Expression of the A antigen (gp57-65) of Marek's disease virus by a recombinant baculovirus

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1 Journal of General Virology (1991), 72, t04. Printed in Great Britain 1099 Expression of the A antigen (gp57-65) of Marek's disease virus by a recombinant baculovirus Masahiro Niikura, 1. Yoshiharu Matsuura, 2 Masakazu Hattori, 1 Misao Onuma I and Takeshi Mikami 3 1 Department of Epizootiology, Faculty of Veterinary Medicine, Hokkaido University, Sapporo 060, 2Department of Veterinary Science, National Institute of Health, Tokyo 141 and 3Department of Veterinary Microbiology, Faculty of Agriculture, The University of Tokyo, Tokyo 113, Japan A recombinant baculovirus expressing the A antigen (A Ag) of Marek's disease virus (MDV) was constructed. In Spodoptera fruiperda (Sf) cells infected with the recombinant virus, A Ag expression was localized to the cell surface. Only a small amount of recombinant A Ag was detected in the culture supernatant of infected Sfcells, but authentic A Ag is mainly secreted into the culture supernatant of MDV-infected chicken embryo fibroblasts. Cell surface-associated recombinant A Ag seemed to be slightly larger than authentic A Ag, whereas the secreted recombinant A Ag seemed to be smaller. The recombinant A Ag was shown to be reactive with the sera of MDV-infected chickens by immunodiffusion studies and ELISA. Sera of chickens immunized with recombinant A Ag formed a precipitin line with the culture supernatant of MDV-infected chicken embryo fibroblasts in an immunodiffusion test. These results indicate that the recombinant A Ag expressed by the recombinant baculovirus retains the antigenic and immunogenic properties of the authentic A Ag. Introduction Marek's disease virus (MDV) is the causative agent of Marek's disease (MD) of chickens, which results in lymphomas and paralysis of the extremities (Calnek & Witter, 1984). Live vaccines developed from antigenically related but non-pathogenic viruses are used in the prevention of this disease (Okazaki et al., 1970; Purchase et al., 1972). The mechanisms of prevention of MD by live vaccines have been extensively studied as it is one of a few diseases induced by viruses, which result in tumour formation, that can be prevented by vaccination. However, the antigen(s) shared by pathogenic and nonpathogenic viruses which confers the protective immunity to vaccinated chickens has not been identified. In previous studies, two prominent antigenic glycoproteins, the A antigen (A Ag; gp57-65) and the B antigen (gpl00, 60, 49), were reported to be shared by pathogenic and non-pathogenic viruses (Okazaki et al., 1970; Witter et al., 1970; Purchase et al., 1972; Velicer et al., 1978; Ikuta et al., 1983, 1984). Of these antigens, the A Ag is the major antigenic protein produced in and secreted from infected cells during infection (Churchill et al., 1969; Van Zaane et al., 1982; Ikuta et al., 1983). The A Ag of MDV has been characterized as a glycoprotein of Mr of 61K to 65K, and its synthesis,_ pr_ocessin_g and. secretion have been studied fully (Long et al., 1975a,b; Glaubiger et al., 1983; Ikuta et al., 1985; Isfort et al., 1986). Moreover, the localization in the genomes of pathogenic and non-pathogenic viruses of the genes that encode A Ags, and their complete nucleotide sequences, have been reported (Isfort et al., 1986, 1987; Coussens & Velicer, 1988; Binns & Ross, 1989). The A Ag of pathogenic viruses has been reported to be lost during attenuation of the virus by serial passage in culture (Churchill et al., 1969; Ikuta et al, 1983), but the role of the A Ag in the course of infection and in the production of protective immunity against MD has not been studied