JOURNAL OF VIROLOGY, OCt. 1992, p. 5744-5751 0022-538X/92/105744-08$02.00/0 Copyright © 1992, American Society for Microbiology

Vol. 66, No. 10

Monoclonal Anti-Idiotypes Induce Neutralizing Antibodies to Enterovirus 70 Conformational Epitopes JAMES A. WILEY,"2 JOSEE HAMEL,' AND BERNARD R. BRODEUR .2* National Laboratory for Immunology, Laboratory Center for Disease Control, Ottawa, Ontario, Canada KIA OL2,' and Department of Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Ottawa, Ontanio, Canada KIH 8MS2 Received 11 May 1992/Accepted 24 June 1992

Monoclonal antibodies (MAbs) directed against the prototype enterovirus 70 (EV-70) strain J670/71 were generated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogate immunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbs recognize conformational epitopes on the virion surface. An EV-70-neutralizing antibody, MAb/ev-12 (MAbl), was selected for the production of MAb2s. Five MAb2s were selected for their capacities to inhibit the interaction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. In addition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that they recognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2s recognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s were specific for private idiotopes. Immunization with each of the MAb2s was carried out for the production of anti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggested that they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well as heterologous Ab3s. Ab3 sera were shown to possess antibodies capable of immunoprecipitating 5S-labeled viral proteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3 neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s to serve as surrogate immunogens for conformational epitopes is substantiated by the results presented in this report.

Enterovirus 70 (EV-70) belongs to the family Picornaviridae, genus Enterovirus, and is the causative agent of acute hemorrhagic conjunctivitis (AHC). In the past 20 years, EV-70 has caused two major pandemics of AHC. Both of these pandemics have been confined to crowded coastal regions lying within what has been referred to as the "AHC belt" of the world (32). AHC, a self-limiting disease, is characterized by such symptoms as an acute onset of edema and hemorrhaging of the subconjunctiva and eyelids as well as the sensation of a foreign body in the eye (23, 38). However, outbreaks of EV-70 AHC have also been linked to neurological complications involving the central nervous system. Symptoms of these sequelae have included a poliomyelitislike paralysis of the limbs and acute isolated cranialnerve palsy (17, 39, 40). Like those of all picornaviruses, the EV-70 virion is composed of a positive-sense single strand of genomic RNA encapsulated by a naked protein shell derived from the interaction of four structural proteins. These four proteins are VP1 (35,000-molecular-weight protein [35K]), VP2 (28K), VP3 (27K), and VP4 (9K) (12). In other picornavi-

Discontinuous or conformational epitopes associated with virus neutralization have already been mapped on the surface of several picornaviruses (22, 26, 35, 41). The objective of this study was to elicit a protective immune response against EV-70 by using anti-idiotypic antibodies (Ab2s) which mimic conformational neutralizing epitopes on the virus. Such epitopes have been shown to be of importance in the pathogenesis of other viral infections (19). Ab2s are antibodies which are directed against the idiotypic determinants of another antibody. Idiotypic determinants are confined to the variable region of the immunoglobulin molecule. Ab2s have been categorized according to the locations of the specific idiotypic determinants they recognize (16). Ab2s which recognize a determinant distal to the paratope of the original antibody (Abl) are referred to as Ab2a. The binding of Ab2a does not inhibit the binding between the Abl and its antigen. If the target idiotope is located proximal to or overlaps with the paratope of the Abl such that the binding of Abl to its antigen is inhibited, then the Ab2 is referred to as Ab2y (10). Ab2s which bind directly to the paratope of Abl are categorized as Ab2I. Not only do Ab2Is inhibit antigen-Abl binding, but they are also said to possess the internal image of the original antigen, since both antigen and Ab2I bind to the same Abl paratope. Since Ab2,s possess the internal image of the original antigen, they have been used to elicit an antigen-specific response. The antigen-specific antibodies which constitute this response are referred to as anti-anti-idiotypic antibodies, or Ab3s. This paper describes the development and characterization of three generations of antibodies synthesized in BALB/c mice by using EV-70 as the original antigen. These

ruses, VP4 is concealed on the inner surface of the virion and appears not to be detected by the immune system. The remaining three proteins are of immunological significance. Their interactions are responsible for the configurations that form the epitopes on the virion surface which are recognized by the immune system and for the formation of cellular attachment sites. Since the configuration of such epitopes is conformationally dependent on the interaction of peptide chains, these epitopes are referred to as discontinuous (28). *

