VIRAL IMMUNOLOGY Volume 5, Number 4, 1992 Mary Ann Lieben, Inc., Publishers Pp. 257-263
Bovine Viral Diarrhea Virus-Specific Neutralizing Antibodies Induced by Anti-Idiotypic Antibodies D.V.
ONISK,1 R. DONIS,
C.L. KELLING, and S. SRIKUMARAN
ABSTRACT Two m urine neutralizing monoclonal antibodies (M Abs), 4D8 and 6D11, recognizing epitopes on gp53, a surface glycoprotein of bovine viral diarrhea virus (BVDV), were used to generate anti-idiotypic antibodies (anti-ids) in a calf. The polyclonal anti-ids were isolated from serum by affinity chromatography on their respective Ab-1-Sepharose columns, followed by repeated adsorption on isotype-matched antibody-Sepharose columns. The anti-ids reacted specifically with their respective Ab-1, but not with isotype-matched controls. They also inhibited the binding of their Ab-1 to BVDV in a concentration-dependent manner. Mice immunized with the two anti-id preparations developed antibodies to BVDV, which neutralized the virus in vitro.
viral diarrhea virus RNA virus that belongs to the genus Pestivirus is a in the It is an of cattle that exerts significant economic impact on cattle production directly, and on the biologies industry via product contamination (21,26). This virus is one of the etiological agents of the bovine respiratory disease complex (20,23), abortion (5,13), congenital defects, and neonatal diarrhea (17). The major problem in controlling BVDV is the ability of the virus to infect the fetus and cause a lifelong persistent infection, and the unavailability of safe and efficacious vaccines. The modified live virus (MLV) vaccines are not safe for use in pregnant animals, and the inactivated virus vaccines do not provide complete protection of the fetus (24). Anti-idiotypic antibodies (anti-ids) have been tested as alternatives to conventional immunogens in a variety of systems. In most of the viral systems studied, the anti-ids elicited humoral and/or cell-mediated immune responses against their respective viruses (4,8,11,15,19,30,33). In some cases, the immune response elicited was strong enough to protect against subsequent challenge with virulent viruses (9,10,25). Suppression of specific immune responses, however, has also been reported in a few cases (18). The parameters influencing the outcome of an anti-id immunization are not well understood, although the dose, isotype, and route of immunization of the anti-id have been suggested to play arole (7). Although the anti-ids
positive-stranded Bovine family Togaviridae.(BVDV)important pathogen
Department of Veterinary Science, University of Nebraska, Lincoln, Nebraska. 'Current Address: United States Department of Agriculture, Agricultural Research Service, North Atlantic Area, Plum Island Animal Disease Center, Post Office Box 848, Greenport, L.I., N.Y. 11944-0848. 257
ONISK ET AL. will not replace conventional vaccines, they may offer an alternative in those systems where conventional vaccines are ineffective or problematic, such as the case with BVDV. In this study, we have examined the potential of anti-ids as immunogens for the induction of neutralizing antibodies to BVDV. Two murine monoclonal antibodies (MAbs), 4D8 and 6D11, that neutralize BVDV (6) were used as antibody 1 (Abl) to generate the anti-ids in a calf. The murine hybridomas secreting these MAbs were kindly provided by Dr. E.J. Dubovi (Cornell University, Ithaca, NY). Both of these MAbs recognize epitopes on gp53 glycoprotein of BVDV (3,6). The isotypes of 4D8 and 6D11 are IgG2b and IgG2a, respectively. Seven other hybridomas were used as the source of BALB/c mouse IgG2a and IgG2b antibodies with irrelevant specificities. The hybridomas MM 105 (IgG2b) and MM 113 (IgG2a), which are specific for bovine herpesvirus 1 (BHV-1; 28), and the hybridomas MM 603 (IgG2b), MM 604 (IgG2b), and MM 605 (IgG2a), which are specific for Pasteurella haemolytica leukotoxin, were developed in our laboratory (12). The hybridoma 1E11 (IgG2a), which is specific for the gl glycoprotein of BHV-1 (31), was kindly provided by Dr. S. Van Drunen Littel-Van Den Hurk of the Veterinary Infectious Diseases Organization, Saskatoon, Canada. The hybridoma 5A5 (IgG2a), which is specific for the E2 glycoprotein of transmissible gastroenteritis virus (TGEV) (14), was a gift of the late Dr. P. Gough of the Iowa State University (Ames, IA). The hybridomas were cultured in protein-free hybridoma medium (PFHM-II, Gibco Laboratories, Grand Island, NY) and the MAbs were isolated from the culture fluid by affinity-chromatography on protein A-Sepharose CL-4B column (Pharmacia Fine Chemicals Inc., Piscataway, NJ). A 6-month-old male Hereford calf was inoculated intramuscularly (i-m) with 500 u.g each of the protein A-purified and alum-precipitated 