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Inoculation of fowlpox viruses coexpressing avian influenza H5 and chicken IL-15 cytokine gene stimulates diverse host immune responses


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Fowlpox virus (FWPV) has been used as a recombinant vaccine vector to express antigens from several important avian pathogens. Attempts have been made to improve vaccine strains induced-host immune responses by coexpressing cytokines. This study describes the construction of recombinant FWPV (rFWPV) strain FP9 and immunological responses in specific-pathogen-free (SPF) chickens, co-expressing avian influenza virus (AIV) H5 of A/Chicken/Malaysia/5858/2004, and chicken IL-15 cytokine genes. Expression of H5 (50 kD) was confirmed by western blotting. Anti-H5 antibodies, which were measured by the haemagglutinin inhibition test, were at the highest levels at Week 3 post-inoculation in both rFWPV/H5-and rFWPV/H5/IL-15-vaccinated chickens, but decreased to undetectable levels from Week 5 onwards. CD3+/CD4+ or CD3+/CD8+T cell populations, assessed using flow cytometry, were significantly increased in both WT FP9-and rFWPV/H5-vaccinated chickens and were also higher than in rFWPV/H5/IL-15-vaccinated chickens, at Week 2. Gene expression analysis using real time quantitative polymerase chain reaction (qPCR) demonstrated upregulation of IL-15 expression in all vaccinated groups with rFWPV/H5/IL-15 having the highest fold change, at day 2 (117±51.53). Despite showing upregulation, fold change values of the IL-18 expression were below 1.00 for all vaccinated groups at day 2, 4 and 6. This study shows successful construction of rFWPV/H5 co-expressing IL-15, with modified immunogenicity upon inoculation into SPF chickens.
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AsPac J. Mol. Biol. Biotechnol. 2019
Vol. 27 (1) : 84-94
Inoculation of fowlpox viruses coexpressing avian influenza H5 and
chicken IL-15 cytokine gene stimulates diverse host immune responses
Abdul Razak Mariatulqabtiaha,b*, Nadzreeq Nor Majidb, Efstathios S. Giotisc, Abdul Rahman Omard,
Michael A. Skinnerc
aLaboratory of Vaccines and Immunotherapeutic, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor, Malaysia.
bDepartment of Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM,
Serdang, Selangor, Malaysia.
cSection of Virology, Faculty of Medicine, Imperial College London, St. Mary’s Campus, Norfolk Place, London W2 1PG United Kingdom.
dDepartment of Veterinary Pathology and Microbiology, Faculty of Veterinary Medicine, Universiti Putra Malaysia, 43400 UPM, Serdang,
Selangor, Malaysia.
Received 27th Nove mber 2018 / Accepted 4th February 2019
Fowlpox virus (FWPV) has been used as a recombinant vaccine vector to express antigens
from several important avian pathogens. Attempts have been made to improve vaccine strains induced-
host immune responses by coexpressing cytokines. This study describes the construction of recombinant
FWPV (rFWPV) strain FP9 and immunological responses in specific-pathogen-free (SPF) chickens, co-
expressing avian influenza virus (AIV) H5 of A/Chicken/Malaysia/5858/2004, and chicken IL-15
cytokine genes. Expression of H5 (50 kD) was confirmed by western blotting. Anti-H5 antibodies, which
were measured by the haemagglutinin inhibition test, were at the highest levels at Week 3 post-inoculation
in both rFWPV/H5- and rFWPV/H5/IL-15-vaccinated chickens, but decreased to undetectable levels
from Week 5 onwards. CD3+/CD4+ or CD3+/CD8+T cell populations, assessed using flow cytometry,
were significantly increased in both WT FP9- and rFWPV/H5-vaccinated chickens and were also higher
than in rFWPV/H5/IL-15- vaccinated chickens, at Week 2. Gene expression analysis using real time
quantitative polymerase chain reaction (qPCR) demonstrated upregulation of IL-15 expression in all
vaccinated groups with rFWPV/H5/IL-15 having the highest fold change, at day 2 (117±51.53). Despite
showing upregulation, fold change values of the IL-18 expression were below 1.00 for all vaccinated
groups at day 2, 4 and 6. This study shows successful construction of rFWPV/H5 co-expressing IL-15,
with modified immunogenicity upon inoculation into SPF chickens.
Keywords: avian influenza virus, fowlpox virus, haemagglutinin, interleukin-15, interleukin-18
Since the late 1980s, recombinant FWPVs
(rFWPV)s based on attenuated FWPV strains
have been developed to express antigens from
several important avian pathogens, including:
avian influenza virus (AIV; (Qian et al., 2012)),
Newcastle disease virus (NDV; (Sun et al., 2008)
and Marek’s disease virus (MDV; (Lee et al., 2003).
*Author for correspondence: Abdul Razak Mariatulqabtiah, Department of
Cell and Molecular Biology, Faculty of Biotechnology and Biomolecular
Sciences, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor,
Malaysia. Email
rFWPVs expressing haemagglutinin (HA) H5
protein of AIV (rFWPV/H5), particularly derived
from A/Turkey/Ireland/83 (H5N9), or
A/Goose/Guangdong/96 (H5N1), have been
used in South East Asia as vaccines against highly
pathogenic avian influenza (HPAI) H5N1.
Despite this preventive measure, HPAI H5N1 is
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
still a major concern due to its ongoing, sporadic
re-emergence. The need to boost existing
eradication efforts to limit the spread and
occurrence of the outbreak has prompted
development of several strategies to improve
readily available avian influenza vaccines. We
describe here one such strategy: to co-express
host cytokines from rFWPV/H5.
In mice, recombinant vaccinia virus (rVACV)
co-expressing gp160 of human immunodeficiency
virus (HIV) and human interleukin 15 (hIL-15)
has been shown to provide a stronger and more
enduring response than rVACV expressing gp160
alone (Oh et al., 2003). Integration of hIL-15 into
rVACV Wyeth strain or Modified VACV Ankara
(MVA) resulted in better survival (Perera et al.,
2007) and enhanced in vivo viral clearance
(Zielinski et al., 2010) in vaccinated athymic nude
mice upon intranasal challenge with virulent
VACV strain Western Reserve, or intravenous
challenge with monkeypox virus strain Zaire 79,
respectively. Enhanced CD4 and CD8 T cell
memory responses, along with reduction in lung
mycobacterial load in lungs, was also observed in
mice infected with Bacille Calmette-Guérin
(BCG), supplemented with IL-7 and IL-15
recombinant proteins, but not IL-1, IL-6 or
interferon (IFN)-α (Singh et al., 2010). In mice
model, it has also been shown that IL-15 offers
potent antiviral effects against rVACV
coexpressing IL-15, with high dependency on the
presence of NK cells and IFNs (Foong et al.,
Almost all of the chicken cytokines that have
been investigated are Th1-like. In a rare avian
study, in ovo plasmid DNA vaccination against an
intestinal coccidial parasite, Eimeria acervulina,
using coccidial gene 3-1E coexpressed with
chicken IL-15, was shown to induce higher serum
antibody levels than immunization with 3-1E
alone. Following challenge with the homologous
parasite, chickens vaccinated with 3-1E plus IL-
15 showed a significant decreased in oocyst
shedding and had an increased body weight,
compared to chickens vaccinated with 3-1E alone
(Lillehoj et al., 2005). Similar results were obtained
whether the construct was given subcutaneously
(Min et al., 2001) or intramuscularly (Ma et al.,
Studies with rFWPV coexpressing HA from
AIV H5N1 and chicken IL-18 (Chen et al., 2011;
Mingxiao et al., 2006) or IL-6 (Qian et al., 2012)
have been described. The results showed that all
chickens vaccinated with rFWPV/H5/IL-18
exhibited reduced virus shedding and replication
(Chen et al., 2011), and had higher levels of cellular
immunity (Mingxiao et al., 2006), compared to
rFWPV/H5 alone. Study of the effect of chIL-15
coexpression by rFWPV/H5 in chickens, as
reported here, is novel.
