Identification of Virus-specific Polypeptides by Monoclonal Antibodies against Serotype 2 Marek's Disease Virus

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1 J. gen. Virol. (1989), 70, Printed in Great Britain 2563 Key words: MDV2/MAbs/polypeptides Identification of Virus-specific Polypeptides by Monoclonal Antibodies against Serotype 2 Marek's Disease Virus ByKAZUHIRO NAKAJIMA, 1. TAKESHI SHIBAYAMA, L MIYA YOKOTA, 1 KAZUYOSHI IKUTA, 1 SHIRO KATO 1 AND KANJI HIRAI 2 t Department of Pathology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565 and 2Department of Virology and Immunology, Medical Research Institute, Tokyo Medical and Dental University, Yushima, Bunkyo-ku, Tokyo 113, Japan (Accepted 15 June 1989) SUMMARY A total of 41 antibody-secreting hybridoma cells against the HPRS24 strain of Marek's disease virus (MDV) type 2 (MDV2) have been isolated. Of these monoclonal antibodies (MAbs), 24 were found by immunofluorescence tests to react specifically with MDV2-infected cells, but not MDV type 1 (MDV1)- or herpesvirus of turkeys (HVT)-infected cells, while eight reacted with MDV1- or MDV2-infected cells and nine with MDVI-, MDV2- or HVT-infected cells. By using these MAbs, seven classes of MDV type-specific or cross-reactive polypeptides were characterized by immunoprecipitation followed by SDS-PAGE. Among them, a 28K/32K glycoprotein differed from the previously identified ga and gb. The 28K/32K glycoprotein was found on the surface of MDV2-infected cells and in the cytoplasm by an immunofluorescence test with MAbs. In addition, a cross-reactive polypeptide of 25K/29K was also detected in MDVl-infected cells with MAbs reactive with the 28K/32K glycoprotein of MDV2. INTRODUCTION Marek's disease virus (MDV) is the aetiological agent of Marek's disease (MD), a highly contagious, neoplastic disease in chickens. Strains immunologically related to MDV have been divided into three serotypes on the basis of serological analysis with an indirect immunofluorescence test and an agar gel precipitation test (yon Billow & Biggs, 1975a, b). Serotype 1 (MDV 1) includes pathogenic strains of MDV and attenuated forms of these strains. Serotype 2 (MDV2) includes naturally occurring apathogenic strains of MDV. Serotype 3 (MDV3) includes the herpesvirus of turkeys (HVT). MD is controlled by a live vaccine based on the three serotypes of MDV, and HVT has been widely employed as a vaccine against MD. Recently very virulent strains (vvmdv) have developed in HVT-vaccinated flocks leading to so-called vaccine breaks (Eidson et al., 1978, 1981 ; Witter et al., 1980; Witter, 1983). Because vvmdv has emerged, improvement of the vaccines is urgently needed. Protection against vvmdv by inoculation of the combination of HVT and MDV2 strain SB1 has been demonstrated in laboratory experiments (Witter, 1982) and in field trials (Calnek et al., 1983; Witter et al., 1984). Further, Witter et al. (1987) examined several vaccine candidates against vvmdv and demonstrated that some of the MDV2 isolates may be good vaccine candidates. The identification of MDV-induced polypeptides, especially cross-reactive glycoproteins, is a prerequisite for studies on vaccinal immunity to MDV. Ikuta et al. (1981) and Van Zaane et al. (1982) have identified more than 40 proteins of MDV1 and HVT, ranging in size from 19K to 350K, by immunoprecipitation with polyvalent sera. Six kinds of polypeptides were also identified by monoclonal antibodies (MAbs) against MDV-related viruses. These proteins are glycoprotein A (ga) (Ikuta et al., 1983; Hirai et al., 1986), glycoprotein B (gb) (Ikuta et al., SGM

2 2564 K. NAKAJIMA AND OTHERS 1984 a; Silva & Lee, 1984; Ono et al., 1985 ; Hirai et al., 1986), a major virus-specific polypeptide (Ikuta et al., 1984b), DNA-binding protein (Nakajima et al., 1986), non-dna-binding proteins (Nakajima et al., 1986) and phosphorylated polypeptides (Ikuta et al., 1985; Naito et al., 1986; Nakajima et al., 1987). Of these the two glycoproteins ga and gb were identified on the surface of infected cells. Ono et al. (1985) showed that immunization of chickens with purified gb resulted in partial protection against MD. Although the isolation of MAbs against MDV2 and their reactivity in immunofluorescence tests have been