Anti-idiotype technique: an alternative approach for immunodiagnosis of bluetongue

Veterinary Immunology and Immunopathology 72 (1999) 237±242

Anti-idiotype technique: an alternative approach for immunodiagnosis of bluetongue En-Min Zhou* National Center for Foreign Animal Diseases, Canadian Food Inspection Agency, 1015 Arlington Street, Winnipeg, Manitoba, Canada R3E 3M4 Accepted 22 September 1999

Abstract In development of a bluetongue alternative immunodiagnostic rest, the polyclonal anti-idiotypic antibodies were generated by the sequential immunization of rabbits with three monoclonal antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies recognize the idiotypes that are located within or near the antigen-combining sites and are associated with both heavy and light chains of the antibodies to VP7 of bluetongue virus. The anti-idiotypic antibodies mimic the VP7 antigen by recognizing the anti-VP7 antibodies from cattle and sheep that were infected with various serotypes of bluetongue viruses. The results indicate that the rabbit anti-idiotypic antibodies may be used as surrogate antigen in serological assays to detect the antibodies from different species of animals infected with various serotypes of bluetongue viruses. # 1999 Elsevier Science B.V. All rights reserved. Keywords: Anti-idiotype; Immunodiagnosis; Bluetongue

1. Introduction Anti-idiotype that recognizes an idiotype and represents an internal image of the antigen have been generated in many systems (Zhou et al., 1987). Their antigenic mimicry makes them valuable as substitutes for certain infectious agents as immunogens. The approach of using anti-idiotype as reagents has several advantages over current immunoassays. Since anti-idiotypes are immunoglobulins and are not infectious they overcome the inherent problems of working with dangerous animal pathogens, facilitate *

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continuous production of the reagents by hybridoma technology, enhance the test reproducibility and standardization, and improve the test specificity. Bluetongue virus (BTV), a member of the genus Orbivirus of the family Reoviridae (Howell and Verwoerd, 1971), causes diseases that have serious economic consequences in ruminants (sheep and cattle) worldwide. Bluetongue virions are non-enveloped, icosahedral-shaped particles consisting of a segmented double-stranded RNA genome encapsidated in a double-layered protein coat. Removal of the outer protein layer activates a virion-associated RNA polymerase that transcribes the 10 genome segments into 10 mRNAs that are in turn translated into seven structural proteins (VP1 to VP7) and three nonstructural proteins (NS1 to NS3). The outer capsid layer of BTV consists of two major polypeptides, VP2 and VP5, and of these, VP2 determines virus serotypes, induces virus-neutralizing antibodies and is involved in cell attachment. To date, at least 24 different serotypes of BTV have been recognized worldwide. The core particle is composed of two major polypeptides, VP3 and VP7, and three minor polypeptides, VP1, VP4 and VP6. Of these, VP3 and VP7 have been identified as the serogroup specific antigens (Huismans et al., 1987) by complement fixation and agar gel immunodiffusion tests and VP7 has been widely used as a diagnostic reagent (Jochim, 1985). It has been suggested that VP7 provide cell-mediated protective immunity in sheep (Roy et al., 1994). Currently the two main approaches employed to control bluetongue infection are vaccination and diagnosis of the disease and slaughter of ruminants in the infected area. Diagnosis of bluetongue infections is based upon virus isolation or demonstration of antivirus antibodies in serum. Various assay systems, including complement fixation, agar gel immunodiffusion, and ELISA, have been developed for the detection of serum antibodies (Afshar, 1994). Traditionally, these assays have required the time-consuming and labourintensive preparation of BTV antigens by conventional methods. Therefore, there is a great need for a less laborious procedure for the production of improved novel diagnostic reagents. In addition to the promising use of recombinant DNA technology for this purpose, the production of certain anti-idiotypic antibodies bearing the internal image of antigen according to Jerne's theory (Jerne, 1974), offers an alternate approach. In this report, rabbit polyclonal anti-idiotypic antibodies were produced in response to monoclonal antibodies (mAbs) specific for the BTV core protein VP7 and showed promising results of being used as bluetongue serodiagnostic reagent. 2. Results and discussion 2.1. Generation and characterization of rabbit polyclonal anti-idiotypic antibodies Three mAbs produced by Appleton and Letchworth (1983) with the specificity for BTV VP7 group-specific epitope(s) have been used in our laboratory in competitive ELISAs for the detection of antibodies to BTV (Afshar et al., 1993). These mAbs were used to generate anti-idiotype in rabbits using sequential immunization (Zhou et al., 1994). The resulting anti-idiotype (designated RAb2s) interacts with all three mAbs and their interaction was inhibited by BTV VP7 antigen (Table 1), which indicates that

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Table 1 RAb2s recognize idiotype within or near the antigen-combining sites of mAb1sa Direct binding (absorbance at 405 nm)b M1875 1.1