Ethical approval.
All animal experiments
performed in this study were in accordance with
the ethical standards of the local Institutional
Animal Care and Use Committee (IACUC) of
Universiti Putra Malaysia (UPM) with reference
number UPM/FPV/PS/
Viruses and cells.
The initial stock of parental
FP9 was from M.A. Skinner laboratory (Imperial
College London, UK). The development of FP9
via 438 serial passages of the wild-type fowlpox
virus HP-1, followed by plaque purification, has
been described (Laidlaw and Skinner, 2004).
Chicken embryonic fibroblast (CEFs) used in this
study were cultured in 2% newborn bovine serum
(NBBS) in DMEM media (both from Gibco).
Construction of recombinant plasmids.
Previously cloned and sequenced cDNA
encoding full-length H5 of influenza strain
A/Ch/Malaysia/5744/2004 (Balasubramaniam et
al., 2011) was amplified by PCR with primers H5-
TGCAAATTCTGC-3’, introducing EcoRV sites
as underlined. Sequence encoding the pentabasic
peptide motif at the protease cleavage site of H5
was replaced with threonine (T) using mutagenic
The assembled amplicon was inserted into the
SmaI site of lac Z-selectable, FWPV
expression/recombination vector pEFL29
(Qingzhong et al., 1994), downstream of a copy of
the vaccinia virus p7.5 early/late promoter. The
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
chicken IL-15 gene (supplied by the late Prof. Dr.
Pete Kaiser from the then Institute for Animal
Health, Compton, UK) was inserted downstream
of a synthetic/hybrid promoter in vector
pEFgpt12S, before being subcloned into vector
pPC1.X (Abd Razak, 2011). Positive
transformants were grown in LB broth (15 mL)
supplemented with appropriate antibiotic(s) (750
µg) at 37°C overnight. The culture (0.5 µL) was
used to provide templates for analytical PCR. The
reaction mixture for a small scale PCR verification
contained 10X PCR buffer (2 µL; Sigma),
JumpStart Taq DNA polymerase (0.5 U; Sigma),
dNTPs (0.5 µL of 10 mM) and oligonucleotide
primers (0.5 µL of each 10 µM stock), in a total
volume of 20 µL. PCR was conducted in 2 steps;
4 cycles of 95°C for 3 min, 95°C for 30 s, 50°C
for 30 s, and 72°C for 1.5 min followed by 26
cycles of 95°C for 30 s, 59°C for 30 s, and 7°C for
1.5 min. Final extension was operated at 7C for
10 minutes. Verification of H5 integration into
FWPV was carried out using H5-F and H5-R
primers, while primers pPC1.X-F: 5’-
GAGG-3’ were used to screen for rFWPV/H5
carrying IL-15 gene.
Recombination/transfection, selection and
purification of recombinant viruses.
detailed protocol for recombination/transfection
has been described (Laidlaw and Skinner, 2014),
with minor modifications i.e. replacing 199 media
with DMEM (Gibco), and polyfect with lipofectin
(Thermo Fisher Scientific), of the same volume.
Successful recovery of rFWPV/H5 carrying a
LacZ gene from pEFL29 into FP9 was
demonstrated by blue plaques upon X-Gal
overlay (at final concentration of 0.4 mg/mL) on
day 4 post-transfection. Further screening for
rFWPV/H5 carrying chIL-15 was done by
mycophenolic acid (MPA) selection of gpt gene
and spontaneous resolving of the gpt gene by a
second crossover event, as described previously
(Laidlaw et al., 1998). The recombinant protein
lysates were prepared by infecting CEFs, with
rFWPV/H5 at a multiplicity of infection (MOI)
of 3, for 48 hours. The cell pellet was subjected to
15% SDS-PAGE. The electro-transferred
nitrocellulose membrane (GE Healthcare) was
incubated with a goat polyclonal primary antibody
against haemagglutinin H5 (Cat. No. ab62587,
Abcam, USA) with the final concentration 1
µg/µL, for 1 hour. The membrane was developed
using a commercial kit using the chromogenic
substance, WesternBreeze (Invitrogen).
Immunofluorescence antibody test (IFAT)
was performed using 80% confluent CEFs. Cells
were either infected with viruses at 0.3 MOI, or
left uninfected (negative control). The infection
was left overnight in 2% NBBS DMEM medium,
before incubation with a rabbit polyclonal primary
antibody against haemagglutinin H5 (Cat. No.
ab70077, Abcam, USA) with the final
concentration 1 µg/µL, for 2 hours. After three
washes with PBS, cells were incubated with
fluorescein-labelled secondary antibodies for 1
hour. Slides were viewed under a fluorescent
microscope (model Leica DMRA II).
Immunization of animals.
One-day old specific
pathogen-free (SPF) chickens were inoculated
subcutaneously with 105 plaque forming unit
(PFU) of parental FWPV FP9 (WT FP9),
rFWPV/H5 or rFWPV/H5/IL-15, diluted in
PBS to a total volume of 100 μL, at the scruff of
the neck, using a 27-G needle. One control group
was mock-treated with 100 μL of PBS. Nine
chickens were assigned for each group. Blood
sampling (for serum) of each chicken was done on
a weekly basis. At Weeks 2 and 5, whole blood
(0.2 mL) of each chicken in each group of nine
was sampled and pooled into 3 groups (0.6 mL in
total), for CD4+ and CD8+ lymphocyte isolation,
followed by flow cytometry analysis. As for IL-15
and IL-18 gene expression analysis, immunization
of 105 PFU of aforementioned vaccine groups
was done on 14-days old SPF chickens; twelve
chickens for each group. At every two consecutive
days’ post immunization, RNA was extracted
from the spleens (four from each group) and
processed for qPCR.
Serological tests.
Haemagglutination inhibition
(HI) tests were performed in U-bottomed 96-well
microtitre plates using 4 HA units/25 μL of
H5N2 virus strain A/Malaysia/Duck/8443/04
(Veterinary Research Institute Ipoh, Malaysia),
and washed chicken erythrocytes (25 μL of 0.8%
v/v). The antigen-antibody was incubated for 1
hour. HI titres were determined as the reciprocal
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
of the highest serum dilution that completely
inhibited haemagglutination.
