Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen

I'eterinary Microbiology, 29 ( 1991 ) 201-212 Elsevier Science Publishers B.V., Amsterdam

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Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen W. Xue a, D.J. Orten a, O.Y. Abdelmagid a, M. Rider~, F. Blecha b, and H.C. Minocha a'~ aDepartments of Laboratoo, Medicine, and bAnatomy & Physiology, Kansas State University. Manhattan, KS 66506, USA (Accepted 17 May, 1991 )

ABSTRACT Xue, W., Omen, D.J., Abdelmagid, O.Y., Rider, M., Blecha, F. and Minocha, H.C., 199 I. Anti-idiotypic antibodies mimic bovine viral diarrhea virus antigen. Vet. Microbiol. 29:201-212. Polyclonal rabbit anti-idiotypic antibodies (anti-ids) against two neutralizing murine monoclonal antibodies (mAbs) specific to a bovine viral diarrhea virus (BVDV)glycoprotein, 53 kDa, were produced, purified, and characterized. Each anti-id inhibited the binding of its respective mAb to BVDV antigen in a competitive ELISA and blocked the immunoprecipitation of the 53 kDa protein by the mAb. The anti-ids also inhibited the virus-neutralizing activity of their homologous mAbs. These results suggest that the anti-ids bear an internal image of a BVDV antigen and mimic neutralizing epitopes on the 53 kDa protein. Treatment of MDBK cells with the anti-ids inhibited BVDV infection, indicating that they block a cellular component, such as a virus receptor, required for virus ,~UaVIVtLUII or ~ntry. ,| _, ,k,:,L, 'u, ~, .' -u- -, -, u --~" , .t,,: Ik_ .! .u . , u , unB u u ~ ,,,~o A L a n d lack O l inhibition O I the heterologous mAb indicate that the anti-ids are specific for the unique antigen-binding sites on the mAbs.

INTRODUCTION

Bovine viral diarrhea virus (BVDV), an enveloped positive-strand RNA virus, is a common pathogen of cattle and causes a wide range of clinical syndromes, depending on the age and immune status of the animal at the time of infection (Howard, 1969; Woods et al., 1973; Potgieter et al., 1984; McClurkin et al., 1985; Baker, 1987; Radostits and Littlejohns, 1988 ). Among the viral proteins that are synthesized by BVDV in cell cultures, the 53 kilodalton (kDa) glycoprotein is thought to be the most important. This protein is involved in virus neutralization (Magar et al., 1988; Xue et al., 1990) and is a potential candidate for a vaccine (Bolin, 1988 ). The majority of neutralizing mAbs produced to BVDV are against the 53 kDa protein (Bolin et al., IAuthor for correspondence.

0378-1135/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.

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1988; Donis et al., 1988; Xue et al., 1990). In healthy calves that were vaccinated with a modified-live BVDV vaccine, the early neutralizing and precipitating antibodies were specific to the 53 kDa protein (Bolin and Ridpath, 1989). In our previous work, we found that two of a group of neutralizing mAbs to the 53 kDa protein, mAbs D89 and E 15, neutralized about 90% of BVDV field strains (Xue et al., 1990). Proteins that mimic viral antigens, i.e., anti-ids, have been used in a number of systems to analyze different aspects of virogenesis (Keay et al., 1988; Fung et al., 1990) and their potential as vaccine candidates is being evaluated (Gaulton et al., 1986; Kennedy et al., 1986a; Finberg and Ertl, 1987; Ertl, 1989). An anti-id by definition binds to an idiotype, i.e., the three-dimensional binding site composed of the variable regions of both the heavy and light chains of an Ig molecule (Kennedy et al., 1986b). Anti-ids have been used to induce a specific antibody against the original antigen (Gell and Moss, 1985; Garmendia et al., 1989; Grieder and Schultz, 1990 ) and also to identify the cellular receptor sites for viruses and other ligands (Kauffman et al., 1983; Co et al., 1985; Marriott et al., 1987; Keay et al., 1989). The purpose of the study reported here was to characterize polyclonal anti-ids to BVDV-neutralizing mAbs D89 and E 15 in vitro. MATERIALS AND METHODS