4D8 and 6D11, emulsified 1:1 in complete Freund's adjuvant (CFA). Booster injections of 150 p.g each of 4D8 and 6D11 in incomplete Freund's adjuvant (ICFA) were given im at 21-day intervals postpriming. Serum was obtained 12 days following booster injections, pooled and stored at -20°C. The bovine anti-ids were isolated by standard affinity chromatography procedures (28). Affinity-purified MAbs 4D8, 6D11, MM 105, and MM 605 (15 mg of each) were individually coupled to CNBr-activated Sepharose 4B (3 g) according to the manufacturer's instructions (Pharmacia Fine Chemicals, Piscataway, NJ). In the initial step, bovine antibodies to 4D8 and 6D11 were purified on 4D8- and 6D11-Sepharose columns, respectively. The anti-ids specific for 4D8 and 6D11 were subsequently separated from the anti-isotypic and anti-allotypic antibodies by multiple passages through the respective isotype-matched MAb-Sepharose columns [MM 105 (IgG2b) for the anti-4D8-ids and MM 605 (IgG2a) for the anti-6Dl 1-ids,
respectively). The specificity of the different anti-id preparations to their respective Ab-1 idiotype was tested by a standard indirect solid-phase radioimmunoassay (RIA, 28). In this assay, the binding of the Abl 4D8and6Dll, or the isotype-matched controls, to the anti-ids adsorbed to microtiter wells was detected by the addition of 125I-labeled sheep antibodies to mouse Ig. Groups of four female BALB/c mice were primed intraperitoneally (i-p) with 50 u.g of alum-precipitated anti-ids emulsified 1:1 in CFA. The mice were given 10 biweekly booster injections, i-p with 25 p-g of alum-precipitated anti-ids in ICFA. Control groups of mice of the same age and sex were injected similarly with normal bovine Ig previously shown to be free of anti-BVDV antibodies. All animals were bled 10 days after the last booster injection. For the culture and propagation of BVDV, bovine turbinate (BT) cells (ATCC, Rockville, MD) were determined to be free of noncytopathic strains of BVDV by immunofluorescence assays using fluorescein isothiocyanate (FITC)-labeled bovine anti-BVDV antibodies [National Veterinary Service Laboratories (NVSL), Ames, I A]. The virus was propagated in BT cells grown in minimum essential medium (MEM) with 10% heat-inactivated horse serum (HS) and 100 pg/ml gentamicin. The cytopathic strain (NADL) of BVDV (NVSL, Ames, IA) was biologically cloned by plaque picking (27) three times prior to use. The noncytopathic strain (NY-1) of BVDV (NVSL, Ames, IA) was cloned twice prior to use by the limiting dilution technique. An indirect immunofluorescence assay (IFA) in microwell plates (22) was used to detect antibodies specific for BVDV.
Virus-neutralizing activity of the antibodies were tested by a standard plaque reduction assay using BT cells described by Cooper (2). The neutralization titer was expressed as the highest dilution of antibody that neutralized 50% of the input virus. as
258
BVDV-SPECIFIC NEUTRALIZING ANTIBODIES Immunization of the calf with 4D8 and 6D11 resulted in the production of anti-4D8 and -6D11 antibodies. Following the fourth booster injection, the titer of these antibodies reached 1:3200 as determined by an RIA (results not shown). The anti-ids isolated from the anti-4D8 and -6D11 antibody preparations were tested for their specificity to their respective Abl by an indirect RIA. The anti-4D8-ids (Fig. 1 A) bound specifically to their Abl (4D8), but not to the isotype-matched (IgG2b) controls (MM 105, specific for BHV-1 glycoprotein gl; MM 603 and MM 604, specific for the Pasteurella haemolytica leukotoxin). Similarly, the anti-6Dl 1-ids (Fig. 1B) reacted specifically with their Abl (6D11 ), but not with the isotype-matched (IgG2a) controls (MM 113, specific for BHV-1 glycoprotein gIV; 5A5, specific for TGEV; 1 El 1, specific for BHV-1 glycoprotein gl; and MM 605, specific for the P. haemolytica leukotoxin). These results indicated that the bovine anti-id preparations were specific for their respective idiotypes. The lack of binding of the anti-ids to the isotype-matched controls also indicated the absence of bovine antibodies to the isotypic and allotypic determinants of Abl in the anti-id preparations. Furthermore, in a plaque reduction assay, the bovine anti-4D8-ids and anti-6D 11-ids inhibited the neutralization of BVDV by the Abl 4D8and6Dl 1, respectively, in a concentration-dependent manner (Fig. 2). The results of the indirect RIA and plaque reduction assay suggested that the bovine anti-ids recognized idiotopes within or close to the paratope of the Ab 1. In order to determine whether these anti-ids could mimic
4D8
MM 105
MM 603
MM 604
Monoclonal Antibodies
CO
rr
Bovine Viral Diarrhea Virus-Specific Neutralizing Antibodies Induced by Anti-Idiotypic Antibodies D.V.