reported (Lee et al., 1983; Silva & Lee, 1984; Hirose et al., 1987), the polypeptides recognized by such MAbs have not been identified. To provide a background for studying immunity to MDV2 in the future, we investigated the MDV2-induced polypeptides with MAbs. The present study was designed to develop MAbs against MDV2 and identify the MDV2-induced polypeptides that contain serotype-specific and serotype-common epitopes. METHODS Virus and cells. The virus strains used were the oncogenic strains BC1 (Spencer et al., 1972), JM (Sevoian et al., 1962) and Md5 (Witter et al, 1980) of MDV1, the non-oncogenic strains C2 (Kato et al., 1970) and CVI988 (Rispens et al., 1972) of MDV2, strains HPRS24 (Biggs & M ilne, 1972) and SB 1 (Schat & Calnek, 1978) of MDV2 and vaccine strains O1 (Ono et al., 1974) and FC126 (Witter et al., 1970) of HVT. These viruses were propagated in primary chicken embryo fibroblasts (CEF) as described previously (Ikuta et al., 1981). Radiolabeliing ofcelis. Mock-infected and infected cells in 60 mm diameter dishes were labelled with 100 ~tci L-[3sS]methionine (1250 Ci/mmol, Amersham) or 100 laci D-[6-3H]glucosamine hydrochloride (30-3 Ci/mmol, Amersham) per ml from 24 to 48 h post-infection as described previously (Ikuta et al., 1983). Preparation of MAbs. The development of hybridomas and screening procedures for MDV-specific antibodies were described in detail elsewhere (Ikuta et al., 1982). In the development of MAbs against MDV2, BALB/c mice were immunized with sonicated MDV2 HPRS24 strain-infected cell suspensions emulsified in Freund's complete adjuvant. After 4 to 8 weeks, the animals received an intraperitoneal booster injection of the virus-infected cells without adjuvant. Spleen cells from immunized animals were fused with P3-X63-Ag8U1 myeloma cells. The methods for cloning of hybridoma cells producing virus-specific antibodies were described in detail elsewhere (Ikuta et al., 1982). Immunoprecipitation and gel electrophoresis. The techniques for immunoprecipitation of virus-specific polypeptides from lysates of infected CEF were as described previously (Ikuta et al., 1983). The immunoprecipitates were analysed by SDS-PAGE (separation gel 10% polyacrylamide, spacer gel 4~) (Nakajima et al., 1986). Immunofluorescence test. The indirect immunofluorescence test was carried out as described previously (Ikuta et al., 1982). RESULTS Isolation of MAbs reactive with MD [I2 antigens We developed hybridomas producing MAbs against MDV2 strain HPRS24. Forty-one such clones were positive as determined by immunofluorescence tests, and their cross-reactivity against cells infected with the other serotype strains of MDV was also examined by the immunofluorescence test. Among the 41 MAbs, 24 specifically reacted with MDV2-specific epitopes; nine antibodies reacted with MDV 1-, MDV2- and HVT-common epitopes and eight reacted with MDV 1 and MDV2 intertypic epitopes. Therefore about 60 % of the MAbs obtained here were found to react with type-specific epitopes. We also investigated the existence of antigens in a Marek's disease lymphoblastoid cell line (MDCC-MSB1) by an immunofluorescence assay with MAbs against MDV2, but the antigens were not detected (results not shown). Immunoprecipitation of polypeptides from cells infected with MD V by MAbs against MDV2 For identification of the polypeptides reactive with these 41 MAbs, immunoprecipitation experiments were carried out. Only 23 of the MAbs immunoprecipitated polypeptides from HPRS24-infected cells labelled with [35S]methionine; they were divided into seven classes on the basis of the size of the polypeptides and their location in infected cells (Table 1). The MAbs belonging to classes 1, 2 or 3 reacted only with MDV2-specific epitopes as determined by the immunofluorescence test and immunoprecipitation assay. The four class 1 antibodies