Virus and cell cultures The NADL strain of BVDV was originally obtained from the National Veterinary Service Laboratory (Ames, Iowa) and was then cultured in our laboratory. The virus was propagated in Madin-Darby bovine kidney (MDBK) cells in minimal essential medium (MEM, GIBCO Laboratories, Grand Island, NY) with 5% fetal bovine serum (FBS) or serum replacer (JRH Biosciences, Lenexa, KS). All cells, media, FBS, and serum plus were BVDV- and antibody-free, as determined by immunofluorescence and neutralization tests in our laboratory prior to use. Characterizations of mAbs The production and characterization of mAbs against BVD virus was described previously (Xue et al., 1990). The mAbs D89 and El5 were used in this study to generate anti-ids. Briefly, these two mAbs are specific to two related neutralizing epitopes on the 53 kDa protein, as shown by their 70% cross inhibition in a competitive radioimmunoassay. Both are of the subisotype IgG2a. These two mAbs were selected because they neutralized about 90% of BVDV field strains. The mAb C 17 was used as a control mAb to test the specificity of anti-ids. C 17 is also directed against the 53 kDa protein but to a different epitope than D89 and E 15.

CHARACTERIZATION OF ANTi-ID TO BVDV

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Production of anti-ids Groups of two New Zealand white rabbits were injected with 300/zg of protein A-sepharose purified mAb (D89 or E 15 ), normal mouse lgG2a (clarified ascites, Sigma Chemical Co., St. Louis, MO), or PBS. Each sample was emulsified in Freund's complete adjuvant and injected subcutaneously at multiple sites along the animal's back. Second and third injections were given intramuscularly at biweekly intervals, usirg 300/tg of mAb, normal mouse lgG2a or PBS in incomplete Freund's adjuvant. Anti-mAb sera were collected periodically throughout the immunization schedule and analyzed for anti-idiotype activity by ELISA using purified mAb- and normal mouse IgG2a-coated plates (Orten et al., 1988 ). The sera containing the highest idiotype-specific activity were pooled and used for purification of anti-ids.

Purification of the anti-ids The specific anti-ids were purified by sequential immunoaffinity chromotography, as described by Marriott et al ( 1987 ). First, the isotype- and allotype-specific antibodies in the sera were removed by repeated adsorption (in 10 mM Tris-HCl, pH 7.4) with BALB/c mouse IgG coupled to Affi-gel 10 (3io-Rad Laboratories, Richmond, CA). Then, the anti-ids were isolated from the column effluent by adsorption onto an Affi-gel Hz column (Bio-Rad) coupled to mAbs D89 and E 15, respectively. The purified anti-ids were eluted with 0.1 M glycine, pH 2.5 and immediately neutralized with 1 M NaHCO3, pH 8.0. Buffer exchange into PBS and concentration of anti-ids were accomplished by ultrafiltration using a PM-30 membrane (Amicon Corp., Lexington, MA), and protein content was determined with a protein assay kit (BioRad). Finally, the specificity of the anti-ids was analyzed by ELISA°

Anti-id inhibition ELISA Tenfold dilutions of anti-ids or control sera were incubated with equal amounts of a predetermined optimum dilution of each mAb and incubated for 1 h at 37°C. The mixture was added to plates coated with Nocidet P-40 extracts of BVDV-infected cells (Xue et al., 1990) in an ELISA as previously described (Orten et al., 1988). The bound mAb was detected with peroxidase-conjugated goat anti-mouse lgGs (Hyclone Laboratories, Logan, Utah ). Results were expressed as the percentage inhibition of mAb binding by antiids, according to the following formula: 100 × ( A c - As )/Ac, where Ac is the absorbance of the mAb control at 405 nm, and As is the absorbance of the sample mixture.

