Nov 27, 1985 - ... the LD50 of PRV Rice. For the experiments in swine, Landrace-Hampshire- ... and challenged with virulent virus (PRV Rice) on day 21. Each animal ... stained with neutral red and the resultant plaques were enumerated.
JOURNAL
OF
Vol. 61, No. 12
VIROLOGY, Dec. 1987, p. 3977-3982
0022-538X/87/123977-06$02.00/0 Copyright C 1987, American Society for Microbiology
Evaluation of Pseudorabies Virus Glycoprotein gp5O as a Vaccine for Aujeszky's Disease in Mice and Swine: Expression by Vaccinia Virus and Chinese Hamster Ovary Cells CARMINE C. MARCHIOLI, ROBERT J. YANCEY, JR., ERIK A. PETROVSKIS, JAMES G. TIMMINS, AND LEONARD E. POST* The Upjohn Company, Kalamazoo, Michigan 49001 Received 11 May 1987/Accepted 18 August 1987 Pseudorabies virus (PRV) is an alphaherpesvirus which causes an economically important disease of swine. One of the PRV glycoproteins, gp5O, was previously identified as the sequence homolog of herpes simplex virus glycoprotein gD (E. A. Petrovskis, J. G. Timmins, M. A. Armentrout, C. C. Marchioli, R. J. Yancey, Jr., and L. E. Post, J. Virol. 59:216-223, 1986). gp5O was evaluated as a PRV subunit vaccine candidate. gpSO protected mice from PRV-induced mortality either when delivered via infection with a recombinant vaccinia virus or when administered as a subunit vaccine produced in a eucaryotic cell line, Chinese hamster ovary (CHO) cells. In addition, gpSO synthesized in CHO cells protected pigs from lethal infection with PRV. This result demonstrates that a single viral glycoprotein could induce a protective immune response in the natural host of a herpesvirus infection.
Pseudorabies virus (PRV) is an alphaherpesvirus of swine. PRV infects adult pigs and establishes a state of latency with minimal symptoms. However, PRV can cause a severe and even lethal disease in young pigs. In other species, the pathogenesis of PRV is usually a lethal infection, regardless of the age of the animal (for a review, see reference 12). Because of the possibility that a PRV glycoprotein may be useful as a subunit vaccine for PRV, considerable attention has been directed to PRV glycoprotein genes. To date, genes have been mapped and sequenced for six PRV glycoproteins: gI (23, 27), gII (31a), gIII (32), gp50 (26, 39), gp63 (27), and the secreted glycoprotein gX (31). The PRV glycoproteins have been found to have partial amino acid sequence homology with herpes simplex virus (HSV) glycoproteins: gI is homologous to HSV gE (27); gII is homologous to HSV gB (31a); gIII is homologous to HSV gC (32); gpSO is homologous to HSV gD (26); gp63 is homologous to the US7 open reading frame of HSV (27); and gX is homologous to HSV-2 gG (22). In addition to sequence homology, the organization of the cluster of glycoprotein genes in the small component of the PRV genome is similar to that of a cluster of genes in the small component of the HSV genome (27). In view of this consistent homology, it might be expected that the functions of the PRV glycoproteins may be similar to those of their HSV counterparts. Of the HSV glycoproteins, gD has received particular attention as a subunit vaccine candidate. Early work showed that gD is the target of neutralizing antibodies that crossreact with the two types of HSV (7). It has been demonstrated that gD induces the most potent neutralizing monoclonal antibodies among those selected for binding of HSV virions (25). In addition to the induction of helper T cells (35), gD has also been reported to induce delayed-type hypersensitivity (34), cytotoxic (40, 41), and suppressor (20) T-cell responses. Purified gD was shown to protect mice from challenge by HSV (6, 10, 14). A secreted form of gD produced by the expression of a modified form of the cloned gD gene was found to protect mice (13) and guinea pigs (3) *