3 (a) MD V-specific polypeptides (c) K/45K K 35K- OO O v~ L~ ~ -38K -36K -34K 38K- Fig. 1. Immunoprecipitation of potypeptides from MDV-related virus-infected cells with MDV2- specific MAbs E3, HP1 and C4 (a to c). CEF were mock-infected (MO) or infected with strain BC1 (BC) of MDV1, strains HPRS24 (HP) or SB1 (SB) of MDV2, or strain O1 (O1) of HVT labelled with [35S]methionine. The Mr values of the polypeptides were calculated by comparison of their mobilities with those of marker proteins from a calibration kit (Pharmacia). Numbers indicate the Mr of virusspecific potypeptides. Non-specific polypeptide bands that were also detected in immunoprecipitates of mock-infected CEF are indicated by arrows without a number (bottom arrow; 33K). Table 1. Summary of viral polypeptides immunoprecipitated with MAbs against MDV2 Cross-reactivity* Immunofluorescence pattern of Class MAb MDV 1 HVT infected cells Polypeptidet 1 E2, E3, E9, El0 - - Cytoplasm 2 HP1, HP2, HP3 - - Cytoplasm 3 C4, C5 - - Cytoplasm 4 B Nucleus B61, El K-45K 41K, 39K, 35K 38K 145K 5 B Nucleus 41K 6 E8 + - Cytoplasm 47K, 54K 7 C46, HP4, HP5, HP6 - - Cell surface and cytoplasm 28K-32K C47, C48, C49, C52, C * -, reactive only with homologous serotype, but not with heterologous serotype; +, cross-reactive. t Polypeptides identified from strain HPRS24 of MDV2 labelled with [35S]methionine. immunoprecipitated 40K to 45K polypeptides from MDV2-infected cells (Fig. 1 a). The class 2 antibodies immunoprecipitated 41K, 39K and 35K polypeptides from HPRS24-infected cells, and 38K, 36K and 34K polypeptides were immunoprecipitated from SBl-infected cells (Fig. 1 b). Since the 33K band from HPRS24-infected cells was, after a longer exposure of the autoradiograph, also detected in mock-infected cells as indicated by the lower arrow in Fig. 1 (b),

4 2566 (a) K. NAKAJIMA AND OTHERS t45k- 41K- -42K 50K/65K I I ~ Q -54K m0 _47 Fig. 2. Immunoprecipitation of the polypeptides from MDV-infected cells with MDV2 MAbs crossreactive with MDV1 or HVT (a, El3; b, B55; c, E8) as described in the legend to Fig. 1. the 33K polypeptide is considered to be non-specific. Among the MAbs obtained, only the class 2 MAbs recognized polypeptides that differed in size between HPRS24-infected cells and SB1- infected cells. The two class 3 antibodies also recognized cytoplasmic antigens as determined by the immunofluorescence test and reacted with MDV2-specific epitopes. These antibodies immunoprecipitated a 38K polypeptide from MDV2-infected cells (Fig. 1 c). We then identified the polypeptide specificity of the MAbs cross-reactive to the three serotypes. For immunoprecipitation studies representative viruses of each serotype were used (MDV1 : BC1; MDV2:HPRS24 and SB1; HVT": O1). Class 4 MAbs B45, B61 and El3 (see Table 1) immunoprecipitated a 145K polypeptide from HPRS24-infected cells as shown in Fig. 2 (a). All these MAbs were found by the immunofluorescence test to recognize nuclear antigens in virus-infected cells. However, B45 recognized MDV2-specific epitopes, whereas B61 and E13 recognized epitopes cross-reactive among three serotypes. The latter two MAbs immunoprecipitated a 145K protein from MDVI-, MDV2- and HVT-infected cells (Fig. 2a). Class 5 antibody B55 immunoprecipitated a 41K protein from HPRS24-infected cells. Further analysis revealed that B55 also i mmunoprecipitated a 41K polypeptide from BC1- or SB 1-infected cells and a 42K polypeptide from Ol-infected cells (Fig. 2b). This antibody recognized nuclear antigens of MDV 1-, MDV2- and HVT-infected cells. Fig. 2 (a) and (b) also show many bands in addition to the 145K band and a 41K or 42K band, respectively, from immunoprecipitates of virus-infected cells. These bands were also detectable in mock-infected cells after a long exposure. The class 6 antibody recognized MDV1 and MDV2 intertypic epitopes. By immunoprecipitation and SDS-PAGE, 47K and 54K polypeptides were detected in cells infected with the HPRS24 and SB1 strains of MDV2, whereas 50K to 65K polypeptides were detected in cells infected with the BC1 strain of MDV1 (Fig. 2c). Identification of a new glycoprotein with MD V2 MAbs Nine antibodies which belonged to class 7 immunoprecipitated 28K to 32K polypeptides from MDV2 HPRS24- and SBl-infected cells. Among them four MAbs reacted with MDV2- specific epitopes, whereas five reacted with MDV1 and MDV2 intertypic epitopes. These antibodies were found by the immunofluorescence test to react with a cytoplasmic antigen of HPRS24-infected cells (Fig. 3e, f). In addition, these MAbs reacted with the surface of cells infected with HPRS24 (Fig. 3i) and SB1 (data not shown) as determined by the membrane immunofluorescence test.