M1877 0.96

Inhibition (%) of mAb1-RAb2s by VP7 antigenc M1886 0.62

M1875 96

M1877 50

M1886 30

a

RAb2s at 4 ug/ml was used to coat in the wells of ELISA plate. Values were generated by mAbs binding at 500 ng/ml. c VP7 antigen was used at 5 ug/ml. b

Table 2 Serological characteristics of rabbit anti-idiotypic antibodies Recognize the idiotypes associated with both heavy and light chains of the mAb1s Inhibit mAb1s binding to VP7 VP7 inhibits its binding to mAb1s Detect the idiotype on anti-VP7 antibodies from mice, cattle and sheep Elicit anti-anti-idiotype responses in mice and sheep

RAb2s recognize idiotype located within or near the antigen-combining sites and they may represent the internal image anti-idiotype. The characteristics of RAb2s were summarised in Table 2. The epitope binding of these mAbs to the VP7 antigen was not identical but closely related (data not shown). Although the expression of an idiotype is controlled by the antigenic specificity of an antibody molecule, antibodies with different antigenic specificity may also express a common idiotype. This phenomenon has been observed in many systems (Oudin and Cazenave, 1971; Germain et al., 1979; Metzger et al., 1980; Liu et al., 1981; Hiernaux and Bona, 1982; Kohno et al., 1982; Kennedy et al., 1983). The studies on the allergen (Zhou et al., 1991) and an influenza virus (Dinca et al., 1993) have demonstrated that mAbs specific for different epitopes possessed the common idiotype. The sequential immunization method has been shown to improve the generation of antiidiotype to a common idiotype, i.e., in favor of elicitation of internal image anti-idiotype, although the mechanism has not been fully elucidated. 2.2. Rabbit anti-idiotype as diagnostic reagents To determine whether RAb2s, generated to murine mAbs, could recognize bovine and sheep antibodies to BTV, an indirect ELISA was employed to test the binding capacity of RAb2s, along with BTV antigens. Six bovine and four sheep were experimentally infected with various serotypes of BTV and serum samples collected before and 14 days after the infection were used to interact with the solid-phase RAb2s or BTV antigens. Results from Table 3 clearly demonstrated that RAb2s detected, as the VP7 antigen, both bovine and sheep anti-BTV antibodies. The specificity of the binding between RAb2s and bovine and sheep anti-BTV antibodies was confirmed in an inhibition assay, in which BTV antigens partially inhibited the binding. Specificity of this test was further confirmed by testing bovine antibodies to epizootic hemorrhagic disease virus of deer, a closely

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Table 3 Detection of anti-BTV antibodies by RAb2s and VP7 antigena Serum samplesb Bovine No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 Sheep No. 1 No. 2 No. 3 No. 4

Days post infection (d.p.i.)

RAb2s

VP7 antigen

0 14 0 14 0 14 0 14 0 14 0 14

0.323 0.72 0.31 0.8 0.25 0.71 0.15 0.68 0.2 0.95 0.31 0.58

0.31 0.68 0.3 0.78 0.27 0.69 0.13 1 0.18 1 0.3 0.78

0 14 0 14 0 14 0 14

0.12 1.7 0.07 0.85 0.16 1.42 0.18 1.68

0.11 2.2 0.13 1.85 0.25 1.43 0.22 1.25

a

In an indirect ELISA, the plate was coated with RAb2s at 4 ug/ml or VP7 antigen at 1 ug/ml; Results represent the mean values of absorbance at 405 nm of triplicate. b Serum samples were collected from cattle and sheep infected with BTV on day 0 (0 d.p.i.) and day 14 (14 d.p.i.). Cattle Nos. 1±6 were infected with BTV-SA-T3, BTV-SA-T6, BTV-SA-T8, BTV-US-T10, BTV-US-T11, and BTV-US-T17, respectively. Sheep was infected with BTV-US-T11.

related virus of BTV (both are members of the Orbiviruse genus of the Reoviridae family) and found only background binding to RAb2s (data not shown). Internal image anti-idiotype has been shown in many systems to cross genetic barriers in recognizing common idiotype shared by antibodies with similar (Bona et al., 1984; Zhou et al., 1987; Mourad et al., 1988) or distinct specificity (Kennedy et al., 1983; Zhou et al., 1987). This property has been one of the major criteria in defining anti-idiotype as internal image antibodies, and prompted studies investigating anti-idiotype as potential vaccine candidates for many infectious organisms (for review, see Zhou et al., 1987) as well as serodiagnostic reagents (for review, see Linthicum et al., 1988). The use of antiidiotype as diagnostic reagent was first reported by Potocnjak et al. (1982) for Plasmodium berghei, a causative parasite for malaria, using an inhibition test in which the radiolabelled anti-idiotype competed with the antigen from binding to the solid-phase antibodies. A more recent study using anti-idiotype as serodiagnostic reagents for bovine cysticercosis employed anti-idiotype directly on the solid-phase of ELISA plates and detected antibodies against Taenia parasite antigens comparable with the use of parasite antigens on the solid phase (Hayunga et al., 1992). RAb2s as the internal image anti-

