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Development of monoclonal neutralizing antibodies against bovine viral diarrhea virus after pretreatment of mice with normal bovine cells and cyclophosphamide
Journal of Virological Methods, 24 (1989) 237-244
237
Elsevier JVM 00870
Short Communication
Qevelopment of monoclonal neutralizing antibodies against bovine viral diarrhea virus after pretreatment of mice with normal bovine cells and cyclophosphamide E. Weiland, H.-J. Thiel, G. Hess and F. Weiland Federal Research Centre for Virus Diseases of Animals,
Tiibingen, F.R.G.
(Accepted 6 January 1989)
Summary After immunization of mice with bovine viral diarrhea virus (BVDV) concentrated by ultracentrifugation only antibodies against normal cellular constituents were detectable. Monoclonal and polyclonal anti-BVDV antibodies were obtained, however, after induction of tolerance against normal bovine cellular antigens by pretreatment of adult mice with bovine cells and cyclophosphamide followed by immunization with BVDV containing material. Bovine viral diarrhea monoclonal antibody
virus; ~mmunotolerance;
Cyclophosphamide;
Neutralizing
Pestivirus is a genus within the family of Togaviridae. The genus comprises hog cholera virus (HCV), bovine viral diarrhea virus (BVDV) and border disease virus of sheep. All pestiviruses are morphologically and serotogically related and are important viral pathogens of their respective host species. Detailed serological analysis of Pestivirw strains and isolates with polyclonal sera is difficult. Therefore there is a need to develop sensitive probes for differentiation within and between Pestivirus species. For this purpose pestivirus-specific monoclonal antibodies (MAb) were prepared in different laboratories. With one exception (Donis et al., 1988) the efficiency to establish stable antibody producing hyCorrespondence
to: E. Weiland, Federal Research Centre for Virus Diseases of Animals, P.0, Box 11 49, D-7400 ‘Ribingen, F.R.G.
238
Fig. 1, MDBK cells examined by IIF with a MAb against cytoplasmic antigen of bovine cells (a) and with a MAb against BVDV antigen (b).
239
bridomas was extremely low. Peters et al. (1986) succeeded in establishing five stable anti-BVDV producing hybridomas; Juntti et al. (1987) isolated only one antiBVDV hybridoma. After immunization with three different BVDV strains Moennig et al. (1987) reported the generation of 20~5~0 hybridoma colonies leading to only 2, 3, and 4 stable anti-BVDV producing hybridomas, respectively. Teyssedou et al. (1987) reported the production of hybridomas secreting anti-cellular antibodies after immunization with BVDV purified on sucrose gradient. These antibodies recognised an antigen expressed on the cell surface of Madin-Darby bovine kidney cells (MDBK) as well as other bovine cells. We attempted to establish anti-BVDV antibody producing hyb~domas to be used in the differentiation of pestiviruses and the characterization of BVDV structural proteins. SP2IO myeloma cells were fused with spleen cells of mice which had been immunized with the NADL strain of BVDV (lo8 TCID,,) by the intraperitoneal route (i.p.). The virus used for immunization was grown in primary bovine kidney cells, concentrated by ultracentrifugation as described by Peters et al. (19861, and adsorbed to aluminium hydroxide according to Wensvoort et al. (1986). Sera of mice which had been immunized three times and hybridoma supernatants were screened for antiviral activity by indirect immunofluorescence (IIF) on BVDV-infected MDBK cells and by a dot immunobinding assay (Hawkes et al., 1982) using cell lysates of BVDV-infected and noninfected MDBK cells. For preparation of cell Iysates monolayer cells were trypsinized, suspended in phosphate buffered saline and freeze-thawed three times. To eliminate insoluble material the cell lysate were centrifuged