ONISK,1 R. DONIS,
C.L. KELLING, and S. SRIKUMARAN
ABSTRACT Two m urine neutralizing monoclonal antibodies (M Abs), 4D8 and 6D11, recognizing epitopes on gp53, a surface glycoprotein of bovine viral diarrhea virus (BVDV), were used to generate anti-idiotypic antibodies (anti-ids) in a calf. The polyclonal anti-ids were isolated from serum by affinity chromatography on their respective Ab-1-Sepharose columns, followed by repeated adsorption on isotype-matched antibody-Sepharose columns. The anti-ids reacted specifically with their respective Ab-1, but not with isotype-matched controls. They also inhibited the binding of their Ab-1 to BVDV in a concentration-dependent manner. Mice immunized with the two anti-id preparations developed antibodies to BVDV, which neutralized the virus in vitro.
viral diarrhea virus RNA virus that belongs to the genus Pestivirus is a in the It is an of cattle that exerts significant economic impact on cattle production directly, and on the biologies industry via product contamination (21,26). This virus is one of the etiological agents of the bovine respiratory disease complex (20,23), abortion (5,13), congenital defects, and neonatal diarrhea (17). The major problem in controlling BVDV is the ability of the virus to infect the fetus and cause a lifelong persistent infection, and the unavailability of safe and efficacious vaccines. The modified live virus (MLV) vaccines are not safe for use in pregnant animals, and the inactivated virus vaccines do not provide complete protection of the fetus (24). Anti-idiotypic antibodies (anti-ids) have been tested as alternatives to conventional immunogens in a variety of systems. In most of the viral systems studied, the anti-ids elicited humoral and/or cell-mediated immune responses against their respective viruses (4,8,11,15,19,30,33). In some cases, the immune response elicited was strong enough to protect against subsequent challenge with virulent viruses (9,10,25). Suppression of specific immune responses, however, has also been reported in a few cases (18). The parameters influencing the outcome of an anti-id immunization are not well understood, although the dose, isotype, and route of immunization of the anti-id have been suggested to play arole (7). Although the anti-ids
positive-stranded Bovine family Togaviridae.(BVDV)important pathogen
Department of Veterinary Science, University of Nebraska, Lincoln, Nebraska. 'Current Address: United States Department of Agriculture, Agricultural Research Service, North Atlantic Area, Plum Island Animal Disease Center, Post Office Box 848, Greenport, L.I., N.Y. 11944-0848. 257
ONISK ET AL. will not replace conventional vaccines, they may offer an alternative in those systems where conventional vaccines are ineffective or problematic, such as the case with BVDV. In this study, we have examined the potential of anti-ids as immunogens for the induction of neutralizing antibodies to BVDV. Two murine monoclonal antibodies (MAbs), 4D8 and 6D11, that neutralize BVDV (6) were used as antibody 1 (Abl) to generate the anti-ids in a calf. The murine hybridomas secreting these MAbs were kindly provided by Dr. E.J. Dubovi (Cornell University, Ithaca, NY). Both of these MAbs recognize epitopes on gp53 glycoprotein of BVDV (3,6). The isotypes of 4D8 and 6D11 are IgG2b and IgG2a, respectively. Seven other hybridomas were used as the source of BALB/c mouse IgG2a and IgG2b antibodies with irrelevant specificities. The hybridomas MM 105 (IgG2b) and MM 113 (IgG2a), which are specific for bovine herpesvirus 1 (BHV-1; 28), and the hybridomas MM 603 (IgG2b), MM 604 (IgG2b), and MM 605 (IgG2a), which are specific for Pasteurella haemolytica leukotoxin, were developed in our laboratory (12). The hybridoma 1E11 (IgG2a), which is specific for the gl glycoprotein of BHV-1 (31), was kindly provided by Dr. S. Van Drunen Littel-Van Den Hurk of the Veterinary Infectious Diseases Organization, Saskatoon, Canada. The hybridoma 5A5 (IgG2a), which is specific for the E2 glycoprotein of transmissible gastroenteritis virus (TGEV) (14), was a gift of the late Dr. P. Gough of the Iowa State University (Ames, IA). The hybridomas were cultured in protein-free hybridoma medium (PFHM-II, Gibco Laboratories, Grand Island, NY) and the MAbs were isolated from the culture fluid by affinity-chromatography on protein A-Sepharose CL-4B column (Pharmacia Fine Chemicals Inc., Piscataway, NJ). A 6-month-old male Hereford calf was inoculated intramuscularly (i-m) with 500 u.g each of the protein A-purified and alum-precipitated 4D8 and 6D11, emulsified 1:1 in complete