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Journal of General Virology (1993), 74, 1201-1206. Printedin Great Britain
1201
Infectious bursal disease virus structural proteins expressed in a
baculovirus recombinant confer protection in chickens
Vikram N. Vakharia, 1,2. David B. Snyder, 2,~ Junkun He, 1'2 Gerard H. Edwards, 2 Peter K. Savage 2
and Stephanie A. Mengel-Whereat 2
1 Center for Agricultural Biotechnology of Maryland Biotechnology Institute and 2 V A - M D Regional College o f
Veterinary Medicine, University of Maryland, College Park, Maryland 20742, U.S.A.
Plasmids were prepared that contained cDNA segments
of the large genomic segment A of infectious bursal
disease virus (IBDV) strain GLS-5. The genes encoding
the IBDV structural proteins (VP2, VP3 and VP4) were
introduced into the baculovirus transfer vector
pBlueBacI to obtain a recombinant baculovirus vIBD-7.
When insect cells were infected with recombinant
viruses, the result was synthesis of IBDV precursor
proteins which were processed by the viral protease. The
recombinant IBDV antigens were characterized by
immunoprecipitation with monoclonal antibodies
(MAbs) and polyclonal antiserum to IBDV, and by
antigen-capture ELISA using a panel of IBDV-specific
MAbs. The recombinant IBDV antigens closely resembled the native IBDV proteins and reacted with all the
GLS-5 strain-specific neutralizing MAbs that recognize
only conformational epitopes of IBDV. When susceptible chickens were inoculated with the recombinant
IBDV antigens, virus-neutralizing antibodies were
induced that conferred up to 79 % protection against
IBDV challenge.
Infectious bursal disease virus (IBDV) is a pathogen of
major economic importance to the world's poultry
industries (reviewed in Kibenge et al., 1988). It causes
severe immunodeficiency in young chickens by destroying the precursors of antibody-producing B cells in the
bursa of Fabricius (Nick et al., 1976). IBDV is a member
of the Birnaviridae family and its genome consists of two
segments of dsRNA (Dobos et al., 1979). The smaller
segment B encodes VP 1, the putative dsRNA polymerase
(Azad et al., 1985; Morgan et al., 1988; Mfiller &
Nitschke, 1987; Spies et al., 1987), whereas the larger
segment A encodes a precursor polyprotein that is
processed into mature VP2, VP3 and VP4 (Hudson et al.,
1986). VP2 and VP3 are the major structural proteins of
the virion. VP2 is the major host-protective immunogen
of IBDV, and contains the antigenic regions responsible
for the induction of neutralizing antibodies (Azad et al.,
1987; Becht et al., 1988; Fahey et al., 1989; Heine et al.,
1991). VP3 contains the structural group-specific antigens (Becht et aI., 1988), whereas VP4 is a protease
involved in the processing of the precursor protein (Azad
et al., 1987; Jagadish et al., 1988).
The principal method of controlling IBDV infection in
young chickens is by vaccination with an avirulent strain
of IBDV or by the transfer of high levels of maternal
antibody induced by the administration of live and killed
IBDV vaccines to breeder hens (Wyeth & Cullen, 1976).
In recent years, virulent strains of IBDV (Delaware and
GLS variants) have been isolated from vaccinated flocks
on the Delmarva peninsula in the U.S.A. (Rosenberger
et al., 1985; Snyder et al., 1988b, c). These variant strains
are antigenically different from the classic strains of
IBDV isolated before 1985, and lack epitope(s) defined
by neutralizing monoclonal antibodies (MAbs) B69 and
R63. As a result of this antigenic shift, chickens
vaccinated with classic strains of IBDV have only partial
protection against variant virus infection (Snyder et al.,
1992).
In an effort to develop a recombinant vaccine for
IBDV in chickens, a number of investigators have
expressed the VP2 antigen of IBDV in yeast (Macreadie
et al., 1990) as well as in a recombinant fowlpox virus
(Bayliss et al., 1991). The yeast-derived antigen afforded
only passive protection in chickens against IBDV
infection, whereas the fowlpox virus-vectored antigen
afforded protection against mortality, but not against
damage to the bursa of Fabricius. In this communication,
we describe the synthesis of the structural proteins of
virulent IBDV strain GLS-5 in a baculovirus expression
system and evaluation of the protective properties of the
recombinant proteins in chickens.
