Veterinary Medical Research Institute of the Hungarian Academy of Sciences, Budapest, Hungary1
Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, Ontario, CanadaN1G 2W12
Author for correspondence: Éva Nagy. Fax +1 519 824 5930. e-mail enagy{at}ovc.uoguelph.ca
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Abstract |
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Introduction |
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Transmissible gastroenteritis virus (TGEV) is a coronavirus that can cause severe and often fatal diarrhoea in young pigs. The presence of TGEV-specific secretory (s)IgA antibodies in the colostrum and milk of the sows is critical for the survival of the infected piglets. Development of mucosal immunity to TGEV is crucial, not only because it protects the sow from infection but also because sIgA and the sIgA-producing plasma cells, which are transferred into the mammary glands, provide protection for suckling piglets as well.
The protective immunity is directed to the spike (S) protein (Garwes et al., 1978 ; Jimenez et al., 1986
) and four major antigenic sites (A, B, C and D) have been described on the amino-terminal domain (Correa et al., 1990
; Delmas et al., 1990
). Virus-neutralizing antibodies are directed mainly to the A epitopes but to a lesser extent the D antigenic site is also involved. Earlier studies indicated that the intact globular N-terminal half of the protein is sufficient to achieve a protective immune response equivalent to that induced by the full S protein (Tuboly et al., 1995a
).
Although there are several commercially available TGEV vaccines, either inactivated or attenuated, these do not fully protect piglets. Several attempts have been made to develop efficacious recombinant TGEV vaccines. The S gene was expressed in prokaryotic expression vectors (Smerdou et al., 1996 ), vaccinia virus (Hu et al., 1985
), baculovirus (Godet et al., 1991
; Tuboly et al., 1994
) and most recently in plants (Gomez et al., 1998
; Tuboly et al., 2000
). The entire S gene and gene fragments of different sizes have also been expressed by human adenovirus (Torres et al., 1996
). Hamsters immunized with these recombinant viruses elicited a strong TGEV-specific immune response. Data on the effectiveness of the viruses in swine are not clear and the results suggest that a species-specific porcine adenovirus vector could be more effective than a human adenovirus vector.
Since porcine adenoviruses (PAdVs) do not generally cause disease in swine and since PAdVs are being considered as virus vector vaccines (Tuboly et al., 1993 ), especially where a mucosal immune response is required, the genomes of several PAdVs have been extensively studied. To date, five PAdV serotypes have been described (Haig et al., 1964
; Clarke et al., 1967
; Kasza, 1966
; Hirahara et al., 1990
). The E3 region, the site most targeted for foreign gene insertion in adenovirus genomes, has been identified and thoroughly analysed for all five serotypes (PAdV-4, Kleiboeker, 1994
; PAdV-3, Reddy et al., 1995
; PAdV-1 and -2, Reddy et al., 1996
; PAdV-5, Tuboly & Nagy, 2000
).
Recently PAdV-3 was developed into helper-dependent (Reddy et al., 1999a ) and -independent expression vectors (Reddy et al., 1999b
; Hammond et al., 2000
). Vaccines of helper-independent virus vectors are more practical. To date, two virus genes have been expressed by PAdV-3. The gD gene of Aujeszkys disease virus was inserted into the E3 region (Reddy et al., 1999b
) and the E2 gene of classical swine fever virus was inserted near the right-hand terminus of the virus genome (Hammond et al., 2000
). Although both recombinant PAdV-3 viruses expressed the inserted foreign gene, the widespread occurrence of PAdV-3 in swine populations may restrict the use of this serotype as a vaccine vector.
In contrast, the virus involved in the present study (PAdV-5) was originally isolated in Japan (Hirahara et al., 1990 ) and there are no further reports on the presence of PAdV-5 elsewhere around the world. Our earlier studies indicated that at least 60% of the E3 region was not essential for virus replication (Tuboly & Nagy, 2000
), increasing the theoretical vector capacity of PAdV-5 to 2·9 kb, which is much larger than the size given for PAdV-3 (Reddy et al., 1999b
).
The objective of this study was to construct stable recombinant PAdV-5 viruses expressing the TGEV S protein responsible for the induction of virus-neutralizing antibodies. Five different types of recombinant viruses were generated carrying either the entire or the amino-terminal half of the S gene. In order to determine the best configuration for the strongest possible expression without including additional promoter sequences, the genes were inserted in both orientations. Viruses with or without a deletion of the E3 region were generated and tested for the expression of the foreign gene. Viruses expressing the S gene were tested for their ability to induce S protein-specific antibodies in pigs, the natural host of both TGEV and PAdV-5.
