Expression of Equine Herpesvirus 1 Glycoprotein ... - Journal of Virology

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Jun 1, 1993 - DARIA N. LOVE,' CHRISTOPHER W. BELL,2 DAVID PYE,3 STIRLING ... GLENDA L. LAWRENCE,2 DAVID BOYLE,4 TONY PYE,4 AND J.
Vol. 67, No. 11

JOURNAL OF VIROLOGY, Nov. 1993, p. 6820-6823 0022-538X/93/1 16820-04$02.00/0 Copyright ©D 1993, American Society for Microbiology

Expression of Equine Herpesvirus 1 Glycoprotein D by Using a Recombinant Baculovirus DARIA N. LOVE,' CHRISTOPHER W. BELL,2 DAVID PYE,3 STIRLING EDWARDS,3 MAI HAYDEN,3 GLENDA L. LAWRENCE,2 DAVID BOYLE,4 TONY PYE,4 AND J. MILLAR WHALLEY2* Department of Veterinary Pathology, University of Sydney, New South Wales 2006,1 School of Biological Sciences, Macquarie University, New South Wales 2109,2 CSL Ltd., Parkville, Victoria 3052,3 and CSIRO Australian Animal Health Laboratory, Geelong, Victoria 3220,4 Australia Received 1 June 1993/Accepted 2 August 1993

Glycoprotein D (gD) of equine herpesvirus 1 (EHV-1) was expressed at the surface of insect cells infected by a recombinant baculovirus. EHV-1 gD was detected as multiple forms (56, 52, and 48 kDa) from 18 to 96 h postinfection. Laboratory animals inoculated with the recombinant EHV-1 gD developed neutralizing antibody responses against both EHV-1 and EHV-4. Envelope glycoproteins of herpesviruses have a major role in the infectious process and in strain variation and are inducers of both humoral and cell-mediated immune responses (23, 25). Herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) elicits probably the strongest antiviral immune responses (4) and, when presented in a variety of purified and recombinant forms, protects animals against lethal infection with HSV-1 (3, 5, 19, 24). GP50, the gD homolog of pseudorabies virus, protects pigs against infection with whole virus (22). A gD gene homolog has been identified and partly characterized in the alphaherpesvirus equine herpesvirus I (EHV-1) (2, 6-8, 20, 27, 28). While the conservation of a gD homolog in EHV-1 suggests similar biological properties and functions, the gD polypeptides of EHV-1 and HSV-1 share only 20% amino acid identity. EHV-1 is an important pathogen of horses and is a cause worldwide of epidemic abortion, respiratory disease, and neurological symptoms (reviewed in reference 1). In common with other alphaherpesviruses, conventional EHV-1 vaccines have proved ineffective. As part of an assessment of the potential of EHV-1 gD as a component of a subunit vaccine, we have used a recombinant baculovirus to express EHV-1 gD in insect cells. This report describes the expression of the recombinant EHV-1 gD as several processed (glycosylated) forms, location within and at the surface of infected cells, and induction of neutralizing antibody in immunized animals. Baculovirus expression was carried out essentially as described by Summers and Smith (26). Recombinant Autographica californica nuclear polyhedrosis viruses (AcNPV) were constructed as follows. The polymerase chain reaction (PCR) was used to generate a 1.27-kbp fragment encompassing the EHV-1 gD open reading frame (27), flanked by 5' PstI and 3' BamHI sites. This PCR product was cloned into the AcNPV transfer vector pVL1392 (21). Polyhedrin-negative (recombinant) baculoviruses were selected after cotransfection of transfer construct and linearized DNA from wild-type virus (Invitrogen, San Diego, Calif.) into Spodoptera frugiperda (IPLB-Sf21-AE) cells and subjected to three rounds of plaque purification. For electrophoresis and immunoblotting, cell extracts and virus preparations were solubilized by boiling for 3 min in buffer containing 50 mM Tris-HCl (pH 6.8), 100 mM dithiothreitol, and 2% sodium dodecyl sulfate (SDS), and polypeptides were separated on SDS-polyacrylamide gels. *

