_Archives
Vi rology
Arch Virol (1994) 137:55-67
© Springer-Verlag 1994
Printed in Austria
Immunogenicity of the S protein of transmissible gastroenteritis
virus expressed in baculovirus
T. Tuboly, 1~. Nagy, J. R. Dennis, and J. B. Derbyshire
Department of Veterinary Microbiology and Immunology,
University of Guelph, Guelph, Ontario, Canada
Accepted March 3, 1994
Summary. Seven fragments of the spike (S) gene cDNA of transmissible
gastroenteritis virus (TGEV), as well as the full length cDNA, were cloned and
expressed in baculovirus vectors. Piglets were immunized with cells infected
with the recombinant viruses. Each of the recombinants induced TGEV-specific
antibodies detected in a fixed cell enzyme immunoassay. The amino terminal
half of the S protein, containing all four major antigenic sites (A, B, C and D),
and encoded by a 2.2 kb fragment of the S gene, induced virus neutralizing
(VN) antibody titers comparable with those induced by the complete S protein.
Recombinant proteins lacking the A antigenic site, or with a deletion including
the putative receptor binding sites and the D antigenic site, were not capable
of inducing levels of VN antibodies similar to those induced by the whole S
protein.
Introduction
Transmissible gastroenteritis (TGE) is an important disease of swine, characterized by severe diarrhoea and vomiting, often fatal to piglets under two weeks
of age. The disease is caused by a porcine coronavirus which, like other members
of the family Coronaviridae, is an enveloped, pleomorphic virus, with a single
stranded RNA genome of positive polarity. The virions are constructed of three
major structural proteins: a phosphorylated nucleocapsid protein (N) and two
glycoproteins, the membrane (M) and the large spike (S) protein. The mature
S protein, after cleavage, which forms the characteristic club-shaped peplomers,
contains 1431 amino acids and is the major inducer of virus neutralizing (VN)
antibodies [13, 18].
Studies of the antigenic structure of the S protein by monoclonal antibodies
(Mabs) revealed at least four major epitope groups, all of them located on the
amino-terminal half of the polypeptide [4, 18]. Those antigenic determinants
that are involved in neutralization were highly conserved among different
56
T. Tuboly et al.
T G E V strains [12] and appeared to be conformation- and glycosylation
dependent[3, 10].
The baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV)
is widely used for the high level expression of foreign genes [203 and the
complete S protein of T G E V (together with a carboxy-terminal truncated
version) has been expressed in a recombinant baculovirus [15]. The complete
S protein induced VN antibodies in rats. The complete and a truncated form
of the T G E V S protein have also been expressed in recombinant vaccinia
viruses, and they were tested in mice, where both were shown to induce T G E V
neutralizing antibodies [17, 25].
Passive protection of neonatal piglets is of primary importance in the control
of T G E and the only efficient way to achieve this is by T G E V specific immunoglobulins (Igs) primarily of the secretory (s) IgA class in the colostrum and milk
of the sow. T G E V strains that are capable of generating sufficient sIgA response
in the sow may also cause the disease in newborn piglets. An alternative
approach to vaccination could be the use of a helper independent porcine
adenovirus vector expressing the S gene and replicating in the intestinal tract,
but the size of the S gene (4341 bases) probably exceeds the predicted packaging
capabilities of such a vector, based on studies with h u m a n adenovirus vectors
El].
The objective of our study was to select the shortest piece of the S protein
capable of inducing a VN antibody response similar to the response to the
whole S protein.
Materials and methods
Cells, viruses and control sera
AcNPV and the recombinant baculoviruses were grown and assayed in monolayers of
Spodopterafrugiperda cells (Sf21) maintained in Grace's medium supplemented with 0.3~o
yeastolate, 0.3% lactatbumin hydrolysate and 10% fetal bovine serum (FBS, SIGMA) at
28 °C [30]. The origin of the AcNPV has been previously described [22]. The tissue culture
adapted Purdue-ll5 (P-115) strain of TGEV, kindly provided by Dr Linda Sail (Ohio
Agricultural Research and Development Centre, Wooster OH, U.S.A.), was propagated in
confluent swine testis (ST) cells [21]. TGEV specific hyperimmune serum was generated
in pigs as described by Welch and Saif [32] with an initial intramuscular dose of the P- 115
strain and subsequent oral dosing with the virulent Purdue strain. The source of AcNPV
specific serum has been described earlier [31].