at 6000 x g for 10 min. No antiviral activity was observed in sera or in hybridoma supernatants using either assay. However, a considerable amount of hybridomas released antibodies that decorated cytoplasmic structures in different noninfected bovine cells (MDBK cells, primary bovine kidney cells, and nasoturbinate cells) as observed by IIF. Two of them secreting antibodies of the immunoglobulin Gl and G2b subclass were successfully cloned by limiting dilution. These antibodies reacted only with bovine cells, not with cells of other species tested such as horse, hamster or rat. In bovine celis these antibodies decorated coarse to fine spots, scattered throughout the cytoplasm (Fig. 1A). The hybridoma antibodies detected those cytoplasmic antigens not only in bovine cells but also in sucrose gradient purified BVDV preparations using the dot immunobinding assay on nitrocellulose. These findings indicate that those cellular components are still present in ‘purified’ viral preparations. The natu e of these T cellular antigens still remains to be elucidated. To eliminate such undesired antibodies against antigens of non-infected bovine cells which may hide immunogenic viral antigenic determinants, mice were treated i.p. with non-infected MDBK cells (1.5 x 10’ freeze-thawed cells/mouse) followed by i.p. infection of cyclophosphamide (100 mg/kg) after 10 min, 24 h and 48 h according to Matthew and Sandrock (1987). This treatment was repeated every two weeks for three times. Eighteen days after the third tolerizing treatment immunization was performed using lysate of BVDV-infected MDBK cells. As shown in
240
HBrr
LDV
Fig. 2. Reactivity of cell extracts from BVDV-infected (+) and non-infected (-) MDBK cells with MAb against BVDV (uWDV), MAb against unidentified cytoplasmic antigen of bovine cells (aXY), MAb against Golgi antigen (aGolgi) and MAb against lactate dehydrogenase virus (aLDV) in a dot immunobinding assay.
241
a dot immunobinding assay (Fig. 2) this cell lysate contained viral antigen as well as strongly reactive cellular antigens. 200 ~1 of the BVDV (10s TCID,,) containing cell lysate were adsorbed to 200 ~1 of aluminium hydroxide and injected i.p. followed by a second immunization 13 days later. Using IIF on BVDV infected MDBK cells as well as serum neutralization assay against 100 TCID,, of BVDV in a standard test on primary bovine kidney cells as indicators antiviral antibodies were detected in serum collected 11 days after the second immunization. Three days after a third immunization using lysate of BVDV-infected cells without adjuvant spleen cells of a seropositive mouse were fused with myeloma cells. The fusion products of a quarter of the spleen were plated into ten 96-well plates. Growth of hybridoma colonies was observed in 302 wells, and 42 hybridomas secreting antiviral antibodies were identified by IIF on BVDV-infected MDBK cells
TABLE Reactivity HB
lb2 2c2 lc6 lc18 2blO 3b5 2bl 1~42 2~6 lc30 la7 lb23 la1 3b2 3cl lb8 lb31 la16 la5 lc17 D5
1 of 21 anti-BVDV NT”
monoclonal
antibodies Indirect
immunofluorescenceb
NADL OREGON VEMIE ELK-2
SINGER
DANMARK
.
+ w + + + +
_ _ _ _ _
_ _
0
+
_
-
. 0
+ +
-
-
0
+
_
_
. . . . 0 4 4 . . 4
+ + + + + + + + + +
_ _ + + + + + w w w
_ _ _ w w + + w
0
+
w
+
. 0 0 . l
“Neutralization of the immunizing BVDV-NADL strain after application of graded doses of MAbs to 100 TCID,, of virus in the absence of complement. Explanation of symbols used: 0, neutralization with ascitic MAb diluted 1:8100 or more; 0, with dilution 1:2700; 0, neutralization not detectable after application of culture fluid but detectable after incubation with ascitic fluid diluted 1:900. bReactivity pattern with BVDV strains of the CPE-biotype on infected MDBK cells; +, strong reactivity detectable; -, no reactivity detectable; w, weak reactivity recognizable.
242
12345
92.5 K69
K$
46
K-
30
K-.