5 M D V-specific polypeptides ~a) 2567 (b) (c) g (/) "g) (h) Fig. 3. Characteristic appearance of immunofluorescence using MAbs. Uninfected CEF (a, b) and virus-infected CEF (strain BC 1 of M D V 1-infected CEF : c, d; strain HPRS24 of MDV2-infected CEF: e,f ; strain O1 of HVT-infected CEF : g, h) at 48 h post-infection were fixed and treated with 1000-fold diluted ascitic fluid containing C47 antibody (a, c, e, g) or HP4 antibody (b, d, f, h). Membrane antigenspecific immunofluorescence was detected for strain HPRS24-infected cells (i) treated with 100-fold diluted ascitic fluid containing C47 antibody. Bar marker represents 100 ~tm.

6 2568 K. NAKAJIMA AND OTHERS HP SB (a) S H S H (b) MO BC HP O1!: ~ i? : ii:: i :~ ~ i ~ iii i ~ :!: :: 28K/32K 25K/29K I 28K/32K Fig. 4. Detection of virus-specific glycoproteins from MDV2-infected cells by MAb C47. CEF infected with strain BCI (BC), HPRS24 (HP), SB1 (SB), Ol (O1) were labelled with [35S]methionine (S) or [3H]glucosamine (H) in (a) and [35S]methionine in (b). The numbers on the left side and the right side of the figure indicate Mr of the polypeptides. Only the 28K to 32K polypeptide showed heterogeneous electrophoretic mobility, which is characteristic of glycoproteins, and was detected on the cell surface. Therefore, infected cells labelled with [3H]glucosamine were immunoprecipitated with C47 antibody. As shown in Fig. 4 (a), the proteins could be labelled with [3H]glucosamine, indicating that they are glycoproteins, which we designate gp28/32. Furthermore cross-reactive polypeptides were detected in cells infected with the BC 1 strain by MDV1 and MDV2 intertypic MAbs against gp28/32 (Fig. 4b). The size of the polypeptide detected in these cells was 25K/29K, which was smaller than that of MDV2. In earlier work, we have identified two kinds of glycoprotein (ga, gb) from MDVI-, MDV2- or HVT-infected cells by using these MAbs. The Mr of the gp28/32 is different from those of the precursor and processed forms of ga or gb, which suggests that gp28/32 is the third viral glycoprotein to be identified by MAbs by immunoprecipitation from MDV-infected cells. DISCUSSION We have isolated 41 MAbs against strain HPRS24 of MDV2 that were positive in immunofluorescence assays using HPRS24-infected cells. Among the MAbs obtained, 24 were reactive only with MDV2-specific epitopes, whereas eight reacted with MDV1- or MDV2- infected cells and nine with MDVI-, MDV2- or HVT-infected cells. In addition to our previously isolated MAbs against MDV1 or HVT, our type-specific MAbs against MDV2 and MAbs recognizing all three serotypes will be useful for the serotyping of MDV. The use of MAbs against MDV1 has demonstrated that many epitopes are shared with HVT proteins. In this report, we have shown that some of the MAbs against strain HPRS24 of MDV2 react with proteins from cells infected with the virus strains belonging to the MDV1 or HVT groups.

