemagglutinin in the
different sonication cycles. The HA yields in different silkworm groups and at the different elution cycles were evaluated on the basis of four scores as follow: +, >1024; ++,>10,000; +++, >100,000; and ++++, >200,000.

the recombinant virus was obtained on day 4. The homogenates
were then subjected to sonication. The results of HA protein
obtained from 40 pupae are shown in Fig. 3B. A total of
16,343,040 HA protein titres were recovered from three cycles ofsonication per 40 pupae. The yield of haemagglutinin was estimated to be 408,576/pupa, showing a high level of haemagglutinin
production (++++).
3.4. Purification and characterization of H7 haemagglutinin throughsucrose density gradient centrifugation

heavy (HF) and light fraction (LF) fractions, suggesting variationsin the produced VLPs. Western blot analysis demonstrated thatthe HA fraction contained a 77-kDa HA molecule (Fig. 4C). Interestingly, large-VLPs and small-sized HA particles were present in the
HF and LF (Fig. 4D–F and G), .respectively. On the basis of HA andprotein analysis pattern, we obtained the attracted result as morethan 90% of HA fraction was present in heavy fractions and furthermore, most of VLP was recovered in heavy fraction as purified
sample.
3.5. Antigenic characterization of H7 Korea VLP

In order to investigate the biological and structural characteristics of the recombinant protein, sucrose gradient samples wereseparated into about 39 fractions and subjected to sucrose concentration (Fig. 4A), HA activity (Fig. 4B), western blot (Fig. 4C), andprotein content analyses (Fig. 4A). As shown in Fig. 4A and B, themajor peaks of HA and protein concentration were separated into

To investigate the immunogenicity of H7 haemagglutinin produced in silkworm pupae, H7 VLP vaccines were subjected to the
HI reaction with antisera against authentic influenza viruses andsilkworm-derived VLP antigens. As shown in Fig. 5A, antiserum
against A/Anhui/1/2013 (H7N9) virus reacted with H7 Korea VLP

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743

Fig. 4. Purification of Korea H7 haemagglutinin produced in pupae. Purification and characteristics were analysed on a 1–50% (w/w) sucrose density gradient centrifugation
at 110,000g for 4 h at 5 °C. Sucrose and protein concentrations (A), HA activity (B), western blot analysis (C), respectively. In B, comparison of HA activity between Heavy andLight fractions were also showed together with western blot analysis. Electron micrography of heavy (D, E, F) and light fractions (G), respectively. Bar = 50 nm.

antigen at a level similar to that of authentic A/Anhui-H7 virus,
yielding reaction titres of 80–160. Additionally, the reaction wasincreased when using potent IgG antibodies targeting silkwormderived H7 VLP HA antigen prepared in chickens.
Consequently, H7 VLP antigen was injected into the muscles ofchickens with up to three doses. As shown in Fig. 5B, the HI titres
were approximately 240 in the single dose group. In contrast,
higher HI antibody production was confirmed in chickens immunized with three doses of the vaccine, yielding HI titres of approximately 500.
3.6. Immune responses in chickens and mice following oral
immunization with avian H5 and H7 VLP vaccine
Since most of avian influenza viruses infect bird species throughthe oral route, the immune responses to avian H5 and H7 VLP were

examined in chickens and mice after oral immunization. Mice or
chickens were orally administered 0.5 mL H5 Fukushima and H7
Korea HA antigens possessing 16,000 HA titres using a specillum,
and immune doses were conducted three times (doses) at
1-week intervals (weeks 0, 1, and 2).
The results are shown in Fig. 5C and 5D together with HI patterns in 96-well plates (Fig. 5E). The HI titres elicited in micetended to increase in accordance with the increase in the dose of
H5 Fukushima and H7 Korea HA antigens (Fig. 5C–E). For example,
after three doses of the vaccine in mice and chickens, HI titres
reached approximately 450 against H5-HA VLP. To confirm the oral
immune responses to the vaccination, fluorescent antibody
analysis and western blotting were performed using the above
antibodies produced in mice. As shown in Fig. 6A and B, H5HABmNPV-infected cells showed clear fluorescence after intraperitoneal injection or intra-oral inoculation with H5 HA VLP vaccines.

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Fig. 5. Immune responses of H5 and H7 VLP haemagglutinin (A and B). (C and D) Antibody production of H5 and H7 VLP in pupae after oral immunization in chickens andmice.