The virulent GLS-5 strain of IBDV was isolated from
the bursae of infected specific pathogen-free (SPF)
chickens as described by Snyder et al. (1988a). The
genomic RNA was extracted from purified virus (bursaderived) and was subjected to 1% low melting tern-
0001-1367 © 1993 SGM
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5' I
1 kb
2 kb
3 kb
I
I
I
I
strain GLS-5. The complete nucleotide sequence of these
clones will be described elsewhere. A full-length cDNA
clone, pGLS-5, was constructed by digesting DNA of the
above plasmids with appropriate restriction enzymes,
and ligating the cDNA fragments (see Fig. 1).
To insert IBDV structural genes into the baculovirus
transfer vector pBlueBacI (Invitrogen), plasmid pCRGLS
was constructed as follows. Oligonucleotides containing
the
NheI
site
and
having
the
sequence
CGATCGCTAGCGATGACAAAC (upstream primer)
and AGACTCCCAGCTAGCCTCACTCAA (downstream primer) were synthesized and used to amplify the
IBDV segment from plasmid pGLS-5 by PCR. Amplification by PCR was carried out according to the protocol
of the supplier (Perkin Elmer-Cetus), except that the final
concentration of each dNTP was 400 gM. Thirty cycles
(95 °C for 1.5 min, 37 °C for 2 min, and 72 °C for 5 rain)
were employed for the amplification of the above
segment. Approximately 150 ng of amplified DNA
product was ligated into the vector pCR1000, supplied in
a PCR cloning kit (Invitrogen). A recombinant plasmid,
pCRGLS, encoding the structural protein genes of IBDV
was selected. The integrity of the pGLS-5 and pCRGLS
constructs was tested by in vitro transcription (using T7
RNA polymerase) and translation. In both cases, the
expected IBDV-specific products were observed after
immunoprecipitation with polyclonal antisera and gel
electrophoresis (results not shown). A 3-05 kb NheI
fragment containing the large open reading frame
sequence was excised from plasmid pCRGLS and
inserted at the unique NheI site of pBlueBacI to form
pGLSBacI. Recombinant virus vlBD-7 was obtained by
cotransfecting pGLSBacI and Autographa californica
nuclear polyhedrosis virus (AcNPV) DNA into Spodoptera frugiperda (Sf9) cells, and plaque-purifying the
3'
I
t_~
VP2
P
t..._..u (BN)
I
S
B
f t
I
pGLS- 1
I
I vP3 p
VP4
• (BN)
pGLS-4
PP
S
I
i I(BN)
I
B
I
B
I
I
pGLS-2
pGLS-3
t
1
pGLS-5
Fig. 1. Schematic presentation of the plasmids encoding IBDV-specific
polyproteins. A map of the IBDV genome with its coding regions is
shown at the top of the figure. Selected restriction sites, important for
the construction of plasmid pGLS-5, are incorporated in the figure: B,
BamHI; N, NsiI; S, SpeI. Parentheses indicate the restriction sites in
the multiple cloning region of the vector pGEM-7Zf( + ). All plasmids
contain a T7 promoter at their 5' end.
perature agarose gel electrophoresis. The large RNA
segment (approx. 3"2 kb) was recovered from the gel and
used for cloning. Complementary DNA segments of
IBDV were synthesized by the method of Gubler &
Hoffman (1983), using a cDNA synthesis kit (Promega),
and specific primers complementary to the 3' end of the
VP2 gene sequence (Hudson et al., 1986) or to the 3' end
of the large genomic segment (Bayliss et al., 1990). The
cDNA produced was ligated with EcoRI adaptors and
inserted into the unique EcoRI site of the pGEM-7Zf(+ )
vector (Promega). Four overlapping cDNA clones,
pGLS-1, -2, -3 and -4, were selected which spanned the
whole coding region of the genome segment A of IBDV
Table 1. Reactivities of MAbs with various IBD Vs and baculovirus-expressed
proteins
AC-ELISA?
IBDV
Expressed proteins:~
MAb
Eliciting IBDV
strain
VN tests*
Classic
E/Del
GLS-5
vlBD-7
B29
8
10
179
B69
BK9
57
STC (Classic)
GLS-5
GLS-5
GLS-5
STC (Classic)
E/Del
GLS-5
+
+
+
+
+
+
+
+
+
+
--
+
+
+
+
-
+
+
+
+
+
+
+
+
+
_
_
+
AcNPV
* A positive result indicates that the M A b neutralized IBDV in VN tests.
