SHORT COMMUNICATION
Immune responses of chickens inoculated with a recombinant
fowlpox vaccine coexpressing glycoprotein B of infectious
laryngotracheitis virus and chicken IL-18
Hong-Ying Chen1, Pei Cui2, Bao-An Cui1, He-Ping Li1, Xian-Qin Jiao1, Lan-Lan Zheng1, Guo Cheng2
& An-Jun Chao1
College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan Province, China; and 2Henan Center for
Animal Disease Control & Prevention, Animal Husbandry Bureau of Henan Province, Zhengzhou, Henan Province, China
IMMUNOLOGY & MEDICAL MICROBIOLOGY
1
Correspondence: Bao-An Cui, College of
Animal Science and Veterinary Medicine,
Henan Agricultural University, Wenhua Road
95#, 450002 Zhengzhou, Henan Province,
China. Tel.: +86 371 63558878; fax:
+86 371 63558878; e-mail:
baoancui@henau.edu.cn
Received 16 January 2011; revised 5 June
2011; accepted 15 July 2011.
Final version published online 26 August
2011.
DOI: 10.1111/j.1574-695X.2011.00850.x
Editor: Willem van Eden
Keywords
chicken interleukin-18; gB gene; infectious
laryngotracheitis virus; recombinant fowlpox
virus.
Abstract
Infectious laryngotracheitis virus (ILTV) is an alphaherpesvirus that causes
severe and economically significant respiratory disease in poultry worldwide.
Herein, the immunogenicity of two recombinant fowlpox viruses (rFPV-gB
and rFPV-gB/IL18) containing ILTV glycoprotein B (gB) and chicken interleukin-18 (IL-18) were investigated in a challenge model. One-day-old specificpathogen-free chickens were vaccinated by wing-web puncture with the two
rFPVs and challenged with the virulent ILTV CG strain. There were differences
in antibody levels elicited by either rFPV-gB/IL18 or rFPV-gB as determined
using ELISA. The ratios of CD4+ to CD8+ in chickens immunized with rFPVgB/IL18 were higher (P < 0.05) than in those immunized with rFPV-gB, and
the level of proliferative response of the T cells in the rFPV-gB/IL18-vaccinated
group was higher (P < 0.05) than that in the rFPV-gB group. All chickens
immunized with rFPV-gB/IL18 were protected (10/10), whereas only eight of
10 of the chickens immunized with the rFPV-gB were protected. The results
showed that the protective efficacy of the rFPV-gB vaccine could be enhanced
by simultaneous expression of chicken IL-18.
Infectious laryngotracheitis (ILT) is a dramatic disease of
the upper respiratory tract in poultry, which is caused by
an alphaherpesvirus, infectious laryngotracheitis virus
(ILTV) (Guy & Bagust, 2003). The clinical signs range
from mild to severe, with mortality rates that reach up to
70% depending on the virulence of the infecting virus
(Davidson et al., 2009). The milder form of ILT is manifested with nasal discharges, conjunctivitis, and decreased
egg production, whereas in severe forms, the clinical signs
include respiratory depression, coughing, expectoration of
bloody mucus, and dyspnea up to suffocation and rapid
mortality. ILTV establishes lifelong latency in sensory
neurons of surviving animals, and its subsequent reactivation can lead to infection of naive chickens (Fuchs et al.,
2007; Jones, 2010). At present, ILT is controlled by widely
utilizing attenuated live viruses derived either by sequential passage in cell cultures or embryonated chicken eggs.
As immunogenicity of ILTV is usually correlated with its
FEMS Immunol Med Microbiol 63 (2011) 289–295
virulence, almost all modified live ILTV vaccines do not
remain sufficiently attenuated and have shown a variety
of side effects including spread of vaccine virus to nonvaccinated animals, occurrence of long-term ‘carrier’
birds, and increasing virulence during in vivo passages
(Dufour-Zavala, 2008). Therefore, attention has recently
turned toward developing novel vaccines with greater efficacy and fewer side effects.
