Heliyon 6 (2020) e04543

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Research article

Interleukin 12-containing inﬂuenza virus-like-particle vaccine elevate its
protective activity against heterotypic inﬂuenza virus infection
Kenichi Maegawa a, Shigeo Sugita b, Youta Arasaki a, Reiko Nerome a, Kuniaki Nerome a, *ab

The Institute of Biological Resources, 893-2, Nakayama, Nago-shi, Okinawa 905-0004, Japan
Equine Research Institute, Japan Racing Association, 1400-4, Shiba, Shimotsuke-shi, Tochigi 329-0412, JapanA R T I C L E I N F O

A B S T R A C T

Keywords:
Biotechnology
Microbiology
Virology
Inﬂuenza
Vaccination
Antibody
Viral disease
Microbial biotechnology
Vaccines
Bombys mori
Eri silkworm
Cytokine
Avian inﬂuenza

To produce monovalent and bivalent inﬂuenza vaccines composed of virus-like particles (VLPs) containing
hemagglutinin (HA), we generated four recombinant Baculoviruses derived from Bombyx mori nuclear polyhedrosis virus (BmNPV) and Autographa california nuclear polyhedrosis virus (AcNPV). Monovalent Fukushima(A/tufted duck/Fukushima/16/2011 [H5N1]) (FkH5) and Anhui (A/Anhui/1/2013 [H7N9]) (AnH7) VLP
inﬂuenza vaccines were produced in silkworm pupae infected with FkH5-BmNPV or AnH7-BmNPV. To produce abivalent FkH5 and AnH7 vaccine, the pupae were simultaneously inoculated with FkH5-BmNPV and AnH7BmNPV. Then, interleukin (IL)-containing bivalent vaccines were produced by Eri silkworm pupae following
triple infection with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV. Fluorescent antibody tests in Sf9 cells tripleinfected with FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV showed coexpression of FkH5, AnH7, and IL-12
antigens, suggesting the presence of VLPs containing all three antigens. We then performed competitive hemagglutination inhibition (CHI) tests to calculate the VLP vaccine constituents. Inoculation with two recombinantviruses led to the production of bivalent vaccines containing very similar amounts of the H5 and H7 antigens,suggesting that our dual infection system can be used to produce bivalent VLP vaccines. Immunisation of micewith our developed monovalent and bivalent VLP vaccines induced the production of HI antibody, which protected against a sublethal dose of inﬂuenza virus. These IL-12-containing vaccines tended to display increased
protection against hetero-subtype inﬂuenza viruses.

1. Introduction
Recent outbreaks of highly pathogenic avian inﬂuenza viruses havethreatened both the poultry industry and human health [1, 2, 3]. In theﬁrst outbreak in 1997, 6 out of 18 avian inﬂuenza-infected patients died
due to multiple organ failure [3, 4]. The characteristics of the isolated
viral genomes indicated that all human infections occurred at chicken
markets in Hong Kong where diseased chickens were introduced from
epizootic poultry farms [3, 5].
Since then, avian inﬂuenza has attracted considerable public attention, and avian inﬂuenza vaccines have been developed using various
technologies. These vaccines have been produced by various methods
including: 1) using temperature-sensitive mutants [6], 2) cell culture [7],3) hemagglutinin (HA)-containing virus-like particles (VLPs) [8, 9], and
4) DNA-based vaccines [10, 11]. With an aim to provide inexpensive,
effective vaccines against avian inﬂuenza, we developed a recombinantbaculovirus system for the large-scale production of H5 and H7