? IBDV antigens derived from bursal homogenates (1:40 dilutions) were evaluated with various
MAbs in AC-ELISA (Snyder et al., 1992). IBDV strains used were STC (Classic), Delaware variant
E (E/Del) and GLS-5.
:~Expressed proteins derived from the recombinant virus (vIBD-7) or the wild-type baculovirus
(AcNPV)-infected cell lysates (1:150 dilutions) were evaluated with various MAbs in AC-ELISA.
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4
1
•
5
5•
6
7
8
9
•••
:~'
VPX - - ~
VP2
--
106K
--
80K
- - 49K
~
--
- - 32K
VP3
VP4-
,~
~.
~
i:~,~
- - 27K
--
18K
Fig. 2. Immunoblot analysis of IBDV proteins expressed in vIBD-7infected ceils. Sf9 cells were infected with either a wild-type or a
recombinant baculovirus and harvested at 72 h p.i. After lysis, cellular
proteins were immunoprecipitated with polyclonal anti-IBDV rabbit
serum and IBDV-specific MAbs. The samples were separated by
S D S - P A G E on a 12.5 % slab gel, blotted onto nitrocellulose, reacted
with polyvalent chicken anti-IBDV serum, and detected with
streptavidin-alkaline phosphatase and naphthol phosphate fast red
colour development reagents. Lane 1, GLS-5-infected CEF lysates;
lane 2, uninfected CEFs; lane 3, uninfected Sf9 cells; lane 4, AcNPVinfected Sf9 cells; lane 5, vlBD-7-infected Sf9 cells, immunoprecipitated
with polyclonal anti-IBDV rabbit serum; lanes 6 to 9, vlBD-7-infected
Sf9 cells, immunoprecipitated with MAbs 8, 10, 57 and 179,
respectively. The positions of the VPX, VP2, VP3 and VP4 (left) and
marker proteins (right) are indicated.
recombinant virus as described (Summers & Smith,
1987; Vialard et al., 1990). AcNPV and recombinant
virus stocks were propagated in Sf9 cells using Hink's
TNM-FH medium (JRH Biosciences) supplemented with
10 % fetal calf serum in either tissue culture flasks or in
spinner flasks at 28 °C (Summers & Smith, 1987). Cells
grown in spinner flasks contained 0.1% polyethylene
polypropylene glycol (' Pluronic F-68', J R H Biosciences)
in addition to the above supplemented medium.
In order to characterize the IBDV proteins expressed
in insect cells, Sf9 cells were infected with either the wild-
1203
type baculovirus or the recombinant virus (vlBD-7) and
harvested at 72 h post-infection (p.i.). The cells were
washed three times in PBS and lysed in a buffer
containing 1% Triton X-100, 1% sodium deoxycholate,
0"5 M-NaC1, 0.5 M-Tris-HC1 pH 7'2, 0.01 M-EDTA and
0"1% SDS. A portion of the cell lysate was immunoprecipitated with GLS-5 strain-specific MAbs 8, 10, 57
and 179 (see Table 1) and a polyclonal rabbit anti-IBDV
serum. Immunoprecipitated proteins were resolved on a
12.5 % SDS-polyacrylamide gel and detected immunologically following Western blotting with polyvalent
chicken anti-IBDV serum (Becht et aL, 1988). The results
are shown in Fig. 2. Cells infected with recombinant
virus expressing IBDV proteins were correctly processed
0ane 5) and comigrated with the marker proteins
VPX/VP2, VP3 and VP4, derived from strain GLS-5infected chicken embryo fibroblast (CEF) lysates (lane 1 ;
the mobility of the VP2 protein is slightly different as the
lane was overloaded). A protein band between VP3 and
VP4 was often seen and is derived from VP3 (Fahey
et al., 1989). In addition, MAbs 8, 10, 57 and 179
immunoprecipitated IBDV structural proteins VPX/VP2
and VP4 (lanes 6 to 9, respectively). IBDV-specific
proteins could be detected in neither uninfected CEFs,
uninfected Sf9 cells nor AcNPV-infected Sf9 cells (lanes
2 to 4, respectively). In previous studies, the precursor
protein of IBDV was correctly processed by VP4 when
the entire cloned IBDV segment A was expressed in
Escherichia coli (Azad et al., 1987; Jagadish et al., 1988)
and in yeast (Macreadie et al, 1990). Similarly, bacterial
expression of the large genomic segment of infectious
pancreatic necrosis virus (IPNV), a prototype of the
Birnaviridae family, also yielded IPNV proteins that
were cleaved by the non-structural protein (Manning et
al., 1990; Manning & Leong, 1990).