Corresponding author. 5744

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ANTI-IDIOTYPE-INDUCED EV-70-NEUTRALIZING ANTIBODIES

antibodies include (i) monoclonal antibodies (MAbs) specifically directed against EV-70, (ii) anti-idiotypic MAbs (MAb2s) specifically directed against an EV-70-reactive MAb, and (iii) Ab3s. We demonstrate that it is possible to elicit an Ab3 immune response which will specifically neutralize the original antigen, EV-70.

MATERIALS AND METHODS viruses. Rhesus monkey kidney cells, LLC-MK2 Cells and derivative (ATCC CCL 7.1), were used throughout this study for propagation of EV-70 and in the assays pertaining to the development of the antibodies produced. All strains of EV-70 used in this study were obtained from the Centers for Disease Control (Atlanta, Ga.). All other picornaviruses were obtained from the Viral Products Division of the Bureau of Biologics, Health and Welfare Canada, Ottawa, Ontario, Canada. EV-70 was purified from cell lysate by cesium chloride equilibrium density gradient centrifugation for 22 h at 260,000 x g in a Beckman SW50 rotor at 21°C. Gradient fractions were assayed for infectious virus. Animals. BALB/c mice were obtained from the Charles River Laboratories (St. Constant, Quebec, Canada). The (BALB/c x Swiss Webster)F1 mice used for ascites fluid production were obtained from the Animal Resources Division, Health Protection Branch, Health and Welfare Canada, Ottawa, Ontario, Canada. Immunization of mice with EV-70. Preimmune serum samples were taken from each mouse prior to immunization with purified EV-70. One hundred microliters of Freund's complete adjuvant-virus solution was injected intraperitoneally. Subsequent injections with Freund's incomplete adjuvant were given every 3 weeks. Trial bleeds were taken 1 week after each boost, and samples were tested by immunofluorescence (IF) and in plaque reduction assays to determine the EV-70-specific antibody titer. When the neutralization titer reached 1:4,000, the mouse in question was given a final boost and exsanguinated 4 days later. The spleen was removed for use in a fusion experiment to produce MAbs. Fusion procedure and MAb production. Spleen cells from immunized mice were fused with nonsecreting plasmacytoma SP2/0 as previously described (6). The hybridomas were cultured in modified Dulbecco's minimal essential media (GIBCO, Burlington, Ontario, Canada) supplemented with 15% bovine calf serum, 2 mM L-glutamine (Sigma Chemical Co., St. Louis, Mo.), and 50 p,g of gentamicin (Sigma) per ml in the presence of hypoxanthine, aminopterin, and thymidine (Sigma) as selective agents. Fourteen days after the fusion, aminopterin was removed from the culture medium. Class and subclass determinations of the MAbs were carried out with the Fisher Biotech classsubclass determination kit in accordance with the manufacturer's instructions (Fisher Scientific, Orangeburg, N.Y.). Selected hybridomas were cloned twice by limiting dilution and then used for ascites production according to the method of Brodeur and Tsang (7). In this study, we also used two EV-70-specific neutralizing MAbs, 72-SE and 73-2F, which were kindly donated by L. J. Anderson (3). Microneutralization assay. Initially, the hybridoma clones were screened for the production of neutralizing antibodies by using a microneutralization assay. Ninety-six-well tissue culture plates were seeded with LLC-MK2 cells and allowed to approach 100% confluence. Duplicate samples of 125 pl of hybridoma culture supernatant were removed from the fusion plates and incubated with 5,000 PFU of EV-70 for 60 min at 37°C. Culture supernatant from a hybridoma produc-

