key: cord-0004963-ecfwnphv
authors: Tuboly, T.; Nagy, É.; Dennis, J. R.; Derbyshire, J. B.
title: Immunogenicity of the S protein of transmissible gastroenteritis virus expressed in baculovirus
date: 1994
journal: Arch Virol
DOI: 10.1007/bf01311173
sha: 9f7c607911ddb87ca96ce7934cace386e59381ff
doc_id: 4963
cord_uid: ecfwnphv

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.

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.

TGEV 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 TGEV (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 TGEV S protein have also been expressed in recombinant vaccinia viruses, and they were tested in mice, where both were shown to induce TGEV neutralizing antibodies [17, 25] .

Passive protection of neonatal piglets is of primary importance in the control of TGE and the only efficient way to achieve this is by TGEV specific immunoglobulins (Igs) primarily of the secretory (s) IgA class in the colostrum and milk of the sow. TGEV 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 human 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.

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 clonin 9

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 

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 [30] ), Three times plaque purified viruses were propagated and the extracellular virus was used in further studies.

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] .

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] .

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.

Eight baculovirus recombinants were generated by the cotransfection of A c N P V D N A and the pVL1393 transfer vector carrying different fragments of the T G E V S gene cDNA. The construction of the recombinant transfer vectors resulted in an identical 8 basepair 5' leader sequence from the T G E V genome upstream of the S gene initiation codon.

BamHI-EcoRI and SalI generated D N A fragment patterns of the purified wild type and recombinant virus D N A s and the corresponding Southern blot hybridization are shown in Fig. 3 . The BamHI-EcoRI double digest confirmed the size of the foreign gene in the recombinant viruses and the SalI RE pattern indicated that these c D N A fragments were within the polyhedrin gene as the size of the 3.07 kb A c N P V SalI band was increased in each recombinant by 60 T. Tuboly et al. (Fig. 3A) . The Southern blot hybridization with the S gene cDNA showed reactions only with the DNA 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).

The recombinant S polypeptide expression was monitored by SDS-PAGE using [35S] methionine labelled lysed infected Sf21 cells (Fig. 4A) , or the proteins immunoprecipitated by TGEV specific hyperimmune serum (Fig. 4B) , and these gels were used for the estimation of the size of the recombinant polypeptides. (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).

Sera collected weekly from pigs immunized with Sf21 cells infected with the recombinant viruses were examined for the presence of TGEV neutralizing antibodies. Figure 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.

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 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 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. None 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 DNA 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.

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Comparison of inactivated Newcastle disease viral vaccines containing different emulsion adjuvants

Antigenic structure of the E2 glycoprotein from transmissible gastroenteritis coronavirus

Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus

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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.

Authors' address: Dr. J. B. Derbyshire, Department of Veterinary Microbiology and Immunology, University of Guelph, Guelph, Ontario NIG 2W1, Canada.Received December 8, 1993