Immunophenotyping analysis.
Fresh, non-
coagulated chicken whole blood was diluted to 1
mL using cold PBS and was carefully layered on 2
mL Ficoll-Paque PLUS (GE Healthcare).
Isolation of peripheral blood mononuclear cells
(PBMC) was done by following the standard
Ficoll-Paque PLUS protocol. Approximately 106
cells were incubated with mouse anti-chicken
CD8a-PerCP-Cy5-conjugated (1 µg/mL), CD3-
PE-conjugated (0.5 µg/mL) and CD4-FITC-
conjugated (0.5 µg/mL) monoclonal antibodies
(all from Southern Biotech), prior to analysis
using a BD FACSCalibur flow cytometer (Becton
Dickinson, San Jose, CA, USA).
Quantitative real time polymerase chain
reaction (qPCR
). The total RNA from chicken
spleens was harvested using TRIzol reagent
(Ambion) according to manufacturer’s
recommendations. The extracted RNA was
reverse-transcribed using Script cDNA synthesis
kit (Jena Bioscience) in a total volume of 20 μL
containing 2.5 μM primers, 1X Script reverse
transcriptase (RT) buffer, 500 μM dNTP, 5 μM
DTT stock, 40 units RNAse inhibitor, 100 units
Script RT and 5 μg RNA template. The reaction
mix was incubated at 42oC for 10 minutes
followed by 50°C for 60 minutes. Primer
sequences for cytokines IL-15 and IL-18, and a
housekeeping gene, glyceraldehyde 3-phosphate
dehydrogenase (GAPDH), were designed from
public databases (Brisbin et al., 2010; Cai et al.,
2009), as shown in Table 1. The qPCR
amplification was performed according to the
KAPA SYBR FAST qPCR kit (KAPA Biosystem)
using Bio-Rad CFX96 real-time system. The data
was imported into the analysis module of the Bio-
Rad CFX Manager. The expression of GAPDH
gene were used as the qPCR normalization
standards. All results are reported as delta-delta
CT (ΔΔCT), relative to the control group.
Statistical analysis.
Data variations between
groups were analysed by one-way ANOVA or
paired-samples T test using SPSS (Version 15)
software. Results were expressed as the mean ±
standard error of the mean (SE). P values less than
0.05 were considered statistically significant in all
Table 1. Forward and reverse primer sequences used for qPCR.
Primer name
Primer sequence (5’ to 3’)
IL-15 - F
IL-15 - R
IL-18 - F
IL-18 - R
F, forward; R, reverse; IL, interleukin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.
PCR amplification of H5 gene.
haemagglutinin (HA) gene of the H5 virus
contains multiple basic amino acids, arginine and
lysine, that allow cleavage by ubiquitous proteases
(furin and PC6) (Horimoto et al., 1994). To
maintain compatibility with recombinant, killed
H5N1 influenza vaccines (J. Wood, personal
communication), and to reduce any potential
biosafety issues, the pentabasic peptide motif
(underlined) at the protease cleavage site of H5, S-
P-Q-R-E-R-R-R-K-K-R was removed and
replaced with threonine (T), leaving a monobasic
arginine (R) at the site (S-P-Q-R-E-T-R). The H5-
F and S(1-R) primers generated the first H5
fragment (1036 bp), while primers H5-R and S(2-
F) generated the second H5 fragment (684 bp).
Full length mutated H5 gene (1695 bp), was
obtained through PCR overlap extension
mutagenesis (Figure 1a).
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
Figure 1. (a) PCR amplification of H5 gene upon removal of polybasic sequence. Lane 1: Negative control;
Lane 2: Full length HA H5 gene (1707 bp); Lane 3: Fragment 1 (1036 bp) and Lane 4: Fragment 2 (684
bp) were generated using mutagenic primers S(2-F) and S(1-R); Lane 5: Re-assembled full length H5
sequence, lacking the polybasic region (1695 bp) was obtained using PCR overlap extension mutagenesis.
(b) Verification for the integration of the IL-15 gene into rFWPV/H5 by PCR after genomic DNA
extraction, with exclusive amplicon of 700 bp which corresponds to the positive control. Lanes 1-8:
Genomic DNA of recombinant clones rFWPV/H5 after second homologous recombination. Lane 9: IL-
15 plasmid positive control. M1: 1 kb ladder marker (New England Biolabs); M2: 100 bp ladder marker
(New England Biolabs). The DNA bands were observed in 1% (w/v) agarose gel.
Verification of H5-recombinant fowlpox
viruses coexpressing chicken IL-15.
successful transfection, the recombinant clones
were verified for presence of the inserted H5 gene
by PCR of extracted FWPV genomic DNA (data
not shown). Positive recombinants (rFWPV/H5)
were subjected to second homologous
recombination of vector pPC1.X carrying chicken
cytokine gene IL-15 at a second non-essential site,
the PC-1 (fpv030) homology region. The cytokine
expression cassettes in pPC1.X/IL-15 were
previously confirmed by restriction digests and
sequencing (data not shown). Screening of
positive recombinant viruses (rFWPV/H5/IL-
15) was done using primers external and internal
to the inserted genes (the latter resulting in PCR
products exclusively for recombinant clones)
(Figure 1b). H5 protein expression was analysed
by western blotting (Figure 2). A faint band at ~50
kD was observed for H5 recombinant, none for
uninfected cell lysate and negative control (WT
FP9). This is the first report on the size of H5
protein from strain A/Ch/Malaysia/5744/2004.
Further analysis using IFAT detected fluorescent
signals only for CEF infected with H5
recombinant, which indicates successful H5
protein expression. No reactivity was observed
for uninfected or WT FP9-infected CEF (Figure
Figure 2. Western blot analysis of CEF cells
infected with rFWPV/H5. Transferred
nitrocellulose membrane was incubated with a
goat polyclonal primary antibody against
haemagglutinin H5 (Cat. No. ab62587, Abcam,
USA) with the final concentration 1 µg/µL, for 1
hour and separated on 12% SDS-PAGE. M:
BenchmarkTM Pre-stained Protein Ladder
(Thermo Fisher Scientific); Lane 1: Uninfected
CEF as negative control; Lane 2: CEF infected
with WT FP9; Lane 3: CEF infected with
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
Figure 3. IFAT analysis for verification of H5
protein expression from rFWPV/H5. CEFs were
(A) uninfected; (B) infected with WT FP9 as
negative control; (C) infected with rFWPV/H5.
Infected cells were incubated with a rabbit
polyclonal primary antibody against
haemagglutinin H5 (Cat. No. ab70077, Abcam,
USA) with the final concentration 1 µg/µL.
Observation was performed under visible light (ii)
or UV light (ii). The images did not represent 80%
of cell confluency due to repeated washing of the
cells without fixation during procedure.
Haemagglutinin inhibition tests for chickens
following rFWPV immunizations.