from HSV. HSV gD expressed by vaccinia virus is protective in mice (8, 24). Even synthetic peptides from the gD sequence are protective (11, 37). To our knowledge, however, none of these forms of gD has been tested as a human vaccine. Since all of these lines of evidence make gD look like a possible subunit vaccine for HSV, the gD homolog in PRV would be a candidate for a PRV subunit vaccine. Wathen and Wathen (39) originally proposed that PRV gpSO may be homologous to HSV gD, and sequencing of the gpSO gene demonstrated this to be true (26). The gpSO glycoprotein is a target of neutralizing antibodies that can protect mice and swine from PRV disease (38; C. C. Marchioli, R. J. Yancey, Jr., J. G. Timmins, L. E. Post, B. R. Young, and D. A. Povendo, submitted for publication). In this report, we describe two different types of experiments for testing gpSO as a subunit vaccine. Both when delivered via infection with a recombinant vaccinia virus and when delivered as a subunit synthesized in a eucaryotic cell line, gpSO was able to protect mice from PRV-induced mortality. In addition, gpSO synthesized in a Chinese hamster ovary (CHO) cell line protected pigs from lethal infection with virulent PRV. This demonstrates that a single herpesvirus glycoprotein could produce a protective immune response in the natural host species. MATERIALS AND METHODS Cell lines and viruses. The virulent strain of PRV (PRV Rice) was originally obtained from D. P. Gustafson of Purdue University, West Lafayette, Ind. Challenge virus stocks were grown in primary rabbit kidney cells obtained from H. E. Renis, The Upjohn Co., Kalamazoo, Mich. The construction of CHO cell lines expressing gpSO (CHO gp5O) has been previously described (26). Clones of transfected CHO cells expressing gpSO were isolated by cloning cylinders and screened for expression by immunofluorescence with anti-gp50 monoclonal antibody 3A-4 (26; Marchioli et al., submitted). The CHO cells expressing human renin, used as a negative control, have been described by Poorman et al. (30).
Corresponding author. 3977
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Vaccinia virus methods, including virus growth, DNA preparation, transfection, and selection of recombinants, were as described by Mackett et al. (17). The plasmid pGS20 (15) was obtained from B. Moss. Methods for the labeling of protein, immunoprecipitations, and gel electrophoresis have been previously described (26). Immunizations and animal experiments. In the animal
immunization studies with vaccinia virus expressing PRV gp50 (Vp50), CF-1 mice (Charles Rivers Breeding Laboratories, Portage, Mich.) were inoculated with either VpS0, wild-type vaccinia virus, or Eagle basal medium (M. A. Bioproducts, Walkersville, Md.). Inoculations were by either the footpad route (50 ,ul per mouse) or tail scarification (25 ,ul per mouse). At 4 weeks after immunization, the mice were challenged intraperitoneally with approximately 10 times the 50% lethal dose (LD50) of PRV Rice. CHO gpSO and CHO renin cells were grown to confluence in roller bottles in Dulbecco modified Eagle medium plus 10% fetal bovine serum and Eagle nonessential amino acid mixture to supply proline. Cells were harvested by rinsing in phosphate-buffered saline (PBS) minus magnesium and calcium, followed by incubation at 37°C with PBS minus magnesium and calcium plus 1 mM EDTA. After approximately 10 min, the cells were suspended in PBS-EDTA by tapping the