Polypeptides were then transferred from gels onto nitrocellulose by a semidry method (18) and exposed first to primary antibody for 6 to 16 h and then to species-specific horseradish peroxidase-linked secondary antibody for 4 to 8 h, all with appropriate washing. The chromogen used for horseradish peroxidase was 4-chloro-1-naphthol. To monitor the expression of EHV-1 gD in insect cells, cultures were infected with approximately 10 PFU of recombinant AcNPV/EHV-1 gD per cell and sampled over a 120-h time period. Western blot (immunoblot) results clearly demonstrated that the EHV-1 gD was expressed in readily detectable antigenic forms. Figure 1 shows the time course of expression of polypeptides detected by EHV-1 gD-specific polyclonal rat serum. EHV-1 gD polypeptides were detected at 18 h postinfection (p.i.) as a single band of 52 kDa and then by 24 h as three main bands of 56, 52, and 48 kDa, possibly reflecting stages of post translational processing. At times later than 24 h, additional bands, possibly arising from degradation, were evident (see also lanes 4 and 7 of Fig. 3). The same expression profile was observed with use of rabbit anti-EHV-1 serum, monoclonal antibody 20C4 to EHV-1 gD (kindly supplied by G. P. Allen, University of Kentucky), and pooled convalescent equine serum (neutralizing titer of 40 to EHV-1)

(not shown). The cellular location of EHV-1 gD in insect cells was determined by immunofluorescence microscopy. Insect cells were seeded in multiwell chambers (Nunc Lab-Tek) at densities of approximately 5 x 104 cells per cm2, and wells were inoculated with recombinant or wild-type baculoviruses at approximately 5 PFU per cell. At 40 h p.i., cells were incubated in blocking buffer (2% bovine serum albumin and 5% rabbit serum in phosphate-buffered saline [PBS]) for 1 h at room temperature, with or without prior fixation in ice-cold methanol, and then washed three times in PBS. Preparations were incubated for 1 h with primary antibody. After three washes in PBS, cells were treated for 1 h at 37°C with appropriate Fluorescein-isothiocyanate-conjugated secondary antibody (1:100 to 1:200 dilution in blocking buffer). Figure 2 shows that both fixed and unfixed insect cells infected with EHV-1 gD recombinant at 40 h p.i. had distinct fluorescence in comparison with cells infected with wild-type baculovirus. When cells were incubated with primary antibody prior to fixation, fluorescence was seen clearly at the cell surface (Fig. 2A), indicating the transport to and presentation of EHV-1 gD at this location. In the case of fixed cells (Fig. 2C), EHV-1 gD

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FIG. 1. Western blot detection of EHV-l gD polypeptides electrophoresed on an SDS-8% polyacrylamide gel. Samples were taken as a time course study over a 120-h period from insect cell cultures infected by recombinant AcNPV/EHV-1 gD. Lanes: 1 to 8, 0, 18, 24, 40, 48, 72, 96, and 120 h p.i.; 9, wild-type 48 h p.i.; M, molecular weight markers. The primary antibody was rat monospecific serum raised against EHV-1 gD ,B-galactosidase fusion product (20).

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proteins were also visualized in the cytoplasm but not in nuclei. Similar results were obtained with rat monospecific gD antiserum, monoclonal antibody 20C4, and pooled convalescent equine serum. The EHV-1 gD gene products synthesized in recombinant baculovirus-infected cells were compared with EHV-1 gD synthesized in a variety of cells and other recombinant vehicles, using gel electrophoresis and Western blotting (Fig. 3). The distinct triple-banded pattern in insect cells was not evident in the mammalian cell preparations used in this blot, although multiple EHV-1 gD bands have been observed by us and others (8, 20). The 18-h band and the upper band of the 24-h recombinant baculovirus product were similar in mobility to EHV-1 gD in infected equine cells and to the EHV-1 gD product expressed by a vaccinia virus recombinant in a human cell line. Other mammalian cells transfected with EHV-1 gD constructs (a constitutively expressing CHO cell line and transiently expressing COS cells) also yielded single or multiple band products of similar mobilities. These data suggest that insect and mammalian cells may process the EHV-1 gD gene product by similar mechanisms. Some information on the glycosylation of EHV-1 gD was obtained by using peptide N-glycosidase F (Boehringer Mannheim), which removes both high-mannose and complex N-linked oligosaccharides. In preparations of 18-h p.i. EHV-1

FIG. 2. Immunofluorescence of insect cells infected with EHV-1 gD recombinant or wild-type baculovirus. (A and C) Cells infected with recombinant virus; (B and D) cells infected with wild-type virus; (A and B) cells treated with primary antibody prior to fixation; (C and D) cells incubated with primary antibody after fixation. Rat monospecific serum was the primary antibody in each case. All magnifications and exposure times were identical. Bar = 10 ,um.

