Immunology 1990 70 284-289
Anti-idiotypic antibodies induce neutralizing antibodies to bovine herpesvirus 1 S. SRIKUMARAN, D. V. ONISK, M. V. BORCA, C. NATARAJ & T. J. ZAMB Department of Veterinary Science, University of Nebraska, Lincoln, Nebraska, U.S.A.
Acceptedfor publication 21 March 1990
SUMMARY A neutralizing murine monoclonal antibody (mAb) of the IgG2a isotype (MM-1 13), specific for bovine herpesvirus I (BHV- 1) glycoprotein gIV, was used to develop anti-idiotypic antibodies (antiId) in a calf. The bovine anti-Id were isolated from the serum of the immunized calf by affinity chromatography on an MM-I 13-Sepharose column, followed by repeated adsorption on a murine IgG2a column. The anti-Id thus obtained specifically reacted with MM-113, but not with isotypematched controls. They also inhibited the binding of MM-113 to BHV-1 in a concentrationdependent manner. Mice immunized with the anti-Id produced neutralizing antibodies to BHV-1. The anti-Id bound to cells permissive to BHV- 1 in a cell-binding radioimmunoassay (RIA).
(Babiuk et al., 1987). Alternate immunization strategies that do not involve live viruses, therefore, should be examined. Viral subunits (Babiuk et al., 1987) and anti-idiotypic antibodies (anti-Id) may emerge as possible alternatives. The potential of anti-Id as immunogens has been examined in a number of viral systems. Anti-Id may induce or suppress specific immune responses (Finberg & Ertl, 1987; Dalgleish & Kennedy, 1988; Hiernaux, 1988). The parameters influencing the outcome of anti-Id immunizations are not clear. The dose, isotype and route of injection of anti-Id may influence the response of the injected animals (Eichmann, 1974; Eichmann &
INTRODUCTION
Bovine herpesvirus 1 (BHV-l) is an alpha herpesvirus and is an important pathogen of cattle. It causes severe respiratory infections as well as vulvovaginitis, abortion, encephalitis and generalized systemic infections (Gibbs & Rweyemamu, 1977). The BHV-1 infection of the respiratory tract can predispose animals to secondary bacterial infection, most commonly by Pasteurella haemolytica, leading to pneumonia and death (Yates, Babiuk & Jerico, 1983). A major problem encountered by vaccination programmes against BHV- 1 (and most other herpes viruses) is that of latent and persistent infections (Rouse & Babiuk, 1978). Not only the wild-type virus, but also the modified live virus (MLV) vaccine strains can establish latent infections with subsequent reactivation and shedding of the virus (Pastoret et al., 1980; Nettleton & Sharp, 1980). A recent report also suggested a possible role for the vaccinal virus in some outbreaks of the disease (Whetstone, Wheeler & Reed, 1986). In light of these observations, the safety of the currently used MLV vaccines has been questioned
Rajewsky, 1975). In the majority of the viral systems studied, the anti-Id elicited humoral and/or cell-mediated immune responses against the respective viruses (Reagan et al., 1983; Sharpe et al., 1984; Gell & Moss, 1985; Uytdehaag & Osterhaus, 1985; Kennedy et al., 1986; Hariharan et al., 1989), and in some cases provided protection against subsequent challenge with virulent viruses (Reagan, 1985; Gaulton et al., 1986; Garmendia, Morgan & Baxt, 1989). In a few cases, the anti-Id were also useful as probes for the viral receptors on permissive cell surfaces (Noseworthy et al., 1983; Co et al., 1985; Marriott, Roeder & Consigli, 1987). Although anti-Id are unlikely to find application in those systems where conventional vaccines have been successful, they may be promising alternatives in those
Abbreviations: ABI, antibody 1; Ab2, antibody 2; Ab3, antibody 3; anti-Id, anti-idiotypic antibodies; AZ, sodium azide; BHV-1, bovine herpes virus 1; BSA, bovine serum albumin; CFA, complete Freund's adjuvant; ELISA, enzyme-linked immunosorbent assay; HSV- I, herpes simplex virus type l; HSV-2, herpes simplex virus type 2; IFA, incomplete Freund's adjuvant; i.m., intramuscularly; i.p., intraperitoneally; mAb, monoclonal antibody; MDBK, Madin-Darby bovine kidney; M LV, modified live virus; PBS, phosphate-buffered saline; RIA,
systems where the use of conventional vaccines are ineffective or problematic. In this study, the induction of neutralizing antibodies to BHV-I by anti-Id is described.
MATERIALS AND METHODS
radioimmunoassay; S/N, signal to noise ratio. Correspondence: Dr S. Srikumaran, Dept. of Veterinary Science, University of Nebraska, Lincoln, NE 68583-0905, U.S.A.
Virus The Cooper strain of BHV- 1 was used in this study. The virus was propagated in Madin-Darby bovine kidney (MDBK) cells
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and purified on potassium tartrate gradients according to the procedures of Misra, Blumenthal & Babiuk (1981).
of incubation, the plates were washed three times, and the wells were removed and counted for bound radioactivity.
Monoclonal antibodies Female BALB/c mice, 8-10 weeks of age, were injected intraperitoneally (i.p.) with 100 pl of a commercial MLV vaccine against BHV-1 (Norden Laboratories Inc., Lincoln, NE). A booster injection of 50 yl of the same vaccine was given i.p. 3 weeks later. Three days following the booster injection, the spleen cells from the immunized mice were fused with the murine cell line SP-2/0. The procedures of Kohler & Milstein (1975) for cell fusion, cloning and stabilization of the hybridomas were adopted with slight modifications. Positive hybridomas were initially selected by an indirect solid-phase radioimmunoassay (RIA) described below. The BHV- 1 glycoprotein specificity of the monoclonal antibodies (mAb) was determined by immunoprecipitation of [3H]glucosamine-labelled BHV-1 proteins followed by SDS-PAGE and autoradiography, as described by Van Drunen Littel-Van Den Hurk et al. (1984). The immunoglobulin (Ig) isotypes of the BHV- 1-specific mAb were determined by a standard enzymelinked immunosorbent assay (ELISA) on BHV- 1-coated microtitre plates using alkaline phosphatase-labelled goat antimurine Ig isotype-specific antibodies (Southern Biotechnology, Birmingham, AL). Three other hybridomas were used as the source of BALB/c mouse IgG2a antibodies with irrelevant specificities. One of these hybridomas, which secretes a mAb (MM-409) specific for porcine rotavirus, was developed at the University of Nebraska. The other hybridoma, which secretes a mAb (1 El 1) specific for the gI glycoprotein of BHV-1 (Van Drunen Littel-Van Den Hurk et al., 1984), was kindly provided by Dr S. Van Drunen Littel-Van Den Hurk of the Veterinary Infectious Diseases Organization, Saskatoon, Canada. The third one, which secretes a mAb (5A5) to the E2 glycoprotein of porcine coronavirus (Hariharan et al., 1989), was the gift of Dr P. Gaugh of the Iowa State University.
