JOURNAL OF VIROLOGY, Dec. 1991, p. 6979-6984 0022-538X/91/126979-06$02.00/0 Copyright © 1991, American Society for Microbiology
Vol. 65, No. 12
A Conserved Coronavirus Epitope, Critical in Virus Neutralization, Mimicked by Internal-Image Monoclonal Anti-Idiotypic Antibodies CARLOS SUN4,1 CRISTIAN SMERDOU,1 INES M. ANTON,1 PILAR ABRIL,2 JUAN PLANA,2 AND LUIS ENJUANESl* Centro de Biologia Molecular, Consejo Superior de Investigaciones Cientificas, Universidad Aut6noma, Canto Blanco, Madrid 28049,1 and Laboratorios Sobrino (Cyanamid), Departamento de Investigaci6n y Desarrollo, Olot, Gerona,2 Spain Received 26 February 1991/Accepted 10 September 1991
Monoclonal antibody (MAb) 6A.C3 neutralizes transmissible gastroenteritis coronavirus (TGEV) and is specific for a conserved epitope within subsite Ac of the spike (S) glycoprotein of TGEV. Six hybridomas secreting anti-idiotypic (Ab2) MAbs specific for MAb 6A.C3 (Abl) have been selected. All six MAbs inhibited the binding of Abl to TGEV and specifically cross-linked MAbl-6A.C3. Four of these hybridomas secreted -y-type anti-idiotypic MAbs. The other two Ab2s (MAbs 9A.G3 and 9C.Ell) were recognized by TGEV-specific antiserum induced in two species. This binding was inhibited by viruses of the TGEV group but not by serologically unrelated coronaviruses. These results indicate that MAb2-9A.G3 and MAb2-9C.E11 mimic an antigenic determinant present on the TGEV surface, and they were classified as ,8-type ("internal-image") MAbs. TGEV-binding Ab3 antiserum was induced in 100% of mice immunized with the two "-type MAb2s and in 25 to 50% of mice immunized with -y-type MAb2. Both ,B- and -y-type Ab2s induced neutralizing Ab3 antibodies in mice that were mainly directed to antigenic subsite Ac of the S protein.
The spike (S) protein of transmissible gastroenteritis coronavirus (TGEV) induces neutralizing antibodies against this virus and, possibly, protection against transmissible gastroenteritis (TGE) (11, 14). Four antigenic sites (A, B, C, and D) have been identified in the S protein of TGEV. Site A is the main inducer of neutralizing antibodies and has been subdivided into three subsites, Aa, Ab, and Ac (6-8). Monoclonal antibody (MAb)-resistant (mar) mutants selected with MAbs binding to these subsites contain amino acid substitutions mapping in residues 538, 591, and 543, respectively. In addition, residue 586 is involved in the formation of subsites Aa and Ab (5, 10, 16). To determine the region of the S protein potentially responsible for the induction of protection against TGE, antigenic sites B, C, and D have been reproduced by sequences selected from random hexapeptides attached to a ,B-galactosidase hybrid protein expressed by vectors of the pEX family (29). In addition, sites C and D have been mimicked by nonapeptides selected by epitope scanning (5, 10, 16, 32). Peptides representing site A were antigenic but not immunogenic (15, 16). Since site A is the main inducer of TGEV neutralizing antibodies, we searched for alternative procedures to represent this site. Antigenic determinants of pathogenic microorganisms can be mimicked by anti-idiotypic antibodies (Ab2s) (26-28, 33, 35). These Ab2s could be useful for inducing protection. Ab2s were originally classified into two subsets (20). Ab2-a is the classical Ab2 which recognizes an idiotope and does not inhibit the binding of the idiotype (Abl) to the antigen. Ab2-P displays an idiotopic structure which looks like an antigen and therefore inhibits the binding of the Abl to the antigen. Afterwards, a third type of Ab2 was introduced, the Ab2--y, which binds to a determinant very close to the paratope of Ab2 and itfhibits the binding of the Abl to the antigen (1, 2). However, in contrast to the Ab2-,B, Ab2-y *
does not mimic an antigenic structure which fits the paratope of Abl. The representation of subsite Ac epitopes by antiidiotypic MAbs (MAb2s) is of interest for the following reasons. (i) Subsite Ac is the main inducer of neutralizing antibodies against TGEV and is structurally complex and glycosylation dependent (5, 10, 16). (ii) Subsite Ac is highly conserved in enteric and respiratory coronaviruses from different species (37). (iii) One of the three MAbs which define this subsite (MAb 6A.C3) neutralizes TGEV infectivity in vitro at least 109-fold (40). (iv) Protection against TGE requires mucosal immunity, which could be induced by Ab2s, since immunoglobulins are actively adsorbed in the gut (31). (v) Sequencing of the hypervariable region of Ab2-,B (internal-image) immunoglobulins may identify sequences responsible for antigen mimicry and may help to design mimotopes of interest. Anti-idiotopes sharing short amino acid sequences with those of nominal antigens have been described for reovirus and poly(Glu-Ala-Tyr) systems (4, 34). For the TGEV system, one previous report (18) has described the production of polyvalent Ab2s inducing TGEV-specific Ab3. In this article, we describe the selection of six monoclonal Ab2s raised against MAb 6A.C3 specific for antigenic subsite Ac. Two MAb2s behave as an internal image of subsite Ac. Mice immunized with a-type MAb2s and two -y-type MAb2s produced TGEV neutralizing antibodies. MATERIALS AND METHODS
