- Email: [email protected]
Mechanisms of neutralization by monoclonal antibodies to different antigenic sites on the bovine herpesvirus type 1 glycoproteins
l&50,260-264
VIROLOGY
Mechanisms
KATSUNORI
(1986)
of Neutralization by Monoclonal Antibodies to Different Antigenic on the Bovine Herpesvirus Type 1 Glycoproteins
OKAZAKI,*
*Department
EIICHI
HONDA,*
TOSHIMI
MINETOMA,~
AND
TETSUO
Sites
KUMAGAI**’
of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology Fuchu 185, Japan, and ~Bacteridogy and Virology Section, Zen-m Institute of Animal Health, Sakum 285, Japan ReceivedJuly
19, 1985;accepted
December
18, 1985
Monoclonal antibodies directed to different antigenie sites on bovine herpesvirus type 1 (BHV-1) glycoproteins were used to study the mechanisms of neutralization of the virus. Three nonoverlapping neutralizing antigenic sites, designated Ia, Ib, and Ic, were defined on gp9’7. Antibodies to site Ia which mediated viral neutralization without complement were effective on inhibition of virus adsorption. Antibodies to a single neutralization site on gp71, designated IIa, were able to neutralize the virus without complement even when they were incubated with the virus which had already adsorbed onto the cells. Antibodies directed against gp117 and antibodies against sites Ib and Icon gp87 required complement fO?!
VirUS
8 1!%6 Academic
UeUtrdiZatiOII.
Press. Inc.
Bovine herpesvirus type 1 (BHV-1) is an important pathogen of cattle producing many types of illness such as rhinotrachitis, conjunctivitis, pustular vulvovaginitis, encephalomyelitis, and fatal systemic infection (1). BHV-1 specifies 25 to 33 polypeptides, some of which are glycosylated and associated with virus envelope and the infected cell membranes (2, 3). Glycoproteins of many enveloped viruses have been reported to play important roles in the adsorption and penetration of the virus (4). Viral glycoproteins are also major targets of the immune response to infections, at both the humoral and cellular level (5, 6). Neutralizing antibody is one of the main arms of protective immunity. However, there is little information about the interaction between antibody and the antigenic determinants of the virus resulting in interference with the infectivity. We report here on the production of a panel of monoclonal antibodies to viral glycoproteins and
i To whom dressed. 0042-6822/86 Copyright All rights
requests
for
reprints
$3.00
(B 19&? hy Academic Press, Inc. of reproduction in any form reserved.
should
describe the use of these antibodies to find out the mechanisms of neutralization of BHV-1 strain Los Angeles. Hybrid cell lines producing antibodies to BHV-1 were obtained following the method of Kida et al. (7). Out of the 24 hybridoma clones producing antibody detectable by ELISA, 12 clones secreted antibodies neutralizing BHV-1 with or without guinea pig complement. High concentrations of monoclonal antibodies were prepared from the ascitic fluid of BALB/c mice bearing intraperitoneal hybridomas. The polypeptide specificity of the neutralizing antibodies was determined by immunoprecipitation of [%]methionine or [3Hlglucosamine labeled viral proteins in infected cell lysates. As illustrated in Fig. 1, the neutralizing antibodies could be divided into three groups: One group comprising seven monoclonal antibodies, 245112, 28216, 248/l, 279/l, 251/l, 292/l, and 200/3, recognized 87 kDa glycoprotein (lane a). Polypeptide of 157 kDa was detected by these antibodies only when the r”S]methionine labeled precipitates were analyzed by prolonged exposure. Labeling of
be ad-
260
SHORT
COMMUNICATIONS
261