Virus neutralization assay BHV-1-neutralizing activity of the hybridoma culture fluids, ascites fluids or the plasma from mice immunized with the antiId was tested by a standard plaque reduction assay as described by Babiuk, Wardley & Rouse (1975). Appropriate negative controls were used as in the RIA described above. The neutralization titre was expressed as the highest dilution of antibody that neutralized 50% of the input virus.
Detection of anti-BHV-i antibodies by RIA A standard indirect solid-phase RIA (Rosenthal, Hayashi & Notkious, 1973) was used to detect anti-BHV-1 activity of the mAb or plasma from mice immunized with the anti-Id. Polyvinyl chloride microtitre plate wells were coated with purified BHV-1 protein in carbonate buffer, pH 9-6 (0-25 pg/ 20 pl/well), and incubated at 370 overnight. The unbound sites in the wells were then blocked by the addition of 2% bovine serum albumin (BSA) in borate buffer, pH 9-6 (50 p1/well). After 1 hr of incubation at room temperature in a humid chamber, the plates were washed three times with phosphate-buffered saline (PBS) containing 01% BSA and 0-01% sodium azide (PBSBSA-AZ). Undiluted hybridoma culture fluids, serially diluted ascites fluids or plasma from mice immunized with the anti-Id or normal bovine Ig, were added to the virus-coated wells (20 pl/ well). Culture fluid from the parent cell line SP-2/0, ascites fluid from mice injected with the SP-2/0 or plasma from mice before immunization with the anti-Id or normal bovine 1g, respectively, were used as negative controls. After 1 hr of incubation at room temperature, the wells were washed three times with PBS-BSAAZ and ['251]-labelled sheep F (ab')2 anti-mouse Ig (specific activity 10 pCi/pg, 20,000 c.p.m./20 pl/well; New England Nuclear Co., Boston, MA was added. Following another hour
Preparation of mAb for the production of anti-Id Ascites tumours of hybridomas were developed in mice according to standard procedures. Ascites fluids were precipitated with 50% ammonium sulphate and dialysed in PBS. The hybridomas also were grown in serum-free medium (KC2000; Hazleton, Biologics Inc., Lenexa, KS) and the mAb in the culture fluids were affinity-purified on a protein A-Sepharose CL-4B column (Pharmacia Fine Chemicals Inc., Piscataway, NJ). Immunization of calf A male 6-month-old Hereford calf was injected intramuscularly (i.m.) with 1 mg of protein A-purified MM-1 13 emulsified 1: 1 in complete Freund's adjuvant (CFA). Booster injections of 500 pg of MM-1 13 in incomplete Freund's adjuvant (IFA) were given i~m. on Days 21, 49 and 77 post-priming. The calf was bled on Days 7, 14 and 21 following each booster injection and the serum was harvested.
Preparation of anti-Id Ammonium sulphate-precipitated MM-113 and MM-409 (18 mg of each) were individually coupled to CNBr-activated Sepharose 4B (3 g) according to the manufacturer's instructions (Pharmacia Fine Chemicals Inc.). Bovine anti-Id to MM-1 13 were isolated by standard affinity chromatography procedures. First, bovine antibodies to MM- 113 (anti-idiotypic, anti-allotypic and anti-isotypic antibodies) were isolated from the serum of the immunized calf using the MM-l 13-Sepharose column. Serum (20 ml) from the immunized calf was passed over the MM-l 13-Sepharose column. Unbound protein was washed away with PBS. The column-bound antibody was eluted with 0-2 M glycine-HCl at pH 2 8 and immediately neutralized with 2 M Tris buffer at pH 9-6. The bovine antibodies to the isotypic and allotypic determinants of MM-1 13 present in this preparation were then removed by adsorption on the MM-409 Sepharose column (mouse IgG2a of a different idiotype). The adsorption procedure was repeated until no antibodies to the isotypic and allotypic determinants of MM- 113 were left in the preparation. The anti-MM-1 13 Id thus recovered were concentrated in Amicon stirred ultrafiltration cells (Amicon Corporation, Danvers, MA).
Characterization of the anti-Id The specificity of the anti-Id to the idiotype of MM-1 13 was tested by an indirect solid-phase RIA and an inhibition RIA (Rosenthal et al., 1973). Indirect solid-phase RIA Polyvinyl chloride 96-well microtitre plates were coated with affinity-purified bovine anti-Id (10 pg/ml in PBS, 20 p1/well) and
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incubated at room temperature in a humid chamber overnight. The unbound sites in the wells were then blocked by the addition of PBS-BSA (50 pl/well). After I hr of incubation in a humid chamber at room temperature, the plates were washed three times with PBS-BSA-AZ. The mAb MM- 113, lEl 1, MM-170, MM-409 or 5A5, in the form of culture fluids, or the culture fluid from the parent cell line SP-2/0 (20 pl/well) were then added to the wells. The plates were washed after an hour of incubation and ['251]-labelled sheep F(ab')2 anti-mouse Ig (specific activity 10 pCi/pg, 20,000 c.p.m./20 pl/well) was added to the wells. Following another hour of incubation, the plates were washed, the wells removed and counted for bound radioactivity.
Inhibition assay The inhibition of binding of MM-1 13 to BHV-1 protein by the anti-Id was determined by a modified RIA. Fifty-microlitre samples of undiluted MM-1 13 culture fluid were mixed with equal volumes of the anti-Id or normal bovine Ig (diluted in PBS-BSA-AZ) so that the final concentration of the anti-Id or bovine Ig ranged from 4 to 512 pg/ml. After incubation at room temperature for 1 hr, duplicate samples of 30 pl of the mixture were transferred to the wells of 96-well microtitre plates coated with BHV-1 protein (0-25 pg/well). Upon incubation at room temperature for 1 hr, the plates were washed three times with PBS-BSA-AZ and ['25I]-labelled sheep F(ab')2 anti-mouse Ig (specific activity 10 pCi/pg, 20,000 c.p.m./20 pl/well) was added. After another hour of incubation, the wells were washed, removed from the plate and counted for bound radioactivity. Immunization of mice A group of four female BALB/c mice was primed i.p. with 50 pg of anti-Id precipitated on alum and emulsified 1: 1 in CFA. These mice were given five bi-weekly booster injections i.p. with 25 pg of alum-precipitated anti-Id in IFA. A control group of four mice of the same age and sex was injected similarly with normal bovine Ig. Beginning with the third booster injection, all animals were bled 10 days after the booster injections.