HA-containing VLP vaccines in silkworms [8, 12]. We also established asimilar system in Eri silkworms [13] that allows the production of HA
titres greater than 30 million per pupa. H7-containing VLP vaccines
produced using different systems elicited strong immune responses inmurine, ferret, and avian models [14, 15, 16, 17].
Multiple recent reports of zoonotic infections caused by H5N8 andH5N6 avian inﬂuenza viruses have raised concerns regarding the threat
to avian and human health [18]. Recent H7 inﬂuenza virus infections inhumans has also elevated the threat of infection in urban environments[19, 20, 21]. These epizootic and epidemic reports have led to thedevelopment of multivalent inﬂuenza vaccines [22, 23] and active
vaccination programmes worldwide [24].
To prevent inﬂuenza virus infections in humans and poultry, we have
implemented the following strategies. First, we attempted to effectively
and rapidly produce inexpensive bivalent and multivalent vaccines insilkworms on a large scale to provide high-quality vaccines for thepoultry and healthcare industries. Then, we inserted membrane-* Corresponding author.
E-mail address: rnerome_ibr@train.ocn.ne.jp (K. Nerome).
https://doi.org/10.1016/j.heliyon.2020.e04543
Received 3 December 2019; Received in revised form 3 January 2020; Accepted 21 July 2020
2405-8440/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

K. Maegawa et al.

Heliyon 6 (2020) e04543(Thermo Fisher Scientiﬁc) supplemented with 10% foetal bovine serum(FBS). Madin-Durby canine kidney (MDCK) cells were cultured in Eagle's
minimum essential medium (MEM) containing 10% FBS.
The P6E strain of Bombyx mori nuclear polyhedrosis virus (BmNPV)
was used to generate FkH5-BmNPV and AnH7-BmNPV recombinant viruses. Autographa californica nuclear polyhedrosis virus (AcNPV) wasused to produce FkH5-AcNPV and AnH7-AcNPV recombinant viruses.
Inﬂuenza virus A/PR/8/34 (H1N1) strain PRH1 was cultivated in
MDCK cells. Recombinant inﬂuenza viruses HkH5 (RG-A/BarnSwallow/
Hong Kong/1161/2010-A/PR/8/34 [R][6 þ 2] [H5N1]) and AnH7 (RGA/Anhui/1/2013-A/PR/8/34[R][6 þ 2] [H7N9]), were kindly provided
by Dr. R.G. Webster (St. Jude Children's Research Hospital, Memphis, TN,
USA). Viruses were propagated in 11-day-old embryonic chicken eggs
and MDCK cells.

anchored interleukin (IL)-12 into VLP vaccines to enhance mucosal immune responses and elicit cellular immune responses [25, 26, 27, 28].
Khan et al. [25] demonstrated that coexpression of membrane-anchored
IL-12 and IL-23 with inﬂuenza vaccines resulted in enhanced mucosal
immune responses. Here, we describe an effective multiple-infection
system that can be used to synthesise bivalent or VLP vaccines containing IL-12 and multiple HA VLP antigens.
2. Materials and methods
2.1. Cells and viruses
Bombyx mori Bm-N cells and Spodoptera frugiperda ovarian Sf9 cells(Sigma Aldrich, Tokyo, Japan) were cultured in Grace's insect medium

Figure 1. Production of VLP vaccines from silkworm
pupae infected with recombinant Baculoviruses.
Monovalent vaccines were produced from Bombyx
mori pupae infected with (A) FkH5-BmNPV- and (B)AnH7-BmNPV. To produce bivalent FkH5þAnH7-VLP
vaccines, B. mori pupae were co-infected with FkH5Bm NPV and AnH7-Bm NPV, and bivalent vaccines
were extracted from the infected pupae (C). Bivalentvaccines containing membrane-anchored IL-12 were
produced by Eri silkworm pupae triple infected with
IL-12-AcNPV, FkH5-AcNPV, and AnH7-AcNPV (D).