Various strategies have been adopted to develop genetically engineered ILT vaccines. Okamura et al. (1994) successfully obtained a stable recombinant ILTV expressing
Lac-Z. Schnitzlein et al. (1995) reported the successful
construction of a thymidine kinase (TK)-negative ILTV
mutant. Recent studies of a gG-deletion mutant of ILTV
in specific-pathogen-free (SPF) chickens have shown that
vaccination with gG-deficient ILTV prevents disease following subsequent challenge with virulent virus (Devlin
et al., 2007, 2008). SPF chickens infected with gG-deficient
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
290
virus had altered tracheal leukocyte populations and
lower serum antibody levels compared with those infected
with the parent virus (Devlin et al., 2010). Pavlova et al.
(2010) reported that the innate and specific immune
responses against ILTV-DeltagC were not reduced, but
enhanced, and surviving chickens were protected completely against challenge infection. Furthermore, ILTVDeltagC might serve as a basis for marker vaccines
permitting differentiation between vaccinated and fieldvirus-infected animals. However, infection with high
doses of ILTV-DeltagC was still fatal for approximately
20% of the animals. To overcome this problem, subunit
preparations containing affinity-purified glycoprotein
were tested as alternatives and were found to be successful (York & Fashey, 1991). However, because of the high
costs of production and delivery, this vaccine is not
suitable for immunization of large chicken flocks. DNA
vaccines (Chen et al., 2010) that could induce specific
immune responses and have shown efficacy against
challenge virus are under development.
Fowlpox virus (FPV) has become widely used as an
effective live viral vector in vaccine development, successfully expressing protective foreign genes from various
poultry pathogens, including Newcastle disease virus
(Shen et al., 2007), avian influenza virus (Qiao et al.,
2009), infectious bronchitis virus (Wang et al., 2009),
ILTV (Tong et al., 2001), and Marek’s disease virus (Lee
et al., 2004). FPV is also easy to produce, and technologies for large-scale production are available. A recombinant fowlpox virus (rFPV) vaccine alone, however, is
limited in that it often generates only weak immune
responses, particularly the cellular response, in the
absence of suitable adjuvants (Leong et al., 1994;
Ma et al., 2008). Some cytokines such as IL-1 (Park et al.,
2006), IL-2 (Saade et al., 2008), IL-12 (Su et al., 2011),
interferon-gamma (IFN-c) (Ma et al., 2008), and granulocyte-macrophage colony-stimulating factor (Tan et al.,
2009), have been reported to be effective immunomodulators in animal models or clinical tests. Cytokine
adjuvants have been widely used to promote the induction
H.-Y. Chen et al.
of immune responses and enhance the immunoprotective
effects of rFPV vaccines (Noll & Autenrieth, 1996; Wang
et al., 2009; Su et al., 2011). Interleukin-18 (IL-18) is one
possible option, and is known as IFN-c-inducing factor
because of its ability to stimulate T helper 1 (Th1) cells to
secrete IFN-c (Schneider et al., 2000). Previous research
indicated that recombinant chicken IL-18 has the positive
adjuvant effect for the rFPV (Mingxiao et al., 2006).
Glycoprotein B (gB) is the most highly conserved herpesvirus structural glycoprotein and has been demonstrated
to be a important target of cell-mediated and humoral
immune responses and to confer protective immunity to
ILTV. York & Fashey (1991) reported a subunit vaccine
made of a 205-kDa complex containing gB protected
100% of chickens against clinical disease and also against
viral replication. Sun et al. (2008) found that their constructed rFPV co-expressing the F and HN genes of Newcastle disease virus and the gB gene of ILTV protected
100% of the chickens from death and 70% of the chickens from respiratory signs against an ILTV challenge.
These studies prove that gB is a major protective immunogen of ILTV. Therefore, two rFPVs expressing the gB
gene of ILTV and co-expressing the gB gene of ILTV and
the chicken IL-18 gene were constructed, and their
immunologic efficacy investigated by immunizing SPF
chickens, respectively.
To generate rFPV-gB and rFPV-gB/IL18, two recombinant plasmids pSY-gB and pSY-gB/IL18 were constructed.