cDNA synthesis and clonin9
The viral genomic RNA was extracted from concentrated TGEV P-115 as described by
Wesley et al. [33]. PolyA mRNA was purified by oligo (dT) spun-column chromatography
(QuickPrep Micro mRNA Purification Kit, Pharmacia) according to the manufacturers'
instructions, from ST cells 6h after infection with TGEV P-115 at a m.o.i, of 10.
The cDNA synthesis was carried out with the RiboClone cDNA Synthesis System
(Promega) using the TGEV genomic RNA and mRNA with three different oligonucteotide
primers (Regional DNA Synthesis Laboratory, Univ. of Calgary) indicated in Fig. 1
tmmunogenicity of the TGEV S protein
S gene
AUG
~Ec
57
UAA
~Ic
4341 b
AU GCC UCU GCU GUU AUA A-3'
TA CGG TGT CGA CAA TAT T-5'
;I~.
UA AGC CAC CUA ACA GUA CUU~'
AT TCG GTG GAT TGT CAT GTT-5'
CAU CUG CUA UAA UAG CAG-3'
Hpal
Hpal
(in pVL1393) I~lc
~1( GTA GAC GAT ArT ATC GTC-5'
<
I ,
~
Xhol
1
Xhol
I
Hindlli
i
Hindlll
I
i
Smal/Hpal
I ~1~
v
T
v
Xhol
'
Hindlll
,
,
Sspl
Kpnl
'
~lcl
v
Blunt end/Sinai
,1' I
pTS
Fig. 1. Synthesis of the TGEV S gene cDNA. The generation of the cDNA fragments with
the primers binding to the indicated bases is shown by the horizontal arrows. The lines
represent the intermediate DNA fragments used for the construction of the full length S
gene cDNA cloned into the pGEM-7 vector, designated pTS. The restriction enzyme sites
used for the generation of pTS and the recombinant baculovirus transfer vectors are
indicated. The initiation and stop sites are marked with asterisks
(5'-TTATAACAGCTGTGGCAT-3', 5'-TTGTACTGTTAGGTGGCTTA-3', 5'-CTGCTATTATAGCAGATG-Y') complementary to the genome [26] downstream of the initiation
site at 1793-1810, 3335-3354 and 4365-4382 bases respectively. The resulting overlapping
cDNA fragments were blunt ended with T4 DNA polymerase, cloned into the SmaI site
of the pGEM-7Zf(+) vector in both orientations: BamHI-EcoRI(+), EcoRI-BamHI(-).
The clones were propagated in DH50~ E. coli cells and analyzed by restriction enzyme (RE)
digestions. All REs were obtained from BRL (Life Technologies, Gaithersburg, MD, U.S.A.).
Generation and analysis of the recombinant baculoviruses
The 1.6kb HpaI cDNA fragment was cloned into the Sinai site of the pVL1393
baculovirus transfer vector (kindly provided by Dr. C. Richardson. Biotechnology Research
Institute, Montreal, Canada) in (+) orientation and the 1.1 kb BamHI-XhoI subfragment
of this clone was used together with the cDNA generated by the other two primers for
the construction of the complete TGEV S cDNA (pTS. Fig. 1). Figure 2 shows the size of
the cDNA fragments selected for the generation of the listed recombinant viruses and their
relation to the positions of the major antigenic sites [3, 9]. The truncated cDNA fragments
in the recombinant baculoviruses expressing the TGEV S gene (rBTS) 1.8, 2.2 and 3.3 were
generated by double RE digestions of pTS (+) with BamHI-SspI, BamHI-KpnI or
BamHI-HindIII and cloned into the BamHI-SmaI (rBTS 1.8), BamHI-KpnI (rBTS 2.2) and
BamHI-HindIII (rBTS 3.3) sites of pGEM-7 respectively. The in phase cDNAs of rBTS
2.2D, 3.3D and 4.4D were generated by deleting the two HaeIII fragments between 529