Freund's adjuvant (CFA). Booster injections of 150 p.g each of 4D8 and 6D11 in incomplete Freund's adjuvant (ICFA) were given im at 21-day intervals postpriming. Serum was obtained 12 days following booster injections, pooled and stored at -20°C. The bovine anti-ids were isolated by standard affinity chromatography procedures (28). Affinity-purified MAbs 4D8, 6D11, MM 105, and MM 605 (15 mg of each) were individually coupled to CNBr-activated Sepharose 4B (3 g) according to the manufacturer's instructions (Pharmacia Fine Chemicals, Piscataway, NJ). In the initial step, bovine antibodies to 4D8 and 6D11 were purified on 4D8- and 6D11-Sepharose columns, respectively. The anti-ids specific for 4D8 and 6D11 were subsequently separated from the anti-isotypic and anti-allotypic antibodies by multiple passages through the respective isotype-matched MAb-Sepharose columns [MM 105 (IgG2b) for the anti-4D8-ids and MM 605 (IgG2a) for the anti-6Dl 1-ids,
respectively). The specificity of the different anti-id preparations to their respective Ab-1 idiotype was tested by a standard indirect solid-phase radioimmunoassay (RIA, 28). In this assay, the binding of the Abl 4D8and6Dll, or the isotype-matched controls, to the anti-ids adsorbed to microtiter wells was detected by the addition of 125I-labeled sheep antibodies to mouse Ig. Groups of four female BALB/c mice were primed intraperitoneally (i-p) with 50 u.g of alum-precipitated anti-ids emulsified 1:1 in CFA. The mice were given 10 biweekly booster injections, i-p with 25 p-g of alum-precipitated anti-ids in ICFA. Control groups of mice of the same age and sex were injected similarly with normal bovine Ig previously shown to be free of anti-BVDV antibodies. All animals were bled 10 days after the last booster injection. For the culture and propagation of BVDV, bovine turbinate (BT) cells (ATCC, Rockville, MD) were determined to be free of noncytopathic strains of BVDV by immunofluorescence assays using fluorescein isothiocyanate (FITC)-labeled bovine anti-BVDV antibodies [National Veterinary Service Laboratories (NVSL), Ames, I A]. The virus was propagated in BT cells grown in minimum essential medium (MEM) with 10% heat-inactivated horse serum (HS) and 100 pg/ml gentamicin. The cytopathic strain (NADL) of BVDV (NVSL, Ames, IA) was biologically cloned by plaque picking (27) three times prior to use. The noncytopathic strain (NY-1) of BVDV (NVSL, Ames, IA) was cloned twice prior to use by the limiting dilution technique. An indirect immunofluorescence assay (IFA) in microwell plates (22) was used to detect antibodies specific for BVDV.
Virus-neutralizing activity of the antibodies were tested by a standard plaque reduction assay using BT cells described by Cooper (2). The neutralization titer was expressed as the highest dilution of antibody that neutralized 50% of the input virus. as
258
BVDV-SPECIFIC NEUTRALIZING ANTIBODIES Immunization of the calf with 4D8 and 6D11 resulted in the production of anti-4D8 and -6D11 antibodies. Following the fourth booster injection, the titer of these antibodies reached 1:3200 as determined by an RIA (results not shown). The anti-ids isolated from the anti-4D8 and -6D11 antibody preparations were tested for their specificity to their respective Abl by an indirect RIA. The anti-4D8-ids (Fig. 1 A) bound specifically to their Abl (4D8), but not to the isotype-matched (IgG2b) controls (MM 105, specific for BHV-1 glycoprotein gl; MM 603 and MM 604, specific for the Pasteurella haemolytica leukotoxin). Similarly, the anti-6Dl 1-ids (Fig. 1B) reacted specifically with their Abl (6D11 ), but not with the isotype-matched (IgG2a) controls (MM 113, specific for BHV-1 glycoprotein gIV; 5A5, specific for TGEV; 1 El 1, specific for BHV-1 glycoprotein gl; and MM 605, specific for the P. haemolytica leukotoxin). These results indicated that the bovine anti-id preparations were specific for their respective idiotypes. The lack of binding of the anti-ids to the isotype-matched controls also indicated the absence of bovine antibodies to the isotypic and allotypic determinants of Abl in the anti-id preparations. Furthermore, in a plaque reduction assay, the bovine anti-4D8-ids and anti-6D 11-ids inhibited the neutralization of BVDV by the Abl 4D8and6Dl 1, respectively, in a concentration-dependent manner (Fig. 2). The results of the indirect RIA and plaque reduction assay suggested that the bovine anti-ids recognized idiotopes within or close to the paratope of the Ab 1. In order to determine whether these anti-ids could mimic
4D8
MM 105
MM 603
MM 604
Monoclonal Antibodies
CO
rr