A DNA fragment encoding the gB of ILTV was amplified
using PCR from the DNA of the ILTV CG strain (GenBank accession No. DQ812546) using the forward primer
5′-GAGGAATTCAATGGCTAGCTTG-3′ and the reverse
primer 5′-GCGTGAATTCTTATTCGTCTTCCTT-3′ (EcoR
I restriction enzyme site is shown by an underline on the
sense and antisense primers). The PCR product was
cloned into the EcoRI site of plasmid pSY538 under the
control of the early-late LP2EP2 promoter of FPV
(Fig. 1a). The LacZ gene fragment with the P11 late
promoter of vaccinia virus from the plasmid pSC11 was
cloned into the SmaI site of the pSY538 containing the
Fig. 1. Schematic representations of FPV expression plasmids (pSY538, P11 and pSY681) and recombinant plasmids pSY-gB and pSY-gB/IL18.
ª 2011 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Immunol Med Microbiol 63 (2011) 289–295
A rFPV vaccine coexpressing gB of ILTV and chicken IL-18
gB gene. The DNA fragment containing the gB expression
cassette and LacZ expression cassette was cloned into the
NotI site between the homologous arms of the poxvirus
gene in the FPV transfer vector pSY681, resulting in
the plasmid pSY-gB (Fig. 1b). For the construction of
plasmid pSY-gB/IL18, chicken IL-18 was amplified using
PCR from the plasmid pGEM-IL-18 reported previously
(GenBank accession No. AY775780) using the primers 5′-CCCGAATTCATGAGCTGTGAAGAGATC-3′ and
5′-CGGGGAATTCTCATAGGTTGTGCCTTT-3′ (EcoRI site
is underlined on the sense and antisense primers), and
cloned into the EcoRI site of pSY538. Finally, the chicken
IL-18 expression cassette was inserted into a NotI site of
the pSY-gB plasmid to pSY-gB/IL18 (Fig. 1c).
The two recombinant plasmids were transfected into
80% confluent chicken embryo fibroblasts (CEF) that had
already been infected with the parental FPV of S-FPV-017
strain at multiplicity of infection of 0.01 2 h before transfection. The viruses were collected after cytopathic effect
appeared, and rFPVs were screened for beta-galactosidase
activity in the presence of 5-bromo-4-chloro-3-indolyl
b-D-galactoside (X-gal) (TaKaRa, Dalian, China). After
eight rounds of blue plaque purification, the two rFPVs
were obtained and cultured in CEF cells. Insertion of the
recombinant gene into the FPV genome was confirmed
using PCR and DNA sequencing performed on the PCR
products, and expression of gB and chicken IL-18 confirmed using RT-PCR, DNA sequencing performed on
the RT-PCR products, and indirect immunofluorescence
assay (data not shown).
Groups of SPF chickens (1-day-old, n = 10 per group)
were immunized using either (1) rFPV-gB/IL18, (2)
rFPV-gB, (3) ILTV attenuated vaccine (GuyMaCher Animal Health Inc., China); S-FPV-017, or (4) PBS. All
groups were done by wing-web puncture with a double
needle used for commercial vaccination of poultry.
Chickens were given with 50 lL 1 9 106 plaque-forming
units (PFU) rFPV-gB/IL18, 50 lL 1 9 106 PFU rFPV-gB,
the recommended dose of ILTV attenuated vaccine,
50 lL 1 9 106 PFU FPV, and 50 lL PBS, respectively. At
weeks 1, 2, 3, 4, 5, and 6 after immunization, antibody
responses in sera were determined by quantitative ELISA
using recombinant gB protein as a coating antigen. Student’s t-test analysis was used to evaluate the statistical
significance of differences among the groups, and a
P < 0.05 was considered to be statistically significant. The
rFPV-gB and rFPV-gB/IL18 induced detectable antibodies to ILTV Ag in chickens 1 week after vaccination
(Fig. 2), and the levels further increased during the
following weeks. There was no specific antibody response
in chickens inoculated with PBS or S-FPV-017. The level
of anti-ILTV antibodies in the animals vaccinated with
rFPV-gB/IL18 was higher, but not significantly different
FEMS Immunol Med Microbiol 63 (2011) 289–295
291
Fig. 2. Detection of antibodies in different vaccine inoculated groups
using ELISA (n = 5, i.e. number of times the test was repeated).