Detection of antibodies to BHV-J in mice immunized with the anti-Id Antibodies to BHV-1 were detected by the standard indirect solid-phase RIA and the neutralization test described earlier. Detection of binding of the anti-Id to BHV-J-permissive cells A cell-binding RIA (Goldsby, Osborne & Engleman, 1979) was used to detect the binding of the anti-Id to cells permissive to BHV-1 infection. Fifty microlitres of the anti-Id or normal bovine Ig (at a concentration of 15, 30 and 60 pg/ml in PBS-BSA-AZ) were added to the wells of polyvinyl chloride microtitre plates containing 5 x 105 MDBK cells in 50 pl of PBS-BSA-AZ. After 1 hr of incubation in a humid chamber at room temperature, the plates were centrifuged at 800 g for 2 min. The supernatant fluid was removed by carefully flicking the plates. The cell pellets in the wells were resuspended by shaking the plates in a microshaker (Dynatech Inc., Alexandria, VA). The unbound antibodies were washed away by three washing cycles. Each cycle consisted of addition of washing buffer (200 pl of PBS-BSA-AZ per well), centrifugation of the plate (800 g for 2 min), removal of the supernatant fluid and resuspension of the cell pellets. At the end of the third washing cycle, ['251]-labelled murine mAb to bovine Ig (Srikumaran et al.,
Table 1. Characteristics of the anti-BHV-l mAb
Reactivity in Designation
MM-I 13 MM-155 MM-170 MM-171 MM-105 MM-130 MM-160
RIA
+ + + + + + +
Neutralizing activity
Glycoprotein specificity
+ +
gIV (gD) gIV (gD) gll (gC) gill (gC) gI (gB)
-
ND ND
Isotype IgG2a IgG2a IgG2a IgGI IgG2b IgGI
IgM
ND, not determined.
1987) was added to the wells (specific activity 10 MCi/pg, 20,000 c.p.m./20 pl/well). After 1 hr incubation and three washing cycles, a drop of 2% agarose was added to the wells and allowed to solidify. The wells were then cut and counted for bound radioactivity. RESULTS Characteristics of mAb Seven hybridomas secreting mAb to BHV-1 were stabilized (Table 1). Two of these secreted mAb (MM-1 13 and MM-155) that neutralized the virus. Both of these mAb immunoprecipitated the glycoprotein gIV. Competitive RIA showed that these two mAb recognized the same epitope or closely juxtaposed epitopes (results not shown) on the glycoprotein gIV. The mAb MM- 113 was selected for the development of anti-idiotypes.
Development and characterization of the anti-Id Immunization of the calf with MM- 113 resulted in the production of anti-MM-1 13 antibodies in high titre. Following the second booster injection, the titre reached 1: 128,000 as determined by RIA. Serum collected on Day 14 following the third booster injection gave the highest yield of bovine anti-MM-113 antibodies (approximately 0 5 mg per ml of serum). In the indirect RIA used to determine the specificity of the anti-idiotypes, MM- 113 bound to the anti-Id (Fig. 1). The mAb l E 11 (specific for gI of BHV- 1), MM- 170 (specific for gIII of BHV-1), MM-409 (specific for porcine rotavirus) and 5A5 (specific for porcine coronavirus) did not bind to the anti-Id. These results indicated that the anti-Id were specific for the idiotype of MM-1 13. The lack of binding of the anti-Id to El 1, MM-170, MM-409 or 5A5 (all are BALB/c IgG2a mAb) also confirmed that there were no bovine antibodies to the isotypic and allotypic determinants of IgG2a left in the anti-Id preparation. In the inhibition assay, the anti-Id inhibited the binding of MM-1 13 to BHV-1 in a concentration-dependent manner, while normal bovine Ig did not inhibit the binding of MM-1 13 to BHV-1 significantly (Fig. 2). Production of antibodies to BHV-1 by mice immunized with the anti-Id The results of the inhibition assay suggested that the anti-Id recognized idiotopes within or close to the paratope of MM-
Anti-idiotypic antibodies
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12 10
E
8 0
on
6
=`
4
300
r
200 100 Ol
1000 4000 16,000 Reciprocal of plasma dilution
MM- ElI MM- MM- 5A5 113 170 409 Monoclonal antibodies
Figure 1. Specificity of the anti-Id to MM-1 13. The binding of MM-1 13 (Ab 1), and the isotype-matched controls I E I 1, MM- 170, MM-409 or 5A5 to the anti-Id, was determined by an indirect solid-phase RIA as described in the Materials and Methods section. The signal to noise ratio (S/N) was calculated by dividing the c.p.m. given by the mAb in the form of culture fluids by the c.p.m. given by the culture fluid from the parent cell line SP-2/0.
113. To determine whether these anti-Id mimic the epitopes of BHV- 1 recognized by MM- 113, BALB/c mice were injected with the anti-Id or with normal bovine Ig. Anti-BHV- 1 antibody activity of the plasma from these mice was tested by an RIA and compared with that of plasma collected before injection. All the mice that received the anti-Id responded with the production of antibodies to BHV-1. The RIA titre (average of four mice) of these antibodies was 1: 1000 after the third booster injection (data not shown), which increased to 1:8000 after the fifth booster injection (Fig. 3). None of the mice in the control group showed any detectable anti-BHV- response. The induction of anti-BHV- antibodies in naive mice by the anti-Id confirmed the presence of internal image anti-Id in the polyclonal anti-Id preparation. The virus-neutralizing activity of plasma from mice injected with the anti-Id was determined by a plaque reduction assay. No neutralizing activity was detectable in the plasma after the third booster injection. After the fifth booster, however, the plasma
80F 70 _
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50
0
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500 400-
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Figure 3. Anti-BHV-l antibody response of BALB/c mice immunized with the anti-Id. Plasma from mice injected with the anti-Id (A) or normal bovine Ig (-) was serially diluted in PBS-BSA-AZ, from 1: 1000 to 1: 32,000, and tested for binding to BHV- I by an indirect solid-phase RIA. The results are presented as the mean of the c.p.m. given by the dilutions of plasma from mice (n = 4). The plasma from the mice before injection (0) was also tested similarly. Table 2. BHV-1-neutralizing activity of plasma from mice injected with the anti-Id Neutralization titre
Mouse no. Before injection After injection 1 2 3 4
1:3 1:3 1:3 1:3
1:16 1:48 1:6 1:12
from these mice neutralized BHV- 1, with titres ranging from 1: 6 to 1:48 (Table 2).
Binding of BHV-l-permissive cells by the anti-Id The induction of neutralizing antibodies to BHV- 1 by the antiId was interpreted to mean that the anti-Id mimicked (at least partially) the epitope recognized by MM-113. Since MM-113 (the antibody 1, Abl) recognized a neutralizing epitope on the glycoprotein gIV, some of the anti-Id mimicking that epitope may recognize the surface protein(s) recognized by the original epitope on the glycoprotein gIV. In the cell-binding RIA used to detect the binding to cell-surface proteins, the anti-Id bound to the MDBK cells strongly while the binding of the normal bovine Ig was at the background level (Fig. 4).
\__AI.i/
20
101l1 4 8
I1 64128256512
16 32 Conc. anti-Id (pug/ml)
Figure 2. Inhibition of binding of MM-1 13 to BHV-1 by the anti-Id. Undiluted MM-1 13 culture fluid was mixed with the anti-Id (A) or normal bovine Ig (-) at varying concentrations (4-512 pg/ml). Following incubation for 1 hr, the binding of MM- 1 13 to BHV- l was detected by an indirect solid-phase RIA, as described in the Materials and Methods.