Inhibition of immunoprecipitation by anti-ids Five/zg of each anti-id was incubated with 10/tl of each mAb ascitic fluid or PBS for 1 h at 37°C in a microfuge tube. Then 200 Ill of 35S-labeled virusinfected cell lysate (Xue et al., 1990) was added to each tube, and the mix-

CHARACTERIZATION OF ANTi-ID TO BVDV

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RESULTS

Production and purification of anti-ids Sera exhibiting high ELISA titers were pooled and used for purification of anti-ids. Anti-D89 sera had high titers on both D89-coated and mouse IgG2acoated plates (Fig. 1 ). After purification by sequential immunoaffinity chromatography, the anti-id only reacted with D89 but not normal mouse IgG2a. The purified anti-El5 anti-id exhibited a similar anti-idiotype specificity. These results dearly indicate that idiotype-speeifie antibodies were induced

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by injection of mAbs D89 and El 5 and that these anti-ids were purified from the whole serum. Inhibition tf mnAbbinding abihty by anti-ids

Inhibitory ELISA and immunoprecipitation tests were performed to demonstrate that the anti-ids would recognize and bind the antigen-binding sites of the mAbs. Figure 2 shows results of the inhibition ELISA. The anti-D89 and anti-E 15 both inhibited the binding of their respective mAb to BVDV antigens. In addition, anti-D89 inhibited E 15 and anti-El 5 inhibited D89, although to a lesser degree compared with the inhibition of their respective mAbs. This finding is another indication of the relatedness of the epitopes for which D89 and E 15 are specific (Xue et al., 1990). Conversely, when mAb C 17, which binds to an unrelated epitope on the 53 kDa glycoprotein (Xue et al., 1990)) was used in the assay, no inhibition by anti-D89 or anti-E 15 was found. As hypothesized, the anti-mouse IgG2a and normal rabbit igG did not inhibit mAb binding to virus antigens. Similar results were obtained in the inhibitory immunoprecipitation (Fig. 3). The ability of D89 and El 5 to precipitate the 53 kDa protein was inhib-

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BVDV (Xue et al., 1990). However, preincubation with anti-D89 reduced the mAb neutralizing ability as much as 83% and 73% for D89 and ElS, respectively (Fig. 4); anti-El 5 inhibited 80% o f E l 5 and 66% of D89 neutralization. C 17 neutralization was not inhibited by either anti-id.

Anti-ids inhibit virus infection Anti-ids have been used in the study of cell components involved in virus attachment and entry (Williams et al., 1988; Strosberg, 1989). For this purpose, anti-ids must be able to block virus infection in susceptible cells. In our study, anti-id treatment and virus infection were performed at 4 °C to prevent entry, of anti-id and degradation by the cells. The infected cells were detected by immunofluorescence. Results showed that treatment of MDBK cells with anti-ids inhibited virus infection by 25% to 30%, compared to less than 5% inhibition by normal rabbit IgG and anti-mouse IgG2a (Fig. 5 ). This inhibition was concentration dependent, but beyond an optimum concentration of anti-ids, the inhibition was reduced. The optimum concentration in this experiment was 5 to 10 llg anti-ids per well. DISCUSSION

We have described the production and characterization of polyclonal antiids to BVDV. BVDV has been grouped under the genus Pestivirus in the family Togaviridae (Westaway et al., 1985), although recent studies suggested that it should be a member of the genus Flavivirus in the family Flaviviridae (Renard et al., 1987; Collett et al., 1988). It has been suggested that final classification of BVDV should be postponed until more data have been ob-