widely. One of the problems in examining the role of the antigen is the difficulty in obtaining the antigenic protein in sufficient quantity in an authentic form, free from contamination with other viral antigens. Vectors derived from the insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) are among the most efficient available for the expression of foreign glycoproteins in eukaryotic cells (Matsuura et al., 1986, 1987; Luckow & Summers, 1988). Foreign proteins expressed by recombinant AcNPVs can be modified post-translationally, for example they can be glycosylated (Matsuura et al., 1987, 1989; Luckow & Summers, 1988; Kuroda et al., 1990). We employed this baculovirus expression systemtoproducetheaagofmdv. As well SGM

2 11 O0 M. Niikura and others as its use in the study of the role of the A Ag, the recombinant A Ag may be useful as an antigen for serological tests because it forms a predominant precipitin line in conventional immunodiffusion tests (Chubb & Churchill, 1968; Sharma, 1980). In this report, the construction of a recombinant AcNPV expressing the A Ag of MDV, and the molecular and immunological characterization of the expressed protein, are described. Methods Cells and viruses. AcNPV and recombinant AcNPV were grown in Spodoptera frugiperda (Sf) cells in Grace's medium (Gibco) supplemented with 10% foetal bovine serum. The plaque assay of AcNPV and recombinant AcNPV was done as described previously (Brown & Faulkner, 1977). MDV (strain Md5) was grown in primary chicken embryo fibroblasts (CEFs) in Eagle's MEM supplemented with 10% calf serum and 3 g/l tryptose phosphate broth (Difco). MDV was passaged three times in CEFs prior to use in this study. DNA manipulations. Plasmid DNA manipulations were performed by the alkali lysis method described by Maniatis et al. (1982). Restriction and other modifying enzymes were purchased from Takara Shuzo. The plasmid containing the BamHI B fragment of MDV (Fukuchi et al., 1984) was kindly provided by Dr K. Hirai (Tokyo University of Medicine and Dentistry, Tokyo, Japan); the AcNPV transfer vector, pacym1, has been described previously (Matsuura et al., 1987). Insertion of the MD V A Ag DNA into the pac YM1 transfer vector. The genomic DNA encoding the A Ag of MDV was excised from a plasmid containing the BamHI B fragment by complete digestion with EcoRI and partial digestion with KpnI. The digested fragments were separated by electrophoresis in an agarose gel and the fragment encoding the A Ag was subcloned into puc 18. The subcloned fragment was excised by digestion with BamHl and EcoRI, isolated by agarose gel electrophoresis and the ends were repaired using the Klenow fragment of DNA polymerase I. Phosphorylated BamHI linkers (0-01 A unit; Takara Shuzo) were then added to the repaired ends using T4 DNA ligase and excess linkers were removed by BamHI digestion and separation in a Quick Spin Column (Boehringer Mannheim). The DNA fragment with BamHI linkers added was inserted into the BamHI site of the pacym 1 vector. The orientation of the inserted fragment was confirmed by nucleotide sequencing of the 5' junction region using a 7-deazaguanosine sequencing kit (Toyobo). Transfection and selection of recombinant virus. Sf cells were cotransfected with a mixture of purified infectious AcNPV DNA (1 ~tg) and transfer vector DNA (25 Ixg) by the calcium phosphate method, as described previously (Matsuura et al., 1986). After 4 days incubation at 28 C, the culture supernatant was subjected to plaque assay. A plaque without polyhedra, identified by transmission light microscopy, was recovered and, after three successive plaque purifications, high titre stocks (> 107 p.f.u./ml) of the recombinant virus were obtained using Sf cell