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Methods |
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Adenovirus DNA was extracted from PAdV-5-infected ST cells by the method of Hirt (1967) when extensive cytopathic effect (CPE) was seen. TGEV S gene cDNA synthesis and cloning have been described elsewhere (Tuboly et al., 1994
).
Transfer vector construction.
Full-length genomic PAdV-5 DNA clones were constructed by homologous recombination in E. coli strain BJ5183 cells (Hanahan, 1983 ) as described (Degryse, 1996
). The strategy for the construction of the recombinant transfer vectors is summarized in Fig. 1
. Plasmid Rpac+ was generated by replacing the 1·9 kb SalIHpaI fragment (spanning part of the pVIII protein and the majority of the E3 coding region) with a unique PacI restriction enzyme (RE) site. The MluI B fragment of PAdV-5 (Tuboly et al., 1995b
) was used for the insertion of the S gene. Five different S gene-containing MluI B fragments were generated. One construct contained the entire E3 region while in the remaining four constructs, a 1·2 kb HincIIHpaI fragment was deleted in the E3 region. The five fragments generated were: (i) MluIB-2.2S, which contains the 2·2 kb 5' end of the 4·4 kb S gene inserted into the HpaI site of the E3 region in left to right (lr) orientation; (ii)
MluIB-2.2Sc, which contains the 1·2 kb HincIIHpaI fragment of the E3 region replaced by the 2·2 kb 5' S fragment in lr orientation; (iii)
MluIB-2.2Sr, which has the same deletion in the E3 as in the
MluIB-2.2Sc construct but with the 2·2 kb S gene inserted in the reverse (rl) orientation; (iv)
MluIB-2x2.2S, which contains the 2·2 kb S gene inserted in both the lr and the opposite rl orientations as contiguous inverted repeats into the partially deleted E3 region; and (v)
MluIB-4.4S, which has the entire 4·4 kb S gene inserted into the partially deleted E3 region in lr orientation.
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DNA transfection and selection of recombinant viruses.
Lipofectin-mediated (Life Technologies) ST cell transfections were performed as previously described (Tuboly & Nagy, 2000 ), following the instructions of the manufacturer. The transfected cells were covered with 0·7% agarose in DMEM supplemented with 10% foetal bovine serum. Plaque formation was monitored daily and ten individual plaques from each transfection were transferred to Eppendorf centrifuge tubes with 1 ml of DMEM on day 7 post-transfection. The tubes were frozen to -70 °C and thawed on ice. The contents were used for the inoculation of duplicate wells of ST cell monolayers in 6-well tissue culture plates.
The cell culture supernatant from each well was collected about 67 days after inoculation and stored at -70 °C until the next round of plaque purification. Cells were harvested for RE analysis of the DNA and for Western blotting. Only those viruses that contained the entire expected S gene insert (one from each lineage) were included in further rounds of plaque purification. Viruses selected after three rounds of such plaque purification were designated RPAdV-2.2S, RPAdV-2.2Sc,
RPAdV-2.2Sr,
RPAdV-2x2.2S and
RPAdV-4.4S and were used for large-scale virus propagation.
Western blot analysis of recombinant S proteins.
Wild-type and recombinant adenovirus-infected cells together with uninfected ST cells were harvested at the peak of CPE formation. The proteins were separated on 10% SDSpolyacrylamide gels as described (Laemmli, 1970 ) and transferred to nitrocellulose membranes (Sambrook et al., 1989
). They were detected with TGEV-specific pig polyclonal antibodies (Tuboly et al., 1994
) at a 1:500 dilution. The reaction was developed by the Boehringer Mannheim chemiluminescent detection kit according to the instructions of the manufacturer.
S gene mRNA time-course.
ST cells grown in 6-well dishes were infected at an m.o.i. of 10 with the recombinant and wild-type viruses. RNA was extracted with the total RNA extraction kit (RNeasy, QIAGEN) every 4 h between 2 and 24 h post-infection (p.i.) and frozen to -70 °C. RNA from mock-infected ST cells was also collected. Equal amounts of the total RNA from each time-point were separated in 1·1% formaldehydeagarose gels, transferred to Nytran membranes (Sambrook et al., 1989 ) and immobilized by UV cross-linking (UV Cross-linker, Fisher Scientific). Prehybridization, hybridization in the presence of 50% formamide and washing of the blots were carried out as described by Sambrook et al. (1989)
. The cloned 2·2 kb TGEV S gene was released from the plasmid, labelled with [32P]dCTP (ICN Pharmaceuticals) by the random primer method (Random primer labelling kit, Life Technologies) and used as a probe.
Animal experiments.