58
T. Tuboly et al.
S protein
m
B
t COOH
NH2 t
B
D
D
C
A (1)
A,B (2)
(antigenic sites)
1447 aa
4341 b
Recombinants
1585 bp
528 aa
rBTS 1.6
1759bp
586 aa
rBTS 1.8
2236 bp
745 aa
rBTS 2.2
3329 bp
rBTS 3.3
1109 aa
4341bp
1447aa
rBTS 4.4
rBTS 2.2D
rBTS 3.3D
Haelll
747bp
1489 bp
496 aa
2582 bp
860 aa
3594 bp
1198 aa
rBTS 4.4D
Fig. 2. The S gene fragments of the recombinant baculoviruses and their relation to the S
protein, The open bar indicates the complete S glycoprotein of the TGEV P-115 strain
with the approximate location of the major antigenic sites (A 538.-591aa, B 97-144aa, C
49-52aa and D 378-395aa) ~l~followed throughout this study, according to Correa et al.
[3] and IZ~after Delmas et al. [9]. The putative receptor binding sites are shown by the
solid bars (92-219aa and 405-465aa). The designations of the recombinant baculoviruses
are indicated on the left. The size of the cDNA fragment (represented by the lines) in each
recombinant virus is given in basepairs together with the number of amino acids encoded
by these fragments. The dotted line indicates the deletion of the HaeIII DNA fragments
and 1276 bp and religating the cohesive ends. These cloned fragments were released from
pGEM-7 by BamHI-EcoRI digestions and cloned into the identical unique sites of the
pVL1393 transfer vector behind the polyhedrin gene promoter. The resulting constructs
were cotransfected with linearized AcNPV DNA into Sf21 cells using cationic liposomes
(AcMNPV Linear DNA Transfection Module, Invitrogen). The recombinant viruses were
selected by plaque morphology and dot blot hybridization probing with [32p]dCTP
(3000 Ci/mmol, ICN) labelled S gene cDNA [30]), Three times plaque purified viruses were
propagated and the extracellular virus was used in further studies.
Analysis of the recombinant virus DNA
The recombinant and wild type virus DNAs were extracted from purified virions [5],
digested with REs and analyzed on 0.8~o agarose gel in the presence of 0.25 gg/ml ethidium
bromide [27]. The DNA was transferred onto Nytran membranes (Schleicher and Schuelt)
and probed with the 32p labelled S gene cDNA [22].
Immunogenicity of the TGEV S protein
59
RadiolabeIling and immunoprecipitation
The viral proteins were radiolabelled as described [15]. Briefly, monolayers of Sf21 cells
were infected with AcNPV or the recombinant viruses at a m.o.i, of 10 and incubated at
28 °C for 36 h. The medium was then replaced by methionine free Grace's medium (BRL)
supplemented with 2% FBS and 100 ~tCi of [35S] methionine (1143 Ci/mmol, TRAN35S Label, ICN) per ml. After incubation for 4 h at 28 °C, the cells were washed with PBS,
lysed with RIPA buffer (150mM NaC1, 1% sodium deoxycholate, 1% Triton X-100, 0.1%
sodium dodecyl sulphate, 10raM Tris-HC1 pH 7.2) and analyzed directly, or after
immunoprecipitation with TGEV specific hyperimmune pig serum and Staphylococcus
aureus protein A (Pansorbin cells, Calbiochem) on 8% SDS-polyacrylamide gels [19].
Labelled uninfected and TGEV infected ST cell lysates were prepared as described by De
Diego et al. [7].
Immunizations
Sf21 cells infected at a m.o.i, of 10 were collected between 60 and 72h p.i., a time frame
providing for similar concentrations of the recombinant proteins (judged by SDS-PAGE),
washed and resuspended in PBS. The samples were aliquoted into 3 x 107 cells/dose and
stored at - 7 0 °C. Four week old crossbred Yorkshire piglets from a TGEV seronegative
herd (confirmed by VN assay) were immunized in groups of three by the intraperitoneal
route with one dose of infected cells emulsified in an oil adjuvant [2] and two doses without
adjuvant at two and four weeks after the first injection. Blood samples were taken at weekly
intervals and the sera were tested after heat inactivation at 56 °C for 30 rain for the presence
of TGEV specific antibodies.