monolayers. Immunofluorescence analysis. Sf cells infected with the recombinant or wild-type AcNPV were harvested 48 h post-infection (p.i.) and incubated with a monoclonal antibody (MAb), M26, specific to the A Ag of MDV (Ikuta et al., 1983), for 30 min at room temperature (RT). The cells were washed with phosphate-buffered saline (PBS) and incubated with fluorescein isothiocyanate-conjugated anti-mouse immunoglobulin (Cappel) for 30 min at RT. The cells were washed again with PBS and examined for fluorescence. Immunoprecipitation analysis. Sf cells (lxl06) were infected with recombinant or wild-type AcNPVs at an m.o.i, of 1 and incubated at 28 C for 36 h. The infected cells were labelled with 50 [.tci TRAN[35S] - LABEL (ICN) in methionine-free medium for 12 h and lysed with lysis buffer (0-5 % NP40, 0-15 M-NaC1, 1 mm-pmsf and 0.02% sodium azide in 50 mm-tris-hc1 ph 8.0; Ikuta et al., 1981). The cell lysates and the supernatants of the infected cells were immunoprecipitated with the MAb and agarose-linked anti-mouse IgG (American Qualex International) and the immune complexes were analysed by SDS-PAGE. CEFs infected with MDV were labelled as described by Ikuta et al. (1983) using TRAN[3~S]-LABEL instead of [35S]methionine. The effect of tunicamycin (TM) on the expressed protein was examined as described previously (Matsuura et al., 1987; Isfort et al., 1986). ELISA. Sf cells infected with recombinant or wild-type AcNPVs were harvested and lysed with lysis buffer at the indicated times p.i. After centrifugation, the supernatants were diluted to 1:300 with carbonate buffer (50 mm, ph 9.6) and then adsorbed to ELISA plates (Sumitomo) overnight at 4 C. The adsorbed A Ag of MDV was detected with the MAb and peroxidase-conjugated goat anti-mouse immunoglobulin (Cappel) as described previously (Nishikawa et al., 1983). To detect the antibodies in the sera of experimentally infected chickens, a lysate of the recombinant virus-infected cells harvested 48 h p.i. was used as antigen. The chicken sera were used at a dilution of 1:2000 and A Ag-specific antibodies were detected using peroxidaseconjugated rabbit anti-chicken IgG (Zymed). Immunodiffusion test. Immunodiffusion was performed essentially as described (Long et al., 1975b). The antigen was prepared by lysing Sf cells infected with recombinant or wild-type AcNPVs at a concentration of 106 cells/50 ~tl. Authentic A Ag was prepared by concentrating the culture supernatant of MDV-infected CEFs as described by Long et al. (1975b). Production of antibodies to recombinant A Ag in a chicken. A chicken showing no serological activity against MDV was given a subcutaneous injection of recombinant AcNPV-infected cells mixed with Freund's complete adjuvant (Difco). After 7 days, the same amount of infected cells was injected intraperitoneally. Sera from the inoculated chicken were collected at 4, 6, 9 and 12 days after the second injection. Experimental infection of chickens. Newborn chicks were infected with the Md5 strain of MDV by intramuscular inoculation and sera were collected from surviving chickens 20 weeks p.i. Results Construction of recombinant virus The baculovirus transfer vector containing the A Ag gene of MDV was constructed as described in Methods. The nucleotide sequence of the 5' junction region of the plasmid indicated that this plasmid contained the A Ag gene in the proper orientation for expression, with 104 bp of non-translating sequence upstream of the initiation codon (Fig. 1). This plasmid was cotransfected into Sf cells with the purified infectious AcNPV DNA. One plaque without polyhedra was selected from the supernatant fluid of the transfected cells and, after plaque purification, stocks of the recombinant virus were obtained.