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respectively; 4 and 7, insect cells infected with recombinant EHV-1 gD baculovirus for 18 and 24 h p.i., respectively; 5, 12, and 13, CHO cells stably transfected with pRC/CMV/EHV-1 gD plasmid and with pRC/ CMV vector (Invitrogen) and untransfected CHO cells, respectively; 9, equine dermis cells; 11, insect cells infected with wild-type baculovirus at 24 h p.i.; M. molecular weight markers. The primary antibody was monoclonal antibody 20C4. Electrophoresis conditions were as for Fig. 1.

with less than 20 for preimmunization sera from the same animals) and recognition in Western blots of the same molecular weight bands in EHV-1 and E. coli fusion product. To investigate whether the EHV-1 gD induced antibody responses to the genetically closely related respiratory pathogen EHV-4 (1), similar plaque reduction assays were carried out on an EHV-4 isolate (HMVI 28) in equine dermis cells. The rabbit serum prepared against the EHV-1 gD baculovirus recombinant product had a titer of 80 against EHV-4, while mouse serum prepared against the same product had a titer of 160. In the same assay, both convalescent horse serum and serum from a rabbit immunized with EHV-1 had titers of greater than 640 against EHV-4. Recombinant baculovirus expression systems have been used previously to express a variety of HSV-1 glycoproteins (7, 9-16), and several of these (gB, gC, gD, and gI) have been shown to protect mice against challenge with HSV-1. HSV-1 gD has also been shown to be expressed at the insect cell surface, in several glycosylated forms, and to induce neutralizing antibody in mice (15, 17). The characteristics of the baculovirus recombinant described here are consistent with a structural and functional conservation between EHV-1 gD and the gD of HSV-1, despite the low level of amino acid sequence homology. The neutralization data provide good support for the potential of recombinant EHV-1 gD in a vaccine against both EHV-1 and EHV-4. The EHV-1 gD recombinant baculovirus also represents a useful source of glycoprotein for vaccine studies and for further analysis of the structure and function of the gD homolog in EHV-1.

gD baculovirus-infected cells suspended in enzyme buffer, only two EHV-1 gD-specific bands of 54 and 52 kDa were evident. By 1 h and up to 8 h after enzyme addition, the 52-kDa gD band had a molecular mass shift to 46 kDa. This value compares with the 42.4 kDa predicted from amino acid sequence for the unglycosylated polypeptide after cleavage of the predicted signal sequence (27). Peptide N-glycosidase F treatment of 24-h p.i. recombinant-infected cells had no effect on the upper (56-kDa) band but resulted in a complicated band pattern due to deglycosylation and possible proteolytic degradation of the 52- and 46-kDa products. EHV-1-infected RK13 cells showed the same pattern of bands as the 18-h p.i. baculovirus preparation did, suggesting that the EHV-1 gD gene products in animal and insect cells were being similarly deglycosylated to forms possibly containing approximately 4 kDa of 0-linked sugars. Endoglycosidase and tunicamycin studies of HSV-1 gD expressed in insect cells have also demonstrated N glycosylation by alteration in electrophoretic mobilities from around 58 to 48 kDa, leaving a small amount of predicted 0-linked sugar residues (15, 17). The ability of baculovirus recombinant EHV-1 gD to induce a neutralizing antibody response was assessed in a standard plaque reduction complement-independent assay for EHV-1 (HVS 25A) plated on RK13 cells. In this assay, serum from a rabbit immunized with baculovirus recombinant gD-infected cell extracts (1 x 106 to 5 x 106 cells per immunization) had a titer of 80, serum from a rabbit immunized with whole EHV-1 had a titer of 320, and pooled convalescent EHV-1 horse serum had a titer of 40. Preimmunization rabbit serum had a titer of less than 10. The baculovirus gD rabbit antiserum recognized a single band at 54 kDa in EHV-1 and a 160-kDa EHV-1 gD Escherichia coli recombinant fusion product (20). Similar responses developed in immunized mice, with neutralization titers against EHV-1 of 160 and 40 to 80 (compared