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2.2. Generation of recombinant viruses
The FkH5 and AnH7 genes were synthesised by GENEWIZ (Saitama,
Japan). The methods used to generate BmNPV- and AcNPV-based recombinant viruses containing these HA genes have been described previously [8, 12].
The IL-12–40/35 genes were also synthesised by GENEWIZ. Brieﬂy,
IL-12–40/35 was constructed by combining two IL-12 subunits (p40 andp35) with a glycine linker (L1) such that p40 was located at the N-terminus, and the membrane anchor region, which consists of the inﬂuenza
strain WSN (H1N1) HA membrane-anchoring domain (Figure 1D), waslocated in the C-terminus [25]. Then, the IL-12–40/35 gene was inserted
downstream of the pFastBac1 polyhedrin promotor. The generated
plasmids were transformed into DH10Bac cells. Subsequently, recombinant Ac-NPV DNA was puriﬁed and transfected into Sf9 cells as described
previously [13]. After 1 week of culture, the supernatant, containing
recombinant Ac-NPV viruses, was harvested and used in subsequent
experiments.
2.3. Production of VLP vaccines in silkworm pupae
B. mori and Eri silkworm pupae were used to produce VLP vaccines by
infecting the silkworms with BmNPV and AcNPV recombinant viruses,
respectively (Figure 1). Baculovirus inoculation and preparation of VLP
vaccines were performed as previously described [13].
2.4. Hemagglutination and hemagglutination inhibition (HI) tests
Hemagglutination and HI tests were performed as described previously [8, 12].
2.5. Competitive HI (CHI) tests to calculate HA antigen content in thebivalent vaccines
CHI tests were performed to calculate the HA antigen content in the
produced multivalent VLP vaccines. Brieﬂy, 25 μl of monovalent FkH5VLP vaccine or AnH7-VLP vaccine were mixed with 0, 5, 10, 15, 20,
25, 30, or 35 μl of homologous anti-FkH5 serum or anti-AnH7 serum, andPBS was added to bring the ﬁnal volume to 60 μl. After mixing, the tubes
were incubated for 30 min at room temperature (20–25  C), and the HA
titres were determined.
Although the addition of 5 μl of serum did not affect the HA titre, theaddition of 10 μl or more of homologous anti-FkH5 serum to the FkH5VLP vaccine resulted in a linear reduction in the HA titre on a semilogarithmic plot (Figure 2A). In contrast, anti-AnH7 serum did not affectthe titre. When both anti-FkH5 and anti-AnH7 sera were added to theAnH7-VLP vaccine, only the anti-AnH7 serum affected the HA titre,
which was linearly reduced in the presence of 10 μl or more of the serum(Figure 2B). These results indicated that the anti-FkH5 and anti-AnH7
antibodies are not cross-reactive. Therefore, when anti-FkH5 serum isadded to bivalent FkH5þAnH7 vaccines, the residual HA titre should
correspond to the AnH7 HA antigen titre. Conversely, the FkH5 antigen
HA titre can be similarly calculated when anti-AnH7 antibody is added.
Therefore, the H5 and H7 antigen contents could be determined using
this CHI test. The residual HA titre, as determined in the presence of antiFkH5 and anti-AnH7 sera was 64 for both antigens (Figure 2C). Therefore, the bivalent vaccine contains the same amounts of H5 and H7
antigens.

Figure 2. CHI tests using anti-FkH5HA and anti-AnH7HA sera. Monovalent
FkH5-VLP vaccine (A) and AnH7-VLP vaccine (B) and bivalent FkH5þAnH7VLP vaccine (C) were evaluated by CHI tests using anti-FkH5HA and antiAnH7HA sera.

2.7. Fluorescent antibody (FA) tests
Bm-N and Sf9 cells were infected with recombinant viruses andincubated in Grace's insect medium supplemented with 10% FBS at 27  C
for 3 days. Then, FA tests were performed as described previously [8, 12].
Antisera against H1HA, H5HA, and H7HA were prepared from mice
immunised with puriﬁed PRHA, HkH5, and AnH7 inﬂuenza viruses (HAtitres: 2,048–4,096), respectively. Goat anti-IL-12 polyclonal antibody
was purchased from Peprotech (USA). The following secondary antibodies were used: Alexa Fluor 488-conjugated goat anti-mouse IgG (Cell
Signaling Technology, USA), Alexa Fluor 555-conjugated goat anti-rabbit
IgG (Cell Signaling Technology), Alexa Fluor 555-conjugated donkey
anti-goat IgG (Abcam, UK), and Alexa Fluor 488-conjugated donkey antimouse IgG (Abcam).
2.8. Analysis of the protective efﬁcacy against intranasal challenge withinﬂuenza viruses
Five groups of 4-week-old female ddY mice purchased from Japan
SLC co., Ltd. (Shizuoka, Japan) were immunised intraperitoneally with
PBS, inactivated PRH1, and IL-12-containing FkH5- and AnH7-AcVLP
vaccines. The VLP vaccines contained HA titres of 4,000–16,800.
Two weeks later, the animals were immunised a second time as

