Veterinary Microbiology, 24 (1990) 193-198 Elsevier Science Publishers B.V., Amsterdam


A complement-dependent neutralizing monoclonal antibody against glycoprotein II of pseudorabies virus Toshihiro Nakamura ~, Takeshi Ihara ~, Taeko Nagata ~, Akira Ishihama 2 and Susumu Ueda 1

~Nippon Institute for Biological Science, Shinmachi 2221-1, Ome, Tokyo 198 (Japan) 2Departrnent of Molecular Genetics, National Institute of Genetics, Yata 1111, Mishima, Shizuoka 411 (Japan) (Accepted 8 February 1990)

ABSTRACT Nakamura, T., Ihara, T., Nagata, T., Ishihama, A. and Ueda, S., 1990. A complement-dependent neutralizingmonoclonal antibody against glycoprotein II of pseudorabies virus. Vet. Microbiol., 24: 193-198. A monoclonal antibody (MAb), 1.21, was produced against pseudorabies virus (PRV). It exhibited virus neutralization activity only in the presence of complement. Immunoblot analysis after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of virions revealed that MAb 1.21 bound with the 230 kilodalton (kD) virus protein only under non-reducing conditions. This protein was purified by immuno-affinitychromatography using MAb 1.21 and was found to be composed of three subunits, 60, 70 and 120 kD polypeptides when analyzed by SDS-PAGE under reducing conditions. This protein is probably glycoprotein II of PRV.


Pseudorabies virus ( P R V ) , a herpesvirus of swine, encodes at least six different glycoproteins. Almost all P R V neutralizing antibodies reported so far recognized gI, gill or gp50 of these glycoproteins (Hample et al., 1984; BenPorat et al., 1986; Mettenleiter et al., 1987; Eloit et al., 1988; Ishii et al., 1988 ). Glycoprotein II (glI), one of the major glycoproteins, has not received much attention as a target antigen for virus neutralization. This paper reports on the production of an anti-glI MAb with neutralizing activity in the presence of complement. 0378-1135/90/$03.50

© 1990 - - Elsevier Science Publishers B.V.





Cells BHK21/13 and RK13 cells were maintained at 37°C for virus growth and for virus neutralization tests, respectively, in a modified Eagle's medium containing 100 units of penicillin and 100 jtg of streptomycin per ml, and supplemented with 10% fetal bovine serum. Virus

Virus stocks of the Indiana S strain of PRV were prepared from infected BHK21/13 monolayer cultures as previously described by Chantler and Stevely ( 1973 ). The virus was purified following the methods of Spear and Roizman (1972).

Immunization and production of monoclonal antibodies BALB/c mice were immunized three times with 0.1 mg of UV-inactivated, purified PRV emulsified with complete Freund's adjuvant. Three days after the last immunization, spleen cells were fused with myelomas (P3)< 63Ag8.653 ). Culture supernatants of hybridomas were screened by enzyme-linked immunosorbent assay (ELISA), using 0.1 mg/ml of purified PRV as antigen and positive colonies were cloned twice by limiting dilution. Staining by MAb PRV infected BHK21/13 and RK13 cells, which had been grown as monolayers in LAB-TEK tissue culture chambers (Miles Lab., Naperville) were incubated with culture supernatants of hybridomas. After washing in phosphate buffered saline (PBS), pH 7.4, infected cells were incubated for 1 h with fluorescein isothiocyanate-labeled anti-mouse immunoglobulins (Igs) rabbit serum (Tago, Burlingame, CA). Following a further wash in PBS, cells were examined under incident fluorescent light. Virus neutralization test Culture supernatants of hybridomas or protein A purified MAbs were incubated for 1 h at 37°C with 100 plaque forming units of PRV, and with 5% rabbit serum as a source of complement with or without heat-inactivation (56 ° C, 30 min). After the incubation, 0.1 ml of each mixture was inoculated to monolayered RK13 cells in a 96-well plate. After 2 days incubation at 37 °C, the virus neutralization titer was determined as the reciprocal of the highest dilution of MAb or culture supernatants that prevented the appearance of a cytopathic effect.



SDS-PAGE and immunoblot Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was carried out following standard techniques. Using culture supernatants of hybridomas as primary antibodies, enzyme-linked immunoelectrotransfer blot (immunoblot) was carried out after SDS-PAGE of virion or purified glI. Peroxidase-conjugated anti-mouse Igs rabbit serum (Tago, Burlingame, CA) was used as a second antibody. For the detection of sugars, a Glycan Detection Kit (Boehringer Mannheim, Mannheim) was used after SDS-PAGE of purified glI. GII purification Hybridomas were injected into BALB/c nu/nu mice and ascites fluids were collected 2 to 3 weeks later. Monoclonal antibodies in these ascites fluids were purified by protein A coupled Sepharose 4B beads (Pharmacia, Uppsala). Purified monoclonal antibodies were coupled to cyanogen bromide-activated Sepharose 4B beads (Pharmacia, Uppsala). Purified PRV was solubilized by 1% Triton X-100 or 1% Nonidet P40 in PBS and applied to 1.21 MAb coupled Sepharose 4B column. Bound proteins were eluted with 0.1 M glycineHC1 buffer, pH 2.3. Eluted proteins were dialyzed against PBS. RESULTS