Fig. 3. Radioimmunoprecipitation analysis of neutraIizing monoclonal antibodies. After metabolic labeling of BVDV-infected cells with %I-glucosamine, the cell extract was precipitated with (1) autologous goat serum against HCV (unpublished data). (2) preimmune goat serum, (3, 4) monoclonal antiBVDV antibodies lb23 and 3~1, respectively. (5) control monoclonal antibody. Precipitates were analyzed on 10% SDS-PAGE and bands visualized after fluorography using X-ray film. Molecular weight markers refer to t4C-phosphorylase b (92.5 kDa). albumin (69 kDa). ovalbumin (46 kDa) and carb(~~~nh~drase (30 kDa).
(Fig. 1B). Twenty-one of them were selected for recloning and preparation of ascitic fluid. All subcloned hybridomas that were found positive in IIF neutralized viral infectivity (Table 1). However, there were marked differences in the potency of neutralization. Four MAbs neutralized BVDV only as ascitic fluid, whereas 17 MAbs neutralized BVDV even as culture supernatant. In order to identify the antigen(s) recognized by the neutralizing monoclonal antibodies, extracts from metabolically labelled BVDV-infected MDBK cells were immunoprecipitated. As shown in Fig. 3, two monoclonal antibodies specifically precipitated a 53 kDa glycoprotein from BVDV-infected cells, which has also been detected by others (Donis et al., 3988). A control monoclonal antibody did not react (Fig. 3, iane 5). Preliminary data indicate that the other neutralizing monoclonal antibodies recognize the same antigen (data not shown). When applied in IIF on MDBK cells infected with six BVDV strains of the cytopathic-biotype (listed in Table 1 and used in a previous study, Hess et al., 1988) 12 MAbs reacted with four of the BVDV strains, three MAbs with five strains and six MAbs with all strains tested.
243
The described procedure led to the efficient isolation of anti-BVDV antibody producing hybridomas. Application of this method may help to overcome problems in producing monoclonal antibodies against other viruses as well, especially in cases where the virus is difficult to purify and/or is closely associated with cellular material. References Donis, R.O.. Corapi, W. and Dubovi, E.J. (1988) Neutralizing monoclonai antibodies to bovine viral diarrhoea virus bind to the 56 K to 58 K glycoprotein. J. Gen. Viral. 69. 77-86. Hawkes, R., Niday, E. and Gordon, J. (1982) A dot immunobinding assay for monoclonal and other antibodies. Analytical Biochemistry 119, 142-147. Hess, R.G., Coulibaly, C.O.Z., Greiser-Wilke, I., Moennig, V. and Liess, B. (1988) Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses. Vet. Microbial. 16, 315-321. Juntti, N., Larsson, B. and Fossum, C. (1987) The use of monoclonal antibodies in enzyme linked immunosorbent assays for detection of antibodies to bovine viral diarrhoea virus. J. Vet. Med. B 34, 3X-363. Matthew, W.D. and Sandrock, A.W. (1987) Cyclophosphamide treatment used to manipulate the immune response for the production of monoclonal antibodies. J. Immunol. Methods 100, 73-82. Moennig, V., Bolin, S.R.. Coulibaly. C.O.Z., Kelso Gourley, N.E., Liess, B., Mateo, A., Peters, W. and Greiser-Wilke, I. (1987) Untersuchungen zur Antigenstruktur von Pestiviren mit Hilfe monoklonaler Antikiirper. Dtsch. TierBrztl. Wochenschr. 94, 572-576. Peters, W., Greiser-Wilke. I., Moennig, V. and Liess, B. (1986) Preliminary serological characterization of bovine viral diarrhoea virus strains using monoclonal antibodies. Vet. Microbial. 12, 195-200. Teyssedou, E., Magar, R., Justewicz, D.M. and Lecomte, J. (1987) Cell-protective monoclonal antibodies to bovine enterovirus-3 and partial or no activity against other serotypes. J. Viral. 61, 2050-2053. Wensvoort, G., Terpstra, C., Boonstra, J., Bloemrad, M. and Van Zaane, D. (1986) Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet. Microbiol. 12. 101-108.