2.6. Hemadsorption tests
Bm-N and Sf9 cells were infected with recombinant viruses. Then, thecells were washed once with PBS and used in hemadsorption tests with
0.5% chicken erythrocytes, as described previously [12].
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Heliyon 6 (2020) e04543

3.3. Hemadsorbing activity of recombinant, co-infected Sf9 cells

described above. Six weeks later, the mice were intranasally challenged
with virulent PRH1, FkH5, and AnH7 viruses. Animal health and survival were observed daily for 10 days. Mouse experiments were carriedout in speciﬁc pathogen-free (SPF) conditions, with the approval of theAnimal Welfare and Animal Care Committee, including the Animal
Ethics Committee of the Institute of Biological Resources, Okinawa,
Japan.

To conﬁrm cell surface localisation of the HA antigens in cells infectedwith recombinant AcNPV, hemadsorption activity was assessed(Figure 5). Hemadsorption assays can be used to verify the biologicalcompleteness of the HA protein on the surface of recombinant virusinfected cells, because erythrocytes are added external to the infected
cells.
No erythrocyte adsorption was observed using IL-12-AcNPV-infected
Sf9 cells (Figure 5A). In contrast, clear hemadsorption was detected using
FkH5-AcNPV- and AnH7-AcNP-infected Sf9 cells, regardless of thepresence of IL-12-AcNPV (Figure 5B–F), indicating the presence of the
FkH5 and AnH7 antigens on the cell surface. Similar hemadsorption wasobserved for IL-12-AcNPV co-infected cells, suggesting that membraneanchored IL-12 does not interfere with the transport of H5 or H7 HA to
the cell surface.

3. Results
3.1. AcNPV-infected Sf9 cells produce recombinant vaccines
We previously established a recombinant AcNPV system in Eri silkworms that produces abundant recombinant proteins in pupae [13]. In
this study, we generated three recombinant AcNPV viruses:
FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV. FkH5-AcNPV and
AnH7-AcNPV have been described previously [13], and they respectively
produced H5- and H7-subtype inﬂuenza virus HA proteins (Figure 3A, B).
IL-12-AcNPV produced membrane-anchored, single-chain IL-12, which
was described previously [25]. Membrane-anchored IL-12 is an immunopotentiating agent for inﬂuenza vaccines; therefore, the efﬁcacy of VLP
vaccines should be improved by introducing IL-12. IL-12 protein production was conﬁrmed by FA tests (Figure 3C). Almost all the Sf9 cells
infected with FkH5-AcNPV, AnH7-AcNPV, or IL-12-AcNPV were positively stained with anti-H5HA serum, anti-H7HA serum, or anti-IL-12
antibody, respectively. Next, we attempted to produce multivalent VLP
inﬂuenza vaccines using these recombinant viruses (Figure 3).

3.4. Large-scale production of mono- and bivalent vaccines
As reported previously, we successfully and separately produced large
amounts of the FkH5- and AnH7-VLP vaccines using B. mori pupae
infected with FkH5-BmNPV and AnH7-BmNPV, respectively [13]. Using
these two recombinant viruses, we attempted to produce bivalent VLP
vaccines containing both FkH5 and AnH7 via dual infection of B. moripupae. To assess co-expression of the two HA antigens, co-infected pupae
were homogenised in PBS containing 0.5% sodium thiosulfate as anantioxidant. Then, the homogenate was puriﬁed by sucrose gradientcentrifugation, as described previously [12]. The isolated
FkH5þAnH7-VLPs were used as a bivalent vaccine. We then quantiﬁed
the H5 and H7 HA antigens in the bivalent vaccine using a simple CHI
test. The isolated bivalent vaccine had an HA titre of 838,808, including
62% H5 antigen (H5 HA titre: 524,144) and 38% H7 antigen (H7 HA
titre: 314,644; Figure 6A).
Finally, we attempted to produce bivalent vaccine containing
membrane-anchored IL-12 by triple infection of Eri silkworm pupae with
FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV. The triple-infected pupae
were homogenised in PBS containing 0.5% sodium thiosulfate, and theantigens were puriﬁed by sucrose density gradient centrifugation. CHI
tests showed that the IL-12-containing VLP bivalent vaccine contained an
HA titre of 9,624,159 (Figure 6B), with 57% FkH5 (5,481629) and 43%
AnH7 (4,142,530). These results indicate that the VLP antigen yield in
Eri silkworms was greater than that in B. mori silkworms as reported
previously [13].
Large-scale production of bivalent vaccines and subsequent analyses
indicated that: 1) large amounts of bivalent H5–H7 VLP vaccines containing nearly equivalent levels of both HA antigens can be easily obtained through dual or triple infection of silkworm pupae by adjusting
the MOI, and 2) CHI tests can clearly distinguish the different HA antigens in bivalent vaccines. These results suggest that CHI tests might be