Several MAbs specific against purified PRV were selected by ELISA. Next, we screened the MAb collection as to crossreaction against envelope glycoproteins. Two MAbs, 1.21 and N.01, strongly stained the cell surface of PRV infected cells (Fig. 1 ). Detailed analysis was then focused on these two MAbs. Culture supernatant of the 1.21 hybridoma had a strong virus neutralization activity in vitro in the presence of complement. Immunoblots using MAb 1.21 after SDS-PAGE of the virion revealed that MAb 1.21 reacted with the 230 kD virus protein only under non-reducing conditions (Fig. 2). On the other hand, MAb N.01 reacted both with 230 kD protein and with other viral proteins (Fig. 2). The virus neutralization activity of MAb N.01 was, however, less than that of MAb 1.21. To examine the nature of the 230 kD protein, an attempt was made to purify it using an affinity column. A SDS-PAGE profile of retained and eluted proteins is shown in Fig. 3. A single species of the major protein with the molecular weight of 230 kD was identified, which was as large as that identified in immunoblot under non-reducing conditions. However, the 230 kD protein gave three bands, 60, 70 and 120 kD, under reducing conditions, indicating that the 230 kD protein was composed of three non-identical subunits, each being linked by a disulphide bond. Sugar moieties were detected by Glycan Detection Kit (Boehringer Mannheim) for all the three subunit bands (data not shown), suggesting that they



Fig. 1. Specific immunofluorescence of unfixed BHK21 / 13 cells infected with PRV stained by MAb 1.21.



! |


~ 2 3 2

- -



- -





Fig. 2. Immunoblot analysis of the purified virus proteins, employing 1.21 (A, C) or N.01 (B, D ) MAbs under non-reducing conditions (A, B) or under reducing conditions (C, D ). Relative molecular weight markers are indicated ( × 103).




B 340

III 170-,

- 97





Fig. 3. SDS-PAGE profile o f the purified glI u n d e r n o n - r e d u c i n g ( A ) or reducing ( B ) conditions. Note t h a t the 230 k D protein ( A ) gave three bands, with a molecular weight of which were 60, 70, a n d 120 kD ( B ) . Relative molecular weight markers are indicated ( × 103 ).

TABLE 1 Characters of two monoclonal antibodies MAb Ig subclass

Minimum concentration of MAbs (/tg/ml) for detection of activity in ELISA t

1.21 N.01

IgG2b, K IgG2b, K

0.01 0.1

VNT 2 with C..I- 3

C _4

0.1 >100

> 100 >100

~Purified glI as an antigen. 2Virus neutralization test. 3Rabbit serum was used as a source of complement. 4Heat-treated rabbit serum was used for inactivated complement.

are glycoproteins. These observations suggest that the 230 kD protein is glI. MAb N.01 also reacted to the purified glI in ELISA and in immunoblot. This binding activity to glI also disappeared under reducing conditions in immunoblot using either purified virus (Fig. 2) or purified glI (data not shown ). The binding activity to purified glI and the virus neutralizing activity of the two MAbs are summarized in Table 1.




MAb 1.21 strongly stained unfixed PRV infected cells in an indirect immunofluorescence assay, suggesting that it recognized major envelope protein of PRV. The molecular weight of the crossreactive protein is 230 kD, and the purified 230 kD protein is composed of three non-identical subunits (Fig. 3 ). These results indicate that this protein is the gII of PRV (Ben-Porat et al., 1986; Robbins et al., 1987). MAb 1.21 no longer reacted to gII under reducing conditions, indicating that it recognizes a conformational epitope on the native gII molecule. MAb N.01 reacted not only against gII but also against other components of PRV in immunoblots. MAb N.01 also failed to bind to gII under reducing conditions. This indicates that MAb N.01 recognizes a conformational epitope on native gII but this epitope differs from the one recognized by MAb 1.21 because of its broad specificity and the lack of neutralizing activity. MAb 1.21 against gII had a PRV neutralizing activity, suggesting that gII plays an important role in the protection from PRV infection.

REFERENCES Ben-Porat, T., DeMarchi, J.M., Lomniczi, B. and Kaplan, A.S., 1986. Role of glycoprotein of pseudorabies virus in eliciting neutralizing antibodies. Virology, 154: 325-334. Chantler, J.K. and Stevely, W.S., 1973. Virus-induced protein in pseudorabies-infected cells. J. Virol., 11: 815-822. Eloit, M., Fargeaud, D., L'Haridon, R. and Toma, B., 1988. Identification of the pseudorabies virus glycoprotein gp50 as a major target of neutralizing antibodies. Arch. Virol., 99: 45-56. Hample, H., Ben-Porat, T., Ehrlicher, L., Habermehl, K. and Kaplan, A.S., 1984. Characterization of the envelope proteins ofpseudorabies virus. J. Virol., 52: 583-590. Ishii, H., Kobayashi, Y., Kuroki, M. and Kodama, Y., 1988. Protection of mice from lethal infection with Aujeszky's disease virus by immunization with purified gVI. J. Gen. Virol., 69:1411-1414. Mettenleiter, T.C., Schreurs, C., Thiel, H. and Rziha, H., 1987. Variability of pseudorabies virus glycoprotein I expression. Virology, 158:141-146. Robbins, A.K., Dorney, D.J., Wathen, M.W., Whealy, M.E., Gold, C., Watson, R.J., Holland, L.E., Weed, S.D., Levine, M., Glorioso, J.C. and Enquest, L.W., 1987. The pseudorabies virus gII gene is closely related to the gB glycoprotein gene of herpes simplex virus. J. Virol., 61: 2691-2701. Spear, P.G. and Roizman, B., 1972. Proteins specified by herpes simplex virus. V. Purification and structural proteins of the herpesvirion. J. Virol., 9:143-159.

A complement-dependent neutralizing monoclonal antibody against glycoprotein II of pseudorabies virus.

A monoclonal antibody (MAb), 1.21, was produced against pseudorabies virus (PRV). It exhibited virus neutralization activity only in the presence of c...
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