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tained (Weiland et al., 1990). Studies on BVDV using anti-ids may provide useful data for its classification. The anti-ids to mAbs D89 and El 5 produced in this study bear internal images of the 53 kDa glycoprotein of BVDV. Both anti-ids competed with the original virus antigens for binding to D89 and El5 (Figs. 2 and 3) in both ELISA and immunoprecipitation. This competitive binding to original antigen is one of the criteria used to differentiate between internal-image and non-internal-image anti-ids (Grieder and Schultz, 1990). Additionally, mAbs D89 and E 15 are against two related epitopes of the 53 kDa protein, and their anti-ids inhibited the neutralizing ability of both mAbs. This indicates that the anti-ids mimic the neutralizing epitope of the viral protein. Another criterion for identifying an internal-image anti-id is its ability to induce an idiotypic immune response that recognizes the original antigen (Grieder and Schu!tz, 1990). The study of the response to BVDV stimulated by anti-ids is in progress in our laboratory. Anti-ids have been succesfully used to specifically prime or stimulate the immune response in other virus systems (Reagan, 1985; Keay et al., 1988; Hariharan et al., 1989). As mentioned above, our mAbs D89 and E 15 are specific for related epitopes on the 53 kDa protein. The anti-ids induced in this study against these two mAbs also share a common structural element. If the inhibition of D89 by anti-D89 is considered to be 100%, then the inhibition of El 5 by anti-D89 is about 80% in both ELISA and immunoprecipitation. Anti-El 5 demonstrated similar degrees of inhibition against the two mAbs. Furthermore, the anti-ids did not inhibit the heterologous mAb C 17 because it is specific for an epitope unrelated to the epitopes for D89 and E 15 (Xue et al., 1990). These findings sugg~.st that the anti-ids have a very high specificity. I indprrn~nn ( ! 973 ) a , , o , . . q h , . a , h , , ,.,.,.,,-....,,, , h ~ , , ~ , . . ~ : ~ a : ~ o:,~ ~ ¢ ~ ,~,.t ~----ibody (Abl) is complementary to the epitope of its antigen (Epl) and, hence, stimulates the binding properties of the receptor for Epl. In turn, an anti-id (Ab2) induced by Abl could be anticipated to have a structure analogous to that of Epl. Numerous studies have been conducted using this concept as the working hypothesis. The anti-ids produced in this study almost completely inhibited mAb neutralization (Fig. 4) and inhibited BVDV infection by 25 to 30% (Fig. 5). This indicates that the anti-ids might bind to cell receptors and inhibit virus attachment. It is important to note that the binding of antiids to cell receptors (or the competition between virus and anti-ids for receptors) varies widely among different virus-cell systems. In a polyomavirus system, anti-id IgG inhibited about 20% of virus attachment when used simultaneously with the virus and almost completely inhibited virus infection when used to pretreat mouse kidney cells (Marriott et al,. 1987 ). However, Grieder and Schultz (1990) found no difference in bluetongue virus infectivity when the monolayer was preincubated with anti-id mAb as compared to preincubation with control antibody. Other work in our laboratory showed that anti•

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ids inhibited bovine herpesvirus-I infection of MDBK cell monolayers by about 30%. In a recent study on rubella virus, another member of famiiy Togaviridae, the anti-ids that were used did not recognize the putative virus receptor on Vero cells (Nath et al., 1989 ). One question arising from this study is why the anti-ids totally inhibit the ability of the parent mAbs to neutralize BVDV, but only partially inhibit BVDV from infecting MDBK cells. One possible explanation for this discrepancy is that the binding affinity of BVDV to the MDBK cell receptors is much higher than the binding affinity of the anti-ids. Another possibility is that there are actually multiple receptors involved in the binding of BVDV to MDBK cells. Recently, Moennig et al. ( 1988 ) described a m ~,b against a bovine cell surface protein which interfered specifically with the infectivity of BVDV and suggested that BVDV uses a single receptor for infection of bovine cells. However, further study showed that this mAb could not completely inhibit BVDV infection and infected cells were detectable in monolayers pretreated with the mAb (Collett et al, 1989). Therefore, it remains unclear whether there are multiple receptors or only a single receptor for BVDV on bovine cells. Additional work is being done in our laboratory using these antiids in an attempt to answer this question. ACKNOWLEDGEMENTS

This work was supported by U.S. Department of Agriculture Special Research Grant #87CRSR-23195. Published as contribution ¢~91-325-J, Kansas Agricultural Experimental Station.

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