237
Elsevier JVM 00870
Short Communication
Qevelopment of monoclonal neutralizing antibodies against bovine viral diarrhea virus after pretreatment of mice with normal bovine cells and cyclophosphamide E. Weiland, H.-J. Thiel, G. Hess and F. Weiland Federal Research Centre for Virus Diseases of Animals,
Tiibingen, F.R.G.
(Accepted 6 January 1989)
Summary After immunization of mice with bovine viral diarrhea virus (BVDV) concentrated by ultracentrifugation only antibodies against normal cellular constituents were detectable. Monoclonal and polyclonal anti-BVDV antibodies were obtained, however, after induction of tolerance against normal bovine cellular antigens by pretreatment of adult mice with bovine cells and cyclophosphamide followed by immunization with BVDV containing material. Bovine viral diarrhea monoclonal antibody
virus; ~mmunotolerance;
Cyclophosphamide;
Neutralizing
Pestivirus is a genus within the family of Togaviridae. The genus comprises hog cholera virus (HCV), bovine viral diarrhea virus (BVDV) and border disease virus of sheep. All pestiviruses are morphologically and serotogically related and are important viral pathogens of their respective host species. Detailed serological analysis of Pestivirw strains and isolates with polyclonal sera is difficult. Therefore there is a need to develop sensitive probes for differentiation within and between Pestivirus species. For this purpose pestivirus-specific monoclonal antibodies (MAb) were prepared in different laboratories. With one exception (Donis et al., 1988) the efficiency to establish stable antibody producing hyCorrespondence
to: E. Weiland, Federal Research Centre for Virus Diseases of Animals, P.0, Box 11 49, D-7400 ‘Ribingen, F.R.G.
238
Fig. 1, MDBK cells examined by IIF with a MAb against cytoplasmic antigen of bovine cells (a) and with a MAb against BVDV antigen (b).
239
bridomas was extremely low. Peters et al. (1986) succeeded in establishing five stable anti-BVDV producing hybridomas; Juntti et al. (1987) isolated only one antiBVDV hybridoma. After immunization with three different BVDV strains Moennig et al. (1987) reported the generation of 20~5~0 hybridoma colonies leading to only 2, 3, and 4 stable anti-BVDV producing hybridomas, respectively. Teyssedou et al. (1987) reported the production of hybridomas secreting anti-cellular antibodies after immunization with BVDV purified on sucrose gradient. These antibodies recognised an antigen expressed on the cell surface of Madin-Darby bovine kidney cells (MDBK) as well as other bovine cells. We attempted to establish anti-BVDV antibody producing hyb~domas to be used in the differentiation of pestiviruses and the characterization of BVDV structural proteins. SP2IO myeloma cells were fused with spleen cells of mice which had been immunized with the NADL strain of BVDV (lo8 TCID,,) by the intraperitoneal route (i.p.). The virus used for immunization was grown in primary bovine kidney cells, concentrated by ultracentrifugation as described by Peters et al. (19861, and adsorbed to aluminium hydroxide according to Wensvoort et al. (1986). Sera of mice which had been immunized three times and hybridoma supernatants were screened for antiviral activity by indirect immunofluorescence (IIF) on BVDV-infected MDBK cells and by a dot immunobinding assay (Hawkes et al., 1982) using cell lysates of BVDV-infected and noninfected MDBK cells. For preparation of cell Iysates monolayer cells were trypsinized, suspended in phosphate buffered saline and freeze-thawed three times. To eliminate insoluble material the cell lysate were centrifuged at 6000 x g for 10 min. No antiviral activity was observed in sera or in hybridoma supernatants using either assay. However, a