DISCUSSION Previous studies have reported the feasibility of inducing immune responses to viral antigens by anti-Id (Reagan et al., 1983; Sharpe et al., 1984; Uytdehaag & Osterhaus, 1985; Kennedy et al., 1986; Hariharan et al., 1989; Reagan, 1985; Gaulton et al., 1986; Garmendia et al., 1989). The outcome of immunization with anti-Id against herpesviruses has been mixed. In the study by Kennedy et al. (1984), mice immunized with an anti-Id against herpes simplex type 2 (HSV-2) were more
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S. Srikumaran et al. 1400 1200 E CLL Q 1000
*0 800_
600 o 400
_200 30 g 60kgConc. anti-Id Figure 4. Binding of the anti-Id to cells permissive to BHV-1. The binding of the anti-Id (-) or normal bovine Ig (0) to MDBK cells was tested by a cell-binding RIA as described in the Materials and Methods. 0
15,Lx
susceptible to infection by HSV-2. Lathey, Martin & Rouse (1987) reported the induction of only mild antibody and cellular responses, and suppression of delayed type hypersensitivity responses against herpes simplex type 1 (HSV-1) following immunization with anti-Id. Gell & Moss (1985), however, generated dose-dependent delayed-type hypersensitivity responses against HSV-1 by prior administration of anti-Id. Reasons for the differences in the outcome of these anti-Id immunizations are not clear. The dose (Gel & Moss, 1985), isotype (Eichman, 1974), and route of injection of anti-Id (Lathey et al., 1987), however, have been thought to influence the outcome of anti-Id immunization. Although the development of anti-Id to BHV-1-specific mAb has been reported previously (Whetstone, Babiuk & Van Drunen Little-Van Den Hurk, 1988; Orten et al., 1988), induction of immune response to BHV-1 by anti-Id was not demonstrated in those studies. This is the first report of the induction of neutralizing antibodies to BHV-1 by anti-Id. The anti-Id (Ab2) developed in this study specifically reacted with the idiotype of MM-113 (Abl) and not with the idiotypes of other isotype-matched controls. The inhibition of binding of AbI to BHV-1 by the anti-Id in a concentration-dependent manner suggests that they may recognize idiotope/s within the paratope of the Abl. This inhibition also could be due to the induction of a conformational change in the Ab1 paratope or creation of a steric obstruction to Abl paratope by the anti-Id binding to the Abl. The induction of anti-BHV-1 antibodies (Ab3) by the anti-Id in naive mice, however, clearly indicates the internal image nature of these anti-Id. These anti-BHV- 1 antibodies (Ab3), like the Abl, neutralized BHV-1 in vitro. The titres of anti-BHV-1 antibodies (Ab3) induced by the anti-Id were not very high. The Ab3 was a polyclonal mixture of antibodies induced by a mixture of different anti-Id, each mimicking the paratope of AbI in a different manner. Some of them may be true internal image Ab2, whereas others may mimic idiotopes outside the paratope of Abl . The concentration of the internal image anti-Id, which determines the induction of Ab3 identical or similar to Abl, is unknown and it is very difficult to enrich the proportion of the same in a polyclonal anti-Id preparation. Nevertheless, the production of BHV-1neutralizing Ab3 indicates that there was true internal image
Ab2 in the anti-Id preparation and also points to the need for the development of monoclonal internal image anti-Id. The potential use of anti-Ids as immunogens against BHV- I may be questioned because of the need for repeated booster injections. The immunogenicity of anti-Id, however, may be enhanced by conjugation to carriers and by the use of different adjuvants. Further experiments are also needed to evaluate the influence of dose, Ig isotype and the route of anti-Id administration on the induction of anti-BHV-l antibody response. These studies, along with the development of monoclonal internal image anti-Id (which is underway in this laboratory), should enhance the potential of anti-Ids as immunogens against BHV- 1. Internal image anti-Id have also been used as probes to identify and isolate the receptors for some viruses (Noseworthy et al., 1983; Co et al., 1985; Marriott et al., 1987). The finding that the anti-Id developed in this study bind to BHV- I permissive cells is very encouraging. Further studies are in progress in this laboratory to determine whether the anti-Id bind to a BHV-I receptor protein on the permissive cells.
ACKNOWLEDGMENTS Published as Journal Series No. 9087, with approval of the University of Nebraska Agricultural Research Division. This research project was partially supported by the USDA Animal Health Grant No. 8834116-3639.
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Anti-idiotypic antibodies GOLDSBY R.A., OSBORNE B.A. & ENGLEMAN E.G. (1979) Identification, isolation and characterization of a human T-cell population by a monoclonal antibody. Curr. Microbiol. 3,141. HARIHARAN K., SRIKUMARAN S., MOXLEY R.A. & OSORIO F.A. (1989) Induction of neutralizing antibodies to transmissible gastroenteritis virus by anti-idiotypic antibodies. Viral. Immunol. 2, 133. HIERNAUX J.R. (1988) Idiotype vaccines and infectious diseases. Infect. Immun. 56, 1407. KENNEDY R.C., ALDER-STORTHZ K., BURNS J.W., HENKEL R.D. & DRESSMAN G.R. (1984) Anti-idiotypic modulation of herpes simplex virus infection leading to increased pathogenicity. J. Virol. 50, 951. KENNEDY R.C., EICHBERG J.W., LANFORD R.E. & DREESMAN G.R. (1986) Anti-idiotypic antibody vaccine for type B viral hepatitis in chimpanzees. Science, 232, 220. KOHLER G. & MILSTEIN C. (1975) Continuous cultures of fused cells secreting antibody of pre-defined specificity. Nature (Lond.), 256,495. LATHEY J.L., MARTIN S. & ROUSE B.T. (1987) Suppression of delayed type hypersensitivity to herpes simplex virus type 1 following immunization with anti-idiotypic antibody: an example of split tolerance. J. Gen. Virol. 68, 1093. MARRIOTT S.J., ROEDER D.J. & CONSIGLI R.A. (1987) Anti-idiotypic antibodies to a polyomavirus monoclonal antibody recognize cell surface components of mouse kidney cells and prevent polyomavirus infection. J. Virol. 61, 2747. MISRA V., BLUMENTHAL R.M. & BABIUK L.A. (1981) Proteins specified by bovine herpesvirus 1 (Infectious bovine rhinotracheitis virus). J. Virol. 40, 367. NETTLETON P.F. & SHARP J.M. (1980) Infectious bovine rhinotracheitis virus excretion after vaccination. Vet. Rec. 107, 379. NOSEWORTHY J.H., FIELDS B.N., DICHTER M.A., SOBOTKA C., PIZER E., PERRY L.L., NEPOM J.T. & GREENE M.I. (1983) Cell receptors for the mammalian reovirus. I. Syngeneic monoclonal anti-idiotypic antibody identifies a cell surface receptor for reovirus. J. Immunol. 131, 2533. ORTEN D.J., BLECHA F., MORRILL J.L. & MINOCHA H.C. (1988) Characterization of anti-idiotype reagents to bovine herpesvirus-l monoclonal antibody. Vet. Immunol. Immunopathol. 20, 1. PASTORET P.P., BABIUK L.A., MISRA V. & GRIEBEL P. (1980) Reactivation of temperature-sensitive and non temperature-sensitive infectious bovine rhinotracheitis virus with dexamethasone. Infect. Immun. 29, 483.