To characterize further the expressed IBDV proteins,
the cell lystates were analysed with a panel of IBDVspecific MAbs in an antigen-capture ELISA (ACELISA) as previously described (Snyder et al., 1992). The
panel of MAbs used in AC-ELISA is shown in Table 1.
The results indicate that the baculovirus-expressed IBDV
antigens bind to MAbs 57, 10, 179 and 8, suggesting that
the expressed IBDV antigens are GLS-5 strain-specific.
Since these neutralizing MAbs are conformation-dependent, the expressed IBDV antigens appear to form
conformationally correct structures.
To prepare an inoculum for immunization, SO cells
(in a 1 litre flask) were infected with either a wild-type or a
recombinant baculovirus at a multiplicity of 5 p.f.u./cell,
and then incubated for 3 to 4 days. The infected cells
were recovered by centrifugation, washed twice with
PBS, and resuspended in 10ml of PBS. The cell
suspension was sonicated three times for 1 min, at 2 rain
intervals. A portion of each cell lysate was tested by AC-
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Table 2. Protection of inoculated chickens against virulent IBD V challenge
Challenge
(CIDs0 of
Group Inoculation GLS-5 strain)
I
II
III
IV
AcNPV
vIBD-7
100
100
100
IBDV
antigen
in bursa
0/14"
14/14
14/14
1/14
Neutralizing
Bursal
BBWR
No. of animals Protection antibody titre
lesions (mean+_s.D,) infected (BBWR)
(%)
after incubation
0/14t
14/14
14/14
5/14
5"22±1"27~
1.36±0-25
1"61±0"35
4"87±1"63
0
14
14
3
0
0
79§
<8
<8
<8
256,1024
* Bursal homogenates, prepared 4 days post-challenge,were evaluated with a panel of IBDV-specificMAbs in AC-ELISA (see
Table 1). The nominator denotes the number of animals positive for IBDV antigen in bursal tissue and the denominator denotes
the number of animals inoculated.
t Bursae, obtained 8 days post-challenge, were analysed for the bursal lesions by histopathological examination. The
nominator indicates the number of animals positive for bursal lesions and the denominator denotes the number of animals
inoculated.
:~BBWR was calculated for each animal (Lucio & Hitchner, 1979). Any value for individually challenged chickens falling
_+2 S.D. from the average of the negative control group was scored as a positive indicator of IBDV infection. The number of
animals infected, as calculated by BBWR method, is shown in the next column.
§See text.
E L I S A to estimate the amount of I B D V proteins present.
Total protein content of the recombinant baculovirusinfected cell lysate was about 4 m g / m l of which
approximately 12 to 13% represented IBDV products.
Fifteen ml of baculovirus- or recombinant baculovirusinfected cell lysate was emulsified with an equal volume
of Freund's incomplete adjuvant and used for inoculation.
To evaluate the protective properties of the recombinant proteins, 2-week-old white leghorn SPF chickens
(SPAFAS, Inc.) were pre-bled and divided into four
groups of 28 animals each. Chickens of groups I and II
received no inoculations and served as negative and
positive controls, respectively. Chickens of groups III
and IV were inoculated intramuscularly with 0.5 ml of
inoculum prepared from wild-type baculovirus- or
recombinant baculovirus-infected cell lysates, respectively. At 4 weeks of age, chickens of groups I I I and IV
were boosted with 0"25 ml of the respective inoculum.
The immune response to the recombinant I B D V proteins
was detected in a virus neutralization (VN) test which
was performed as previously described (Snyder et al.,
1988 a). In addition, the sera were tested for the presence
of anti-IBDV antibody using a commercially available
IBDV antibody E L I S A test kit (Kirkegaard & Perry),
and by the agar gel precipitin test (AGPT) using
undiluted antisera to diffuse against a 50 % (w/v) bursal
homogenate that was prepared from chickens infected
with IBDV.