3 Expression of MD V A Ag by baculovirus 1101 A Ag of MDV i ~ i I - - "1 BarnHI B I i i i i MDV A Ag ~ i i i i J EcoR1 digest Kpnl partial digest Ligated into puci8 digested with EcoRl and Kpnl BamHL EcoRI digest Repaired using Klenow fragment of DNA polymerase ] Add BamH1 linkers Ligated into pacymi digested with BamHl 5' CTATAAATAGGATCCCGGGTACCAATCGG-90 bp- CTCATGCTC 3' BamHI KpnI Met Leu AcNPV ~ ~MDV *A Ag Fig. 1. Construction of the transfer vector containing DNA encoding the A Ag of MDV. Details of the preparation of the recombinant transfer vector are given in Methods. 0"8 I I I I I I infected, unfixed Sf cells (data not shown), indicating that the recombinant AcNPV expresses the A Ag. The kinetics of expression of the recombinant A Ag were examined by ELISA (Fig. 2). When Sf cells were infected with the recombinant AcNPV at a multiplicity of 1, the recombinant A Ag appeared 12h p.i. and reached a plateau 36 h p.i. The molecular characterization of the expressed protein by immunoprecipitation analysis is shown in Fig. 3. MAb M26 mainly precipitated a protein of Mr 58K to 68K from the lysate of infected Sf cells (Fig. 3 a), whereas it precipitated a 46K to 54K band from the supernatant of infected Sf cells, although the band was significantly fainter than the 58K to 68K band. The former protein was slightly larger than the authentic A Ag precipitated from the supernatant of MDV-infected cells (52K to 62K) and the latter was smaller. The 43K and 63K bands seen when MDV-infected CEFs and recombinant AcNPV-infected Sf cells, respectively, were analysed, were thought to be non-specific precipitates because these bands were present in negative controls. Moreover, immunoprecipitation analysis also showed that the recombinant A Ag was localized mainly in the lysate of infected cells rather than the culture supernatant, although the A Ag of MDV exists mainly in the culture supernatant. The effect of TM on the recombinant A Ag is shown in Fig. 3 (b). TM treatment of infected cells reduced the size of the precipitated band to 52K. 0-6.~ "0 I I l I I I I Time p.i. (h) Fig. 2. Time course studies on the expression of the A Ag in Sf ceils by recombinant AcNPV. Sf cells infected with recombinant AcNPV at a multiplicity of 1 were harvested, lysed at 12 h intervals and adsorbed onto microtitre plates. The adsorbed A Ag was detected with MAb M26 by the procedure described in Methods. Background A4o5 values of cells infected with wild-type AcNPV were subtracted. Antigenicity of the recombinant A Ag The antigenicity of the recombingnt A Ag was examined by immunodiffusion and ELISA. Precipitin lines were formed between the infected cell lysate and four sera from individual MDV-infected chickens, but not with normal chicken sera. No precipitin line was observed between the lysate of wild-type AcNPV-infected cells and sera of chickens infected or uninfected with MDV (data not shown). In ELISA, the sera of MDV-infected chickens reacted with the lysate of recombinant AcNPV-infected cells, but not that of wild-type AcNPV-infected cells (Fig. 4). Normal chicken serum did not react with lysates of either type of cell. Expression of the A Ag by recombinant A cnp V To confirm the expression of the A Ag in recombinant AcNPV-infected Sf cells, immunofluorescence analysis was performed; the MAb reacted with the surface of Immunogenicity of the recombinant A Ag The immunogenicity of the recombinant A Ag was examined by immunodiffusion using sera from the chicken inoculated with recombinant AcNPV-infected