Cells and viruses. The virus strains PUR46-MAD-CC120, porcine respiratory coronavirus (PRCV) HOL87, feline infectious peritonitis virus (FIPV) ATCC VR-2004, mouse hepatitis virus (MHV) A59, and human coronavirus (HCV)
229E and the cell types to grow them have been described previously (37). Procedures for neutralization, radioimmunoassay (RIA), and virus purification have been described
Corresponding author. 6979
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previously (6). [35S]methionine-labeled TGEV was grown and purified as described previously (40). Isolation of MAb2s. MAb 6A.C3 was used as an Abl (MAbl-6A.C3) (5, 6, 21) to induce Ab2s. Immunoglobulins were purified from 6A.C3 ascitic fluids by high-pressure liquid chromatography (HPLC) by using a TSK DEAE-5 PW column (7.5 by 75 mm; Bio-Rad), as described by Deschamps et al. (9). To elicit an Ab2 response, mice were subcutaneously immunized with 75 ,g of MAbl-6A.C3 coupled to keyhole limpet hemocyanin (KLH; Calbiochem) with glutaraldehyde (3 x 104 immunoglobulin molecules per KLH molecule, with a medium KLH molecular mass of 5.5 x 106 Da). Purified MAb (1 mg/ml) was dialyzed against phosphate-buffered saline (PBS) and mixed with an equal weight of KLH. Coupling was initiated by adding 1 ml of 0.05% glutaraldehyde per mg of immunoglobulin; the mixture was stirred at room temperature for 15 min. The reaction was stopped with 0.05 M lysine and incubation at room temperature for 2 h; the solution was then dialyzed against PBS for 3 days at 4°C. Mouse myeloma cells (X63Ag 8.653) (22) were fused with spleen cells from BALB/c mice immunized with MAbl-6A.C3. Fusions were made as previously described (38). Hybridomas were screened by a solid-phase RIA (sandwich assay) based on the binding of Abl by Ab2, similar to the method described by Thanavala et al. (41). Briefly, each well of a 96-well flat-bottom polyvinyl plate (Dynatech Laboratories) was coated with 1 Rg of purified idiotype MAbl-6A.C3 immunoglobulin in 50 ,u of carbonate-bicarbonate buffer, pH 9.6, overnight at room temperature. Nonspecific binding sites were blocked by incubating the plates for 90 min at 37°C in the presence of 5% bovine serum albumin (BSA) in PBS, pH 7.2. Blocking solution was removed, and 50 ,ul of hybridoma supernatants (or mouse serum diluted in 0.1% BSA in PBS) was added to each Abl-coated well and incubated for 60 min at 37°C. Supernatants were removed, and the wells were washed three times with PBS containing 0.1% BSA before 50 RI of 12511 labeled idiotype (5 x 104 cpm; specific activity, 107 cpm/Ig of immunoglobulin) (17) was added per well. Incubation was carried out for 2 h at 37°C. Wells were then washed six times with PBS containing 0. 1% BSA and 0.1% Tween 20 and dried, and individual wells were counted in a y-spectrometer. MAb2 isotype was determined by immunodiffusion (30) with antiserum specific for mouse immunoglobulin heavy and light chains (Research Products, Inc.). Inhibition of idiotype (Abl) binding to TGEV by MAb2s. Each well of a 96-well polyvinyl plate was coated with 1 jig of HPLC-purified MAb1-6A.C3 in 50 IL of carbonate-bicarbonate buffer (pH 9.6) and incubated overnight at room temperature. Nonspecific binding sites were blocked by incubation in the presence of PBS containing 5% BSA for 90 min at 37°C. Blocking solution was removed, and 50 RI of 35S-labeled purified TGEV (8 x 104 cpm; 1.2 x 104 cpm/Ig), in PBS with 0.1% BSA, was added per well in the presence of the indicated concentrations of HPLC-purified MAb2. Plates were incubated for 120 min at 37°C and washed six times with PBS containing 0.1% BSA and 0.1% Tween 20. Radioactivity bound to the wells was solubilized with 100 pI of 0.1% NaOH and neutralized with 0.1% HCl. Bray's liquid scintillation fluid (3) was added, and the samples were counted. Binding of antibodies against TGEV to MAb2. The binding of TGEV-specific antiserum to MAb2 was determined by a solid-phase RIA identical to the one described for the screening of MAb2, but purified MAb2 instead of MAbl was used. Antisera against TGEV were diluted in PBS with 0.1% BSA. Murine antisera numbers 1 and 2 had RIA titers against TGEV of 106 and 5 x 104, respectively. Porcine