Serology
VN antibodies were assayed by standard methods in 96 well microtiter plates of ST cells
using t00 TCIDso of the TGEV P-115 strain. The sera were also tested in a TGEV specific
fixed cell enzyme immunoassay (FCEIA) as described by Cubero et al. [6] after an overnight
preincubation of each sample with uninfected ST cells at 4 °C. The horseradish peroxidase
labelled anti-swine lgG used in this assay was purchased from Kirkegaard and Perry
Laboratories.
Results
Construction of the recombinants
Eight baculovirus r e c o m b i n a n t s were generated by the cotransfection of
A c N P V D N A a n d the pVL1393 transfer vector carrying different fragments of
the T G E V S gene c D N A . The c o n s t r u c t i o n of the r e c o m b i n a n t transfer vectors
resulted in an identical 8 basepair 5' leader sequence from the T G E V g e n o m e
u p s t r e a m of the S gene initiation codon.
BamHI-EcoRI a n d SalI generated D N A fragment patterns of the purified
wild type a n d r e c o m b i n a n t virus D N A s and the c o r r e s p o n d i n g S o u t h e r n blot
hybridization are s h o w n in Fig. 3. The BamHI-EcoRI double digest c o n f i r m e d
the size of the foreign gene in the r e c o m b i n a n t viruses and the SalI R E p a t t e r n
indicated that these c D N A fragments were within the p o l y h e d r i n gene as the
size of the 3.07 kb A c N P V SalI b a n d was increased in each r e c o m b i n a n t by
60
T. Tuboly et al.
Fig. 3. Analysis of the DNA extracted from the recombinant baculoviruses. A The
BamHI-EcoRI (B-E) and SalI (S) generated DNA fragment patterns of the wild type and
the recombinant baculoviruses. Arrows show the different S gene eDNA bands. The size
of some of the DNA markers (1 kb DNA ladder) in M is indicated on the left of the gel.
I is the wild type AcNPV, 2 rBTS 1.6 3 rBTS 1.8, 4 rBTS 2.2, 5 rBTS 3.3, 6 rBTS 4.4,
7 rBTS 2.2D, 8 rBTS 3.3D, 9 rBTS 4.4D. B Southern blot hybridization of the same get
with the cloned S gene eDNA
the size of the cloned D N A (Fig. 3A). The Southern blot hybridization with
the S gene c D N A showed reactions only with the D N A bands which
were expected to carry the homologous S sequences (Fig. 3B). The orientation
of the foreign gene in the recombinant viruses was determined by extensive RE
analysis (data not shown).
Expression of T G E V S polypeptides in Sf21 cells
The recombinant S polypeptide expression was monitored by S D S - P A G E
using [35S] methionine labelled lysed infected Sf21 cells (Fig. 4A), or the proteins
immunoprecipitated by T G E V specific hyperimmune serum (Fig. 4B), and these
gels were used for the estimation of the size of the recombinant polypeptides.
The glycosylated proteins expressed by rBTS 1.6, 2.2 and 2.2D were larger
(99.4 kD, 130 k D and 97.4 k D respectively) than the size predicted by the length
of the cloned fragments, as these coding sequences were in frame with the
downstream polyhedrin gene sequences. The size of the glycoproteins expressed
Immunogenicity of the TGEV S protein
61
Fig. 4. A Autoradiogram of the [35S] methionine labelled recombinant proteins analyzed
by 8% SDS-PAGE. Each lane represents a lysate of 1 x 104 infected Sf21 cells. Arrows
indicate the glycosylated S proteins expressed by the different constructs. B Autoradigram
of the labelled recombinant proteins of 3 x 104 infected Sf21 cells, immunoprecipitated
with TGEV specific pig antiserum. The size of the 14C-labelled protein markers in M is
indicated on the left. I AcNPV, 2 rBTS 1.6, 3 rBTS 1.8, 4 rBTS 2.2, 5 rBTS 3.3, 6 rBTS
4.4, 7 rBTS 2.2D, 8 rBTS 3.3D, 9 rBTS 4.4D, 10 TGEV infected ST cells
by rBTS 1.8, 3.3, 4.4, 3.3D and 4.4D were in agreement with the expected size
(86kD, 154kD, 184kD, 121.8kD and 165.4kD repectively). The expressed
proteins were found mainly intracellularly, however those without the anchor
region were also detected in the cell culture supernatants (data not shown). The
maximum rate of protein synthesis was detected between 24 and 36 h p.i. but
the accumulation of these peptides continued up to 72h p.i. as measured in
Coomassie blue stained polyacrylamide gels (data not shown).