considerable amount of hybridomas released antibodies that decorated cytoplasmic structures in different noninfected bovine cells (MDBK cells, primary bovine kidney cells, and nasoturbinate cells) as observed by IIF. Two of them secreting antibodies of the immunoglobulin Gl and G2b subclass were successfully cloned by limiting dilution. These antibodies reacted only with bovine cells, not with cells of other species tested such as horse, hamster or rat. In bovine celis these antibodies decorated coarse to fine spots, scattered throughout the cytoplasm (Fig. 1A). The hybridoma antibodies detected those cytoplasmic antigens not only in bovine cells but also in sucrose gradient purified BVDV preparations using the dot immunobinding assay on nitrocellulose. These findings indicate that those cellular components are still present in ‘purified’ viral preparations. The natu e of these T cellular antigens still remains to be elucidated. To eliminate such undesired antibodies against antigens of non-infected bovine cells which may hide immunogenic viral antigenic determinants, mice were treated i.p. with non-infected MDBK cells (1.5 x 10’ freeze-thawed cells/mouse) followed by i.p. infection of cyclophosphamide (100 mg/kg) after 10 min, 24 h and 48 h according to Matthew and Sandrock (1987). This treatment was repeated every two weeks for three times. Eighteen days after the third tolerizing treatment immunization was performed using lysate of BVDV-infected MDBK cells. As shown in
240
HBrr
LDV
Fig. 2. Reactivity of cell extracts from BVDV-infected (+) and non-infected (-) MDBK cells with MAb against BVDV (uWDV), MAb against unidentified cytoplasmic antigen of bovine cells (aXY), MAb against Golgi antigen (aGolgi) and MAb against lactate dehydrogenase virus (aLDV) in a dot immunobinding assay.
241
a dot immunobinding assay (Fig. 2) this cell lysate contained viral antigen as well as strongly reactive cellular antigens. 200 ~1 of the BVDV (10s TCID,,) containing cell lysate were adsorbed to 200 ~1 of aluminium hydroxide and injected i.p. followed by a second immunization 13 days later. Using IIF on BVDV infected MDBK cells as well as serum neutralization assay against 100 TCID,, of BVDV in a standard test on primary bovine kidney cells as indicators antiviral antibodies were detected in serum collected 11 days after the second immunization. Three days after a third immunization using lysate of BVDV-infected cells without adjuvant spleen cells of a seropositive mouse were fused with myeloma cells. The fusion products of a quarter of the spleen were plated into ten 96-well plates. Growth of hybridoma colonies was observed in 302 wells, and 42 hybridomas secreting antiviral antibodies were identified by IIF on BVDV-infected MDBK cells
TABLE Reactivity HB
lb2 2c2 lc6 lc18 2blO 3b5 2bl 1~42 2~6 lc30 la7 lb23 la1 3b2 3cl lb8 lb31 la16 la5 lc17 D5
1 of 21 anti-BVDV NT”
monoclonal
antibodies Indirect
immunofluorescenceb
NADL OREGON VEMIE ELK-2
SINGER
DANMARK
.
+ w + + + +
_ _ _ _ _
_ _
0
+
_
-
. 0
+ +
-
-
0
+
_
_
. . . . 0 4 4 . . 4
+ + + + + + + + + +
_ _ + + + + + w w w
_ _ _ w w + + w
0
+
w
+
. 0 0 . l
“Neutralization of the immunizing BVDV-NADL strain after application of graded doses of MAbs to 100 TCID,, of virus in the absence of complement. Explanation of symbols used: 0, neutralization with ascitic MAb diluted 1:8100 or more; 0, with dilution 1:2700; 0, neutralization not detectable after application of culture fluid but detectable after incubation with ascitic fluid diluted 1:900. bReactivity pattern with BVDV strains of the CPE-biotype on infected MDBK cells; +, strong reactivity detectable; -, no reactivity detectable; w, weak reactivity recognizable.
242
12345
92.5 K69
K$
46
K-
30
K-.