5745

ing antibody specific for cytomegalovirus was tested as a control. LLC-MK2 cell culture fluid was replaced with the virus-hybridoma culture supernatant mixture. Plates were incubated for 5 days at 33°C. On days 3, 4, and 5, each well was examined for cytopathic effect (CPE). The hybridomas corresponding to those wells which did not display any CPE relative to controls were retained for further testing. Plaque reduction assay. For all further characterizations of the antibody neutralizing activity, plaque reduction assays were carried out as previously described (42). A modification of this assay was used to show inhibition of neutralization by MAb2. A MAb dilution giving 90% neutralization was incubated with MAb2 for 60 min at 37°C prior to the addition of the virus (approximately 200 PFU). Following this, the assay was performed as described above. All plaque reduction and inhibition-of-neutralization assays were done in duplicate and repeated several times. Neutralization activity was considered present if 50% or more of the input PFU were neutralized. IF assay. Indirect IF assays were carried out as previously described (42). A modification of this assay was used to show inhibition of fluorescence by MAb2. A minimum amount of any EV-70 MAb giving a strong fluorescence reaction was preincubated with dilutions of MAb2 for 60 min at 37°C. The mixture was then applied to the wells, and the assay was completed as described above. RIP assay. All radioimmunoprecipitation (RIP) assays were performed as follows. LLC-MK2 cells were infected at a multiplicity of infection of 0.1 when they reached 90% confluence. A mock-infected culture served as a control. When infected cells reached + 1 CPE, all cultures were incubated in methionine-free medium (ICN Biochemicals Canada, Mississauga, Ontario, Canada) for 90 min. Following methionine starvation, 200 WCi of Tran (35S]methionine (ICN Biochemicals Canada) was added to each culture. The mock-infected control and one infected culture were then allowed to incubate for 3 h. A remaining infected culture was allowed to proceed to +4 CPE. Following the 3-h incubation, cells were scraped from the mock- and EV-70-infected cultures. Cells were washed three times in RIP buffer (50 mM Tris-HCl, 150 mM NaCl [pH 7.2]) at 4°C and then lysed in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1% sodium dodecyl sulfate [SDS], 0.5% sodium deoxycholate, 2% Triton X-100) at 4°C. The nuclei were pelleted at 16,000 x g for 60 s (Eppendorf centrifuge model 5415C), and supernatants were frozen at -20°C for future use. The culture, which was allowed to proceed to +4 CPE, was frozen and thawed three times, and the virus was purified by cesium chloride gradient

centrifugation.

One milliliter of antibody solution (ascites fluid at 1:1,000, serum-free tissue culture fluid at 1:2, or Ab3 serum at 1:10) was added to each radiolabeled sample; all samples contained equal amounts of [35Slmethionine. These mixtures were allowed to incubate for 1 h at 37°C or overnight at 4°C with constant agitation. Protein A-Sepharose beads (Pharmacia, Montreal, Quebec, Canada) were hydrated in phosphate-buffered saline (PBS) and incubated in 0.05% bovine serum albumin (BSA) for 1 h at 37°C to block nonspecific binding sites. Beads were washed three times in RIP buffer and suspended in serum-free medium to a final concentration of 30% (vol/vol). The suspension was then aliquoted in equal volumes to each of the samples. Samples were incubated for 1 h at 37°C or overnight at 4°C with constant agitation. The beads were then pelleted and washed three times in RIP buffer. Following the final wash, dissociation buffer (0.3 mM Tris [pH 6.81, 5% [wt/vol] SDS, 50% [vol/vol] glycerol,

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WILEY ET AL.