(Table 1). The binding of enzyme-conjugated antibody 245/12 precipitating gp87 was inhibited by the homologous antibody as well as by antibody 282/6, indicating c their specificities to identical or topographically overlapping epitopes, designated site Ia. However, none of the other antibodies competed with 245112 conjugate, indicating their specificities to antigenie sites nonoverlapping with site Ia. Similarly, two other nonoverlapping neutralizing antigenic sites were defined on gp87 (sites Ib and Ic), and a single neutralizing site (IIa) was detected on gp71. 436.5 In these assays, antibodies 251/l and 292/l enhanced the binding of antibody 245/12, and antibody 232/6 enhanced that FIG. 1. Polypeptide specificities of monoclonal an- of antibody 248/l. We used monoclonal antibodies in the tibodies to BHV-1. MDBK cells monolayers were laand virus adsorption inhibeled with [asS]methionine (56 &i/ml) (A) or neutralization bemjglucosamine (25 &i/ml) (B) from 3 or 6 hr to 24 bition assays to clarify the relation hr postinfection with l-10 m.o.i. of virus. Extracts tween these neutralizing antigenic sites were prepared from the infected cells using RIPA and biological functions of the antibodies buffer (0.05 M Tris-HCl, pH 8.0,0.15 MNaCI, 1% Tri(Table 2). Two monoclonal antibodies 2451 ton X-100,1% sodium deoxycholate) and then centri12 and 282/6 reactive with site Ia on gp87 fuged for 1 hr at 10,000 g. The supernatant was used neutralized the virus infectivity without as antigens in immunoprecipitation assays (7) which complement and blocked essentially the were subsequently analyzed by SDS-PAGE under rebinding of virus to cells. On the other hand, ducing (lanes a-c) or nonreducing (lane d) conditions. antibodies against site IIa on gp71 had only Lane a, polypeptides precipitated by antibody 245/12; activity against virus adlane b, 9/6; lanes c and d, lW2. The positions of the weak blocking molecular weight markers X10-’ are shown in the sorption although they effectively neutralright-hand margin. ized the infectivity without complement. Other monoclonal antibodies against sites Ib and Ic and gp117 molecule, required infected cells with [3Hlglucosamine failed complement for virus neutralization and to render the 157 kDa polypeptide. The did not show significant interference with second group comprising three monoclonal virus adsorption. antibodies, 9/6, 19711, and 264/3, precipiTo obtain further evidence suggesting tated a single glycoprotein of 71 kDa (lane the mechanisms of neutralization of the b), and the third group comprising two an- virus, a postadsorption neutralization test tibodies, 18512 and 26/l, reacted with a was carried out with the two monoclonal complex of glycoproteins of doublet 117- antibodies, 245/12 and 264/3, which were 111 kDa, doublet 70-64 kDa, and doublet directed to gp87 (site Ia) and gp71 (site IIa), 51-47 kDa (lane c). Only the 117 kDa gly- respectively, both neutralizing without coprotein was detected when the immucomplement. After 2 hr of virus adsorption, noprecipitates were analyzed under non- using ca. 100 PFU/well, serial dilutions of reducing conditions (lane d), showing that purified antibodies were placed on the cells, the 70-64 and 51-47 kDa glycoproteins ap- and assayed for plaque reduction (Fig. 2). peared to be components of a disulfideMonoclonal antibody 264/3 directed to linked complex of 117 kDa. gp71, neutralized the virus infectivity Competitive binding assays were carried completely. The amount of IgG required for out in order to identify separate neutral50% plaque reduction was 0.6 pg. In conizing antigenic sites on the glycoproteins trast to this complete neutralization, A
abed
B abc
262
SHORT
COMMUNICATIONS TABLE
1
COMPETITWE BINDING ASSAYS BETWEEN MONOCLONAL ANTIBODIES TO BHV-1 a Unlabeled antibody Antigp87
245/12
245/12
2b
248/l 279/l 251/l 292/l
2b 2a 2a 2a
200/3
2b
282/6
2b
Anti-gp71 9/6
197/l 264/3
% Inhibition to binding of HRPO-antibody
IgG subclass
1 2a 2b
100 88
248/l -60
9
251/l 10
-48
82
-77
84
88
35
24
9/6
197/l
26413
100 -11 17
80
44
100 63
23 100
6
Ia
-16
94 98 100
5
7
100 101
Antigenic site
Ib
Ic
IIa
a Monoclonal antibodies were purified using protein A (8) and labeled with horseradish peroxidase (HRPO) by the two-step glutaraldehyde coupling procedure (9). The assays were carried out by a modification of the technique described previously (10). The degree of competition binding was expressed by the perce-rtage of competition estimated by the formula 100 (A - X)/(A - B) where A, B, and X are ODa in the absence of competitor, in the presence of homologous antibody (5 pg), and in the presence of competitor (5 a), respectively. Minus indicates enhancement of the conjugate binding.