Values are expressed as mean optical density ± SE. A value 2.1 was
considered as positive by calculating the absolute ratio of post/naı̈ve
serum. Statistically significant differences (P < 0.05) are indicated by
*(compared with S-FPV-017 or PBS).
(P > 0.05) than that of chickens immunized with rFPVgB alone.
Five peripheral blood samples from each group were
collected via wing vein puncture at weeks 1, 2, 3, 4, 5,
and 6 after immunization. Peripheral blood mononuclear
cells (PBMC) were isolated from each blood sample using
Ficoll–Hypaque density gradient centrifugation. Fifty
microliters of the resuspended cells (1 9 106 cells) were
incubated for 20 min at 4 °C in the dark with 10 lL
mouse anti-chicken CD3-Spectral Red (SPRD) and 10 lL
mouse anti-chicken CD4-R-phycoerythrin (R-PE), or
10 lL mouse anti-chicken CD8a-R-PE (Southern Biotech,
Birmingham, AL), and 10 lL mouse IgG1-FITC and
10 lL mouse IgG1-PE, respectively. The percentages of
CD3+, CD3+CD4+, and CD3+CD8+ lymphocytes in the
PBMC suspension were determined using flow cytometry
(model EPICSXL; American Beckman Coulter, Fullerton,
CA). The results showed that the percentages of CD3+,
CD4+CD3+, and CD8+CD3+ T-lymphocytes were significantly higher (P < 0.05) in chickens immunized with rFPVgB/IL18 than in those of the rFPV-gB group (data not
shown). The ratios of CD4+ to CD8+ lymphocytes in rFPVvaccinated groups were significantly higher (P < 0.01) than
in groups inoculated with S-FPV-017 or PBS from the first
week after vaccination. The ratios of CD4+ to CD8+ lymphocytes in chickens immunized with rFPV-gB/IL18 were
significantly higher (P < 0.05) than in those immunized
with rFPV-gB (Table 1).
To determine whether T-cell proliferation response to
the rFPV vaccine encoding the gB gene may be boosted by
chicken IL-18, the PBMCs from the vaccinated chickens on
day 28 after immunizations were examined for antigen-specific T-cell proliferation. PBMCs (3 9 105 cells per well)
were seeded in a 96-well plate in triplicate and stimulated
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H.-Y. Chen et al.
292
Table 1. Ratio of CD4+ : CD8+ T-lymphocytes after vaccination*
Week post vaccination
Group
rFPV-gB/IL18
rFPV-gB
S-FPV-017
PBS
1
2.05
1.79
1.32
1.28
2
±
±
±
±
a
0.27
0.15b
0.11c
0.06c
2.11
1.94
1.42
1.33
3
±
±
±
±
a
0.33
0.25b
0.09c
0.17c
1.98
1.77
1.29
1.23
4
±
±
±
±
a
0.25
0.19b
0.06c
0.08c
2.13
1.86
1.35
1.37
5
±
±
±
±
a
0.31
0.26b
0.12c
0.17c
1.89
1.72
1.25
1.19
6
±
±
±
±
a
0.20
0.08b
0.07c
0.08c
2.15
1.90
1.49
1.41
±
±
±
±
0.32a
0.15b
0.18c
0.21c
Data with the same superscript letter are not significantly different (P > 0.05).
*Number of times the test was repeated is 5. Data are expressed as mean ratios ± SE.
under various conditions at 37 °C for 60 h in a humid
atmosphere with 5% CO2; these conditions included treatment with 5 lg mL 1 concanavalin A (Con A; positive
control), 5 lg mL 1 purified gB antigen (specific antigen),
5 lg mL 1 bovine serum albumin (irrelevant antigen), or
medium alone (negative control). A 20-lL aliquot of
CellTiter 96 Aqueous One Solution Reagent (Promega) was
added into each well according to the manufacturer’s protocol. After a 4-h incubation at 37 °C, the absorbance was
read at 490 nm. Proliferative activity was estimated using
the stimulation indexes (SI) that were defined as the mean
OD 490 of the antigen-containing wells divided by the
mean OD 490 of the wells without antigen. As shown in
Fig. 3, an enhanced T-cell proliferative response to the gB
protein was clearly observed in the groups immunized with
rFPVs when stimulated with purified ILTV gB protein, and
the level of T-cell proliferative response in the group
immunized with rFPV-gB/IL18 was significantly higher
than that in the group immunized with rFPV-gB
(P < 0.05), whereas the chickens vaccinated with S-FPV017 or PBS did not respond to gB. The Con A control sample showed a SI of 4–5. This result indicated that higher
Fig. 3. Peripheral blood T lymphocyte proliferation assay (n = 5, i.e.