0.05% bromophenol blue, 10% [vol/vol] j-mercaptoethanol) added, and the samples were heated for 5 min in boiling water and centrifuged at 16,000 x g (Eppendorf centrifuge model 5415C) for 45 s. Supernatants were analyzed by electrophoresis on an SDS-polyacrylamide gel (18). The gel was dried and exposed to X-ray film (Cronex 4; Dupont, Wilmington, Del.) at -70°C for 18 h. Purification of antibody and preparation of F(ab)'2 fraction. The MAbs were purified from ascites fluid by passage through a protein A- or protein G-Sepharose column (Pierce Chemicals, Rockford, Ill.), depending on the isotype of the MAb to be purified, and eluted with 0.1 M glycine (pH 3.0). Purity of the preparation was assessed by SDS polyacrylamide gel electrophoresis (PAGE), and the biological activity was tested by IF and neutralization assays. The F(ab)'2 preparations were made by pepsin digestion of the purified immunoglobulin fractions according to the method described by Parham (25). Briefly, 25 mg of antibody was digested for 3 h at 37°C in 20 mM sodium acetate buffer, pH 4.1, by using 1.0 mg of pepsin immobilized on Sepharose beads (Pierce Chemicals). The digestion was stopped by the addition of 500 ,ul of 1.0 M Tris (pH 7.5). Beads were removed by centrifugation, and the supernatant was dialyzed against PBS (pH 7.2) for 16 h. The undigested immunoglobulin fraction was separated from the F(ab)'2 fraction by passage through a protein A-Sepharose column. The purity of the F(ab)'2 fraction was assessed by SDS-PAGE, and the biological activity was confirmed by IF. The F(ab)'2 preparation was titrated for use in enzyme immunoassay (EIA). Immunization with MAbs for production of Ab2s and Ab3s. MAbs were coupled to keyhole limpet hemocyanin (Sigma) at a 1:1 (wt/wt) ratio for 90 min at room temperature with constant agitation in the presence of 0.05% (final concentration) glutaraldehyde. The sample was then dialyzed against PBS (pH 7.2) for 24 h. Prior to the first injection, a sample of preimmune serum was taken from each mouse. Injections (80 ,ug of conjugate) were given intraperitoneally with Freund's adjuvant or subcutaneously with Quil A (Cedarlane Laboratories, Toronto, Ontario, Canada) (25 p,g per mouse) at 4-week intervals. Serum samples were taken 10 days after each injection and assessed for the presence of Ab2 by EIA and inhibition of neutralization. In the case of MAb2 production, if a sufficient Ab2 serum titer was detected, a final intravenous boost without Quil A was given. Four days later, the mouse was exsanguinated and the spleen was removed for hybridoma production. EIA screening for MAb2. The screening of MAb2 hybridoma supernatant was performed as described elsewhere (5, 15). In brief, EIAs were standardized so that each well was coated with 0.13 jig of the F(ab)'2 fraction of MAb/ev-12 in 100 ,ul of PBS (pH 7.2) overnight at room temperature. The plates were washed once in PBS-0.02% Tween 20 (PBSTween). Any remaining binding sites were blocked with 200 ,ul of 1% BSA in PBS (pH 7.2) for 1 h at 37°C followed by three washes with PBS-Tween. One hundred microliters of hybridoma supernatant was added to each well, and plates were incubated for 1 h at 37°C. Following three PBS-Tween washes, 100 ,ul of alkaline phosphatase-conjugated antimouse immunoglobulin G (IgG), Fc specific (Cappel-Organon Teknika, West Chester, Pa.), in 3% BSA-PBS (pH 7.2) was added to each well. The plates were incubated for 1 h at 37°C and then washed three times with PBS-Tween. Seventy-five microliters of 10% (vol/vol) diethanolamine (pH 9.8) containing 1 mg of p-nitrophenylphosphate (Sigma) per ml

TABLE 1. Characteristics of MAbs directed against EV-70

was

TiteiA MAb

MAb/ev-1 MAb/ev-6 MAb/ev-12 72-5EC 73-2Fc MAb/P2-4e

IF

8,000 8,000 200 500 500 0

g

Neutralization

isotype

0 50

16,000 >8,000 >8,000

3(K) 3(K) 2a(K) 3 2b

0

2a(K)

Specifici

All strains All strains Prototype J670/71 All strainsd

All strainsd No strains

a Reciprocal endpoint dilution of ascites fluid giving + 1 fluorescence (IF) or 50% reduction of 100 PFU per well (neutralization). I For other EV-70 strains tested in this report. c Anti-EV-70 MAb obtained from L. J. Anderson (3). d Tested by L. J. Anderson (3).

Anti-Haemophilus influenzae type b MAb (13).