monoclonal antibody 245/12 directed to gp87, caused no neutralization even when 50 pg of IgG was applied. This report demonstrates that several neutralizing antigenic sites were distributed on at least three glycoproteins of BHV-1. Three nonoverlapping neutralizing antigenic sites, designated Ia, Ib, and Ic, were defined on gp87. Antibodies 245/12 and 282/6 against site Ia inhibited virus adsorption and neutralized the virus without complement. Therefore, these antibodies are considered to cause neutralization as a result of hindrance of virus attachment to the cells. Site Ia on gp87 of BHV-1 may be overlapped with or very close to the critical site on the virion for cell attachment. Alternatively, the antibodies might induce conformational changes in the receptor binding site on gp87, and result in virus neutralization. Such conformational changes were suggested by the fact that one of the antibodies (282/6) enhanced the binding of antibody
248/l against site Ib in competitive binding assays. The monoclonal antibodies to site IIa on gp71 were effective in neutralization when
ANTIBODY
(re)
FIG. 2. Postadsorption neutralization assays by monoclonal antibodies to BHV-1. After 2 hr adsorption of the virus (100-200 PFU/well) at 4O, monolayers were washed with cold PBS. Purified monoclonal antibodies (0.05 to 50 pg in 200 ~1 MEM) were plated onto the virus-adsorbed cells, followed by further incubation for 2 h at 4’. The monolayers were washed again, and assayed residual infectivity by plaque counting. 0,245/12; 0,264/3.
SHORT
COMMUNICATIONS
263
TABLE 2 BIOLOGICAL FUNCTIONSOF MONOCLONAL ANTIBODIES TO BHV-1” 50% Neutralization titer Antibody
Antigenic
Anti-gp87 245112 232/6 248/l 279/l 251/l 292/l 20013 Anti-gp71 916 197/l 264/3
site
50% Adsorption inhibition titer
C-
c+
Ia Ia Ib Ib Ib Ib IC
100 150 cl0 t10
2,800 2,500 230 400 250 720 3,400
2560 2560 10 NTb 40 NT
IIa IIa IIa
2ooo 480 8100
20@-3 18,200 25,600
Anti-117 185/2 26/l
3,100
2,2(@
a The virus neutralizing capacity of ascitic fluid containing monoelonal antibody was quantified by a plaque redaction assay. Serial dilutions of ascitic fluid were mixed with virus suspension (100-200 PFWwell) and the mixture was plated on a 24-well plate of MDBK cells subsequent to 1 hr incubation at 37”. Complementenhanced neutralization was determined by the addition of guinea pig sera (final concentration of 2%) to the virus-antibody mixture. The adsorption inhibition assays were carried out using [S6Sjmethionine-labeled BHV1. The virus was purified by differential centrifugation and two successive sedimentations through a 10-50% potassium tartrate gradient. The purified radiolabeled virus (8,000-10,000 cpm) was mixed with serially diluted ascitic fluid and incubated for 1 hr at 3Y. The virus-antibody mixture was added to a flexible 96-well plate of MDBK cells. The plate was incubated and gently agitated at 4” for 2 hr to allow adsorption of virus to cells. After washing the cells, each well was cut off from the plate and assayed for the count per minute by liquid scintillation counting. The percentage inhibition was estimated graphically from the sigmoid courves of bound cpm. b Not tested.