3.2. Sf9 cells infected with multiple recombinant AcNPVs coexpress HA
and IL-12
Since the goal of this study was to produce VLP vaccines containing
multiple antigens by co-infecting multiple recombinant AcNPVs, it was
important to examine the expression of these antigens. To address this,Sf9 cells were simultaneously infected with multiple recombinant viruses
and analysed in FA tests. Sf9 cells were dually infected with IL-12-AcNPVand either FkH5-AcNPV or AnH7-AcNPV, and then co-expression of both
IL-12 and HA was detected by antibody co-labelling (Figure 4A, B). The
results clearly demonstrated the coexpression of multiple antigens andthe feasibility of multiple-antigen production by VLP vaccines using a coinfection method.
We hypothesised that IL-12-containing VLP vaccines could be produced in silkworm pupae by triple infection with FkH5-AcNPV, AnH7AcNPV, and IL-12-AcNPV. Because dual antigen expression was observedin dual-infected cells, we next tried to examine whether triple-infected
cells expressed IL-12 and dual HA antigens. Indeed, when Sf9 cells
were triple infected with IL-12-AcNPV, FkH5-AcNPV, and AnH7-AcNPV,
both HA antigens (H5 and H7) and IL-12 were expressed (Figure 4C, D).

Figure 3. Expression of FkH5HA, AnH7HA, and IL-12
antigens in recombinant AcNPV-infected Sf9 cells. Sf9cells were infected with FkH5-AcNPV (A), AnH7AcNPV (B), and IL-12-AcNPV (C). Then, the infected
cells were ﬁxed with methanol and stained with antiFkH5 mouse serum (A), anti-AnH7 mouse serum (B),or anti-IL-12 goat polyclonal antibody (C). Antibody
staining was detected with Alexa Fluor 488-conjugated anti-mouse IgG (A, B) and Alexa Fluor 555-conjugated anti-goat IgG (C).

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Heliyon 6 (2020) e04543

Figure 4. Conﬁrmation of FkH5, AnH7, and IL-12
antigen expression in triple-infected Sf9 cells. Sf9cells were co-infected with FkH5-AcNPV and IL-12AcNPV (A), AnH7-AcNPV and IL-12-AcNPV (B), or
FkH5-AcNPV, AnH7-AcNPV, and IL-12-AcNPV (C, D).
Infected cells were ﬁxed with methanol and stainedwith anti-IL-12 goat polyclonal antibody (A, B, C, D),anti-FkH5 mouse serum (A, C), or anti-AnH7 mouseserum (B, D). Stained cells were then labelled with
Alexa Fluor 488-conjugated anti-mouse IgG and Alexa
Fluor 555-conjugated anti-goat IgG. Double-positive
cells expressing two antigens are indicated by arrows.