Serological response of pigs immunized with the recombinant S polypeptides
Sera collected weekly from pigs immunized with Sf21 cells infected with the
recombinant viruses were examined for the presence of T G E V neutralizing
antibodies. Figure 5A summarizes the VN results obtained with the full length
62
T. Tuboly et al.
A
10
8
(n
C
.o
~56
E
=:
2
B T
5
==4
o
0
1
2
3
4
5
weeks of immunization
rBTS 1.6 ~ rBTS 1.8 ~; rBTS 2.2 ~ rBTS 3.3 i rBTS 4.4
Fig. 5. A Mean TGE VN titers of sera from pigs immunized with the complete
recombinant S protein or the recombinant proteins truncated at the carboxy-terminal half.
Serum dilutions start at 1:2. B The mean antibody titers measured by the TGEV specific
FCEIA. Serum dilutions start at 1:25. Standard deviations within groups were tess than 5%
recombinant S protein and the proteins truncated at the carboxy-terminus.
rBTS 1.6 did n o t induce VN antibodies and rBTS 1.8 had only a limited VN
inducing capability. Those recombinant viruses which encoded not only the B,
C and D antigenic sites but also the entire A region (rBTS 2.2 and 3.3) induced
a VN response similar to that induced by the recombinant expressing the
complete S potypeptide (rBTS 4.4). The same sera were tested in a F C E I A
Immunogenicity of the TGEV S protein
63
7
6
5
tO
4
13
E
-!
~ffl 3
0
-- 2
0
?',7/1
7/ill
0
1
2
3
weeks of immunization
rBTS 2.2D ~
rBTS 3.3D •
4
5
rBTS 4.4D
Fig. 6. TGEV specific mean FCEtA titers of the pig sera obtained after immunization with
the proteins expressed by recombinant baculoviruses carrying a deletion at the 5' terminal
half of the S gene. Dilutions start at 1:25
(Fig. 5B) in which they all reacted with the TGEV antigen by the third week
post-vaccination, and the highest titres were generally produced in response
to the larger proteins.
The three recombinant viruses with a deletion in the amino-terminal half
coding sequences of the S gene did not induce any detectable VN antibodies
although they all contained the A site coding sequences. When these serum
samples were tested in the FCEIA, the results resembled those obtained by the
recombinant viruses without the HaeIII deletion (Fig. 6).
Sera from the pigs immunized with the wild type baculovirus, used as
negative control in both assays, did not show any TGEV specific activity.
Discussion
The antigenic structure of the TGEV S glycoprotein has been well characterized
by monoclonal antibodies [3, 18] or by expressing fragments of the S gene in
prokaryotic systems [4, ! 1] and with the use of synthetic oligopeptides [14, 24].
Due to the limitations of the prokaryotic expression system and the use of
64
T. Tuboly et al.
nonglycosylated oligopeptides, the immunogenicity of defined regions of the S
protein in pigs is unclear.
The baculovirus expression system has, however, not only the advantage
of strong foreign gene expression under the control of the polyhedrin gene
promoter but also that the recombinant polypeptides are usually expressed in
a soluble form allowing the protein to fold into the proper structure. Insect
cells also carry most of the enzymes necessary for the posttranslational
modifications of such proteins [20].