antibody was plated onto cells on which the virus had already become adsorbed. These data suggest that the antibodies could inhibit the infection process after adsorption, probably penetration of the virus. The binding of the antibodies to site IIa on gp71 is considered to affect the critical site, possibly by means of masking or conformational changes. In our experiment, antibodies to site IIa on gp71 showed a neutralizing titer much higher than that of antibodies to site Ia on gp87 in the test without complement. Recently, Fuller and Spear (11) have shown with herpes simplex virus that, although some antibodies can
neutralize the virus by blocking adsorption, a more important mechanism of neutralization by antibodies may be interference with a step subsequent to adsorption. It is expected that in order to be effective, a BHV-1 vaccine would have to induce antibodies to gp71. Antigenic sites Ib and Ic on gp87 and gp117 molecule were recognized by monoclonal antibodies requiring complement for virus neutralization. Complement-dependent neutralization has been demonstrated for a number of enveloped viruses (12-14). Further studies comparing complementdependent and complement-independent
264
SHORT
COMMUNICATIONS
neutralizing antibodies would be useful to elucidate the diverse mechanisms of BHV1 neutralization. ACKNOWLEDGMENTS This work was supported in part by Grant-in-Aid for Developmental Scientific Research 58369030, from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. KAHRS,
S. F., J. Amer. Vet. Med ASSOC. 117,10551064 (1977). 2. MISRA, V., BLUMENTHAL, R. M., and BABIUK, L. A., J. Vird 40,367-3’78 (1981). 8. BOLTON, D. C., ZEE, Y. C., and ARDANS, A. A., Vet. Microbid 8,57-68 (1983). 4. CHOPPIN, P. W., and SCHEID, A., Rev. In&e&. LG. 2,40-61 (1980).
5. PERRIN, L. H., TISHON, A. J., and OLDSTONE, M. B. A., J. Immund 118,282-290 (1977). 6. SPEAR, P. G., In “Cell Membranes and Viral Envelopes” (H. A. Blough, and J. M. Tiffany, eds.), Vol. 2, pp. 709-750. Academic Press, New York, 1989. 7. KIDA, H., BROW, L. E., and WEBSTER, R. G., virology 122,38-47 (1982). 8. EY, P. L., PROWSE, S. J., and JENKIN, C. R., Immunochemistry l&429-436 (1978). 9. AVRAMEAS, S., and TERNYNCK, T., Immunochemistry 8,1175-1179 (1971). 10. KIMURA-KURODA, J., and YASUI, K., J. vird 45, 124-132 (1983). 11. FULLER, A. O., and SPEAR, P. G., J. V+oL 55,475482 (1985). 12. YOSHINO, K., and TANIGUCHI, S., Vi~obgy 22,193201 (1964). 18. RADWAN, A. I., BURGER, D., and DAVIS, W. C., Vzrology 53,372-378 (1973). 14 POTGIETER, L. N. D., Canad J. Ccnnp. Med 39,427433 (1975).
VIROLOGY
Mechanisms
KATSUNORI
(1986)
of Neutralization by Monoclonal Antibodies to Different Antigenic on the Bovine Herpesvirus Type 1 Glycoproteins
OKAZAKI,*
*Department
EIICHI
HONDA,*
TOSHIMI
MINETOMA,~
AND
TETSUO
Sites
KUMAGAI**’
of Veterinary Medicine, Faculty of Agriculture, Tokyo University of Agriculture and Technology Fuchu 185, Japan, and ~Bacteridogy and Virology Section, Zen-m Institute of Animal Health, Sakum 285, Japan ReceivedJuly
19, 1985;accepted
December
18, 1985
Monoclonal antibodies directed to different antigenie sites on bovine herpesvirus type 1 (BHV-1) glycoproteins were used to study the mechanisms of neutralization of the virus. Three nonoverlapping neutralizing antigenic sites, designated Ia, Ib, and Ic, were defined on gp9’7. Antibodies to site Ia which mediated viral neutralization without complement were effective on inhibition of virus adsorption. Antibodies to a single neutralization site on gp71, designated IIa, were able to neutralize the virus without complement even when they were incubated with the virus which had already adsorbed onto the cells. Antibodies directed against gp117 and antibodies against sites Ib and Icon gp87 required complement fO?!
VirUS
8 1!%6 Academic
UeUtrdiZatiOII.
Press. Inc.
Bovine herpesvirus type 1 (BHV-1) is an important pathogen of cattle producing many types of illness such as rhinotrachitis, conjunctivitis, pustular vulvovaginitis, encephalomyelitis, and fatal systemic infection (1). BHV-1 specifies 25 to 33 polypeptides, some of which are glycosylated and associated with virus envelope and the infected cell membranes (2, 3). Glycoproteins of many enveloped viruses have been reported to play important roles in the adsorption and penetration of the virus (4). Viral glycoproteins are also major targets of the immune response to infections, at both the humoral and cellular level (5, 6). Neutralizing antibody is one of the main arms of protective immunity. However, there is little information about the interaction between antibody and the antigenic determinants of the virus resulting in interference with the infectivity. We report here on the production of a panel of monoclonal antibodies to viral glycoproteins and
i To whom dressed. 0042-6822/86 Copyright All rights
requests
for
reprints
$3.00
(B 19&? hy Academic Press, Inc. of reproduction in any form reserved.