None of the
nine control chickens inoculated with PBS or WT
FP9 showed any evidence of HI antibody
responses. Mean HI titres, in log2, of all groups
were calculated for general comparison (Table 2).
H5 antibodies reached detectable levels in
chickens vaccinated with rFWPV/H5/IL-15 one
week earlier than those vaccinated with
rFWPV/H5 but, thereafter, there was no
significant difference between the two groups.
Responses were highest at Week 3 in both groups
of recombinant vaccine-treated chickens.
However, the antibodies were undetectable based
on HI tests that have been carried out from Week
5 onwards.
CD3+/CD4+ and CD3+/CD8+ T cells
population following rFWPV immunizations.
The levels of CD3+/CD4+ T cells in the control
group remained relatively constant at Weeks 2 and
5. Samples from groups vaccinated with WT FP9
or rFWPV/H5/IL-15 demonstrated increases in
CD3+/CD4+ T cell population levels over time,
of 2.06 and 3.16 point percentages, respectively.
The rFWPV/H5 vaccinated group showed a
significantly higher CD3+/CD4+ T cell
population relative to control at Week 2 (P≤0.05)
but had returned to control levels by Week 5. No
statistically significant difference in CD3+/CD4+
T cell levels was observed for other groups at
either sampling point (Figure 4).
Animal experiments also revealed a relatively
constant CD3+/CD8+ T cell population for
control chickens. The same was true for the slight
to somewhat higher levels observed in
rFWPV/H5/IL-15-, WT FP9- and rFWPV-
vaccinated birds (significant for WT- and
rFWPV/H5- but not rFWPV/H5/IL-15-
vaccinated birds), although a fall to control levels
was observed in rFWPV/H5-vaccinated birds at
Week 5.
Table 2. Mean of HI titre, log2, of sera from immunized chickens.
Vaccine group
HI titre
Weeks, post immunization
ND indicates undetected titre. Each value represents the means±SE of nine birds.
No significant difference was observed between rFWPV/H5 and rFWPV/H5/IL-15 at any time point (P≥0.05).
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
Figure 4. Immunophenotyping of CD3+/CD4
(a) and CD3+/CD8+ (b) lymphocytes from
chickens after mock-treatment with PBS
(control), or vaccination with WT FP9,
rFWPV/H5 or rFWPV/H5/IL-15. Each value
represents the mean percentages of T
lymphocytes sub-population ± SE, from PBMC
samples of nine chickens pooled in threes (n=3),
sampled at Weeks 2 and 5. Significant differences
between vaccinated and control groups (*), were
determined by one-way ANOVA (P≤0.05).
Significant differences within the same group at
different points were determined by paired-
samples T-test (P≤0.05).
Gene expression analysis of IL-15 and IL-18
IL-15 expression in all vaccinated groups showed
upregulation on day 2, notably in those vaccinated
with rFWPV/H5/IL-15, which overexpress
chicken IL-15 (Figure 5). WT FP9- and
rFWPV/H5-vaccinated birds expressed 7- to 19-
fold more IL-15 than control birds; over-
expression by rFWPV/H5/IL-15 boosted IL-15
levels to 120 fold more than control. Expression
of IL-15 dropped to control levels by day 4, for all
tested groups.
IL-18 expression was lower in all of the
FWPV-infected groups (two to five fold lower for
WT FP9- and rFWPV/H5-vaccinated groups,
possibly up to ten fold lower for rFWPV/H5/IL-
15) and this decreased expression was extended
out to 6 days.
Figure 5. Relative expression level, by qPCR, of
IL-15 (a) and IL-18 genes (b) in inoculated SPF
chickens compared to control SPF chickens. The
expression was expressed as fold change (2 log -
∆∆CT) to that of the unvaccinated controls after
normalization of expression to GAPDH. The
standard errors were calculated from the result of
three replicates. Significant differences between
vaccinated groups and WT FP9 (*) were
determined by paired-samples T-test (P≤0.05).
The most important component of host immune
response that confers protection in chickens
against AIV is the humoral response against HA
(Swayne, 2007). To achieve this, several different
types of vaccines have been developed, e.g.
inactivated AIV vaccines (Bublot et al., 2007; Tian
et al., 2010), DNA vaccines (Lim et al., 2012), and
virus-like particles (Hendin et al., 2017). In this
study, a safe, lab-adapted FWPV-based vector
expressing the H5 of AIV was modified to co-
express a chicken IL-15 cytokine gene to test if it
would enhance the host cell mediated immune
response, which may be critical in clearance of
AIV during infection (Foong et al., 2009).
However, we did not perform protective or
challenge studies for AIV, nor did we evaluate
protection against FWPV.
Vaccination with rFWPV/H5/IL-15
produced HA antibody titres comparable to
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
vaccination with rFWPV/H5. This finding
contrasts with several studies conducted in mice,
including that by Perera et al. (2007) which
reported induction of two-fold higher VACV-
neutralizing antibody titres in hIL-15-expressing
recombinant VACV. The group also showed that
recombinant VACV strain Wyeth, expressing five
heterologous influenza virus genes, induced
stronger neutralizing antibodies against AIV H5
when adjuvanted with hIL-15 (Poon et al., 2009).
The inconsistencies in HA antibody titres
between our study and those by Perera et al. (2007)
and Poon et al. (2009) might be due to the usage
of heterologous (instead of a homologous) H5N2
virus strain A/Malaysia/Duck/8443/04 antigens
against the H5 antibodies from our rFWPV
recombinants. Heterologous antigens used to
assay H5 antibodies induced by rFWPV were
shown to produce either low (Taylor et al., 1988),
highest (Bublot et al., 2010) or inconsistent
(Swayne et al., 2007) HI titres. Although these
studies did not use homologous antigens, which
might be more suitable for their HI testing, the
results provide useful comparisons of HI
antibody levels elicited by rFWPV/H5 and
rFWPV/H5/IL-15. Several studies have shown
rFWPV expressing H5 can provide complete or
nearly complete protection against lethal
challenge, even when achieving pre-challenge HI
titres of as low as 3 log2 (Bublot et al., 2010;
Webster et al., 1991). Post-vaccination protection
of chickens against AIV may not be dependent
entirely on HI antibodies but also on non-HI
antibodies and possibly also on cell-mediated
rFWPV/H5/IL-15 did not increase CD4+ T
cell populations, compared to rFWPV/H5,
following vaccination. This finding is consistent
with previous reports that IL-15 only has
profound effects on the proliferation and survival
of memory CD8+ T cells, not on CD4+ T cells
(Marks-Konczalik et al., 2000; Zhang et al., 1998),
although a significant increment of CD4+ T cell
populations was observed in a DNA vaccine
coexpressing H5 and chicken IL-15 genes (Lim et
al., 2012). It is not known whether inherent
molecular patterns of, or immunomodulatory
proteins expressed by, FWPV FP9 can influence
IL-15 levels in vaccinated chickens. It has been
reported that IL-15 can only activate CD4+ T cell
proliferation when at high concentration presence
(Kanegane and Tosato, 1996). Niedbala et al.