This research was supported in part by a grant from the Australian Research Council. We thank George Allen for supply of monoclonal antibody, Kirsten Vandenberg and Bronwyn Pollock for immunization of mice, Robert Love for immunization of rabbits, Ron Oldfield for assistance with microscopy, and Jenny Norman for photography. REFERENCES 1. Allen, G. P., and J. T. Bryans. 1986. Molecular epizootiology, pathogenesis and prophylaxis of equine herpesvirus-1 infections. Prog. Vet. Microbiol. Immunol. 2:78-144. 2. Audonnet, J.-C., J. Winslow, G. Allen, and E. Paoletti. 1990. Equine herpesvirus type 1 unique short fragment encodes glycoproteins with homology to herpes simplex virus type 1 gD, gI and gE. J. Gen. Virol. 71:2969-2978. 3. Berman, P. W., T. Gregory, D. Crase, and L. A. Lasky. 1985. Protection from genital herpes simplex virus type 2 infection by vaccination with cloned type 1 glycoprotein D. Science 227:14901492. 4. Blacklaws, B. A., and A. A. Nash. 1990. Immunological memory to herpes simplex virus type 1 glycoproteins B and D in mice. J. Gen. Virol. 71:863-871. 5. Chan, W. 1983. Protective immunisation of mice with specific HSV-1 glycoproteins. Immunology 49:343-352. 6. Elton, D. M., I. W. Halliburton, R. A. Killington, D. M. Meredith, and W. A. Bonass. 1992. Identification of the equine herpesvirus type-1 glycoprotein 17/18 as a homologue of herpes simplex virus glycoprotein D. J. Gen. Virol. 73:1227-1233. 7. Flowers, C. C., E. M. Eastman, and D. J. O'Callaghan. 1991. Sequence analysis of a glycoprotein D gene homolog within the unique short segment of the EHV-1 genome. Virology 180:175184. 8. Flowers, C. C., and D. J. O'Callaghan. 1992. Equine herpesvirus 1 glycoprotein D: mapping of the transcript and a neutralization epitope. J. Virol. 66:6451-6460. 9. Ghiasi, H., R. Kaiwar, A. B. Nesburn, S. Slanina, and S. L. Wechsler. 1992. Baculovirus-expressed glycoprotein E (gE) of herpes simplex virus type-I (HSV-1) protects mice against lethal intraperitoneal and lethal ocular HSV-1 challenge. Virology 188: 469-476.

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FIG. 3. Western blot detection of EHV-1 gD polypeptides expressed in eukaryotic expression systems. Lanes: 1 and 15; inclusion body preparation of I-galactosidase EHV-1 gD fusion product expressed in E. coli; 2, 6, and 8, equine dermis cells infected with EHV-1 (100,000 x g, 1-h pellet); 3 and 10; 143B cells infected with recombinant EHV-1 gD/vaccinia virus and with wild-type vaccinia virus,