an important index for evaluating the progression of pneumonia of test
mouse, we ﬁrst examined the body weight of test mice, and the results areshown in Figure. 8A–C. Resultant body weights of each mouse group
comprising of unimmunised and non-challenged group increased
constantly during 10 days (Figure 8A). In contrast, a slight decrease ofbody weight was determined in the mice immunised with PBS andchallenged with the HkH5 virus until 5 days after infection. Interestingly,
the two groups of mice immunised with either FKH5-VLP or IL-12 þ
FkH5 VLP vaccine did not show any reduction in body weights after
infection with HkH5 (Figure 8A). However, although a slight decrease inbody weight was seen in FkH5-VLP vaccinated group, this tendency
recovered after 7 days. It was evident that the changes of body weight oftest mice immunised with IL-12 þ FkH5 VLP were not inﬂuenced with H5virus infection (Figure 8A). In a same experiment, unimmunised andPRH1 virus-infected mouse group showed an obvious decrease of bodyweight, suggesting the occurrence of severe pneumonia (Figure 8C). This
result led us to the following experiment using different VLP vaccines.
Thus, we immunised three groups with PBS, IL12 þ FkH5-VLP vaccine
and IL12 þ FkH5þAnH7-VLP vaccine. After 6 weeks, the above test
groups were challenged with HkH5 virus. As a result, in unimmunised
control mice, two in unimmunized group were found to be died at 8 days
after infection (Figure 8B). It was a sharp contrast that all mice immunised with IL12 containing both vaccines survived during 10 days-entire
test period. This result showed that both monovalent and divalent VLP
vaccines containing IL12 induce protective immunity against H5 inﬂuenza virus (Figure 8B). When compared the above result, it was of interest to examine the protective activity of VLP vaccines against
antigenically heterologous inﬂuenza virus. In next experiment, fourgroups were immunised with PBS (negative control), inactivated whole
PRH1 virus vaccine (positive control), IL12-containing FkH5-VLP and
IL12 containing FkH5- and AnH7-VLP vaccines. And 6 weeks latter, allmouse groups were intranasally infected with PRH1 virus. As a result,dead mice were not conﬁrmed in any group until 6 days after challenge
infection (Figure 8D). However, at 7 days after infection, four mice were
found to be dead in PBS control group (80% mortality and 20% survival).
In contrast, all mice immunised with inactivated PRH1 vaccine survived
during entire test period. However, 2 mice immunised with FkH5 andAnH7 vaccines were dead at 7 days after infection and subsequently,

useful for antigenic differentiation of other bivalent, trivalent, or multivalent vaccines.
3.5. Antibody production in mice immunised with mono- or divalent VLP
vaccines
To examine the immunogenicity of the developed VLP vaccines, wemeasured HI titres in the sera of mice immunised with the vaccines.
Vaccination with FkH5-VLP or AnH7-VLP led to an increase in the serum
HI titre. The HI titres in FkH5-VLP-vaccinated animals were 83, 467, and
640 at 2, 4, and 6 weeks post-immunisation, respectively (Figure 7A),
while those in AnH7-VLP-vaccinated mice were 614, 1638, and 1843 at
2, 4, and 6 weeks post-immunisation, respectively (Figure 7B). Mice
immunised with the AnH7-VLP vaccine showed 3–7 times higher HI titres than mice immunised with the FkH5-VLP vaccine, although theamounts of antigens used for immunisation were the same (HA titre:
16,220).
Although IL-12-containing VLP vaccines are easily produced via
multiple infection, the immunogenicity of these vaccines might bedifferent from that of vaccines prepared from singly infected pupae.
Although IL-12 was included in the vaccine, its adjuvant effect was notdetermined in this study. However, mice immunised with the FkH5-VLP
vaccine exhibited an H5 HI titre of 467 at 4 weeks post-immunisation,
which was similar to that of mice immunisation with the IL-12containing FkH5 vaccine (Figure 7A–C).
Mice immunised with a bivalent vaccine possessing containing HAtitres of 9,324 (FkH5) and 7,060 (AnH7) had similar HI titres against
HkH5 and AnH7 viruses, respectively (Figure 7D). 247–248: Additionally, in a control experiment, mice immunised with IL-12 alone did not
produce FkH5 and AnH7 antigens (Figure 7E).
3.6. Evaluation of the protective efﬁcacy of IL-12-VLP vaccines
Antibody production with different VLP vaccines led us to evaluate
the VLP vaccines for protective efﬁcacy against challenge inﬂuenza viruses. So, we immunised three groups comprising of 5 mice with either
PBS, IL12 þ FkH5-VLP vaccine, or IL12 þ FkH5þAnH7-VLP vaccine
including one unimmunized control. Since the change of body weight is5