(2002) showed that 2 to 4 fold higher
concentrations of IL-15 are required to achieve
optimal CD4+ T cell proliferation than to
promote CD8+ T cell response.
The co-stimulatory effects of IL-15 on CD8
cells have been studied widely, especially with
regard to proliferation and survival of memory
CD8+ T cells. IL-15 has been found to directly
stimulate purified CD8+ memory cells in vitro
(Zhang et al., 1998). Transgenic mice which
constitutively expressed a significant level of IL-
15 in the serum had higher numbers of memory
CD8+ T cells (Marks-Konczalik et al., 2000;
Yajima et al., 2002). In our study, chickens
vaccinated with WT FP9 or rFWPVs showed low
to moderate increase in levels of CD8+ T cells.
The increases, at 1.6 to 2 fold, were significant for
WT FP9 or rFWPV/H5 respectively but, at 1.25
fold increment, was insignificant from the
rFWPV/H5/IL-15. These results suggest that
FWPV enhances chicken CD8+ T cells
stimulation and possibly that IL-15 has the
opposite effect.
Although hIL-15 has been shown to stimulate
CD8+ T cells population and promote the
maintenance of CD8+ CD44hi memory T cells,
the responsiveness of CD8+ T cells to IL-15
might depend on the cytokine background
(Niedbala et al., 2002; Oh et al., 2003).
Unfortunately, in this study, we did not measure
the levels of IL-15, secreted by cells infected with
an initial dose of 105 PFU rFWPV/H5/IL-15, in
peripheral blood prior to flow analysis. Since a
strong synthetic/hybrid promoter was used for
IL-15 co-expression, levels of expression might
have been inconsistent with generation of the
desired immune responses.
We observed elevation of the CD4+ T cell
population and sustained CD8+ T cell population
from WT FP9 and rFWPV/H5/IL-15 inoculated
groups. However, rFWPV/H5 inoculated group
showed a consistent decreasing pattern for both
T cells. By way of comparison, an in vivo study
examining T cell populations in the peripheral
blood of rhesus macaques treated with rhesus IL-
15, where the level of CD4+ and CD8+ memory,
but not naïve, T cells peaked at Weeks 1 to 2 and
returned to baseline by Weeks 3 to 4 (Picker et al.,
Acting synergistically, IL-15 and IL-18 can
AsPac J. Mol. Biol. Biotechnol. Vol. 27 (1), 2019 Coexpression of AIV H5 and IL-15 in fowlpox viruses
perpetuate Th1 responses (Gracie et al., 1999) and
enhance IL-12 stimulation of NK cell to produce
IFN gamma (French et al., 2006). A DNA vaccine
co-expressing H5 and chicken IL-15, induced a
significant increase in IL-15, but not IL-18, levels
post-vaccination (Lim et al., 2012). Our results are
comparable, where enhanced expression of host
IL-15 and reduced expression of host IL-18 are
mediated directly by infection with WT FP9 or
rFWPV/H5 co-expressing exogenous IL-15
(mediated by a strong synthetic poxvirus
The dramatic drop of IL-15 levels from day 2
to day 4 in all FWPV-infected groups might be
due to clearance of these attenuated viruses by
NK cells, their cytolytic activity potentially
augmented by the ability of IL-15 (expressed
endogenously by the host or exogenously by the
recombinant FWPV) to enhance IFN expression
and increase poxvirus clearance (Foong et al.,
2009). However, we cannot currently explain the
concomitant drop in IL-18 mRNA expression
during FWPV infection but FWPV appears to
express one or more IL-18 binding proteins
(Laidlaw and Skinner, 2004), which might reduce
steady-state levels of circulating IL-18 in a similar
manner to the host-encoded regulator IL-18BP
(Harms et al., 2017). It is possible therefore that
the virus encodes additional mechanisms to
down-regulate expression of IL-18 mRNA.
rFWPV/H5 and rFWPV/H5/IL-15 inoculated
groups elicited the highest levels of anti-H5
antibodies at Week 3 post-inoculation.
CD3+/CD4+ or CD3+/CD8+ T cell
populations were significantly increased in both
WT FP9- and rFWPV/H5-, higher than in
rFWPV/H5/IL-15-vaccinated chickens, at Week
2 post-inoculation. IL-15 and IL-18 expressions
were upregulated in all vaccinated groups at day 2
post-inoculation. These diverse immunogenicity
findings may contribute to the limited exploration
of chicken IL-15 in vaccine developments.
This study was supported by the Ministry of
Energy, Science, Technology, Environment and
Climate Change (MESTECC) with project
number 02-01-04-SF1641, and the Ministry of
Education through Institute of Bioscience,
Higher Institution Centre of Excellence (IBS
HICoE) with project number 6369101, from the
Government of Malaysia. N.N.M. was funded by
MyBrain15 of Ministry of Education, Malaysia,
and Graduate Research Fellowship (GRF) of
Universiti Putra Malaysia. M.A.S. and E.S.G. are
supported by the Biotechnology and Biological
Sciences Research Council via Strategic LoLa
BB/K002465/1 (“Developing Rapid Responses
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... Due to this ease of observing and propagating embryonated chicken eggs and embryo fibroblast (CEF) monolayer cultures, vaccines against FWPV were among the first to be licensed . The attenuated, live FWPV vaccine strains, which were developed during the 1920s, have paved the way for their manipulation as recombinant vectors expressing antigens of significant avian pathogens, such as avian influenza virus (AIV; (Mariatulqabtiah et al., 2019;Majid et al., 2020) and Newcastle disease virus (Sun et al., 2008). Recombinant FWPV expressing the H5 gene of AIV (rFWPV-H5) has been commercially used in chickens since the 1990s. ...
... The initial stock of attenuated, lab-adapted WT FWPV strain FP9 was obtained from Dr. Mike Skinner (GenBank Accession No. AJ581527.1), while the rFWPV-H5 was constructed previously (Mariatulqabtiah et al., 2019). Both viruses were propagated in chorioallantoic membranes (CAMs) of 10-day-old specific-pathogen-free (SPF) chicken embryos and harvested 7 days later. ...
... AJ581527 (Laidlaw et al., 2013) and the H5 primer set (5′-ATG GAG AAA ATA GTG CTT TTT G-3′ and 5′-TTA AAT GCA AAT TCT GCA TTG TAA CG-3′; GenBank Accession no. DQ320934 (Mariatulqabtiah et al., 2019), respectively, from the extracted viral DNA. PCR amplification was performed based on the manufacturer's instructions from the MyTaq™ Red Mix (Bioline, Taunton, MA, USA). ...
... Chicken IL-12 gene was inserted downstream of a synthetic/hybrid promoter in vector pEFgpt12S, before being subcloned into vector pPC1.X (pPC1.X/IL-12). Selection of positive transformants using analytical PCR [28] was performed using primer sets: pEFL29-F (5′-CGGAGAC-CATATCCATACGC-3′) and pEFL29-R (5′-CGTA AAAG TAGA AAAT ATATTC-3′); and pPC1.X-F (5′-ATGA AAAA TAGT ACCA CTATGG-3′) and pPC1.X-R (5′-ATCC GATA CTAG TATT AGGTTAGC-3′). Cultures that yielded positive PCR amplicons were subjected to plasmid DNA isolation (QIAGEN Minipreparation Kit), restriction enzyme digestion and sequencing (data not shown). ...