was added. After 30 min at room temperature, the A410 was read with a Dynatech MR7000 Microplate Reader. A modification of this procedure was performed as a competition assay with Ab3 antisera. Inhibition of MAb2MAbW binding was assessed by prior incubation of MAb2 hybridoma supernatant with Ab3 antiserum for 1 h at 37°C. The mixture was then added to the F(ab)'2-coated plates for 1 h at 37°C. Following this, the assay was completed as described above. Inhibition of binding of MAb/ev-12 to EV-70 by MAb2. The dot immunobinding inhibition assay used in this study was modified from that described by Brodeur et al. (4). Virus was harvested, partially purified by removal of cellular debris, and pelleted. The virus suspension was diluted 1:10 in blotting buffer (10 mM Tris-HCl [pH 7.6], 150 mM NaCl) and aliquoted at 50 ,ul per well, and the plates were incubated at either 37°C for 1 h or 4°C overnight. The wells were blocked with 3% nonfat dry milk for 1 h at 37°C and then washed three times with PBS-Tween. One-hundred microliters of MAb/ev-12 hybridoma supernatant and 100 pu1 of an appropriate dilution of MAb2 hybridoma supernatant were incubated for 1 h at 37°C and then applied to the membrane. After a 60-min incubation at 37°C, the membrane was removed from the dot blot apparatus and blocked again with 3% nonfat dry milk for 30 min. "I-labeled anti-mouse IgG (0.1 ,uCi/ml; ICN Biochemicals Canada) was added, and the incubation was continued for 1 h at 37°C. The membrane was then washed, dried, and exposed to X-ray film (Cronex 4; Dupont) at -70°C for 18 h.

RESULTS Characterization of EV-70-specific MAbs. From four fusion experiments, a panel consisting of 12 EV-70-specific MAbs was made. MAb/ev-1 through MAb/ev-11 displayed either low or nonexistent neutralization titers against EV-70, whereas MAb/ev-12 showed a high EV-70 neutralization titer when tested by both microneutralization and plaque reduction assays (Table 1). The former group all carried the IgG3 isotype, and MAb/ev-12 was of the IgG2a isotype. All of the MAbs possessed kappa light chains. MAb/ev-1 and MAb/ev-6 were further characterized for this study. Both of these MAbs demonstrated strong IF reactivity with the prototype EV-70 strain J670/71 as well as with a selection of other EV-70 strains (KW97, R6, RU3875, 1604, and V1205) as previously described (42). However, MAb/ev-12 showed IF activity and neutralizing activity only against the prototype strain. Further evidence that target epitopes for MAb/

ANTI-IDIOTYPE-INDUCED EV-70-NEUTRALIZING ANTIBODIES

VOL. 66, 1992 a

b

c

d

e

f

9

h

i

j _oo

TABLE 2. Inhibition of MAb/ev-12 neutralizing activity by MAb2s

-68

Antibodiesa added

-43

to EV-70

-

29

-18

-14

FIG. 1. Autoradiogram of EV-70 proteins immunoprecipitated by anti-EV-70 MAbs. 35S-labeled mock-infected cell lysate, EV-70infected cell lysate, or purified EV-70 virus was immunoprecipitated with MAbs and then analyzed by SDS-PAGE as described in Materials and Methods. Lanes show MAb/ev-1 with mock-infected cell lysate (lane a), EV-70-infected cell lysate (lane b), or purified EV-70 virus (lane c); mock-infected cells alone (lane d); labeled molecular mass markers (in kilodaltons; lane e); purified EV-70 virus alone (lane f); MAb/ev-12 with purified EV-70 (lane g), EV-70-infected cell lysate (lane h), or mock-infected cell lysate (lane i); and control MAb/P2-4 with purified EV-70 virus (lane j).

ev-1 and MAb/ev-6 differed from that for MAb/ev-12 was provided by experiments showing that preincubation of the prototype strain with either of the former MAbs did not block neutralization by MAb/ev-12. None of the anti-EV-70specific MAbs showed IF reactivity against a panel of picornaviruses exclusive of EV-70. This panel consisted of coxsackievirus serotypes Bi and B3; echovirus serotypes 1, 9, and 23; poliovirus Sabin serotypes 1, 2, and 3; and human rhinovirus serotype 14. Western immunoblot and RIP assays were carried out with each of the MAbs. In Western immunoblot assays, no reactivity could be detected (data not shown). In RIP assays, the three major viral proteins were precipitated by all MAbs, suggesting the recognition of a conformational viral epitope. Representative RIP assay results are presented in Fig. 1. No reaction by the MAbs was observed with the mock-infected cell lysate. The reactivity of the MAbs for EV-70 was demonstrated by this lack of activity against the cellular components and by the corresponding lack of activity of an unrelated MAb against the purified virus. Characterization of MAb2s. The production of an Ab3neutralizing viral response by using MAb2s relies on mimicry of the virion neutralization epitope by the paratope of the MAb2. Since MAb/ev-12 possessed a high neutralization titer, it was chosen as antigen for production of MAb2s. Fusion experiments yielded several clones which, by ETA, bound F(ab)'2 fragments of MAb/ev-12. Five clones, all IgGl, were chosen for further study on the basis of their stability and levels of antibody secretion. In order to determine whether MAb2s were directed against paratope-associated idiotopes of MAb/ev-12, various blocking assays were performed. All of the MAb2s were capable of inhibiting both the IF and the neutralizing activity of MAb/ev-12, suggesting the paratope specificity of the MAb2s. Table 2 presents data representative of the ability of each of the MAb2s to inhibit the EV-70-specific neutralization capacity of MAb/ev-12. In the absence of a MAb2, MAb/ev-12 neutralized 90% of the PFU challenge. Incubation of MAb/ev-12 with any of the