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REAGAN K.J. (1985) Modulation of immunity to rabies virus induced by anti-idiotypic antibodies. Curr. Topics. Microbiol. Immunol. 119, 15. REAGAN K.J., WUNNER W.H., WIKTOR T.J. & KoPROWSKI H. (1983) Anti-idiotypic antibodies induce neutralizing antibodies to rabies virus glycoprotein. J. Virol. 48, 660. ROSENTHAL J.D., HAYASHI K. & NOTKIous A.L. (1973) Comparison of direct and indirect micro-radioimmunoassays for the detection of viral antigens and animal antibody. Appl. Microbiol. 25, 567. RouSE B.T. & BABIUK L.A. (1978) Mechanisms of recovery from herpesvirus infection. A Review. Can. J. Comp. Med. 42,414. SHARPE A.H., GAULTON G.N., McDADE K.K., FIELDS B.B. & GREENE M.I. (1984) Syngeneic monoclonal anti-idiotype can induce cellular immunity to reovirus. J. exp. Med. 160, 1195. SRIKUMARAN S., GOLDSBY R.A., GUIDRY A.J., HAGUE B., ONISK D.V. & SRIKUMARAN P. (1987) Library of monoclonal bovine immunoglobulins and monoclonal antibodies to bovine immunoglobulins. Hybridoma, 6, 527. UYTDEHAAG F.G.C.M. & OSTERHAUS A.D.M.E. (1985) Induction of neutralizing antibody in mice against polio virus type II with monoclonal anti-idiotypic antibody. J. Immunol. 134, 1225. VAN DRUNEN LITTEL-VAN DEN HURK S., VAN DEN HURK J.V., GILCHRIST J.E., MISRA V. & BABIUK L.A. (1984) Interactions of monoclonal antibodies and bovine herpesvirus type I (BHV- I) proteins: characterization of their biochemical and immunological properties. Virology, 135, 466. WHETSTONE C.A., BABIUK L.A., VAN DRUNEN LITTLE-VAN DEN HURK S. (1988) The production and characterization of anti-idiotypic antibodies directed against a monoclonal anti-bovine herpesvirus I antibody. In: Anti-Idiotypes, Receptors, and Molecular Mimicry (eds D. S. Linthicum and N. R. Farid), p. 203. Springer-Verlag, New York Inc. WHETSTONE C.A., WHEELER J.G. & REED D.E. (1986) Investigation of possible vaccine-induced epizootics of infectious bovine rhinotracheitis, using restriction endonuclease analysis of viral DNA. Am. J. Vet. Res. 47, 1789. YATES W.D.G., BABIUK L.A. & JERICO K.W.F. (1983) Viral-bacterial pneumonia in calves: duration of the interaction between bovine herpesvirus 1 and Pasteurella haemolytica. Can. J. Comp. Med. 47, 257.
Anti-idiotypic antibodies induce neutralizing antibodies to bovine herpesvirus 1 S. SRIKUMARAN, D. V. ONISK, M. V. BORCA, C. NATARAJ & T. J. ZAMB Department of Veterinary Science, University of Nebraska, Lincoln, Nebraska, U.S.A.
Acceptedfor publication 21 March 1990
SUMMARY A neutralizing murine monoclonal antibody (mAb) of the IgG2a isotype (MM-1 13), specific for bovine herpesvirus I (BHV- 1) glycoprotein gIV, was used to develop anti-idiotypic antibodies (antiId) in a calf. The bovine anti-Id were isolated from the serum of the immunized calf by affinity chromatography on an MM-I 13-Sepharose column, followed by repeated adsorption on a murine IgG2a column. The anti-Id thus obtained specifically reacted with MM-113, but not with isotypematched controls. They also inhibited the binding of MM-113 to BHV-1 in a concentrationdependent manner. Mice immunized with the anti-Id produced neutralizing antibodies to BHV-1. The anti-Id bound to cells permissive to BHV- 1 in a cell-binding radioimmunoassay (RIA).
(Babiuk et al., 1987). Alternate immunization strategies that do not involve live viruses, therefore, should be examined. Viral subunits (Babiuk et al., 1987) and anti-idiotypic antibodies (anti-Id) may emerge as possible alternatives. The potential of anti-Id as immunogens has been examined in a number of viral systems. Anti-Id may induce or suppress specific immune responses (Finberg & Ertl, 1987; Dalgleish & Kennedy, 1988; Hiernaux, 1988). The parameters influencing the outcome of anti-Id immunizations are not clear. The dose, isotype and route of injection of anti-Id may influence the response of the injected animals (Eichmann, 1974; Eichmann &
INTRODUCTION
Bovine herpesvirus 1 (BHV-l) is an alpha herpesvirus and is an important pathogen of cattle. It causes severe respiratory infections as well as vulvovaginitis, abortion, encephalitis and generalized systemic infections (Gibbs & Rweyemamu, 1977). The BHV-1 infection of the respiratory tract can predispose animals to secondary bacterial infection, most commonly by Pasteurella haemolytica, leading to pneumonia and death (Yates, Babiuk & Jerico, 1983). A major problem encountered by vaccination programmes against BHV- 1 (and most other herpes viruses) is that of latent and persistent infections (Rouse & Babiuk, 1978). Not only the wild-type virus, but also the modified live virus (MLV) vaccine strains can establish latent infections with subsequent reactivation and shedding of the virus (Pastoret et al., 1980; Nettleton & Sharp, 1980). A recent report also suggested a possible role for the vaccinal virus in some outbreaks of the disease (Whetstone, Wheeler & Reed, 1986). In light of these observations, the safety of the currently used MLV vaccines has been questioned
Rajewsky, 1975). In the majority of the viral systems studied, the anti-Id elicited humoral and/or cell-mediated immune responses against the respective viruses (Reagan et al., 1983; Sharpe et al., 1984; Gell & Moss, 1985; Uytdehaag & Osterhaus, 1985; Kennedy et al., 1986; Hariharan et al., 1989), and in some cases provided protection against subsequent challenge with virulent viruses (Reagan, 1985; Gaulton et al., 1986; Garmendia, Morgan & Baxt, 1989). In a few cases, the anti-Id were also useful as probes for the viral receptors on permissive cell surfaces (Noseworthy et al., 1983; Co et al., 1985; Marriott, Roeder & Consigli, 1987). Although anti-Id are unlikely to find application in those systems where conventional vaccines have been successful, they may be promising alternatives in those
Abbreviations: ABI, antibody 1; Ab2, antibody 2; Ab3, antibody 3; anti-Id, anti-idiotypic antibodies; AZ, sodium azide; BHV-1, bovine herpes virus 1; BSA, bovine serum albumin; CFA, complete Freund's adjuvant; ELISA, enzyme-linked immunosorbent assay; HSV- I, herpes simplex virus type l; HSV-2, herpes simplex virus type 2; IFA, incomplete Freund's adjuvant; i.m., intramuscularly; i.p., intraperitoneally; mAb, monoclonal antibody; MDBK, Madin-Darby bovine kidney; M LV, modified live virus; PBS, phosphate-buffered saline; RIA,
systems where the use of conventional vaccines are ineffective or problematic. In this study, the induction of neutralizing antibodies to BHV-I by anti-Id is described.