Fifteen Yorkshire piglets from a TGEV- and PAdV-5-seronegative herd were weaned 21 days after birth and divided into five groups and housed separately. One group received uninfected ST cell supernatant, one group was immunized with wild-type PAdV-5 and three groups were immunized with the selected recombinant viruses (RPAdV-2.2S, RPAdV-2.2Sc and
RPAdV-2.2Sr). Each pig received a single oral dose of 1 ml with a virus titre of 5x106 p.f.u./ml. Blood samples were collected weekly and the clinical signs were monitored daily. The pigs were euthanized after 3 weeks and subjected to post-mortem examination. Contents from the small intestine and parts of the lung were collected and processed as described (Tuboly et al., 1993
) and then tested for the presence of virus and sIgA antibodies. For antibody detection, the serum samples and the filtered intestinal and lung contents were heat-inactivated at 56 °C for 1 h. Samples were tested in a TGEV-specific IgG or IgA ELISA as described by Tuboly et al. (1993)
and in a TGEV-specific VN microtitre assay (Tuboly et al., 2000
). The PAdV-5-specific antibodies were also determined with a VN assay (Tuboly et al., 1993
).
Rectal swabs were collected daily to monitor virus shedding. The swabs were processed as described (Tuboly et al., 1995a ) and the virus titres were determined on 96-well plates with ST cells. The viruses isolated at day 5 p.i. were pooled in each group and propagated in ST cells for DNA extraction and RE analysis of the virus DNA.
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Results |
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Construction and selection of recombinant PAdV
Recombinant PAdVs were plaque-purified three times and the presence and orientation of the S gene were confirmed by RE analysis of the virus DNA (data not shown) after each round of plaque purification. The expression of the recombinant S protein was monitored by Western blot analysis. Table 1 summarizes the stability data of each recombinant virus in tissue culture.
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Transfection with the vector containing two duplicate 2·2 kb S genes inserted in opposite orientations, carrying altogether a 4·4 kb foreign DNA insert, yielded eight positive plaques in the first round of purification, all of which expressed the S protein and the insert and S gene expression remained stable during subsequent plaque purification.
Only 20% of the viruses in which the complete S gene was inserted into the PAdV E3 region (RPAdV-4.4S) retained the S gene after transfection and the ratio remained low throughout further plaque purifications. In contrast with the rest of the recombinant viruses, the RE digests of the DNA always indicated that only part of the virus population from a single plaque carried the entire S gene, smaller DNA fragments also appeared even after the third plaque purification (data not shown). Similarly, many bands were observed in the Western blots of cells infected with these virus clones.
Expression of the S gene and S protein
S gene expression was monitored by Northern blot analysis of total RNA extracted at 2 h p.i. and every 4 h thereafter from recombinant virus-infected cells and blots were probed with radioactively-labelled 2·2 kb S gene DNA.
RPAdV-2.2S and RPAdV-2.2Sc expressed TGEV S gene-specific mRNA at approximately the same level. The S gene mRNA synthesis in RPAdV-2.2S-infected cells was undetected during early times of virus replication and could be detected only at 18 h p.i., whereas S gene-specific mRNA appeared somewhat earlier in
RPAdV-2.2Sc-infected cells, at 14 h p.i. (Fig. 2
).
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For Western blot analysis, cells infected with the different recombinant viruses were collected at 24 h p.i. RPAdV-2.2S and RPAdV-2.2Sc expressed the S protein of the expected 110 kDa size (Fig. 3
, lanes 2 and 3) and a similar result was obtained with the
RPAdV-2x2.2S recombinant virus (Fig. 3
, lane 4). No S protein was detected in cells infected with
RPAdV-2.2Sr virus. S protein-specific bands with a wide range of sizes (30220 kDa) were seen on the blots of samples collected from
RPAdV-4.4S-infected cells (Fig. 3
, lane 5).
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ELISA and VN assays were conducted to detect TGEV- and PAdV-5-specific antibodies in the infected pigs. The results are summarized in Table 3.
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Discussion |
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PAdV-3 carrying the gD gene of Aujeszkys disease virus (Reddy et al., 1999b ) and the E2 gene of classical swine fever virus (Hammond et al., 2000
) has already been developed as a recombinant virus vector. However, the widespread prevalence of PAdV-3 may be a limiting factor in their use as recombinant vaccines because of widespread pre-existing PAdV-3-neutralizing antibodies.
In contrast, PAdV-5, to our knowledge, is not present in pig populations and has been reported only once in Japan (Hirahara et al., 1990 ). The development of PAdV-5 into a recombinant TGEV vaccine is described in this paper. Five helper-independent recombinant porcine adenoviruses have been constructed and tested for their stability and their ability to express the entire or the 5' 2·2 kb half of the TGEV S gene. Although the genome size of RPAdV-2.2S was, together with the 2·2 kb foreign gene, 106·6% of the original wild-type genome neither the insert nor parts of the E3 region were lost during the plaque purifications or the several virus replication cycles in the pig intestine. The insertion and stable maintenance of such a large foreign DNA is in accordance with the findings of Hammond et al. (2000)
, who increased the genome size of PAdV-3 to 106·8% of the original, despite earlier findings of a maximum of 105% for human adenoviruses (Bett et al., 1993
).