should
describe the use of these antibodies to find out the mechanisms of neutralization of BHV-1 strain Los Angeles. Hybrid cell lines producing antibodies to BHV-1 were obtained following the method of Kida et al. (7). Out of the 24 hybridoma clones producing antibody detectable by ELISA, 12 clones secreted antibodies neutralizing BHV-1 with or without guinea pig complement. High concentrations of monoclonal antibodies were prepared from the ascitic fluid of BALB/c mice bearing intraperitoneal hybridomas. The polypeptide specificity of the neutralizing antibodies was determined by immunoprecipitation of [%]methionine or [3Hlglucosamine labeled viral proteins in infected cell lysates. As illustrated in Fig. 1, the neutralizing antibodies could be divided into three groups: One group comprising seven monoclonal antibodies, 245112, 28216, 248/l, 279/l, 251/l, 292/l, and 200/3, recognized 87 kDa glycoprotein (lane a). Polypeptide of 157 kDa was detected by these antibodies only when the r”S]methionine labeled precipitates were analyzed by prolonged exposure. Labeling of
be ad-
260
SHORT
COMMUNICATIONS
261
(Table 1). The binding of enzyme-conjugated antibody 245/12 precipitating gp87 was inhibited by the homologous antibody as well as by antibody 282/6, indicating c their specificities to identical or topographically overlapping epitopes, designated site Ia. However, none of the other antibodies competed with 245112 conjugate, indicating their specificities to antigenie sites nonoverlapping with site Ia. Similarly, two other nonoverlapping neutralizing antigenic sites were defined on gp87 (sites Ib and Ic), and a single neutralizing site (IIa) was detected on gp71. 436.5 In these assays, antibodies 251/l and 292/l enhanced the binding of antibody 245/12, and antibody 232/6 enhanced that FIG. 1. Polypeptide specificities of monoclonal an- of antibody 248/l. We used monoclonal antibodies in the tibodies to BHV-1. MDBK cells monolayers were laand virus adsorption inhibeled with [asS]methionine (56 &i/ml) (A) or neutralization bemjglucosamine (25 &i/ml) (B) from 3 or 6 hr to 24 bition assays to clarify the relation hr postinfection with l-10 m.o.i. of virus. Extracts tween these neutralizing antigenic sites were prepared from the infected cells using RIPA and biological functions of the antibodies buffer (0.05 M Tris-HCl, pH 8.0,0.15 MNaCI, 1% Tri(Table 2). Two monoclonal antibodies 2451 ton X-100,1% sodium deoxycholate) and then centri12 and 282/6 reactive with site Ia on gp87 fuged for 1 hr at 10,000 g. The supernatant was used neutralized the virus infectivity without as antigens in immunoprecipitation assays (7) which complement and blocked essentially the were subsequently analyzed by SDS-PAGE under rebinding of virus to cells. On the other hand, ducing (lanes a-c) or nonreducing (lane d) conditions. antibodies against site IIa on gp71 had only Lane a, polypeptides precipitated by antibody 245/12; activity against virus adlane b, 9/6; lanes c and d, lW2. The positions of the weak blocking molecular weight markers X10-’ are shown in the sorption although they effectively neutralright-hand margin. ized the infectivity without complement. Other monoclonal antibodies against sites Ib and Ic and gp117 molecule, required infected cells with [3Hlglucosamine failed complement for virus neutralization and to render the 157 kDa polypeptide. The did not show significant interference with second group comprising three monoclonal virus adsorption. antibodies, 9/6, 19711, and 264/3, precipiTo obtain further evidence suggesting tated a single glycoprotein of 71 kDa (lane the mechanisms of neutralization of the b), and the third group comprising