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J. gen. Viral. (1988), 69, 1411-1414. Printed in Great Britain 1411 Key words: ADV[immunity/gVl Protection of Mice from Lethal Infection with Aujeszky's Disease Virus by Immunization with Purified gVI By H I R O S H I I S H I I , * Y U I C H I K O B A Y A S H I , M A S A H I K O K U R O K I YOSHIKATU KODAMA Ghen Corporation, Gifu Laboratory, Sano, Gifu-City 501-11, Japan AND (Accepted 3 March 1988) SUMMARY Virus envelope glycoprotein gVI (gp50) of Aujeszky's disease virus (ADV) was purified from a Nonidet P40-solubilized lysate of ADV-infected Vero cells by immunoaffinity chromatography using a monoclonal antibody against gVI. Mice immunized by the purified gVI produced virus-neutralizing antibody and were successfully protected against subsequent challenge with ADV. Aujeszky's disease virus (ADV), an alphaherpesvirus, causes great economic loss in the swine industry. It is known that herpesvirus glycoproteins are the principal antigens which induce host immune responses (Choppin & Scheid, 1980; Glorioso et al. 1984; Norrild, 1980; Weis et al., 1983), and can be used as protective antigens for subunit vaccines (Ashley et al., 1985; Roberts et al., 1985). These observations encouraged us to try to prepare a vaccine for ADV. In ADV, at least six structural glycoproteins [gI, gII, gIII, gIV, gV and gVI (gp50)] have been identified (Hampl et al., 1984; Wathen & Wathen, 1984). Several monoclonal antibodies (MAbs) against gI, gIII and gVI have been shown to neutralize ADV (Hampl et al., 1984; Mettenleiter et al., 1987; Wathen & Wathen, 1984). Therefore, these three glycoproteins must have epitopes which induce the production of virus-neutralizing antibodies, and these antigens are candidates for useful subunit vaccines. Among these glycoproteins, however, the ability of the antigens to induce a protective immune response has not been fully examined. Moreover, antigenic drift of gI and gIII has been observed among different virus strains (Ben-Porat et al., 1986). Thus, we focused on the glycoprotein gVI, which has several similarities to glycoprotein gD (Petrovskis et al., 1986; W athen & Wathen, 1984), a type-common antigen of herpes simplex virus (Cohen et al., 1978), and has at least one epitope that induces synthesis of a virusneutralizing antibody (Wathen & Wathen, 1984). The YS-81 virulent strain of ADV (Fukusho et al., 1981) was plaque-purified three times and propagated in monolayer culture of a swine kidney cell line. Virions were purified according to the method of Spear & Roizman (1972). MAbs against ADV structural proteins were prepared as follows. Spleen cells of BALB/c mice, immunized with heat-inactivated (60 °C, 2 h) purified virus (200 rag), were fused with P3-NS/1-Ag4-1 (NS-1) myeloma cells in the presence of polyethylene glycol (Galfr6 & Milstein, 1981). Screening of antibody production in the supernatants of hybridomas was performed by immunofluorescence assay using cold acetonefixed ADV-infected Veto cells as antigens. Positive cells were cloned four times by limiting dilution and expanded in the peritoneal cavity of mice. It has been confirmed that the MAbs 38-1, J-68 and J-50 recognize gVI, gII and gI glycoprotein, respectively, by immunoprecipitation methods using a [3H]glucosamine-labelled cell lysate (Fig. 1) (Lamb et al., 1978). MAb 38-1, which reacts with gVI, neutralized ADV in vitro and the neutralizing effect was enhanced when fresh rabbit serum (2%) was added as a source of complement. Moreover, if mice were injected intraperitoneally, 3 h before virus infection with the ascitic fluid of this clone (0.5 ml/mouse) they were protected from lethal challenge with ADV. The other MAbs did not show virusneutralizing activity in vitro. 0000-8172 © 1988 SGM Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 07:35:51 Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 07:35:51 B 2 3 31K- 45K- 66K- 92K - 116K- 200K- 1 2 3 ,0 e 1 2 50K-i dl~ (b) 3 (c) 1 2 3 4 Fig. 2. SDS-PAGE (a) and immunoblotting (b, c) analyses of purified gVI and anti-gVI polyclonal antibody. (a) SDS-PAGE of purified glycoprotein gVI. Coomassie Brilliant Blue staining (lanes 1 and 2) and periodic acid-Schiff's staining (lane 3). Lane 1, NP40-solubilized lysate of virus-infected Vero cells. Lanes 2 • " , cent swine serum. Lane 1, NP40-solubilized lysate of virus-infected Vero cells; lane 2, purified d 3 urified VI. (b) Immunoblottmgof purified gV~ ,using conva|e~_.. . ) a d irnmunoprecipitation(lane 4) using sera of mice ]mmumzed g~I" laPr~e3, Nl~'0-solubilized lysate of mock-infecteo vero cells. (c)h ...... noblottmg (lanes 1 to 3, n with puri~ed gVI. Lane l, purified virions; lane 2, NP40-solubilized lysate of virus-infected Vero cells; lane 3, purified gVI; lane 4, [3H]gl~cosamine'labelledcell lysate was immunoprecipitated by the pooled mouse serum immtmized with gVI. Fig. 2 Fig. 1 Fig. 1. SDS-PAGE of glycoproteins gVI, gI1 and gI of ADV after immunoprecipitation with MAbs. Vero cells were grown in 3.5 cm plastic dishes and infected with ADV (YS-81) at an m.o.i, of 50. After adsorption for 1 h at 37 °C, the cells were washed twice with glucose-free medium, and incubated for 8 h. The medium was replaced with glucose-free medium containing [3H]glucosamine at 0.5 rnCi/ml, and incubated for 3 h. Radiolabelled cells were washed with PBS and lysed with RIPA buffer, The lysates were immunoprecipitated with MAbs 38-1 (lane 1), J-68 (lane 2) and J-50 (lane 3) which recognize gVI, glI and gI, respectively. 