Fluorescence was not observed in infected cells treated with antiserum to JEV (negative control). Interestingly, a 77-kDa peptide
specific for HA was detected after treatment with antisera against
FLAG monoclonal antibody, mouse antibody prepared withintraperitoneal immunization, and antibody obtained from oral
immunization. Furthermore, H7-specific antibodies were identified
in fluorescent antibodies produced by intraperitoneal and intraoral immunization (Fig. 6E and F). In addition to FLAG antibodies,
the above two antibodies were also used in western blot analysis(Fig. 6G). Our findings showed that one weak peptide was observedin anti-FLAG antibody-treated Korea H7 recombinant infected

BmN cells. However, antibody administered by intraperitoneal
injection revealed two peptides larger than the anti-FLAG antibody. In contrast, antibodies produced in mice with oral immunization yielded one sharp peptide Unlike normal HI antibodies
produced by intraperitoneal immunization, two peptides larger
than the normal HA peptide appeared in western blot analysis ofsamples from intra-oral antibody administration. Although thereason for this phenomenon is still unclear, the positive FA of antibody produced in mice increased the possibility that oral immunization with avian influenza virus HA could elicit antibodies
specific to influenza virus.

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745

Fig. 6. Confirmation of anti-H5 antibody production in mice with intraperitoneal (A) or intra-oral immunization (B). (C) Detection of H5 Fukushima HA polypeptides with
antibodies against FLAG. Peritoneal cavity (PC), oral immunization (Oral), negative control of JEV (JE). (D and H) Negative control of JEV (JE). (E and F) FA reaction in Korea H7
recombinant infected BmN cells treated with antibodies against intraperitoneal (E) and intra-oral (F) immunization. (G) Western blot analysis with antibodies to FLAG.
Intraperitoneal immunization (PC), oral immunization (Oral), and JEV antigen (JEV) in the H7 Korea recombinant infected BmN cells.

4. Discussion
Through a series of recombinant baculoviruses expressing HA,
including H5 Fukushima, H7 Korea, and H7 Anhui HA genes ofavian influenza virus, we compared the yields of HA protein produced in silkworm systems on the basis of HA titration and mRNA
synthesis. As a result, we found that the production level of HA
proteins with each recombinant appeared to be influenced by not
only the codon-optimized effect but also the characteristic structure of the HA gene. For example, despite the use of the samecodon-optimized DNAs, the HA titre and number of mRNA copies
of each recombinant were different from each other. Moreover,although remarkable advancements have been made in the fieldof vaccine development in recent years [7,15,16,19–24], it wasinteresting that the influenza VLP vaccine could elicit broader
immune responses than the intact virion vaccine [21], suggesting
potential applications as a future VLP vaccine. Since we don’t haveauthentic H7 influenza virus, comparative immune responses
between silkworm VLP and authentic virus vaccine may be donein near future. Furthermore, an influenza VLP vaccine of H7N9

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virus containing the surface HA, neuraminidase, and internal M1
protein has been reported to produce protective immune
responses in mice [22]. Recent VLP vaccine development is focusedon H7 HA, and various HA VLP subtypes have been shown to protect a wide variety of epidemic or epizootic viruses [23,24].
Based on these previous works, the VLP vaccines developed
herein may be obtained by large-scale production with a relatively
low cost and rapid production, as is needed in the case of pandemic
outbreaks. Therefore, a genomic system for the large scale production of vaccines should be established as soon as possible. Indeed,
we could produce a high level of Korea H7 VLP in silkworm pupae,
showing HA titres of 408,576 per pupa. However, the yields
obtained in this study were still lower than that (approximately
1,000,000 HA titre) obtained for the H5 Fukushima recombinant[7]. In contrast, recombinant containing HA DNA of wild H5
Fukushima did not show meaningful HA activity. In any case, wewere surprised to find that oral immunization with H5 and H7
VLP in chickens and mice resulted in significant increases in antibody titres according to the vaccine dose. For example, antibody
titres in mice and chickens immunized with H5 VLP increased
gradually with increasing doses of the vaccine. These antibodies
were confirmed in further fluorescent antibody tests and western
blot analyses. In order to elicit a strong antibody increase throughbroad mucomembranous stimulation, a large amount of VLP antigen may be necessary. When considering this immune condition,a large amount of VLP antigen may be produced in silkworms at
a low cost.
In this study, we determined that avian HA vaccine protein elicited the HI antibody with oral immunization, suggesting further
detail investigation. Additionally, it was also effect to establish auseful recovery system of HA vaccine from larvae and pupae ofsilkworm.
Conflict of interest
We are applying a patent based on the results described in thepresent report.
Acknowledgments
Authors are grateful to Ms. Emiko Kobayashi for her excellent
techniques of electron microscopy. This work was supported by agrant from Okinawa Communicable Disease Research Hub Formation Promotion Project by Okinawa Prefectural Government Commissioned Projects for fiscal year 2015.
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