two an- virus, a postadsorption neutralization test tibodies, 18512 and 26/l, reacted with a was carried out with the two monoclonal complex of glycoproteins of doublet 117- antibodies, 245/12 and 264/3, which were 111 kDa, doublet 70-64 kDa, and doublet directed to gp87 (site Ia) and gp71 (site IIa), 51-47 kDa (lane c). Only the 117 kDa gly- respectively, both neutralizing without coprotein was detected when the immucomplement. After 2 hr of virus adsorption, noprecipitates were analyzed under non- using ca. 100 PFU/well, serial dilutions of reducing conditions (lane d), showing that purified antibodies were placed on the cells, the 70-64 and 51-47 kDa glycoproteins ap- and assayed for plaque reduction (Fig. 2). peared to be components of a disulfideMonoclonal antibody 264/3 directed to linked complex of 117 kDa. gp71, neutralized the virus infectivity Competitive binding assays were carried completely. The amount of IgG required for out in order to identify separate neutral50% plaque reduction was 0.6 pg. In conizing antigenic sites on the glycoproteins trast to this complete neutralization, A
abed
B abc
262
SHORT
COMMUNICATIONS TABLE
1
COMPETITWE BINDING ASSAYS BETWEEN MONOCLONAL ANTIBODIES TO BHV-1 a Unlabeled antibody Antigp87
245/12
245/12
2b
248/l 279/l 251/l 292/l
2b 2a 2a 2a
200/3
2b
282/6
2b
Anti-gp71 9/6
197/l 264/3
% Inhibition to binding of HRPO-antibody
IgG subclass
1 2a 2b
100 88
248/l -60
9
251/l 10
-48
82
-77
84
88
35
24
9/6
197/l
26413
100 -11 17
80
44
100 63
23 100
6
Ia
-16
94 98 100
5
7
100 101
Antigenic site
Ib
Ic
IIa
a Monoclonal antibodies were purified using protein A (8) and labeled with horseradish peroxidase (HRPO) by the two-step glutaraldehyde coupling procedure (9). The assays were carried out by a modification of the technique described previously (10). The degree of competition binding was expressed by the perce-rtage of competition estimated by the formula 100 (A - X)/(A - B) where A, B, and X are ODa in the absence of competitor, in the presence of homologous antibody (5 pg), and in the presence of competitor (5 a), respectively. Minus indicates enhancement of the conjugate binding.
monoclonal antibody 245/12 directed to gp87, caused no neutralization even when 50 pg of IgG was applied. This report demonstrates that several neutralizing antigenic sites were distributed on at least three glycoproteins of BHV-1. Three nonoverlapping neutralizing antigenic sites, designated Ia, Ib, and Ic, were defined on gp87. Antibodies 245/12 and 282/6 against site Ia inhibited virus adsorption and neutralized the virus without complement. Therefore, these antibodies are considered to cause neutralization as a result of hindrance of virus attachment to the cells. Site Ia on gp87 of BHV-1 may be overlapped with or very close to the critical site on the virion for cell attachment. Alternatively, the antibodies might induce conformational changes in the receptor binding site on gp87, and result in virus neutralization. Such conformational changes were suggested by the fact that one of the antibodies (282/6) enhanced the binding of antibody
248/l against site Ib in competitive binding assays. The monoclonal antibodies to site IIa on gp71 were effective in neutralization when
ANTIBODY
(re)
FIG. 2. Postadsorption neutralization assays by monoclonal antibodies to BHV-1. After 2 hr adsorption of the virus (100-200 PFU/well) at 4O, monolayers were washed with cold PBS. Purified monoclonal antibodies (0.05 to 50 pg in 200 ~1 MEM) were plated onto the virus-adsorbed cells, followed by further incubation for 2 h at 4’. The monolayers were washed again, and assayed residual infectivity by plaque counting. 0,245/12; 0,264/3.