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Heliyon 6 (2020) e04543

Figure 5. Conﬁrmation of VLP HA antigens on the surface of Sf9 cells infected with recombinant Baculoviruses. Sf9 cells were infected with IL-12-AcNPV (A, D, E, F),
FkH5-AcNPV (B, D, F), or AnH7-AcNPV (C, E, F). Then, the hemadsorption activity of the infected cells was examined.

and humans. Although new inﬂuenza viruses may appear in the future,
our system should allow the rapid production of strain-speciﬁc vaccines
against these viruses.
One of greatest communicable disease threats is a pandemic outbreakof avian inﬂuenza, which includes infections caused by the H5, H7, andH9 strains. Indeed, the World Bank recently reported an economic loss of$5.5 trillion due to the 2005 pandemics in developed countries (Japanesenewspaper, Ryukyu Simpo, p.3, 14 May 2017). Recent inﬂuenza outbreaks caused by avian H7N9 have occurred over all the world [21]. In
winter 2016, an outbreak of a virulent avian H7N6 strain in northern
Japan led to the killing of 340,000 chickens in Aomori and Niigata toprevent further outbreaks. As preventative measures, 2,750,000

further 2 mice in the FkH5-VLP group died at 8 days. These results
indicated that IL12 containing VLP vaccine partly elevate protective activity against even distinct HA subtype of inﬂuenza A virus. However, as
shown in Figure 8E, we did not evaluate protective activity of thesedifferent VLP vaccines against AnH7 virus due to failure of growth of
AnH7 virus in mouse lung (Figure 8E).
4. Discussion
We developed a method for large-scale production of divalent vaccines containing IL-12. Vaccines produced using this method may reduce
the threat of highly pathogenic avian inﬂuenza virus infection in birds

Figure 6. Determination of antigen content by CHI
test. (A) Twenty B. mori pupae co-infected with FkH5BmNPV and AnH7-BmNPV yielded 12 mL of bivalent
VLP vaccine (HA titre: 838,808). CHI tests showedthat the bivalent vaccine contained HA titres of524,144 for H5 antigen and 314,664 for H7 antigen.
(B) Ten B. mori pupae triple-infected with IL-12AcNPV, FkH5-AcNPV, and AnH7-AcNPV yielded 5
mL of IL-12-containing bivalent VLP vaccine with an
HA titre of 9,624,159. CHI tests showed that thebivalent vaccine contained titres of 5,481,629 for H5
antigen and 4,142,530 for H7 antigen.

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Figure 7. Evaluation of immune responses byassessment of HI antibody production induced by VLP
vaccines. Time course of the changes in the HI titres inthe sera of mice (n ¼ 5 per group) immunised withmonovalent FkH5-VLP (A) and AnH7-VLP (B) vaccines. HI antibody production at 4 weeks after
immunisation with IL-12-containing FkH5-VLP vaccine (C) or IL-12-containing FkH5þAnH7-VLP vaccine(D). HI antibody production in mice immunised with
an IL-12-VLP vaccine (E). Data represent the means SEM.

be able to treat 131 young adult chickens. Bivalent vaccines are highly
potent, inexpensive vaccines that can be used in birds and humans.
Bivalent vaccines with HA titres of several thousand could elicit the
production of thousands of HI antibodies. Because of the low cost of
production in silkworms, the antigenic quantities of these vaccines could
be increased ten-fold, resulting in even higher HI antibody production.
It may be difﬁcult to increase the protective immunity against someinﬂuenza A HA subtypes. Therefore, we conjugated IL-12 to H5 and H7
VLP antigens. These conjugated viruses elicited elevated HI antibody
production. IL-12-containing bivalent H5 and H7 vaccines enhanced thesurvival of mice challenged with HkH5 and H1N1 subtype (PRH1)
inﬂuenza. These results suggest that the introduction of certain cytokines
into VLP vaccines may be a useful method for producing a universal
vaccine, although this requires additional studies.
The introduction of cytokines into VLP vaccines is not limited tosilkworm-derived VLP vaccines, as cytokines have been used in different
inﬂuenza virus vaccines. For example, a trivalent H5, H7, and H9 vaccine
produced from a single DNA plasmid with cytokines displayed greater
protective efﬁcacy [23, 24]. The results of this study represent a step
closer towards enhanced vaccine development.
As described in previously [26], co-immunisation with IL-12 cDNA
and Coccidioides protective antigen cDNA enhanced immunity against