5747

% Inhibition of neutralization

PFU/wellb PUwl"

0 15, 16 + medium 100 150, 130 + MAb2-2 100 160, 150 + MAb2-7 93 120, 140 + MAb2-9 89 + MAb2-13 126, 123 100 150, 150 MAb/ev-12 + MAb2-15 0 16, 16 MAb/ev-12 + MAb2/3C5C 100 146, 141 Medium + MAb2/3C5 a MAb2 and MAb/ev-12 were preincubated for 1 h at 37°C. They were then

MAb/ev-12 MAb/ev-12 MAb/ev-12 MAb/ev-12 MAb/ev-12

challenged with virus, and the mixture was incubated for 1 h at 37°C. The plaque assay procedure was then followed. b Residual PFU per well following a challenge of 140 PFU per well. Tested in duplicate. c MAb2 against anti-cytomegalovirus Bi MAbl (29).

MAb2s blocked the neutralization of EV-70. An unrelated MAb2 had no effect on the ability of MAb/ev-12 to neutralize EV-70 or on the viral challenge itself. In addition, none of the MAb2s alone neutralized EV-70 to any extent, nor did they react with noninfected LLC-MK2 cells in IF tests (data not shown). It is noteworthy that none of the MAb2s were able to prevent infection of cell monolayers. The specificities of the MAb2s for the paratope of MAbW ev-12 were also demonstrated by their abilities to block MAb/ev-12 binding to EV-70 in dot immunobinding inhibition assays (Fig. 2). Twofold titration of the MAb2s resulted in a dose-responsive decrease in this inhibition. In order to further investigate the specificities of the MAb2s for MAb/ev-12, two EV-70-specific neutralizing MAbs from another laboratory (3) plus MAb/ev-1 and MAb/ ev-6 were tested for recognition by each of the MAb2s. No recognition of these MAbs by any of the MAb2s was observed in IF inhibition and/or competition EIAs. This suggested that the idiotype of each of these four MAbs differed from that of MAb/ev-12. Characterization of Ab3 response. Each MAb2 was used as MAb2-2

0

MAb2-7

*

MAb2-9

*

MAb2-13

9

MAb2-15

*

MAb2/3C5 * 1

*

,

S a

*

9

9

9

2

3

4

5

6

7

FIG. 2. Autoradiogram of dot immunobinding assay of inhibition of MAb/ev-12 binding to EV-70 by MAb2s. MAb/ev-12 hybridoma supernatant was incubated with twofold dilutions of hybridoma supernatant of each MAb2 or an unrelated MAb2 (MAb2/3C5). The antibody preparations were then reacted with the semipurified EV-70 preparation bound to the nitrocellulose membrane, and MAb/ev-12 binding was revealed with '25I-labeled anti-mouse IgG. Viral material was omitted when control MAb2/3C5 was diluted 1:32. Lanes show MAb/ev-12 alone (lane 1); MAb/ev-12 preincubated with MAb2 diluted 1:2 (lane 2), 1:4 (lane 3), 1:8 (lane 4), 1:16 (lane 5), or 1:32 (lane 6); and MAb2 alone diluted 1:2 (lane 7).