4 1102 M. Niikura and others (a) !!!!!i!i~!!!!!iii 67K-- : " ~. iiiiiiiiiiiiii~ iiiiiiiiiiiii iiiiiiiiii!i!!i iiiiiiiiiiii!i! :+:+:+::?!~!~?~!~!~!i!!?~ ::::::::::::::: iiiiiiiiiiiiiii i!i!i!~!!!i!i!:::.:,:+:.:.:,: :.,, :+:+>. 43K- 0-2 :~:!:!:~:~:~:!' :::::::::::::::::::::::: Tfsfff :::::::::::::: -~ :I:I:I:I:I:Y ::~((::((: i:i:i:i:i:i:k q K -- ~ "; ~i 0.0 A B Chicken serum D N -i (b) 1 2 Fig. 4. Application of the recombinant A Ag in an ELISA. Specific antibodies in sera of individual MDV-infected chickens (A, B and D) were detected by ELISA using the recombinant A Ag as an antigen. Normal chicken serum (N) was also included as a control. The specificity of the reaction was confirmed using both the lysate of recombinant AcNPV-infected cells (black bars) and that of wild-type AcNPV-infected cells (open bars) as antigens. Discussion Fig. 3. Radioimmunoprecipitation of A Ag expressed by recombinant AcNPV. (a) SfceUs were infected with the recombinant (lanes 3 and 7) or wild-type (lanes 4 and 8) AcNPVs and incubated for 36 h. After labelling with 50~Ci 35S-labelled amino acid mixture for 12 h, the ceils (lanes 1 to 4) and culture supernatants (lanes 5 to 8) were subjected to immunoprecipitation using MAb M26. CEFs infected with MDV (lanes 1 and 5) or uninfected (lanes 2 and 6) were also subjected to immunoprecipitation. The recombinant and authentic A Ags are indicated by black and open arrowheads, respectively. (b) Sf cells infected with recombinant AcNPV in the presence (lane 1) or absence (lane 2) of TM were subjected to immunoprecipitation. The positions of M r markers (Electrophoresis Calibration Kit for low Mr proteins; Pharmacia) are indicated on the left of each panel. cells. The sera collected 4, 6 and 9 days post-inoculation formed a precipitin line with the concentrated culture supernatant of MDV-infected cells, but not with that of mock-infected cells (data not shown). We constructed a recombinant AcNPV that expresses the A Ag of MDV. The expressed A Ag was localized to the cell surface, but unexpectedly only a small amount was secreted into the supernatant of the infected cells; the A Ag has been reported to be present mainly in the culture supernatant of infected cells (Churchill et al., 1969; Van Zaane et al, 1982; Ikuta et al., 1983). The amino acid sequence of the A Ag contains a short (18 residues) hydrophobic sequence near its C terminus, even though it is secreted (Coussens & Velicer, 1988). However, Binns & Ross (1989) have shown that a long (33 residues) hydrophobic domain is present near the C terminus of the A Ag, in contrast to Coussens & Velicer (1988). The reason the recombinant A Ag expressed in Sf cells was not secreted efficiently might be due to the possible C-terminal hydrophobic sequence of the A Ag effectively anchoring the protein to the Sf cell membrane, although several other secretory proteins which have been expressed using recombinant baculovirus are secreted into the culture supernatant (Maeda et al., 1985; Smith et al., 1985; Jarvis & Summers, 1989). It is interesting to note that the sizes of the secreted A Ag of MDV, the secreted recombinant A Ag and the cellassociated recombinant A Ag were all different. The post-translational modification and secretion of A Ag have been reported to be complicated processes (Isfort et al., 1986). Further studies are needed to clarify the reasons for this size variation, although they may be explained in part by strain differences (the MDV used in this study was of the Md5 strain and the DNA used to

5 Expression of MD V A Ag by baculovirus 1103 construct the recombinant AcNPV was from the GA strain) or differences in post-translational modification in CEFs and Sf cells. TM treatment reduced the apparent size of the recombinant A Ag to 52K, which is relatively close to that of unglycosylated A Ag calculated from the amino acid sequence (53757 when the signal peptides are removed; Coussens & Velicer, 1988); this indicates that the recombinant A Ag is glycosylated. However, this result does not correspond to the previous observation of the reduction in size to 44K of the A Ag from MDVinfected CEFs treated with TM (Isfort et al., 1986). Unknown modification(s) of the A Ag produced in CEFs infected with MDV may occur; we obtained an identical reduction in A Ag size by TM treatment (data not shown). Binns & Ross (1989) also suspected the involvement of mechanisms other than those shown by Isfort et