non-diseased chickens were killed in Japan from 2004 to 2014 (Japanesenewspaper, Asahi Shimbun, p.28, 29 November 2015). Nation-wideinﬂuenza vaccination programmes are used in several countries to protect chickens [22]. Vaccine coverage is particularly high in China, Egypt,
Hong Kong, Mongolia, and Vietnam. Widespread vaccination and thedevelopment of newer vaccines may prevent inﬂuenza outbreaks inhuman populations and protect several bird species.
To improve the response to new inﬂuenza strains, we have developed
new methods by focusing on the following: 1) providing large amounts ofavian inﬂuenza vaccines to many parts of the world, including low- andmiddle-income countries; 2) developing highly effective vaccines that
protect bird species, including chickens; 3) developing and massproducing effective, inexpensive vaccines; and 4) developing a complete, multivalent vaccine production system prior to the appearance of
new viruses transmitted to humans from birds, using the 16 inﬂuenza A
subtypes that are currently circulating in avian populations.
The monovalent and divalent vaccines and the methods developed inthis study may satisfy these above conditions. Monovalent vaccines
produced in silkworm pupae showed high yields (HA titre: 6,583,673 perpupa). Assuming 90% loss during puriﬁcation, the resultant productshould contain 658,367 HA per silkworm. Based on this yield and theadministration of 5,000 HA per chicken, a single silkworm pupa should

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K. Maegawa et al.

Heliyon 6 (2020) e04543

Figure 8. Evaluation of the protective effect ofdifferent VLP vaccines in mice. (A) Change (%) of
body weight in control mice and test mice immunised with PBS, FkH5-VLP and IL12 þ FkH5-VLP
vaccines. (B) Survival rate of mice immunised with
PBS, IL12 þ FkH5-VLP, or IL12 þ FkH5þAnH7-VLP
vaccines following infection with HkH5 virus. (C)Body weight changes (%) in control and immunisedwith PBS, IL12 þ FkH5-VLP vaccine following
infection with PRH1 virus. (D) Survival rates of mice
immunised with four different vaccines and subsequently with PRH1 virus infected mouse group. (E)Tentative evaluation of each VLP vaccine against
AnH7 virus infection, but failed its evaluation due tonon-growth characteristic of a recombinant An H7virus.

Competing interest statement

Coccidioides immitis. The effect of IL-2 and granulocyte macrophagecolony-stimulating factor (GM-CSF) on the efﬁcacy of inﬂuenza vaccines
was investigated previously [27]. In addition, IL-23 is known to play arole in mucosal immunity [25], and adult mice mucosally immunisedwith IL-12- and IL-23-containing inﬂuenza vaccines produced
virus-speciﬁc nasal IgA, leading to reduced viral titres in lung tissue after
challenge. Cytokines such as IL-1-6, IL-12, and IL-15 show promise as
vaccine adjuvants [27], because they may increase the rates of survival
against different virus infections. Therefore, we suggest combining thesecytokines with VLP inﬂuenza vaccines in future vaccine development.

The authors declare no conﬂicts of interest.
Additional information
No additional information is available for this paper.
Acknowledgements
We thank Dr. K. Kuroda (Nihon University School of Medicine) and
Dr. T. Imagawa (The Institute of Biological Resources) for their excellent
assistance with preparation of the manuscript.

Declarations
Author contribution statement

References
Kenichi Maegawa, Shigeo Sugita, Youta Arasaki: Performed the experiments; Contributed reagents, materials, analysis tools or data.
Reiko Nerome: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data.
Kuniaki Nerome: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote thepaper.

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Funding statement
This research did not receive any speciﬁc grant from funding agencies
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