MATERIALS AND METHODS
radioimmunoassay; S/N, signal to noise ratio. Correspondence: Dr S. Srikumaran, Dept. of Veterinary Science, University of Nebraska, Lincoln, NE 68583-0905, U.S.A.
Virus The Cooper strain of BHV- 1 was used in this study. The virus was propagated in Madin-Darby bovine kidney (MDBK) cells
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and purified on potassium tartrate gradients according to the procedures of Misra, Blumenthal & Babiuk (1981).
of incubation, the plates were washed three times, and the wells were removed and counted for bound radioactivity.
Monoclonal antibodies Female BALB/c mice, 8-10 weeks of age, were injected intraperitoneally (i.p.) with 100 pl of a commercial MLV vaccine against BHV-1 (Norden Laboratories Inc., Lincoln, NE). A booster injection of 50 yl of the same vaccine was given i.p. 3 weeks later. Three days following the booster injection, the spleen cells from the immunized mice were fused with the murine cell line SP-2/0. The procedures of Kohler & Milstein (1975) for cell fusion, cloning and stabilization of the hybridomas were adopted with slight modifications. Positive hybridomas were initially selected by an indirect solid-phase radioimmunoassay (RIA) described below. The BHV- 1 glycoprotein specificity of the monoclonal antibodies (mAb) was determined by immunoprecipitation of [3H]glucosamine-labelled BHV-1 proteins followed by SDS-PAGE and autoradiography, as described by Van Drunen Littel-Van Den Hurk et al. (1984). The immunoglobulin (Ig) isotypes of the BHV- 1-specific mAb were determined by a standard enzymelinked immunosorbent assay (ELISA) on BHV- 1-coated microtitre plates using alkaline phosphatase-labelled goat antimurine Ig isotype-specific antibodies (Southern Biotechnology, Birmingham, AL). Three other hybridomas were used as the source of BALB/c mouse IgG2a antibodies with irrelevant specificities. One of these hybridomas, which secretes a mAb (MM-409) specific for porcine rotavirus, was developed at the University of Nebraska. The other hybridoma, which secretes a mAb (1 El 1) specific for the gI glycoprotein of BHV-1 (Van Drunen Littel-Van Den Hurk et al., 1984), was kindly provided by Dr S. Van Drunen Littel-Van Den Hurk of the Veterinary Infectious Diseases Organization, Saskatoon, Canada. The third one, which secretes a mAb (5A5) to the E2 glycoprotein of porcine coronavirus (Hariharan et al., 1989), was the gift of Dr P. Gaugh of the Iowa State University.
Virus neutralization assay BHV-1-neutralizing activity of the hybridoma culture fluids, ascites fluids or the plasma from mice immunized with the antiId was tested by a standard plaque reduction assay as described by Babiuk, Wardley & Rouse (1975). Appropriate negative controls were used as in the RIA described above. The neutralization titre was expressed as the highest dilution of antibody that neutralized 50% of the input virus.
Detection of anti-BHV-i antibodies by RIA A standard indirect solid-phase RIA (Rosenthal, Hayashi & Notkious, 1973) was used to detect anti-BHV-1 activity of the mAb or plasma from mice immunized with the anti-Id. Polyvinyl chloride microtitre plate wells were coated with purified BHV-1 protein in carbonate buffer, pH 9-6 (0-25 pg/ 20 pl/well), and incubated at 370 overnight. The unbound sites in the wells were then blocked by the addition of 2% bovine serum albumin (BSA) in borate buffer, pH 9-6 (50 p1/well). After 1 hr of incubation at room temperature in a humid chamber, the plates were washed three times with phosphate-buffered saline (PBS) containing 01% BSA and 0-01% sodium azide (PBSBSA-AZ). Undiluted hybridoma culture fluids, serially diluted ascites fluids or plasma from mice immunized with the anti-Id or normal bovine Ig, were added to the virus-coated wells (20 pl/ well). Culture fluid from the parent cell line SP-2/0, ascites fluid from mice injected with the SP-2/0 or plasma from mice before immunization with the anti-Id or normal bovine 1g, respectively, were used as negative controls. After 1 hr of incubation at room temperature, the wells were washed three times with PBS-BSAAZ and ['251]-labelled sheep F (ab')2 anti-mouse Ig (specific activity 10 pCi/pg, 20,000 c.p.m./20 pl/well; New England Nuclear Co., Boston, MA was added. Following another hour
Preparation of mAb for the production of anti-Id Ascites tumours of hybridomas were developed in mice according to standard procedures. Ascites fluids were precipitated with 50% ammonium sulphate and dialysed in PBS. The hybridomas also were grown in serum-free medium (KC2000; Hazleton, Biologics Inc., Lenexa, KS) and the mAb in the culture fluids were affinity-purified on a protein A-Sepharose CL-4B column (Pharmacia Fine Chemicals Inc., Piscataway, NJ). Immunization of calf A male 6-month-old Hereford calf was injected intramuscularly (i.m.) with 1 mg of protein A-purified MM-1 13 emulsified 1: 1 in complete Freund's adjuvant (CFA). Booster injections of 500 pg of MM-1 13 in incomplete Freund's adjuvant (IFA) were given i~m. on Days 21, 49 and 77 post-priming. The calf was bled on Days 7, 14 and 21 following each booster injection and the serum was harvested.
Preparation of anti-Id Ammonium sulphate-precipitated MM-113 and MM-409 (18 mg of each) were individually coupled to CNBr-activated Sepharose 4B (3 g) according to the manufacturer's instructions (Pharmacia Fine Chemicals Inc.). Bovine anti-Id to MM-1 13 were isolated by standard affinity chromatography procedures. First, bovine antibodies to MM- 113 (anti-idiotypic, anti-allotypic and anti-isotypic antibodies) were isolated from the serum of the immunized calf using the MM-l 13-Sepharose column. Serum (20 ml) from the immunized calf was passed over the MM-l 13-Sepharose column. Unbound protein was washed away with PBS. The column-bound antibody was eluted with 0-2 M glycine-HCl at pH 2 8 and immediately neutralized with 2 M Tris buffer at pH 9-6. The bovine antibodies to the isotypic and allotypic determinants of MM-1 13 present in this preparation were then removed by adsorption on the MM-409 Sepharose column (mouse IgG2a of a different idiotype). The adsorption procedure was repeated until no antibodies to the isotypic and allotypic determinants of MM- 113 were left in the preparation. The anti-MM-1 13 Id thus recovered were concentrated in Amicon stirred ultrafiltration cells (Amicon Corporation, Danvers, MA).