number of times the test was repeated). Values are expressed as
mean counts ± SE. Statistically significant differences (P < 0.05) are
indicated by *(compared with S-FPV-017 or PBS) or **(compared with
rFPV-gB alone).
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levels of antigen-specific T-cell proliferative responses
could be elicited by immunization with rFPV-gB/IL18 than
by immunization with rFPV-gB alone.
For challenge studies to determine protective efficacy
against lethal infection on day 43 after immunization, all
chickens were inoculated intralaryngeally with 100 EID50
of the virulent ILTV strain CG.
Chickens inoculated with either S-FPV-017 or PBS
started to show clinical signs of viral infection beginning
on day 2 after challenge, and developed coughing, nasal
discharge, and dyspnea. The mortality rates in chicken
inoculated with S-FPV-017 or PBS were 70% and 80%
(Table 2) at 14 days after challenge, respectively. None of
chickens immunized with 106 PFU rFPV-gB/IL18 had
clinical signs of ILTV infection or died after challenge
with ILTV, and none of 10 of the chickens immunized
with rFPV-gB died, but one chicken immunized with
rFPV-gB had clinical signs of ILTV infection on day 3
after challenge.
To better characterize the protection afforded by vaccination with rFPVs, laryngeal swabs were collected from
chickens euthanized at 14 days post-infection. Virus DNA
in the laryngeal swabs was extracted using SDS-proteinase
K-phenol and subjected to PCR using primers directed
to the TK gene (forward primer: 5′-GGGAAACTTGAA
TGTCGGGAG-3′; reverse primer: 5′-TGGATTATACGCC
GTGCCTGT-3′). These studies indicated that 20% of
birds vaccinated with rFPV-gB had detectable virus in
their laryngeal swabs. All chickens inoculated with either
S-FPV-017 or PBS had detectable virus in their laryngeal
swabs. None of chickens vaccinated with rFPV-gB/IL18
had detectable virus in their laryngeal swabs. Protection
was defined as the absence of detectable virus in the
laryngeal swabs. Chickens immunized with rFPV-gB/IL18
were protected (10/10); whereas eight of 10 of the chickens immunized with rFPV-gB were protected.
As a vaccine adjuvant and an immunomodulatory
molecule, IL-18 has been shown to regulate the immune
response toward a Th1 type (Nakanishi et al., 2001), and
chicken IL-18 enhances the immune responses in vaccines
(Mingxiao et al., 2006). Therefore, we chose to test IL-18
as an adjuvant for the gB antigen expressed from a FPV
FEMS Immunol Med Microbiol 63 (2011) 289–295
293
A rFPV vaccine coexpressing gB of ILTV and chicken IL-18
Table 2. Mortality and protection rate after challenge with the virulent CG strain of ILTV
Groups
Morbidity (%)*
Mortality (%)†
Detectable ILTV in laryngeal‡
Protection rate (%)§
rFPV-gB/IL18
rFPV-gB
ILTV attenuated vaccine
S-FPV-017
PBS
0 (0/10)
0 (0/10)
0/10
100
10 (1/10)
0 (0/10)
2/10
80
10 (1/10)
0 (0/10)
3/10
70
100 (10/10)
70 (7/10)
10/10
0
100 (10/10)
80 (8/10)
10/10
0
*Morbidity was recorded for each day after challenge, and is presented as total number of chickens with any clinical signs in each group.
†
Mortality was recorded for each day after challenge, and is presented as total number of dead chickens in each group.
‡
Detectable ILTV determined using PCR in the laryngeal swabs samples from dead and euthanized chickens’ tracheas was positive bird. Detectable ILTV in the laryngeal swabs samples was determined using PCR positive bird from dead and euthanized chickens’ tracheas.