antisera numbers 1 and 2 had RIA titers of 5 x 105 and 840, respectively. Control sera were from nonimmune animals. Specificity of the binding was tested by incubating the cross-linker antibody in the presence of virus. Induction and analysis of Ab3s. MAb2s were self-aggregated and coupled to the carrier protein KLH by chemical cross-linking in the presence of glutaraldehyde as described above. Eight-week-old BALB/c mice were subcutaneously inoculated four times. Antigen doses of 75 SLg of Ab2 (3 x 104 immunoglobulin molecules per KLH molecule; see above) were administered at weekly intervals. The two first doses consisted of KLH-coupled MAb2 emulsified in complete Freund adjuvant. The other two doses were administered in saline. Animals were bled 7 days after the last immunization. Antiserum was decomplemented (56°C for 30 min) and tested in RIA and neutralization assays. Specificities of pools of positive Ab3 antisera were determined by binding and neutralization, using the strain PUR46-MAD-CC120 of TGEV and, when indicated, MAb-resistant (mar) mutants escaping the neutralization by MAbs specific for subsites Aa (mar 1G.A7), Ab (mar 1D.E7), and Ac (mar 1B.B5) of the TGEV S glycoprotein. mar mutants have been described previously (6, 16, 21, 40). After selection of mar mutants, the neutralization titers of the MAbs used in the selection decreased from 4.8 to less than 0.5. Virus neutralization assays were carried out with 103 PFU per well. Ab3 antisera (1:4 dilutions) were mixed with an equal volume (50 Ll) of virus. The mixtures were incubated for 1 h at 37°C. Aliquots (50 [L) were plated onto an ST cell monolayer in 24-well culture dishes (Costar) and then incubated at 37°C for 1 h. Cells were overlayed with agarose, and plaque assays were developed as described previously (6). RESULTS Isolation of Ab2s. The procedure used to screen MAb2s was a sandwich RIA in which Abl coupled to the solid phase was bound to soluble 125I-labeled Abl by Ab2 from hybridoma supernatants. This procedure was specific, since Ab2s only interacted with the Abl used in the immunization (Fig. 1A and B). In addition, only immunoglobulins from mice immunized with this idiotype bound the Abl (Fig. 1A and B). Nonimmune sera of murine, porcine, feline, canine, ovine, or human origin did not bind Abl (results not shown). The highest production of Ab2 involved subcutaneous immunization with Abl immunoglobulins coupled to KLH. Administration of the two last antigen doses within the 24 h preceding the fusion also improved the results (data not shown). A total of 2,455 hybridomas were screened for the production of Ab2. Six hybrid cell lines (0.24%) secreted MAb2 specific for the immunizing idiotype (Table 1). Binding of 35S-labeled purified TGEV to MAbl-coated wells was inhibited by purified Ab2 immunoglobulins of the six Ab2s, as well as by unlabeled TGEV (Fig. 2), but not by a control immunoglobulin of the same isotype. The inhibition was not the result of the binding of MAb2 to virus (data not shown). These results show that MAb2 binds to MAbl close to its antigen-binding site, indicating that they were either of the -y or , type (12, 28). If the anti-idiotypes were behaving as an internal image, then, like the antigen, they should bind polyclonal anticoronavirus antisera which recognize the same subsite Ac as MAbl (37). Murine and porcine TGEV-specific antisera bound MAb2-9A.G3 and MAb2-9C.E11 but not the other four anti-idiotypic MAb2s (Table 2). Similar results were obtained with polyclonal antisera raised against a deletion
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Ab2s MIMICKING CORONAVIRUS EPITOPES
10 2560 640 1 /SERUM DILUTION
FIG. 1. Specificity of idiotype '25I-labeled MAbl-6A.C3 binding
to solid-phase-bound MAbl by anti-idiotypic antisera. Ninety-sixwell U-bottom polyvinyl plates were coated with 1 Kg of MAbl-
6A.C3 (A) or a control MAbl (P3X63Ag8) (22) (B) of the same isotype per well. Dilutions from immune mice serum (full symbols) and preimmune serum (empty symbols) were added in duplicate to wells. The plates were extensively washed, and the amount of bound antibodies was determined by using 251I-labeled MAbl6A.C3, as described in the text.