Characterization of the anti-Id The specificity of the anti-Id to the idiotype of MM-1 13 was tested by an indirect solid-phase RIA and an inhibition RIA (Rosenthal et al., 1973). Indirect solid-phase RIA Polyvinyl chloride 96-well microtitre plates were coated with affinity-purified bovine anti-Id (10 pg/ml in PBS, 20 p1/well) and
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incubated at room temperature in a humid chamber overnight. The unbound sites in the wells were then blocked by the addition of PBS-BSA (50 pl/well). After I hr of incubation in a humid chamber at room temperature, the plates were washed three times with PBS-BSA-AZ. The mAb MM- 113, lEl 1, MM-170, MM-409 or 5A5, in the form of culture fluids, or the culture fluid from the parent cell line SP-2/0 (20 pl/well) were then added to the wells. The plates were washed after an hour of incubation and ['251]-labelled sheep F(ab')2 anti-mouse Ig (specific activity 10 pCi/pg, 20,000 c.p.m./20 pl/well) was added to the wells. Following another hour of incubation, the plates were washed, the wells removed and counted for bound radioactivity.
Inhibition assay The inhibition of binding of MM-1 13 to BHV-1 protein by the anti-Id was determined by a modified RIA. Fifty-microlitre samples of undiluted MM-1 13 culture fluid were mixed with equal volumes of the anti-Id or normal bovine Ig (diluted in PBS-BSA-AZ) so that the final concentration of the anti-Id or bovine Ig ranged from 4 to 512 pg/ml. After incubation at room temperature for 1 hr, duplicate samples of 30 pl of the mixture were transferred to the wells of 96-well microtitre plates coated with BHV-1 protein (0-25 pg/well). Upon incubation at room temperature for 1 hr, the plates were washed three times with PBS-BSA-AZ and ['25I]-labelled sheep F(ab')2 anti-mouse Ig (specific activity 10 pCi/pg, 20,000 c.p.m./20 pl/well) was added. After another hour of incubation, the wells were washed, removed from the plate and counted for bound radioactivity. Immunization of mice A group of four female BALB/c mice was primed i.p. with 50 pg of anti-Id precipitated on alum and emulsified 1: 1 in CFA. These mice were given five bi-weekly booster injections i.p. with 25 pg of alum-precipitated anti-Id in IFA. A control group of four mice of the same age and sex was injected similarly with normal bovine Ig. Beginning with the third booster injection, all animals were bled 10 days after the booster injections.
Detection of antibodies to BHV-J in mice immunized with the anti-Id Antibodies to BHV-1 were detected by the standard indirect solid-phase RIA and the neutralization test described earlier. Detection of binding of the anti-Id to BHV-J-permissive cells A cell-binding RIA (Goldsby, Osborne & Engleman, 1979) was used to detect the binding of the anti-Id to cells permissive to BHV-1 infection. Fifty microlitres of the anti-Id or normal bovine Ig (at a concentration of 15, 30 and 60 pg/ml in PBS-BSA-AZ) were added to the wells of polyvinyl chloride microtitre plates containing 5 x 105 MDBK cells in 50 pl of PBS-BSA-AZ. After 1 hr of incubation in a humid chamber at room temperature, the plates were centrifuged at 800 g for 2 min. The supernatant fluid was removed by carefully flicking the plates. The cell pellets in the wells were resuspended by shaking the plates in a microshaker (Dynatech Inc., Alexandria, VA). The unbound antibodies were washed away by three washing cycles. Each cycle consisted of addition of washing buffer (200 pl of PBS-BSA-AZ per well), centrifugation of the plate (800 g for 2 min), removal of the supernatant fluid and resuspension of the cell pellets. At the end of the third washing cycle, ['251]-labelled murine mAb to bovine Ig (Srikumaran et al.,
Table 1. Characteristics of the anti-BHV-l mAb
Reactivity in Designation
MM-I 13 MM-155 MM-170 MM-171 MM-105 MM-130 MM-160
RIA
+ + + + + + +
Neutralizing activity
Glycoprotein specificity
+ +
gIV (gD) gIV (gD) gll (gC) gill (gC) gI (gB)
-
ND ND
Isotype IgG2a IgG2a IgG2a IgGI IgG2b IgGI
IgM
ND, not determined.
1987) was added to the wells (specific activity 10 MCi/pg, 20,000 c.p.m./20 pl/well). After 1 hr incubation and three washing cycles, a drop of 2% agarose was added to the wells and allowed to solidify. The wells were then cut and counted for bound radioactivity. RESULTS Characteristics of mAb Seven hybridomas secreting mAb to BHV-1 were stabilized (Table 1). Two of these secreted mAb (MM-1 13 and MM-155) that neutralized the virus. Both of these mAb immunoprecipitated the glycoprotein gIV. Competitive RIA showed that these two mAb recognized the same epitope or closely juxtaposed epitopes (results not shown) on the glycoprotein gIV. The mAb MM- 113 was selected for the development of anti-idiotypes.
Development and characterization of the anti-Id Immunization of the calf with MM- 113 resulted in the production of anti-MM-1 13 antibodies in high titre. Following the second booster injection, the titre reached 1: 128,000 as determined by RIA. Serum collected on Day 14 following the third booster injection gave the highest yield of bovine anti-MM-113 antibodies (approximately 0 5 mg per ml of serum). In the indirect RIA used to determine the specificity of the anti-idiotypes, MM- 113 bound to the anti-Id (Fig. 1). The mAb l E 11 (specific for gI of BHV- 1), MM- 170 (specific for gIII of BHV-1), MM-409 (specific for porcine rotavirus) and 5A5 (specific for porcine coronavirus) did not bind to the anti-Id. These results indicated that the anti-Id were specific for the idiotype of MM-1 13. The lack of binding of the anti-Id to El 1, MM-170, MM-409 or 5A5 (all are BALB/c IgG2a mAb) also confirmed that there were no bovine antibodies to the isotypic and allotypic determinants of IgG2a left in the anti-Id preparation. In the inhibition assay, the anti-Id inhibited the binding of MM-1 13 to BHV-1 in a concentration-dependent manner, while normal bovine Ig did not inhibit the binding of MM-1 13 to BHV-1 significantly (Fig. 2). Production of antibodies to BHV-1 by mice immunized with the anti-Id The results of the inhibition assay suggested that the anti-Id recognized idiotopes within or close to the paratope of MM-
Anti-idiotypic antibodies
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12 10
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on
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=`
4
300
r
200 100 Ol
1000 4000 16,000 Reciprocal of plasma dilution
MM- ElI MM- MM- 5A5 113 170 409 Monoclonal antibodies
Figure 1. Specificity of the anti-Id to MM-1 13. The binding of MM-1 13 (Ab 1), and the isotype-matched controls I E I 1, MM- 170, MM-409 or 5A5 to the anti-Id, was determined by an indirect solid-phase RIA as described in the Materials and Methods section. The signal to noise ratio (S/N) was calculated by dividing the c.p.m. given by the mAb in the form of culture fluids by the c.p.m. given by the culture fluid from the parent cell line SP-2/0.