§
A bird that was not showing any clinical signs and a negative result for PCR was defined as a protected one. Percent protection was determined
by the number of unaffected chickens/total number of chickens.
vector vaccine. rFPV-gB/IL18 and rFPV-gB were constructed, inoculated into chickens, and tested in a protection-challenge experiment. The results showed that
vaccination with the rFPV-gB/IL18 can induce stronger
immune responses than vaccination with rFPV-gB or ILTV
attenuated vaccine. Compared with some earlier descriptions of FPV recombinants expressing ILTV gB (Tong
et al., 2001; Sun et al., 2008), cytokine chicken IL-18 and
different ILTV strains were used. Tong et al. reported
that the SPF and commercial chickens immunized with
rFPV-ILTVgB were all 100% protected from death and
three 12-week-old SPF chickens immunized by eye-drop
with rFPV-ILTVgB had clinical signs of ILTV infection
after challenge with an ILTV WG virus. A FPV (rFPV-F/
HN/gB) co-expressing F, HN genes of Newcastle disease
virus and gB gene was constructed by Sun et al. (2008).
One hundred per cent of SPF chickens immunized with
rFPV-F/HN/gB were protected from death and 70% of SPF
chickens were protected from respiratory signs after
challenge with an ILTV WG virus. In our study, none of
chickens immunized with rFPV-gB/IL18 had clinical signs
of ILTV infection or died after challenge with the virulent
ILTV CG strain. Our results showed that the protective
efficacy of the rFPV-gB vaccine could be enhanced by
simultaneous expression of chicken IL-18.
The activation and the proliferation of lymphocytes play
a critical role in both the humoral and cellular immune
responses induced by vaccination. Therefore, we also evaluated whether vaccination with rFPV-gB in the presence or
the absence of chicken IL-18 could influence the antigenspecific T-cell proliferation response. Our results showed
that the T cells of chickens immunized with rFPV-gB alone
exhibited a proliferative response. However, the level of
proliferative response of the T cells in the rFPV-gB/IL18
group was significantly higher than that in the rFPV-gB
group alone (P < 0.05). This suggested that chicken IL-18
protein was able to stimulate T-cell proliferation.
In this study, protection against ILTV laryngeal challenge was increased by vaccination of rFPV-gB/IL18 than
FEMS Immunol Med Microbiol 63 (2011) 289–295
by vaccination of rFPV-gB alone. Vaccination of rFPVgB/IL18 caused a decrease in the incidence of PCR
positive results for the presence of ILTV in the trachea;
furthermore, the protection rate was improved. This
protective immunity might be attributed to enhanced
cell-mediated immunity, which is interpreted as increased
splenocyte proliferation and increased CD4+ to CD8+
ratios, resulting from vaccination of rFPV-gB/IL18. The
results demonstrated that the group inoculated with
rFPV-gB/IL18 displayed stronger cell-mediated immune
responses and had better protection against virus challenge than rFPV-gB vaccinated group.
The Th1-type immune response is known to be significantly involved in the protective response against ILTV
infection. In this study, T-lymphocyte proliferation responses suggest that chicken IL-18 enhances the induction
of immune responses by promoting a Th1-dominant
response. Similar results were also reported by Shen et al.
(2007) and Ma et al. (2008). Moreover, IL-18 expression
has been shown to have a positive effect upon the magnitude and breadth of the immune response after successive
vaccination, particularly with respect to the generation
of significant numbers of antigen-specific CD4+ and CD8+
T cells (Nakanishi et al., 2001; Marshall et al., 2006).
Therefore, IL-18 appears to be a broadly effective Th1 adjuvant that could be useful in development of ILTV vaccines.
The present study demonstrated that rFPV-gB/IL18 vaccine
may be an effective approach to increasing rFPV-gB vaccine immunogenicity.
Acknowledgement
This work was supported by a grant from the National
Key Project of Scientific and Technical Supporting Programs of China (2008BADB2B01).
Authors’ contribution
H-Y.C. and P.C. contributed equally to this work.
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Published by Blackwell Publishing Ltd. All rights reserved
294
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