... Cultures that yielded positive PCR amplicons were subjected to plasmid DNA isolation (QIAGEN Minipreparation Kit), restriction enzyme digestion and sequencing (data not shown). The detailed protocol for recombination/transfection has been described elsewhere [28,29]. Stocks of recombinant viruses were titrated using plaque assay prior to use. ...
... At weeks 2 and 5, whole blood (0.2 ml) of each chicken was sampled and pooled into three groups (0.6 ml in total for each tube). Isolation of peripheral blood mononuclear cells (PBMC) was done by following the standard protocol (GE Healthcare) and the pelleted cells were transferred into flow tubes for CD4+ and CD8+ immunophenotyping analysis using a BD FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA, USA) [28]. All chickens were weighed at week 1 until week 4. Differences between groups of chicken were analysed by one-way ANOVA pairedsample t-test using SPSS (version 15) software. ...
Full-text available
In comparison to the extensive characterization of haemagglutinin antibodies of avian influenza virus (AIV), the role of neuraminidase (NA) as an immunogen is less well understood. This study describes the construction and cellular responses of recombinant fowlpox viruses (rFWPV) strain FP9, co-expressing NA N1 gene of AIV A/Chicken/Malaysia/5858/2004, and chicken IL-12 gene. Our data shows that the N1 and IL-12 proteins were successfully expressed from the recombinants with 48 kD and 70 kD molecular weights, respectively. Upon inoculation into specific-pathogen-free (SPF) chickens at 105 p.f.u. ml-1, levels of CD3+/CD4+ and CD3+/CD8+ populations were higher in the wild-type fowlpox virus FP9 strain, compared to those of rFWPV-N1 and rFWPV-N1-IL-12 at weeks 2 and 5 time points. Furthermore, rFWPV-N1-IL-12 showed a suppressive effect on chicken body weight within 4 weeks after inoculation. We suggest that co-expression of N1 with or without IL-12 offers undesirable quality as a potential AIV vaccine candidate.
... Each deletion mutant virus was screened by PCR with flanking primers (giving PCR products of specific sizes for wild-type and knocked-out genes), one flanking primer and one primer internal to the deleted sequences (detecting only the wild-type gene) or one flanking primer and one primer specific for the GPT gene (detecting the insertion of GPT into the wild-type gene The FPV184 gene was amplified by PCR with the primers M2952 and M2951. The product was digested with XmaI and SacII (within M2952 and M2951, respectively) and cloned into the expression/transfer vector pEFgpt12S-CvectorEGFPmyc, which was derived from the vector pEFgpt12S [29][30][31] that was previously cloned with the coding sequence of EGFP (enhanced green fluorescent protein) from the pEGFP-C1 vector (Molecular Probes, Eugene, OR, USA) [29,31]. The cloning of the FPV184 gene into pEFgpt12S-CvectorEGFPmyc produced pCVecEGFP184 (EGFP at the N-terminus). ...
Full-text available
The avian pathogen fowlpox virus (FWPV) has been successfully used as a vaccine vector in poultry and humans, but relatively little is known about its ability to modulate host antiviral immune responses in these hosts, which are replication-permissive and nonpermissive, respectively. FWPV is highly resistant to avian type I interferon (IFN) and able to completely block the host IFN-response. Microarray screening of host IFN-regulated gene expression in cells infected with 59 different, nonessential FWPV gene knockout mutants revealed that FPV184 confers immunomodulatory capacity. We report that the FPV184-knockout virus (FWPVΔ184) induces the cellular IFN response as early as 2 h postinfection. The wild-type, uninduced phenotype can be rescued by transient expression of FPV184 in FWPVΔ184-infected cells. Ectopic expression of FPV184 inhibited polyI:C activation of the chicken IFN-β promoter and IFN-α activation of the chicken Mx1 promoter. Confocal and correlative super-resolution light and electron microscopy demonstrated that FPV184 has a functional nuclear localisation signal domain and is packaged in the lateral bodies of the virions. Taken together, these results provide a paradigm for a late poxvirus structural protein packaged in the lateral bodies, capable of suppressing IFN induction early during the next round of infection.
Pigeons (Columba livia) have been associated with humans for a long time now. They are raised for sport (pigeon race), exhibition (display of fancy breeds), food, and research. Most of the pigeons kept are Racing Homers, trained to compete in the pigeon race. Other breeds, such as Rollers, Nose Divers, Doneks are bred for their aerial abilities. Incorporation of a good preventive medicine program is one of the most critical factors in averting infectious diseases in pigeon flocks. This review summarizes the common bacterial, viral, and parasitic infections in pigeons. The different clinical signs, symptoms, diagnostic strategies, prevention, and treatments were described in this review. Current researches, molecular diagnostic assays, and treatment strategies such as vaccines and drug candidates were included. The information found in this review can provide insights for veterinarians and researchers studying pigeons to develop effective and efficient immunoprophylactic and diagnostic tools for pigeon diagnosis and therapeutics.
Full-text available
The cytokine interleukin (IL)-18 is a crucial amplifier of natural killer (NK) cell function. IL-18 signaling is regulated by the inhibitory effects of IL-18 binding protein (IL-18BP). Using mice deficient in IL-18BP (IL-18BPKO), we investigated the impact of mismanaged IL-18 signaling on NK cells. We found an overall reduced abundance of splenic NK cells in the absence of IL-18BP. Closer examination of NK cell subsets in spleen and bone marrow using CD27 and CD11b expression revealed that immature NK cells were increased in abundance, while the mature population of NK cells was reduced. Also, NK cells were polarized to greater production of TNF-α, while dedicated IFN-γ producers were reduced. A novel subset of IL-18 receptor α− NK cells contributed to the expansion of immature NK cells in IL-18BPKO mice. Splenocytes cultured with IL-18 resulted in alterations similar to those observed in IL-18BP deficiency. NK cell changes were associated with significantly reduced levels of circulating plasma IL-18. However, IL-18BPKO mice exhibited normal weight gain and responded to LPS challenge with a >10-fold increase in IFN-γ compared to wild type. Finally, we identified that the source of splenic IL-18BP was among dendritic cells/macrophage localized to the T cell-rich regions of the spleen. Our results demonstrate that IL-18BP is required for normal NK cell abundance and function and also contributes to maintaining steady-state levels of circulating IL-18. Thus, IL-18BP appears to have functions suggestive of a carrier protein, not just an inhibitor.
Full-text available
The construction of deletion-knockout poxviruses is a useful approach to determining the function of specific virus genes. This protocol is an adaptation of the transient dominant knockout selection protocol published by Falkner and Moss (1990) for use with vaccinia virus. The protocol makes use of the dominant selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) gene (Mulligan and Berg, 1981), under the control of an early/late poxvirus promoter. The deletion viruses that are produced no longer contain a selectable marker, which may be preferable for the production of vaccines.