roller bottle. The suspension (approximately 107 cells per ml) was frozen, thawed, sonicated briefly, and stored at -80°C. CF-1 mice were immunized with either CHO gp50 or CHO renin cells. The concentrations of the CHO cell lysates were adjusted so that each mouse received 106 disrupted cells per dose. A total of three doses of each preparation was given to mice on days 0, 10, and 21. The first dose was given subcutaneously and was composed of either one of the CHO cell lysates mixed in an equal volume with complete Freund adjuvant (CFA; GIBCO Laboratories, Grand Island, N.Y.), incomplete Freund adjuvant (IFA; GIBCO), aluminum hydroxide, or saline. The second and third doses were given intraperitoneally and were composed of CHO cell lysates suspended in PBS. Mice were chal-
lenged on day 28 by the footpad route with approximately 20 times the LD50 of PRV Rice. For the experiments in swine, Landrace-HampshireYorkshire pigs, 5 to 6 weeks old, were obtained from J. Gilmore Enterprises, Richland, Mich. These animals were housed in a confinement facility located at the Upjohn Agricultural Station and were randomly allotted into rooms, each room containing six pigs. Pigs were given intramuscular immunization with CHO cell preparations on days 0 and 14 and challenged with virulent virus (PRV Rice) on day 21. Each animal received 2 x 107 disrupted CHO gpSO or CHO renin cells per dose. The first dose was mixed with CFA, and the second dose was suspended in saline. A third group of pigs were immunized with a commercially available, live attenuated vaccine (PR-Vac; Norden Laboratories, Lincoln, Nebr.), according to the instructions of the manufacturer. All pigs were challenged at day 21 with approximately 40 times the LD50 (1.1 x 105 PFU per pig) of virulent PRV Rice by intranasal administration. The experiment was termi-
nated at 14 days postchallenge. Serum neutralization assay. Serum samples were assayed for the presence of virus-neutralizing antibody by a microneutralization assay as modified from published procedures (1). Briefly, pooled sera from mice or individual serum samples from pigs were obtained by venipuncture. Sera were heat inactivated and diluted in microtiter plates (Costar, Cambridge, Mass.). Each dilution was mixed with approximately 1,000 PFU of PRV Rice in the presence or absence of
J. VIROL.
10% rabbit complement (Cedarlane Laboratories Ltd., Hornby, Ontario, Canada) and incubated for 3 h at 37°C in the case of mouse sera or 1 h at 37°C in the case of pig sera. Approximately 20,000 porcine kidney-15 cells (PK-15; ATCC CCL 33) were then added to each well. After 48 h of incubation at 37°C, the cells were stained with crystal violet and the neutralization titer of each serum sample was expressed as the reciprocal of the highest dilution of serum which protected greater than 50% of the cells from cytopathic effects. Virus isolation from nasal swabs. In the pig experiments, the presence of PRV was assayed from nasal swabs. Swabs were placed in 1 ml of basal minimal Eagle medium (GIBCO) supplemented with 3% fetal bovine serum and antibiotics. These samples were stored at -70°C until assayed for virus. After the samples were thawed, the swabs were discarded and the media were tested for the presence of PRV. Aliquots of each sample were inoculated onto PK-15 cell monolayers and incubated for 1 h at 37°C to allow virus adsorption. An overlay of medium 199 supplemented with 4% fetal bovine serum, antibiotics, and 1% agar was placed on the infected cell monolayers. After 3 days, the infected monolayers were stained with neutral red and the resultant plaques were enumerated.