al. (1986) for the secretory nature of A Ag. By using the recombinant A Ag, MDV-specific antibodies in experimentally infected chickens could be detected both by immunodiffusion and ELISA. This indicates that the recombinant A Ag may be useful in the detection of MDV-specific antibodies for serological identification. Serum antibodies against MDV are conventionally detected by immunodiffusion or an indirect immunofluorescence assay (Sharma, 1980). For both assays, a low passage number MDV stock and a rather cumbersome procedure are necessary to prepare the antigen because the A Ag disappears from infected cells after serial passage in vitro (Churchill et al., 1969; Ikuta et al., 1983) and the antigens are produced at low levels by infected cells. The high expression of the recombinant A Ag by recombinant AcNPV, in combination with ELISA, may provide a more efficient method for the detection of antibodies to MDV. The immunogenicity of the recombinant A Ag was confirmed by immunodiffusion. Several A Ag epitopes appeared to be conserved on the recombinant A Ag. This conservation of epitopes indicates that the recombinant A Ag will be useful in studies on the rote of the A Ag in the course of MDV infection and in protection against MD, including its possible role in immune evasion (Isfort et al., 1986). This work was supported by grants from the Ministry of Education, Science and Culture of Japan. References BINNS, M. M. & Ross, N. L. J. (1989). Nucleotide sequence of the Marek's disease virus (MDV) RB-1B A antigen gene and the identification of the MDV A antigen as the herpes simplex virus-i glycoprotein C homologue. Virus Research 12, BROWN, M. & FAULKNER, P. (1977). A plaque assay for nuclear polyhedrosis viruses using a solid overlay. Journal of General Virology 36, CALNEK, B. W. & WITTER, R. L. (1984). Marek's disease. In Diseases of Poultry, pp Edited by M. S. Hofstad, H J. Barnes, W. M. Reid & H. W. Yodel Jr, Ames: Iowa State University Press. CHUBB, R. 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6 1104 M. Niikura and others MATSUURA, Y., MIYAMOTO, M., SATO, T., MORITA, C. & YASU, K. (1989). Characterization of Japanese encephalitis virus envelope protein expressed by recombinant baculoviruses. Virology 173, NISHIKAWA, K., ISOMURA, S., SUZUKI, S., WATANABE, E., HAMAGUCHI, M., YOSHIDA, T. & NAGAI, Y. (1983). Monoclonal antibodies to the HN glycoprotein of Newcastle disease virus. Biological characterization and use for strain comparison. Virology 130, OKAZAKI, W., PURCHASE, H. G. & BURMESTER, B. R. (1970). Protection against Marek's disease by vaccination with herpesvirus of turkeys. Avian Disease 14, 413~,29. PURCHASE, H. G., OKAZAKI, W. & BURMESTER, B. R. (1972). Long-term field trials with the herpesvirus of turkeys vaccine against Marek's disease. Avian Disease 16, SHARMA, J. M. (1980). Marek's disease. In Isolation andldentification of Avian Pathogens, pp Edited by S. B. Hitchner, C. H. Domermuth, H. G. Purchase & J. E. Williams. Texas: American Association of Avian Pathologists. SMITH, G. E., Ju, G., ERICSON, B. L., MOSCHERA, J., LAHM H.-W., CmZZONITE, R. & SUMMERS, M. D. (1985). Modification and secretion of human interleukin 2 in insect cells by a baculovirus expression vectcr. Proceedings of the National Academy of Sciences, U.S.A. 82, VAN ZAANE, D., BRINKHOF, J. M. A., WESTENBRINK, F. & GIELKENS, A. L. J. (1982). Molecular-biological characterization of Marek's disease virus. I. Identification of virus-specific polypeptides in infected cells. Virology 121, VELICER, L. F., YAGER, D. R. & CLARK, J. L. (1978). Marek's disease herpesviruses. III. Purification and characterization of Marek's disease herpesvirus B antigen. Journal of Virology 27, WITTER, R. L., NAZERIAN, K., PURCHASE, H. G. & BURGOYNE, G. H. (1970). Isolation from turkeys of a cell-associated herpesvirus antigenically related to Marek's disease virus. American Journal of Veterinary Research 31, (Received 16 October 1990; Accepted 5 February 1991)