VOL. 67, 1993 10. Ghiasi, H., R. Kaiwar, A. B. Nesburn, and S. L. Wechsler. 1992. Baculovirus-expressed glycoprotein G of herpes simplex virus type-1 partially protects vaccinated mice against lethal HSV-1 challenge. Virology 190:233-239. 11. Ghiasi, H., R. Kaiwar, A. B. Nesburn, and S. L. Wechsler. 1992. Baculovirus-expressed glycoprotein H of herpes simplex virus type-1 (HSV-1) induces neutralizing antibody and delayed type hypersensitivity responses, but does not protect immunized mice against lethal HSV-1 challenge. J. Gen. Virol. 73:719-722. 12. Ghiasi, H., R. Kaiwar, A. B. Nesburn, and S. L. Wechsler. 1992. Baculovirus expressed herpes simplex virus type-1 glycoprotein C protects mice from lethal HSV-1 infection. Antiviral Res. 18:291302. 13. Ghiasi, H., R. Kaiwar, A. B. Nesburn, and S. L. Wechsler. 1992. Expression of herpes simplex virus type-1 glycoprotein B in insect cells-initial analysis of its biochemical and immunological properties. Virus Res. 22:25-39. 14. Ghiasi, H., R. Kaiwar, A. B. Nesburn, and S. L. Wechsler. 1992. Expression of herpes simplex virus type 1 glycoprotein I in baculovirus: preliminary biochemical characterization and protection studies. J. Virol. 66:2505-2509. 15. Ghiasi, H., A. B. Nesburn, R Kaiwar, and S. L. Wechsler. 1991. Immunoselection of recombinant baculoviruses expressing high levels of biologically active herpes simplex virus type-1 glycoprotein-D. Arch. Virol. 121:163-178. 16. Ghiasi, H., A. B. Nesburn, and S. L. Wechsler. 1991. Cell surface expression of herpes simplex virus type-1 glycoprotein H in recombinant baculovirus-infected cells. Virology 185:187-194. 17. Krishna, S., B. A. Blacklaws, H. A. Overton, D. H. Bishop, and A. A. Nash. 1989. Expression of glycoprotein D of herpes simplex virus type 1 in a recombinant baculovirus: protective responses and T cell recognition of the recombinant-infected cell extracts. J. Gen. Virol. 70:1805-1814. 18. Kyhse-Andersen, J. 1984. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J. Biochem. Biophys. Methods 10:203-209. 19. Long, D., T. J. Madara, M. Ponce de Leon, G. H. Cohen, P. C. Montgomery, and R. J. Eisenberg. 1984. Glycoprotein D protects

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mice against lethal challenge with herpes simplex virus types 1 and 2. Infect. Immun. 43:761-764. Love, D. N., C. W. Bell, and J. M. Whalley. 1992. Characterization of the glycoprotein D gene products of equine herpesvirus-1 using a prokaryotic cell expression vector. Vet. Microbiol. 30:387-394. Luckow, V. A., and M. D. Summers. 1989. High level expression of nonfused foreign genes with Autographa californica nuclear polyhedrosis virus expression vectors. Virology 170:31-39. Marchioli, C. C., R J. Yancey, Jr., E. A. Petrovskis, J. G. Timmins, and L. E. Post. 1987. Evaluation of pseudorabies virus glycoprotein gpSO as a vaccine for Aujeszky's disease in mice and swine: expression by vaccinia virus and Chinese hamster ovary cells. J. Virol. 61:3977-3982. Norrild, B. 1985. Humoral responses to herpes simplex infections, p. 69-86. In B. Roizman and C. Lopez (ed.), The herpesviruses, vol. 4. Plenum Press, New York. Paoletti, E., B. R Lipinskas, C. Samsonoff, S. Mercer, and D. Panicali. 1984. Construction of live vaccines using genetically engineered poxviruses: biological activity of vaccinia virus recombinants expressing the hepatitis B virus surface antigen and the herpes simplex virus glycoprotein D. Proc. Natl. Acad. Sci. USA 81:193-197. Spear, P. G. 1985. Glycoproteins specified by herpes simplex viruses, p. 315-356. In B. Roizman (ed.), The herpesviruses, vol. 3. Plenum Publishing Corp., New York. Summers, M. D., and G. E. Smith. 1987. A manual of methods for baculovirus vectors and insect cell culture procedures. Tex. Agric. Exp. Stn. Bull. 1555:1-55. Whalley, M., G. Robertson, C. Bell, D. Love, M. Elphinstone, L. Wiley, and D. Craven. 1991. Identification and comparative sequence analysis of a gene in equine herpesvirus-1 with homology to the herpes simplex virus glycoprotein D gene. Virus Genes 5:313-325. Whittaker, G. R., L. A. Taylor, D. M. Elton, L. E. Giles, W. A. Bonass, I. W. Halliburton, R A. Killington, and D. M. Meredith. 1992. Glycoprotein 60 of equine herpesvirus type-1 is a homologue of herpes simplex virus glycoprotein D and plays a major role in penetration of cells. J. Gen. Virol. 73:801-809.