RESULTS Expression vectors. For evaluation of gpSO as a subunit vaccine, two different expression systems were used to produce gpSO. In the first of these, a CHO cell line was constructed that produced gp5O. Expression plasmid pDIE50PA was previously described (26). Briefly, the plasmid consists of the gp5O gene with a BamHI linker inserted at the NarI site 35 base pairs upstream from the gp5O initiation codon and an EcoRI linker inserted at the MaeIII site 41 base pairs downstream from the gp5O termination codon (Fig. la), the cytomegalovirus Towne major immediate-early promoter upstream from the gp5O gene, and the bovine growth hormone polyadenylation signal downstream from the gp5O gene, cloned into the pSV2dhfr vector to provide the dihydrofolate reductase selectable marker. This plasmid was transfected into CHOdhfr- cells, and the cells were shown to produce gp5O (26). A clone of the cells, CHOgp50-17, was isolated for use in the immunization of mice. For insertion of the gp5O gene into vaccinia virus, the vector pGS20 (22) was used. The structure of plasmid pVV50, which contains the gp5O gene inserted into pGS20, is shown in Fig. lb. Important features of pVV50 are that the gp5O gene has a BamHI linker inserted at the NarI site 35 base pairs upstream from the gp5O initiation codon; the gp5O gene is downstream from the 7.5-kilodalton promoter that has been shown to function both early and late in vaccinia infection (15); and the entire gp5O expression unit interrupts sequences coding for the vaccinia thymidine kinase gene. Plasmid pVV50 was transfected into vaccinia-infected cells, and thymidine kinase-negative recombinant viruses were selected with bromodeoxyuridine on 143 cells. The resulting viruses were demonstrated to contain PRV DNA by Southern blot analysis of viral DNA (data not shown). One of these recombinant viruses, VpSO, was selected for further analysis. Cells infected with VpSO produced a protein that was immunoprecipitated with monoclonal antibody 3A-4, and that protein had approximately the same molecular weight as gpSO from PRV-infected cells (Fig. 2). Although precise quantitation has not been done, cells infected with
VOL. 61, 1987
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FIG. 1. Construction of vaccinia virus gp5O (Vp5O). (a) A BamHI cleavage map of the PRV Rice genome is shown (31), with the inverted repeats of the genome indicated by boxes and the arrows showing sites of BamHI cleavage. The expanded map shows several restriction enzyme cleavage sites in the region of the gpSO gene (26). The direction of transcription of the gpSO gene is from left to right. (b) Plasmid pVV50 was used for construction of Vp5O. The NarI cleavage site upstream from the gpS0 gene was converted to a BamHI site with a linker (26), and the BamHI-MaeIII fragment containing the gpSO gene was inserted between the BamHI and SmaI sites of pGS20 (15). P7.5, Promoter from the gene for the vaccinia virus 7.5-kilodalton protein; V-TK, DNA coding for the vaccinia virus thymidine kinase gene; N, N-terminal sequences; C, Cterminal sequences; bp, base pairs; BUdRF, bromodeoxyuridine resistant.
Vp5O apparently produced more gp5O than did cells infected with PRV Rice. Protection of mice from virulent PRV with Vp5O and CHO gpSO. The results of protection tests with Vp5O, the vaccinia recombinant expressing gpSO, are summarized in Table 1. Mice inoculated by a single administration either by the footpad route or tail scarification were protected from the virulent PRV Rice. The animals also developed neutralizing antibody to PRV which was detected with or without complement supplementation in the assay procedure, although the titer with complement was considerably higher. Control animals given either wild-type vaccinia virus or medium by tail scarification did not respond with neutralizing antibody and were not protected from challenge with virulent virus, although a few mice in these groups survived infection. Results from a study analyzing protection of mice by gp5O expressed in a mammalian cell line, CHO-gp5O, are summarized in Table 2. Mice were immunized with disrupted CHO-gp5O or CHO-renin cells in various adjuvants or saline. Mice administered any of the CHO gpSO preparations were protected from challenge with virulent PRV compared with
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FIG. 2. Expression of gp5O from Vp5O. Infected cells were labeled with [3H]glucosamine. Lanes labeled PRV Rice are proteins from PRV-infected Vero cells. Lanes labeled Vp5O are proteins from HeLa S3 cells infected with Vp5O. Lanes labeled Vlac represent a recombinant with E. coli P-galactosidase inserted into pGS20 and recombined into the vaccinia virus genome (J. G. Timmins, unpublished data); +, proteins immunoprecipitated with anti-gpSO monoclonal antibody 3A-4; -, total proteins in the extract before immunoprecipitation. A fluorogram of a 12% sodium dodecyl sulfatepolyacrylamide electrophoresis gel is shown.
the CHO renin-treated or untreated controls. The neutralizing titers were higher and developed earlier in animals given adjuvant preparations. No neutralizing titers were detected (