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idiotype of VP7 antigen not only detected bovine and sheep antibodies against various serotypes of BTV but elicited anti-BTV antibody responses in mice and sheep. The antiBTV antibodies induced by RAb2s prevent partially the BTV infection (data not shown). Acknowledgements I am grateful to Drs. Wei Huang, Min Lin, Ahmad Afshar and Robert Heckert for their valuable suggestions and comments and Mrs. Barbara Leung, Mr. Robert Vogrig and Mrs. Maria Chan for their technical assistance. References Afshar, A., Dulac, G.C., Dubuc, C., Pearson, J.E., Gustafson, G.A., 1993. Competitive ELISA for serodiagnosis of bluetongue: a refinement. J. Vet. Diagn. Invest. 5, 614±616. Afshar, A., 1994. Bluetongue: laboratory diagnosis. Comp. Immun. Microbiol. Infect. Dis. 17, 221±242. Appleton, J.A., Letchworth, G.J., 1983. Monoclonal antibody analysis of serotype-restricted and unrestricted bluetongue viral antigenic determinants. Virology 124, 286±299. Bona, C.A., Victor-Kobrin, C., Manheimer, A.J., Bellon, B., Rubinstein, L.J., 1984. Regulatory arms of the immune network. Immunol. Rev. 79, 25±44. Dinca, L., Neuwirth, S., Schulman, J., Bona, C., 1993. Induction of antihemagglutinin antibodies by polyclonal antiidiotype antibodies. Viral. Immunol. 6, 75±84. Germain, R.N., Ju, S.T., Kipps, T.J., Benacerraf, B., Dorf, M.E., 1979. Shared idiotypic determinants on antibodies and T-cell-derived suppressor factor specific for the random terpolmer L-glutamic acid60-Lalanine30-L-tyrosin10. J. Exp. Med. 149, 613±622. Hayunga, E.G., Sumner, M.P., Duncan Jr., J.F., Chakrabarti, E.K., Webert, D.W., 1992. Production of antiidiotypic antibodies as potential immunoreagents for the serological diagnosis of bovine cysticercosis. Tropical Vet. Med. 653, 178±183. Hiernaux, J., Bona, C., 1982. Shared idiotypes among monoclonal antibodies specific for different immunodominant sugars of lipopolysaccharide of different Gram-negative bacteria. Proc. Natl. Acad. Sci. USA 79, 1616±1620. Howell, P., Verwoerd, D., 1971. Bluetongue virus. In: Hess, W.R., Howell, P.G., Verwoerd, D.W. (Eds.), African Swine Fever Virus, Bluetongue Virus, Springer, New York, pp. 35±74. Huismans, H., Van der Walt, N.T., Cloete, M., Erasmus, B.J., 1987. Isolation of a capsid protein of Bluetongue virus that induces a protective immune response in sheep. Virology 157, 172±179. Jochim, M.M., 1985. An overview of diagnostics for bluetongue. In: Barber, T.L., Jochim, M.M., Osburn, B.I. (Eds.), Bluetongue and Related Orbiviruses. Alan R. Liss, Inc., New York, pp. 423±433. Jerne, N.K., 1974. Towards a network theory of the immune system. Ann. Immunol. 125C, 373±389. Kennedy, R.C., Adler-Storthz, K., Henkel, R.D., Dressman, G.R., 1983. Characteristics of a shared idiotypes by two IgM anti-herpes simplex virus monoclonal antibodies that recognize different determinants. J. Immunol. 130, 1943±1946. Kohno, Y., Berkower, I., Minna, J., Berzofsky, J.A., 1982. Idiotypes of antimyoglobulin antibodies: shared idiotypes among monoclonal antibodies to distinct determinants of sperm whale myoglobin. J. Immunol. 128, 1742±1748. Linthicum, D.S., Kussie, P.H., Combs, S., Yatko, D., 1988. Idiotypes and anti-idiotypes: significance in immunoassays. J. Clin. Immunol. 11, 31±36. Liu, Y.N., Bona, C.A., Schulman, J.L., 1981. Idiotype of clonal responses to influenza virus hemagglutinin. J. Exp. Med. 154, 1525±1538. Metzger, D.W., Miller, A., Sercarz, E.E., 1980. Sharing of idiotypic marker by monoclonal antibodies specific for distinct regions of hen lysozyme. Nature 287, 540±542.

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