Full-text available
Background: DNA vaccines offer several advantages over conventional vaccines in the development of effective vaccines against avian influenza virus (AIV). However, one of the limitations of the DNA vaccine in poultry is that it induces poor immune responses. In this study, chicken interleukin (IL) -15 and IL-18 were used as genetic adjuvants to improve the immune responses induced from the H5 DNA vaccination in chickens. The immunogenicity of the recombinant plasmid DNA was analyzed based on the antibody production, T cell responses and cytokine production, following inoculation in 1-day-old (Trial 1) and 14-day-old (Trial 2) specific-pathogen-free chickens. Hence, the purpose of the present study was to explore the role of chicken IL-15 and IL-18 as adjuvants following the vaccination of chickens with the H5 DNA vaccine. Results: The overall HI antibody titer in chickens immunized with pDis/H5 + pDis/IL-15 was higher compared to chickens immunized with pDis/H5 (p < 0.05). The findings revealed that the inoculation of the 14-day-old chickens exhibited a shorter time to achieve the highest HI titer in comparison to the inoculation of the 1-day-old chickens. The cellular immunity was assessed by the flow cytometry analysis to enumerate CD4+ and CD8 + T cells in the peripheral blood. The chickens inoculated with pDis/H5 + pDis/IL-15 demonstrated the highest increase in CD4+ T cells population relative to the control chickens. However, this study revealed that pDis/H5 + pDis/IL-15 was not significant (P > 0.05) in inducing CD8+ T cells. Meanwhile, with the exception of Trial 1, the flow cytometry results for Trial 2 demonstrated that the pDis/H5 + pDis/IL-18 inoculated group was able to trigger a higher increase in CD4+ T cells than the pDis/H5 group (P < 0.05). On the other hand, the pDis/H5 + pDis/IL-18 group was not significant (P > 0.05) in modulating CD8+ T cells population in both trials. The pDis/H5 + pDis/IL-15 inoculated group showed the highest IL-15 gene expression in both trials compared to other inoculated groups (P < 0.05). Similar results were obtained for the IL-18 expression where the pDis/H5 + pDis/IL-18 groups in both trials (Table 8) were significantly higher compared to the control group (P < 0.05). However, the expressions of other cytokines remained low or undetected by GeXP assay. Conclusions: This study shows the diverse immunogenicity of pDis/H5 co-administered with chicken IL-15 and IL-18,with pDis/H5 + pDis/IL-15 being a better vaccine candidate compared to other groups.
Interleukin-15 (IL-15), a product of monocytes and other cells, has biological activities similar to those of IL-2, including growth stimulation of activated T cells, induction of cytolytic effector cells, and B-cell costimulation for proliferation and lg production. We report that IL-15 at optimal concentrations rapidly induced memory (CD45RO+) CD4+ and CD8+ T cells and naive (CD45RO-) CD8+ T cells to express the CD69 activation marker followed by proliferation. By contrast, IL-15 failed to induce naive (CD45RO-) CD4+ T cells to express CD69 or to proliferate. Similar findings were obtained with IL- 2. Unlike the other T-cell subsets, CD4+ T cells with a naive phenotype expressed little or no IL-2R beta chain, a shared component of the IL-2 and IL-15 receptors required for receptor function. A monoclonal antibody to the IL-2R beta chain, Mik beta 1, reduced CD69 expression and proliferation in CD4+ memory, CD8+ memory, and CD8+ naive T cells activated by IL-15. These results confirm the biological similarities of IL-2 and IL-15. They further document that the pool of naive CD4+ cells, unlike the pool of memory CD4+, memory CD8+, and naive CD8+ cells, is not regulated directly by the T-cell growth factors IL-2 or IL-15.
Possessing a large double stranded DNA genome up to 300 kb, fowlpox virus (FWPV) has been developed to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to coexpress host cytokines from such recombinants. This thesis describes the strategy to construct H5N1-recombinant FWPV (rFWPV) coexpressing chicken Interleukin 12 (IL-12) or Interleukin 15 (IL-15), and discusses the immunogenicity of the recombinants following inoculation into specificpathogen-free (SPF) chickens. Previously cloned and sequenced cDNAs encoding full-length H5 and N1 of influenza strain A/Chicken/Malaysia/5858/2004 genes were amplified by PCR and inserted into plasmid pEFL29, under the control of a copy of the vaccinia virus p7.5 early/late promoter. The expression cassettes were recombined into the genome of the FP9 strain of FWPV at the fpv002 locus. Recombinant viruses were produced by transfection of the plasmid into chicken embryo fibroblasts (CEFs) after infection with FP9, and isolated by six fold plaque purification on CEFs using X-Gal selection. Chicken IL-12 or IL-15 genes, under control of a synthetic/hybrid poxvirus promoter, were inserted into a ‘transient dominant selection’ recombination plasmid, pPC1.X. The cytokine expression cassettes were then recombined, at the fpPC1 (fpv030) locus, into rFWPV already carrying AIV genes. Following three rounds of passage in CEFs in the presence of mycophenolic acid (MPA), recombinant viruses carrying the gpt gene were isolated. These unstable recombinants were plaque-purified in the absence of MPA until they lost the gpt gene spontaneously, verified by their failure to replicate in the presence of MPA. Recombinant proteins were successfully detected using western blotting and indirect immunofluorescence assay (IFAT). Parental and rFWPV (105 PFU) were inoculated subcutaneously into one-day-old SPF chickens. Sera from chickens immunized with rFWPV/H5 and rFWPV/H5/IL-15 demonstrated viral neutralizing activities, based on the haemagglutation inhibition (HI) test, in which reached a peak at Week 3. A competitive enzyme-linked immunosorbent (ELISA) assay detected N1-specific antibodies induced by rFWPV/N1 and rFWPV/N1/IL-12 at Weeks 4 and 5. Non-specific cellular immune responses were assessed by flow cytometric analysis to enumerate CD4+ and CD8+ T-lymphocytes in peripheral blood. Results of Experiment 2 showed chickens vaccinated with rFWPV/H5, rFWPV/H5/IL-15, rFWPV/N1 and rFWPV/N1/IL-12 demonstrated a higher increase in CD8+ than CD4+ T cell population, relative to control and chickens vaccinated with parental FWPV. Weekly weighing showed that chickens vaccinated with rFWPV/H5/IL-15 had the highest body weight compared to other groups, while the rFWPV/N1/IL-12 group showed the significantly lowest body weight. In summary, this study showed diverse immunogenicity of H5N1-rFWPV coexpressing IL-12 or IL-15. It also demonstrated a weight sparing effect of co-expressing IL-15 in rFWPV vaccines. The results provide the basis for future homologous challenge studies, using live H5N1 virus to evaluate the protective efficacy of the rFWPV vaccines.