SHORT
COMMUNICATIONS
263
TABLE 2 BIOLOGICAL FUNCTIONSOF MONOCLONAL ANTIBODIES TO BHV-1” 50% Neutralization titer Antibody
Antigenic
Anti-gp87 245112 232/6 248/l 279/l 251/l 292/l 20013 Anti-gp71 916 197/l 264/3
site
50% Adsorption inhibition titer
C-
c+
Ia Ia Ib Ib Ib Ib IC
100 150 cl0 t10
2,800 2,500 230 400 250 720 3,400
2560 2560 10 NTb 40 NT
IIa IIa IIa
2ooo 480 8100
20@-3 18,200 25,600
Anti-117 185/2 26/l
3,100
2,2(@
a The virus neutralizing capacity of ascitic fluid containing monoelonal antibody was quantified by a plaque redaction assay. Serial dilutions of ascitic fluid were mixed with virus suspension (100-200 PFWwell) and the mixture was plated on a 24-well plate of MDBK cells subsequent to 1 hr incubation at 37”. Complementenhanced neutralization was determined by the addition of guinea pig sera (final concentration of 2%) to the virus-antibody mixture. The adsorption inhibition assays were carried out using [S6Sjmethionine-labeled BHV1. The virus was purified by differential centrifugation and two successive sedimentations through a 10-50% potassium tartrate gradient. The purified radiolabeled virus (8,000-10,000 cpm) was mixed with serially diluted ascitic fluid and incubated for 1 hr at 3Y. The virus-antibody mixture was added to a flexible 96-well plate of MDBK cells. The plate was incubated and gently agitated at 4” for 2 hr to allow adsorption of virus to cells. After washing the cells, each well was cut off from the plate and assayed for the count per minute by liquid scintillation counting. The percentage inhibition was estimated graphically from the sigmoid courves of bound cpm. b Not tested.
antibody was plated onto cells on which the virus had already become adsorbed. These data suggest that the antibodies could inhibit the infection process after adsorption, probably penetration of the virus. The binding of the antibodies to site IIa on gp71 is considered to affect the critical site, possibly by means of masking or conformational changes. In our experiment, antibodies to site IIa on gp71 showed a neutralizing titer much higher than that of antibodies to site Ia on gp87 in the test without complement. Recently, Fuller and Spear (11) have shown with herpes simplex virus that, although some antibodies can
neutralize the virus by blocking adsorption, a more important mechanism of neutralization by antibodies may be interference with a step subsequent to adsorption. It is expected that in order to be effective, a BHV-1 vaccine would have to induce antibodies to gp71. Antigenic sites Ib and Ic on gp87 and gp117 molecule were recognized by monoclonal antibodies requiring complement for virus neutralization. Complement-dependent neutralization has been demonstrated for a number of enveloped viruses (12-14). Further studies comparing complementdependent and complement-independent
264
SHORT
COMMUNICATIONS
neutralizing antibodies would be useful to elucidate the diverse mechanisms of BHV1 neutralization. ACKNOWLEDGMENTS This work was supported in part by Grant-in-Aid for Developmental Scientific Research 58369030, from the Ministry of Education, Science and Culture, Japan.
REFERENCES 1. KAHRS,
S. F., J. Amer. Vet. Med ASSOC. 117,10551064 (1977). 2. MISRA, V., BLUMENTHAL, R. M., and BABIUK, L. A., J. Vird 40,367-3’78 (1981). 8. BOLTON, D. C., ZEE, Y. C., and ARDANS, A. A., Vet. Microbid 8,57-68 (1983). 4. CHOPPIN, P. W., and SCHEID, A., Rev. In&e&. LG. 2,40-61 (1980).
5. PERRIN, L. H., TISHON, A. J., and OLDSTONE, M. B. A., J. Immund 118,282-290 (1977). 6. SPEAR, P. G., In “Cell Membranes and Viral Envelopes” (H. A. Blough, and J. M. Tiffany, eds.), Vol. 2, pp. 709-750. Academic Press, New York, 1989. 7. KIDA, H., BROW, L. E., and WEBSTER, R. G., virology 122,38-47 (1982). 8. EY, P. L., PROWSE, S. J., and JENKIN, C. R., Immunochemistry l&429-436 (1978). 9. AVRAMEAS, S., and TERNYNCK, T., Immunochemistry 8,1175-1179 (1971). 10. KIMURA-KURODA, J., and YASUI, K., J. vird 45, 124-132 (1983). 11. FULLER, A. O., and SPEAR, P. G., J. V+oL 55,475482 (1985). 12. YOSHINO, K., and TANIGUCHI, S., Vi~obgy 22,193201 (1964). 18. RADWAN, A. I., BURGER, D., and DAVIS, W. C., Vzrology 53,372-378 (1973). 14 POTGIETER, L. N. D., Canad J. Ccnnp. Med 39,427433 (1975).