The approach we used has proven to be useful for the study of the $1 spike
glycoprotein subunit of the bovine coronavirus [34] and the TGEV S protein,
although Mabs showed a reduced reactivity towards the truncated form of the
TGEV S protein [15]. Our results of the pig immunization experiments showed
that the bacuIovirus-expressed S polypeptides were capable of inducing TGEV
specific antibodies of the IgG class (measured in the FCEIA, Figs. 5B and 6)
and a positive correlation between the size of the recombinant protein and the
amount of antibodies could be observed. This phenomenon is probably due to
the higher number of antigenic determinants on the larger proteins and not
because of differences in the immunogenicity of these antigens, since similar
levels of VN activity were found in the sera of pigs immunized with rBTS 2.2,
3.3 and 4.4. Although the S polypeptides of rBTS 1.6, 2.2 and 2.2D were
expressed as fusion proteins, their immunogenicity, as judged by the FCEIA,
did not seem to be influenced by the coexpressed part of the polyhedrin protein.
Considering that the A region, which has been divided into subsites Aa,
Ab and Ac [3] is the immunologically most dominant region, as shown by De
Diego et al. [7] the absence of TGE specific VN antibody response to rBTS
1.6 (lacking site A) was not totally unexpected, although sites B and D (encoded
by rBTS 1.6) are also thought to be involved in virus neutralization to some
extent. The reason for the inability of this recombinant protein to induce a VN
response is not clear unless the D site does not generate neutralizing antibodies
in pigs, in contrast to mice. This result is in agreement with the observation
that not all Mabs binding to the 376 to 389 amino acid sequence of S have
neutralizing activity [24] and that pig polyclonal TGEV specific antibodies
did not recognize the sequence FFSYGE which is essential in TGEV neutralization with D site specific Mabs [25].
The baculovirus recombinant rBTS 1.8 that carries part of the A site coding
sequences, together with B, C, and D, induced only low levels of VN antibodies
and the neutralization even at the lowest serum dilution was incomplete (data
not shown). Antibodies with complete TGE VN activity were induced by
immunization with the recombinant proteins that contained all major antigenic
regions (rBTS 2.2, 3.3 and 4.4) and there was no significant difference in the
VN titres obtained with these antigens although the titers were considerably
lower than those obtained by TGEV immunizations [16]. Based on these results
it might be assumed that sites B, C and D are not necessary to generate a VN
response in pigs and that expression of the essential A site in a vector vaccine
may be sufficient to protect against TGE. However, the data obtained for those
Immunogenicity of the TGEV S protein
65
recombinant proteins which were encoded by the recombinant viruses with a
747 bp deletion at the 5'-terminal half of the S gene (rBTS 2.2D, 3.3D, and 4.4D)
refute this assumption. N o n e of these recombinants was able to induce a VN
response in pigs despite their intact A site coding sequences, and increasing
the size of the cloned gene from 1489bp to 2582bp or 3594bp in rBTS 3.3D
and 4.4D did not improve their VN generating capability. This result can be
explained by the fact that the deletion of these HaeIII D N A fragments not
only eliminated the D site coding region but also affected the two putative
receptor binding sites of the S protein [28, 29]. This deletion also may have
altered the proper folding of the site A epitopes. Our findings do not exclude
the possible utility of shorter deletions or deletions of different parts of the
Y-terminal half of the S gene as occurs naturally among strains of the porcine
respiratory coronavirus [28] although the effect of such deletions on the
globular structure [8] and on the immunogenicity of the amino terminal half
of the S protein is difficult to predict.
The results reported in this paper indicate that the amino acid residues
encoded by the Y-terminal half of the S gene do not affect the VN response
induced in pigs and that the amino-terminal half of the S protein is sufficient
to generate a VN response as effectively as the whole S protein, shown by the
comparison of rBTS 2.2 and 4.4. By reducing the size of the S gene from 4.3 kb
to 2.2 kb it may be possible to generate a helper independent recombinant
porcine adenovirus vaccine expressing the part of the S glycoprotein necessary
to induce protective immunity in pigs.
Acknowledgements
This study was supported by the Natural Sciences and Engineering Research Council of
Canada, the Ontario Ministry of Agriculture and Food and the Ontario Pork Producers'
Marketing Board. We thank Mr. P. Huber for photographic assistance, Dr. L. Jordan, and
Dr. S. Reddy for assistance with the experimental piglets, and Mr. D. Bridle and his staff
for their skilled animal care.
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Authors' address: Dr. J. B. Derbyshire, Department of Veterinary Microbiology and
Immunology, University of Guelph, Guelph, Ontario NIG 2W1, Canada.
Received December 8, 1993