variant (PRCV) of TGEV which conserves sites A and D but not C and B (36, 37) (data not shown). Interestingly, the binding of polyclonal anti-TGEV sera to Ab2 was inhibited by native TGEV, PRCV, and FIPV, but not by partially
denatured TGEV in which subsite Ac had been inactivated (16) or by serologically unrelated coronaviruses MHV and HCV-229E (Fig. 3). In addition, mutant mar 1B.B5, which has a modified Ac subsite (16), inhibited the binding of the anti-TGEV-specific sera to Ab2 eightfold less than mar TABLE 1. Detection of hybridomas producing Ab2s by RIA based on the binding of 251I-labeled MAbl to the solid-phase-bound idiotype
Radioactivity (cpm/well)c bound to plates
coated with: Normal mouse immunoIdiotype
0.8 0.032 0.0012 COMPETITOR ANTI-IDIOTYPE, pg/well
IgGl(K) IgGl(K) IgGl(K) IgGl(K) IgGl(K) IgG2b(K)
7,064 9,364 4,790 2,238 7,018 7,434
149 151 172 180 192 190
Abl control 0.1% BSA in PBS Anti-idiotype serum diluted 1:20d Normal serum diluted 1:20e
101 117 7,673
181 145 151
a Undiluted hybridoma supernatants were used in the assays. b Determined by immunodiffusion with chain-specific antiserum. IgGl, immunoglobulin Gl. c Mean values of triplicate experiments are shown. The standard deviation was always less than 10o and is not shown. d Serum from mice immunized with MAb1-6A.C3 (see Materials and Methods). e Serum from nonimmune BALB/c mice.
FIG. 2. Inhibition of 35S-labeled TGEV binding to MAbl-6A.C3 by virus and MAb2. Binding of labeled TGEV to plates coated with MAbl-6A.C3 (see Materials and Methods) was inhibited by unlabeled TGEV (@); MAb2-2A.G3 (A), -9A.G3 (V), -9B.E6 (-), -9C.E11 (A), -12A.D12 (*), and -12G.H7 (O); and control MAbl (P3X63Ag8) (0).
mutants which had altered Aa or Ab subsites (results not shown). Binding of either one of the two putative MAb2-ps (MAb2-9A.G3 and MAb2-9C.E11) selected to the idiotype (MAb 6A.C3) was extensively inhibited by the other five MAb2s selected (Fig. 4), indicating that all Ab2s recognized closely located idiotopes. Induction of Ab3 by Ab2s. Mice immunized with an MAb exhibiting differential specificity produced no antiviral antibody (Fig. 5G). In contrast, all mice immunized with the 3-type MAb2-9A.G3 and MAb2-9C.Ell raised antibodies TABLE 2. Binding of MAb2 by TGEV-induced antisera in a solid-phase RIA
125I-labeled MAb2 (cpm/well)a bound by the following:
Ab2-2A.G3 Ab2-9A.G3 Ab2-9B.E6 Ab2-9C.Ell Ab2-12A.D12 Ab2-12G.H7
MAb2-9A.G3 MAb2-9C.E11 MAb2-12G.H7 MAb2-2A.G3 MAb2-9B.E6 MAb2-12A.D12 MAbl-P3
4,800 1,993 70
31 79 66 36 52 44 40
688 678 NDd ND ND
52 60 50
42 41 40 43
64 61 80 63 56
71 44 45 46 39
44 34 89 48
a The cross-linking of Ab2 bound to polyvinyl plates and soluble 125I_ labeled Ab2 by polyvalent antiserum elicited by TGEV was determined as described in Materials and Methods. Mean values of triplicate experiments are shown. The standard deviation was always less than 10% and is not shown. b The characteristics of the antisera are described in Materials and Methods. c Sera from nonimmune animals. d ND, not done.
SUNE ET AL.
A *1 Ab2 Abl ATor R K Ab otTGEV b2
9 or Ab oK TGEV
) E. 15 E "