7 MD V-specific polypeptides 2569 We also have tested the ability of the 41 MAbs to immunoprecipitate the viral polypeptides. Twenty-three of the MAbs immunoprecipitated viral polypeptides from strain HPRS24- infected cells and seven classes of virus-induced polypeptides were thus identified in infected cells. Our study is the first to identify MDV2-induced polypeptides using MAbs against MDV2. We previously reported six kinds of virus-induced polypeptides which were immunoprecipitated with MAbs against MDV1 or HVT. All seven MDV2 polypeptides characterized here were different in size from these six kinds. Among them, the Mr of the polypeptide immunoprecipitated from strain HPRS24-infected cells with class 4 antibodies was the same as that of the non-dna-binding protein. However the Mr of the polypeptide immunoprecipitated from HVT-infected cells was different. Wyn-Jones & Kaaden (1979) isolated glycoproteins from a membrane fraction of HVTinfected cells which has been used successfully to vaccinate chickens against MD. Such studies show that the glycoproteins that are probably associated with infected cell membranes appear to be important in terms of vaccinal immunity. In addition, the common MDV1-MDV2 antigens and MDV 1-HVT antigens have stimulated interest because they might be responsible for the protection afforded by vaccine strains. In an effort to understand vaccinal immunity more completely, we have investigated the glycoproteins common to MDV1 and vaccine strains and have studied the cross-reactive polypeptides of pathogenic MDV and vaccine viruses by MAbs. Among them ga and gb are candidates for vaccine protection, because they were expressed on the surface of infected cells. In an in vivo experiment, purified gb conferred partial protection against MD (Ono et al, 1985). In MDV2-vaccinated birds common MDV 1-MDV2 antigens are thought to be important for immunity, because they might be responsible for the protection afforded by vaccine strains. Among the seven classes of polypeptides identified in this study, the polypeptides belonging to classes 4, 5, 6 and 7 were cross-reactive between pathogenic MDV1 and MDV2. In particular, the gp28/32 identified with class 7 MAbs may be important, because this antigen was identified as a glycoprotein and was found on the surface of infected cells. This is the third virus-specific glycoprotein that has been detected in MDV-infected cells by MAbs against MDV but only one, gb, has been reported to mediate virus neutralization. Further studies are needed to clarify the role of the new MDV2 glycoprotein in MDV2 vaccine-induced immunity. Although the genome structure of MDV resembles that of herpes simplex virus, the lack of nucleotide and protein sequence data has prevented a detailed molecular analysis. Buckmaster et al. (1988), recently investigating the possible collinearity of the MDV and varicella-zoster virus genomes by comparison of partial nucleotide sequence data, suggested that additional glycoproteins are encoded within the Us region of MDV DNA; gp28/32 may be one of these. Further studies on the identification of the biological function of gp28/32 in vaccinal immunity to MD and on identification of the coding regions of the MDV2 genome are in progress. We thank Dr B. W. Calnek for providing strain SBI. This work was supported in part by a Grant for Cancer Research from the Ministry of Education, Science and Culture of Japan, REFERENCES BIGGS, P. M. & MILNE, B. S. (1972). Biological properties of a number of Marek's disease virus isolates. In Oncogenesis and tterpesvirus I, pp Edited by P. M. Biggs, G. de-the & L. N. Payne. Lyon: International Agency for Research on Cancer. BUCKMASTER, A. E., SCOTT, S. D., SANDERSON, M. l., BOURSNELL, M. E. G., ROSS, N. L. J. & BINNS, M. M. (1988). Gene sequence and mapping data from Marek's disease virus and herpesvirus of turkeys: implications for herpesvirus classification. Journal of General Virology 69, CALNEK, B. W., SCHAT, K. A., PECKHAM, i. C. & FABRICANT, J. (1983). Field trials with a bivalent vaccine (HVT and SB-1) against Marek's disease. Avian Diseases 27, EIDSON, C. S., PAGE, R. K. & KLEVEN, S. H. (1978). Effectiveness of cell-free or cell-associated turkey herpesvirus vaccine against Marek's disease in chickens as influenced by maternal antibody, vaccine dose, and time of exposure to Marek's disease virus. Avian Diseases 22, EIDSON, C. S., ELLIS, M. N. & KLEVEN, S. H. (1981). Reduced vaccinal protection of turkey herpesvirus against field strains of Marek's disease herpesvirus. Poultry Science 60,

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9 MD V-specific polypeptides 2571 WITTER, R. L., SHARMA, J. M. & FADLY, A. M. (1980). Pathogenicity of variant Marek's disease virus isolants in vaccinated and unvaccinated chickens. Avian Diseases 24, WITTER, R. L., SHARMA, J. M. & LEE, L. F. (1984). Field trials to test the efficacy of polyvalent Marek's disease vaccines in broilers. Avian Diseases 28, WITTER, R. L., SILVA, R. F. & LEE, t. F. (1987). New serotype 2 and attenuated serotype 1 Marek's disease vaccine viruses: selected biological and molecular characteristics. Avian Diseases 31, WYN-JONES, A. P. & KAADEN, O.-R. (1979). Induction of virus-neutralizing antibody by glycoproteins isolated from chicken cells infected with herpesvirus of turkeys. Infection and Immunity 25, (Received 22 February 1989)