113. To determine whether these anti-Id mimic the epitopes of BHV- 1 recognized by MM- 113, BALB/c mice were injected with the anti-Id or with normal bovine Ig. Anti-BHV- 1 antibody activity of the plasma from these mice was tested by an RIA and compared with that of plasma collected before injection. All the mice that received the anti-Id responded with the production of antibodies to BHV-1. The RIA titre (average of four mice) of these antibodies was 1: 1000 after the third booster injection (data not shown), which increased to 1:8000 after the fifth booster injection (Fig. 3). None of the mice in the control group showed any detectable anti-BHV- response. The induction of anti-BHV- antibodies in naive mice by the anti-Id confirmed the presence of internal image anti-Id in the polyclonal anti-Id preparation. The virus-neutralizing activity of plasma from mice injected with the anti-Id was determined by a plaque reduction assay. No neutralizing activity was detectable in the plasma after the third booster injection. After the fifth booster, however, the plasma
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0
.
:
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Figure 3. Anti-BHV-l antibody response of BALB/c mice immunized with the anti-Id. Plasma from mice injected with the anti-Id (A) or normal bovine Ig (-) was serially diluted in PBS-BSA-AZ, from 1: 1000 to 1: 32,000, and tested for binding to BHV- I by an indirect solid-phase RIA. The results are presented as the mean of the c.p.m. given by the dilutions of plasma from mice (n = 4). The plasma from the mice before injection (0) was also tested similarly. Table 2. BHV-1-neutralizing activity of plasma from mice injected with the anti-Id Neutralization titre
Mouse no. Before injection After injection 1 2 3 4
1:3 1:3 1:3 1:3
1:16 1:48 1:6 1:12
from these mice neutralized BHV- 1, with titres ranging from 1: 6 to 1:48 (Table 2).
Binding of BHV-l-permissive cells by the anti-Id The induction of neutralizing antibodies to BHV- 1 by the antiId was interpreted to mean that the anti-Id mimicked (at least partially) the epitope recognized by MM-113. Since MM-113 (the antibody 1, Abl) recognized a neutralizing epitope on the glycoprotein gIV, some of the anti-Id mimicking that epitope may recognize the surface protein(s) recognized by the original epitope on the glycoprotein gIV. In the cell-binding RIA used to detect the binding to cell-surface proteins, the anti-Id bound to the MDBK cells strongly while the binding of the normal bovine Ig was at the background level (Fig. 4).
\__AI.i/
20
101l1 4 8
I1 64128256512
16 32 Conc. anti-Id (pug/ml)
Figure 2. Inhibition of binding of MM-1 13 to BHV-1 by the anti-Id. Undiluted MM-1 13 culture fluid was mixed with the anti-Id (A) or normal bovine Ig (-) at varying concentrations (4-512 pg/ml). Following incubation for 1 hr, the binding of MM- 1 13 to BHV- l was detected by an indirect solid-phase RIA, as described in the Materials and Methods.
DISCUSSION Previous studies have reported the feasibility of inducing immune responses to viral antigens by anti-Id (Reagan et al., 1983; Sharpe et al., 1984; Uytdehaag & Osterhaus, 1985; Kennedy et al., 1986; Hariharan et al., 1989; Reagan, 1985; Gaulton et al., 1986; Garmendia et al., 1989). The outcome of immunization with anti-Id against herpesviruses has been mixed. In the study by Kennedy et al. (1984), mice immunized with an anti-Id against herpes simplex type 2 (HSV-2) were more
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S. Srikumaran et al. 1400 1200 E CLL Q 1000
*0 800_
600 o 400
_200 30 g 60kgConc. anti-Id Figure 4. Binding of the anti-Id to cells permissive to BHV-1. The binding of the anti-Id (-) or normal bovine Ig (0) to MDBK cells was tested by a cell-binding RIA as described in the Materials and Methods. 0
15,Lx
susceptible to infection by HSV-2. Lathey, Martin & Rouse (1987) reported the induction of only mild antibody and cellular responses, and suppression of delayed type hypersensitivity responses against herpes simplex type 1 (HSV-1) following immunization with anti-Id. Gell & Moss (1985), however, generated dose-dependent delayed-type hypersensitivity responses against HSV-1 by prior administration of anti-Id. Reasons for the differences in the outcome of these anti-Id immunizations are not clear. The dose (Gel & Moss, 1985), isotype (Eichman, 1974), and route of injection of anti-Id (Lathey et al., 1987), however, have been thought to influence the outcome of anti-Id immunization. Although the development of anti-Id to BHV-1-specific mAb has been reported previously (Whetstone, Babiuk & Van Drunen Little-Van Den Hurk, 1988; Orten et al., 1988), induction of immune response to BHV-1 by anti-Id was not demonstrated in those studies. This is the first report of the induction of neutralizing antibodies to BHV-1 by anti-Id. The anti-Id (Ab2) developed in this study specifically reacted with the idiotype of MM-113 (Abl) and not with the idiotypes of other isotype-matched controls. The inhibition of binding of AbI to BHV-1 by the anti-Id in a concentration-dependent manner suggests that they may recognize idiotope/s within the paratope of the Abl. This inhibition also could be due to the induction of a conformational change in the Ab1 paratope or creation of a steric obstruction to Abl paratope by the anti-Id binding to the Abl. The induction of anti-BHV-1 antibodies (Ab3) by the anti-Id in naive mice, however, clearly indicates the internal image nature of these anti-Id. These anti-BHV- 1 antibodies (Ab3), like the Abl, neutralized BHV-1 in vitro. The titres of anti-BHV-1 antibodies (Ab3) induced by the anti-Id were not very high. The Ab3 was a polyclonal mixture of antibodies induced by a mixture of different anti-Id, each mimicking the paratope of AbI in a different manner. Some of them may be true internal image Ab2, whereas others may mimic idiotopes outside the paratope of Abl . The concentration of the internal image anti-Id, which determines the induction of Ab3 identical or similar to Abl, is unknown and it is very difficult to enrich the proportion of the same in a polyclonal anti-Id preparation. Nevertheless, the production of BHV-1neutralizing Ab3 indicates that there was true internal image
Ab2 in the anti-Id preparation and also points to the need for the development of monoclonal internal image anti-Id. The potential use of anti-Ids as immunogens against BHV- I may be questioned because of the need for repeated booster injections. The immunogenicity of anti-Id, however, may be enhanced by conjugation to carriers and by the use of different adjuvants. Further experiments are also needed to evaluate the influence of dose, Ig isotype and the route of anti-Id administration on the induction of anti-BHV-l antibody response. These studies, along with the development of monoclonal internal image anti-Id (which is underway in this laboratory), should enhance the potential of anti-Ids as immunogens against BHV- 1. Internal image anti-Id have also been used as probes to identify and isolate the receptors for some viruses (Noseworthy et al., 1983; Co et al., 1985; Marriott et al., 1987). The finding that the anti-Id developed in this study bind to BHV- I permissive cells is very encouraging. Further studies are in progress in this laboratory to determine whether the anti-Id bind to a BHV-I receptor protein on the permissive cells.
ACKNOWLEDGMENTS Published as Journal Series No. 9087, with approval of the University of Nebraska Agricultural Research Division. This research project was partially supported by the USDA Animal Health Grant No. 8834116-3639.
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