45K- 66K- 92K- 116K- 200K- 1 (a) t-~ Short communication 1413 Table 1. Efficacy of active immun&ation of mice with the purified g VI and protection against lethal infection with A D V Immunogen Neutralizingantibody* gVI (1 i-tg) gVI (5 ~tg) PBS + CFA <2, 2, 2, 4, 4 8, 8, 16, 16, 16 Challenge d o s e (p.f.u./mouse) Number of survivors/ total number 104 10" 105 104 103 102 4/10 10/10 0/4 0/4 1/4 3/4 *Each of the serial twofold dilutions of serum was mixed with an equal volume of virus solution (100 TCIDs0/0"I ml). After incubation at 37 °C for 1 h, each mixture (0.2 ml) was inoculated onto a Vero cell culture in a microplate well. Four wells were used for each dilution. The titres were expressedas the reciprocalsof the highest serum dilution that neutralized virus in more than 50% of the wells. In order to examine the efficacy of gVI as a subunit vaccine, the glycoprotein was purified from virus-infected Veto cells using an immunoabsorbent column, and then injected into mice. In practice, Vero cells were infected with the virus at an m.o.i, of0-1 and maintained at 37 °C for 18 h. The cells were washed twice in phosphate-buffered saline (PBS pH 7.4) and were scraped into PBS containing 0.5~ Nonidet P40 (NP40) using a rubber policeman. The solubilized material was stirred at 4 °C for 10 rain and centrifuged at 20000 r.p.m, for 2 h. The supernatant was applied to a column of Sepharose 4B (Pharmacia) to which MAb 38-1 had been coupled. Absorbed material was washed with four column volumes of PBS containing 0-5~ NP40 and then with the same volume of PBS. Elution was performed with 3 M-potassium thiocyanate (pH 7.2; Sigma), and eluted material was dialysed against 50 mM-PBS containing 0.05~ NP40. Following SDS-PAGE (Laemmli, 1970), the material showed one band stained with Coomassie Brilliant Blue and with periodic acid-Schiff's staining (Zacharius et al., 1969) (Fig. 2a). After immunoblotting, as described by Towbin et al. (1974), this glycoprotein also reacted with convalescent serum from a pig inoculated intranasally with ADV (Fig. 2 b). These results suggest that the purified protein is viral glycoprotein gVI. Eight-week-old BALB/c mice were immunized with 1 lag or 5 lag of purified gVI emulsified in complete Freund's adjuvant (CFA), and boosted 2 weeks later by the same injection. Ten days after the second immunization, five mice of each group were examined for serum antibody titre and 10 mice were challenged with a lethal dose of ADV. Control mice were immunized with PBS emulsified in CFA. As shown in Table 1, all the mice which had been immunized with 5 lag of gVI were protected from lethal infection, whereas all control mice died within 5 days postinfection. The ability to protect against ADV infection, however, was lower in mice immunized with 1 lag of gVI. The titres of virus-neutralizing antibody were 2 to 4 and 8 to 16 in animals immunized with 1 lag and 5 lag of gVI, respectively. Irnmunoprecipitation and immunoblotting experiments were conducted to examine the specificity of pooled sera of mice immunized with gVI. As shown in Fig. 2 (c), only gVI was immunoprecipitated from [3H]glucosamine-labelled ADV. Immunoblotting analysis, using purified virions, NP40-solubilized cell lysate and purified gVI as antigens, also revealed that gVI specifically reacted with the antiserum. However, a few low M r or diffuse protein bands were also detected with purified virions and NP40-solubilized lysate. These bands may represent different glycosylated forms of gVI or degradation products. The results suggest that glycoprotein gVI might be used as an effective antigen in swine since, in mice, it induces virus-neutralizing antibody. However, cell-mediated immunity also plays an important role in preventing herpesvirus infection (Glorioso et al., 1980; Lawman et al., 1980; Vestergaard, 1980). It will therefore be necessary to elucidate the mechanisms of protective immune responses induced in swine by gVI from the standpoints of cell-mediated immunity as well as humoral immunity, if an effective subunit vaccine is to be developed. Downloaded from www.microbiologyresearch.org by IP: 88.99.165.207 On: Fri, 12 May 2017 07:35:51 1414 Short communication REFERENCES ASHLEY,R., MERTZ, G., CLARK, H., SCHICK,M., SALTER,D. & COREY, L. (1985). Humoral immune response to herpes simplex virus type 2 glycoproteins in patients receiving a glycoprotein subunit vaccine. Journalof Virology 56, 475-481. BEN-PORAT,T., DEMARCHI,J. M., LOMNICZI,B. & KAPLAN,A. S. (1986). Role of glycoproteins of pseudorabies virus in eliciting neutralizing antibodies. Virology 154, 325-334. CHOPPIN, P. W. & SCHEID,A. (1980). The role of viral glycoproteins in adsorption, penetration, and pathogenicity of viruses. Reviews of Infectious Diseases 2, 40-61. COHEN, G. H., KATZE, M., HYDREAN-STERN,C. & EISENBERG, R. J. 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