Our results indicate that the recombinant proteins
induced a significant neutralizing antibody response in
chickens and immunoprecipitated anti-IBDV antibodies
from a convalescent serum in an A G P T . The average V N
titre of the vaccinated chickens was 1024 which is
comparable to that obtained from the unprimed chickens
vaccinated with inactivated virus. However, the VN titre
of naturally infected chickens was much higher and
ranged from 16 384 to 32 768. Sera of vaccinated chickens
also immunoprecipitated I B D V antigens in an A G P T
and had an average E L I S A titre of 3 (on a scale of 0 to
9), which correlated with the V N titre (see Table 2). N o
anti-IBDV response was elicited in the group of chickens
inoculated with wild-type baculovirus-infected cell
lysates.
To assess the ability of the recombinant I B D V antigens
to induce a protective immunity, 7-week-old chickens of
groups II, III and IV were challenged with 100 50%
chick infective doses (CIDso) of virulent GLS-5 strain by
the ocular route. Four days post-challenge, 14 chickens
from each group were sacrificed and their bursae were
removed. Each bursa was processed and analysed for the
presence of I B D V antigen by A C - E L I S A using a panel of
IBDV-specific MAbs (see Table 1). Eight days p.i. the
remaining chickens in all groups were sacrificed and
weighed. The bursa of Fabric±us from each chicken was
carefully excised and also weighed. Bursa to body weight
ratio (BBWR) was calculated for each chicken as
described by Lucio & Hitchner (1979) and each bursa
was assessed for lesions by histopathological examination.
The results of the challenge experiment are shown in
Table 2. All the non-vaccinated group (positive controls)
and AcNPV-vaccinated group of chickens showed the
presence of I B D V antigen, gross bursal lesions and a
drastic reduction in their B B W R (due to damage of the
lymphoid follicles), all indicating I B D V infection (Table
2). However, in the vIBD-7-vaccinated group, only one
of 14 animals ( 7 % ) showed the presence of I B D V
antigen; five of 14 animals (36%) showed mild to
moderate bursal lesions; only three animals (21%) had
a BBWR lower than 2 s.o. below the average of the
unchallenged group, suggesting I B D V infection. N o
IBDV infection was detected in the animals of the
unchallenged group (negative controls).
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In recent studies, the host-protective VP2 antigen of
IBDV has been expressed in yeast (Macreadie et al.,
1990) as well as in a recombinant fowlpox virus (Bayliss
et al., 1991). When microgram quantities of yeastderived antigens were injected into chickens, they
induced high titres of virus-neutralizing antibodies that
were capable of passively protecting young chickens
against IBDV infection. In contrast, when chickens were
vaccinated with a recombinant fowlpox virus expressing
the VP2 antigen, it actively protected chickens against
mortality but not against bursal atrophy. The majority
of vaccinates had severe lymphocyte depletion following
virulent IBDV challenge. On the basis of AC-ELISA,
BBWR and histopathological analysis, our results indicate that 79% of the vaccinated chickens were
protected against virulent IBDV challenge (Table 2).
Protection was defined as: (i) the absence of IBDV
antigen in the bursa as detected by A C - E L I S A ; (ii) no
detectable bursal damage upon histopathological examination; (iii) no decrease in BBWR of 2 S.D. under the
average of the unchallenged group. Failure to achieve
complete protection may be accounted for by an
insufficient amount of IBDV antigens used for immunization (approx. 125 gg) to give complete protection.
In conclusion, expression in Sf9 cells of a construct
containing the entire coding region of IBDV structural
proteins (VP2, VP3 and VP4) resulted in the synthesis
and processing of the IBDV precursor protein and its
correct processing. The recombinant IBDV proteins
were antigenically similar to the native IBDV proteins
and were immunoprecipitated with IBDV-specific MAbs
and polyclonal antiserum to IBDV. The recombinant
IBDV proteins induced a neutralizing antibody response
in chickens and actively protected chickens against
virulent IBDV challenge. Work is underway to determine
the amount of IBDV antigens needed for complete
protection in chickens.
We thank Dr Mark Goodwin, Georgia Poultry Laboratory,
Oakwood, Ga., U.S.A for histopathological analysis of bursal
specimens, Sarah Milczanowski and Elena Newport for technical
assistance, and Helen Phillips for preparing the manuscript. This
research was supported in part by grants from the U.S. Department of
Agriculture (no. 90-37153-5557) and Maryland Agricultural Experiment Station. Scientific article no. A6372, contribution no. 8556, of
the Maryland Agricultural Experiment Station.
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(Received 21 September 1992; Accepted 19 January 1993)
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