One of our goals was to test whether it was necessary to include foreign gene promoter sequences upstream of the insert or if the native PAdV promoters were sufficient to express the gene. In one construct (RPAdV-2.2S) no E3 sequences were removed and the 2·2 kb S gene fragment was inserted in a lr orientation near the 3' end of the E3 region, more than 1·8 kb downstream of the putative E3 promoter (Tuboly & Nagy, 2000 ). S gene-specific transcripts were detected in Northern blots from 18 h p.i., reaching a peak between 18 and 24 h p.i. The S protein was detected in Western blots, indicating that the native adenovirus promoters were sufficient for foreign gene expression (Torres et al., 1996
).
As a result of the 1·2 kb deletion of the E3 region (Tuboly & Nagy, 2000 ), the rest of the recombinant viruses carried the S gene closer to the E3 promoter than in RPAdV-2.2S. Those recombinant viruses that had the insert in the lr orientation (
RPAdV-2.2Sc,
RPAdV-2x2.2S and
RPAdV-4.4S) started to express the gene at the end of the early replication stages, between 14 and 18 h p.i., as detected by Northern blot analysis, whereas the virus with the S gene in reverse orientation (
RPAdV-2.2Sr) showed no signs of S gene expression either in vitro (Northern and Western blots) or in vivo, as judged by the lack of TGEV-specific antibodies in the immunized pigs.
Those RPAdVs carrying a single copy of the 2·2 kb S gene appeared to be stable immediately after the transfection and all plaques tested had the inserted gene of the expected size and at the expected position.
RPAdV-2x2.2S, with two sets of the 3'-truncated S gene, produced 7 out of 10 plaques that carried both inserts right after the transfection and became stable during further rounds of plaque purification. The virus did not lose any of the inserts or the PAdV sequences, as detected by RE analysis (data not shown). The size of the genome of this recombinant virus was 109·6% of the original genome size, exceeding the expected maximum of 106·8% (Hammond et al., 2000
) described for PAdV-3. The
RPAdV-4.4S virus with the full-length S gene did not yield a stable lineage, despite several rounds of plaque purification of the positive viruses. The expected genome size of this virus was also 109·6% of the wild-type genome but unlike the
RPAdV-2x2.2S, parts or all of the insert or the PAdV genome were constantly being lost during virus replication. This phenomenon raised questions about current theories of adenovirus genome stability. According to our experiments, the size of the insert may not be the only important factor influencing the stability of the genome, the nature of the foreign transcript and protein may also play a role.
The recombinant viruses were analysed by Western blotting to determine the size of the recombinant proteins. All of the viruses with the gene in a lr orientation expressed a protein of the predicted size. The estimated size of the S protein in RPAdV-2.2S, RPAdV-2.2Sc and
RPAdV-2x2.2S was 110 kDa. The
RPAdV-4.4S virus preparation also expressed the expected 200 kDa protein but smaller S protein fragments were also detected.
Direct measurement of the amount of recombinant proteins was not carried out, but from comparisons to known amounts of baculovirus- and transgenic plant-expressed S genes (Tuboly et al., 1994, 2000 ), it was estimated that approximately 510 µg S protein/106 cells was obtained at 24 h p.i. This amount is in accordance with that of Torres et al. (1996)
for the expression of TGEV S gene and gene fragments in a human adenovirus vector without the help of additional external promoters.
Three recombinant viruses were tested for their ability to induce TGEV-specific immune responses in pigs. Those viruses that carried the 2·2 kb S gene in a lr orientation induced a TGEV-specific immune response. It was concluded that a single oral dose of the recombinant virus was sufficient to induce both a systemic and a local humoral immune response. The antibodies induced by the recombinant viruses neutralized both PAdV-5 and TGEV. The presence of TGEV-specific IgA antibodies in the small intestine indicated that a local immune response, particularly important against TGEV, was also induced.
Although challenge experiments were not carried out, we conclude that recombinant PAdV-5 carrying the 2·2 kb S gene fragment could be a useful tool in the protection of swine herds against TGEV. It remains important to test the ability of these viruses to protect pigs from TGEV in field trials involving different age groups of pigs and TGEV strains of differing virulence.
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Acknowledgments |
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References |
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Received 18 May 2000;
accepted 22 September 2000.