Fig. 3. Radioimmunoprecipitation analysis of neutraIizing monoclonal antibodies. After metabolic labeling of BVDV-infected cells with %I-glucosamine, the cell extract was precipitated with (1) autologous goat serum against HCV (unpublished data). (2) preimmune goat serum, (3, 4) monoclonal antiBVDV antibodies lb23 and 3~1, respectively. (5) control monoclonal antibody. Precipitates were analyzed on 10% SDS-PAGE and bands visualized after fluorography using X-ray film. Molecular weight markers refer to t4C-phosphorylase b (92.5 kDa). albumin (69 kDa). ovalbumin (46 kDa) and carb(~~~nh~drase (30 kDa).
(Fig. 1B). Twenty-one of them were selected for recloning and preparation of ascitic fluid. All subcloned hybridomas that were found positive in IIF neutralized viral infectivity (Table 1). However, there were marked differences in the potency of neutralization. Four MAbs neutralized BVDV only as ascitic fluid, whereas 17 MAbs neutralized BVDV even as culture supernatant. In order to identify the antigen(s) recognized by the neutralizing monoclonal antibodies, extracts from metabolically labelled BVDV-infected MDBK cells were immunoprecipitated. As shown in Fig. 3, two monoclonal antibodies specifically precipitated a 53 kDa glycoprotein from BVDV-infected cells, which has also been detected by others (Donis et al., 3988). A control monoclonal antibody did not react (Fig. 3, iane 5). Preliminary data indicate that the other neutralizing monoclonal antibodies recognize the same antigen (data not shown). When applied in IIF on MDBK cells infected with six BVDV strains of the cytopathic-biotype (listed in Table 1 and used in a previous study, Hess et al., 1988) 12 MAbs reacted with four of the BVDV strains, three MAbs with five strains and six MAbs with all strains tested.
243
The described procedure led to the efficient isolation of anti-BVDV antibody producing hybridomas. Application of this method may help to overcome problems in producing monoclonal antibodies against other viruses as well, especially in cases where the virus is difficult to purify and/or is closely associated with cellular material. References Donis, R.O.. Corapi, W. and Dubovi, E.J. (1988) Neutralizing monoclonai antibodies to bovine viral diarrhoea virus bind to the 56 K to 58 K glycoprotein. J. Gen. Viral. 69. 77-86. Hawkes, R., Niday, E. and Gordon, J. (1982) A dot immunobinding assay for monoclonal and other antibodies. Analytical Biochemistry 119, 142-147. Hess, R.G., Coulibaly, C.O.Z., Greiser-Wilke, I., Moennig, V. and Liess, B. (1988) Identification of hog cholera viral isolates by use of monoclonal antibodies to pestiviruses. Vet. Microbial. 16, 315-321. Juntti, N., Larsson, B. and Fossum, C. (1987) The use of monoclonal antibodies in enzyme linked immunosorbent assays for detection of antibodies to bovine viral diarrhoea virus. J. Vet. Med. B 34, 3X-363. Matthew, W.D. and Sandrock, A.W. (1987) Cyclophosphamide treatment used to manipulate the immune response for the production of monoclonal antibodies. J. Immunol. Methods 100, 73-82. Moennig, V., Bolin, S.R.. Coulibaly. C.O.Z., Kelso Gourley, N.E., Liess, B., Mateo, A., Peters, W. and Greiser-Wilke, I. (1987) Untersuchungen zur Antigenstruktur von Pestiviren mit Hilfe monoklonaler Antikiirper. Dtsch. TierBrztl. Wochenschr. 94, 572-576. Peters, W., Greiser-Wilke. I., Moennig, V. and Liess, B. (1986) Preliminary serological characterization of bovine viral diarrhoea virus strains using monoclonal antibodies. Vet. Microbial. 12, 195-200. Teyssedou, E., Magar, R., Justewicz, D.M. and Lecomte, J. (1987) Cell-protective monoclonal antibodies to bovine enterovirus-3 and partial or no activity against other serotypes. J. Viral. 61, 2050-2053. Wensvoort, G., Terpstra, C., Boonstra, J., Bloemrad, M. and Van Zaane, D. (1986) Production of monoclonal antibodies against swine fever virus and their use in laboratory diagnosis. Vet. Microbiol. 12. 101-108.