Introduction: The recent emergence of avian influenza strains has fuelled concern about pandemic preparedness since vaccines targeting these viruses are often poorly immunogenic. Weak antibody responses to vaccines have been seen across multiple platforms including plant-made VLPs. To better understand these differences, we compared the in vitro responses of human immune cells exposed to plant-made virus-like particle (VLP) vaccines targeting H1N1 (H1-VLP) and H5N1 (H5-VLP). Methods: Peripheral blood mononuclear cells (PBMC) from healthy adults were stimulated ex vivo with 2-5µg/mL VLPs bearing the hemagglutinin (HA) of either H1N1 (A/California/7/2009) or H5N1 (A/Indonesia/5/05). VLP-immune cell interactions were characterized by confocal microscopy and flow cytometry 30min after stimulation with dialkylaminostyryl dye-labeled (DiD) VLP. Expression of CD69 and pro-inflammatory cytokines were used to assess innate immune activation 6h after stimulation. Results: H1- and H5-VLPs rapidly associated with all subsets of human PBMC but exhibited unique binding preferences and frequencies. The H1-VLP bound to 88.7±1.6% of the CD19(+) B cells compared to only 21.9±1.8% bound by the H5-VLP. At 6h in culture, CD69 expression on B cells was increased in response to H1-VLP but not H5-VLP (22.79±3.42% vs. 6.15±0.82% respectively: p<0.0001). Both VLPs were rapidly internalized by CD14(+) monocytes resulting in the induction of pro-inflammatory cytokines (i.e.: IL-8, IL-1β, TNFα and IL-6). However, a higher concentration of the H5-VLP was required to induce a comparable response and the pattern of cytokine production differed between VLP vaccines. Conclusions: Plant-made VLP vaccines bearing H1 or H5 rapidly elicit immune activation and cytokine production in human PBMC. Differences in the VLP-immune cell interactions suggest that features of the HA proteins themselves, such as receptor specificity, influence innate immune responses. Although not generally considered for inactivated vaccines, the distribution and characteristics of influenza receptor(s) on the immune cells themselves may contribute to both the strength and pattern of the immune response generated.
Eight chicken cytokine genes (IL-1β, IL-2, IL-8, IL-15, IFN-α, IFN-γ, TGF-β4, lymphotactin) were evaluated for their adjuvant effect on a suboptimal dose of an Eimeria DNA vaccine carrying the 3-1E parasite gene (pcDNA3-1E). Chickens were given two subcutaneous injections with 50μg of the pcDNA3-1E vaccine plus a cytokine expression plasmid 2 weeks apart and challenged with Eimeria acervulina 1 week later. IFN-α (1μg) or 10μg of lymphotactin expressing plasmids, when given simultaneously with the pcDNA3-1E vaccine, significantly protected against body weight loss induced by E. acervulina. Parasite replication was significantly reduced in chickens given the pcDNA3-1E vaccine along with 10μg of the IL-8, lymphotactin, IFN-γ, IL-15, TGF-β4, or IL-1β plasmids compared with chickens given the pcDNA3-1E vaccine alone. Flow cytometric analysis of duodenum intraepithelial lymphocytes showed chickens that received the pcDNA3-1E vaccine simultaneously with the IL-8 or IL-15 genes had significantly increased CD3+ cells compared with vaccination using pcDNA3-1E alone or in combination with the other cytokine genes tested. These results indicate that the type and the dose of cytokine genes injected into chickens influence the quality of the local immune response to DNA vaccination against coccidiosis.
A novel fusion cytokine was constructed by replacing signal peptide (SP) of chicken IL-15 (ChIL-15) with SP of chicken IL-2 (ChIL-2). The fusion cytokine (NChIL-15) was cloned into the expression vector pcDNA3.1(+) to generate pcDNA-NChIL-15. An animal experiment was carried out to evaluate the adjuvant effects of NChIL-15 on DNA vaccine pcDNA-3-1E against Eimeria acervulina challenge. The mRNA profiles of ChIL-2 and ChIFN-γ in spleen were characterized by means of real-time PCR. The recombinant positive eukaryotic expression plasmid pcDNA-NChIL-15 were constructed successfully. The protective effects provided by co-immunization with 100μg pcDNA-3-1E and 50μg pcDNA-NChIL-15, measured by relative body weight gain (BWG), average lesion score in duodenum and oocyst decrease ratio, showed no significant difference with 50μg pcDNA-ChIL-15 as an adjuvant on day 6 post infection (PI). However, chickens co-immunized with pcDNA-3-1E and pcDNA-NChIL-15 exhibited significant upregulated level of ChIL-2 and ChIFN-γ transcripts in spleen. Our original data suggests the constructed novel cytokine NChIL-15 could be a potential adjuvant used to enhance the immune protective effects, although the optimized dosage need to be explored further.
A cloned Eimeria acervulina gene (3-1E) was used to vaccinate chickens in ovo against coccidiosis, both alone and in combination with genes encoding interleukin (IL)-1, IL-2, IL-6, IL-8, IL-15, IL-16, IL-17, IL-18, or interferon (IFN)-gamma. Vaccination efficacy was assessed by increased serum anti-3-1E antibody titers, reduced fecal oocyst shedding, and enhanced body weight gain following experimental infection with E. acervulina. When used alone, anti-3-1E antibody titers were transiently, but reproducibly, increased at 2 wk and 3 wk posthatching in a dose-dependent manner. Similarly, significantly reduced oocyst shedding and increased weight gain were observed at relatively high-dose 3-1E vaccinations (>= 25 mu g/egg). Combined immunization with the 3-1E and IL-1, IL-2, IL-15, or IFN-gamma genes induced higher serum antibody responses compared with immunization with 3-1E alone. Following parasite infection, chickens hatched from embryos given the 3-1E gene plus the IL-2 or IL-15 genes displayed significantly reduced oocyst shedding compared with those given 3-1E alone, while 3-1E plus IL-15 or IFN-gamma significantly increased weight gain compared with administration of 3-1E alone. Taken together, these results indicate that in ovo immunization with a recombinant Eimeria gene in conjunction with cytokine adjuvants stimulates protective intestinal immunity against coccidiosis.
Ducks have played an important role in the emergence of H5N1 subtype of highly pathogenic avian influenza (HPAI), and the development of an effective vaccine against HPAI in ducks is a top priority. It has been shown that a recombinant fowlpox virus (FPV)-vectored vaccine can provide protection against HPAI in ducks. In this study, a recombinant fowlpox virus (rFPV-AIH5AIL6) coexpressing the haemagglutinin (HA) gene of the H5N1 subtype of the avian influenza virus (AIV) and chicken interleukin 6 gene was constructed and tested in Gaoyou and cherry valley ducks to evaluate the immune response in ducks. These animal studies demonstrated that rFPV-AIH5AIL6 induced a higher anti-AIV HI antibody response, an enhanced lymphocyte proliferation response, an elevated immune protection, and a reduction in virus shedding compared to a recombinant fowlpox virus expressing the HA gene alone (rFPV-SYHA). These data indicate that rFPV-AIH5AIL6 may be a potential vaccine against the H5 subtype of avian influenza in ducks and chicken interleukin 6 may be an effective adjuvant for increasing the immunogenicity of FPV-vectored AIV vaccines in ducks.