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TABLE 3. Reactivity of Ab3s with MAb2 as tested by inhibition EIAW % inhibition of MAb/ev-12-MAb2 binding with Ab3 antiserumb MAb2

MAb2-2 MAb2-7 MAb2-9 MAb2-13 MAb2-15

Anti-MAb2-2

Anti-MAb2-7

Anti-MAb2-9

Anti-MAb2-13

Anti-MAb2-15

94 95 88 93 93

57 92 51 65 87

71 87 91 82 86

3 69 66 93 58

33 86 18 61 96

a MAb2 and Ab3 antisera (at 1:500) were preincubated together, and the mixture was added to MAb/ev-12-F(ab)'2-coated plates. The inhibition of MAb2 binding relative to that of controls was then determined by the addition of anti-mouse Fc-specific conjugate. b Values are means of six values: two tests per mouse and three mice per MAb2 group.

an antigen in the immunization of 15 mice for the production of Ab3 antisera. In competition EIAs, serum obtained following the third injection demonstrated that each mouse had made an Ab3 response to its MAb2 challenge. As shown in Table 3, each homologous MAb2-MAb interaction was inhibited by more than 90% by the corresponding Ab3 antiserum at a 1:500 dilution of Ab3. This level of inhibition gradually subsided to 50% or less at 1:10,000 (data not shown). The inhibition levels observed in the heterologous MAb2-MAb competitions varied over a wide range, which implied the cross-reactivity of each Ab3 serum with most of the other MAb2s. Two exceptions to this were that Ab3 serum raised to MAb2-13 did not recognize MAb2-2, and Ab3 serum raised to MAb2-15 did not recognize MAb2-2 and MAb2-9. Since MAb2s had clearly stimulated immune responses in these mice, we next wanted to determine whether the Ab3 antisera could neutralize the infectivity of EV-70. As illustrated in Table 4, the immunization of syngeneic mice with MAb2s elicited a neutralizing immune response specific for EV-70. Mice which received MAb2-2 produced the best neutralizing response. Sera from the three mice in this group showed more than 50% neutralization activity at an Ab3 serum dilution of 1:25. Of the mice which received MAb2-9 and MAb2-15, two of the three in each group displayed a 50% or greater neutralization activity at this serum dilution. Of the mice which received MAb2-7 and MAb2-13, only one mouse from each group displayed a 50% neutralization activity at an Ab3 dilution of 1:25. In an effort to assess whether higher neutralization titers could be generated, a group of mice were immunized with MAb2-2 or MAb2-15 and either Freund's adjuvant or Quil A. However, no differences in the neutralization titers were observed following four injections in a 12-week immunization regimen. The highest 50% neutralization titers were

from a mouse which received MAb2-15 with Freund's adjuvant (1:200) and from a mouse which received MAb2-2 with Quil A (1:100) (data not shown). Serum from the mouse which produced the highest Ab3 neutralization titer was tested against the same heterologous EV-70 strains as MAb/ev-12 had been tested against. Neither the Ab3 serum nor MAb/ev-12 demonstrated any neutralizing activity against these heterologous EV-70 strains. This suggested that the specificities of the two preparations were identical. The MAb/ev-12-like specificity of the Ab3s for EV-70 proteins was further assessed by RIP. Serum from a mouse immunized with MAb2-15 in Freund's adjuvant and serum from a mouse immunized with MAb2-2 in Quil A were tested. As seen in Fig. 3, the same viral bands were precipitated with anti-MAb2-15 antiserum (lane d) and antiMAb2-2 antiserum (lane g) as were observed with MAb/ ev-12 (lane a). The reactivity of the Ab3 antiserum for EV-70 was supported by the absence of these viral bands when an unrelated Ab3 antiserum was used (lane b) and by the fact that no cellular material reacted with either of the two antisera (lanes c and h). Evidence that each of the MAb2s was capable of inhibiting the neutralizing activity of MAb/ev-12 was presented above

a

b

d

e

f

9

h

-97 _68

_

29

_14 TABLE 4. Neutralizing activity of Ab3 antiserum against EV-70 % Neutralization' at Ab3 serum dilution of: Ab3 antiserum

Preimmune Anti-MAb2-2 Anti-MAb2-7 Anti-MAb2-9 Anti-MAb2-13 Anti-MAb2-15

0 86, 59, 70, 54, 82,

1:25

1:50

77, 34, 61, 40, 54,

ND 54, 45, 14 12, 12, 0 14, 0, 0 15, 0, 0 35, 0, 0

67 18 27 12 33

a Results are relative to results for preimmune sera of 15 mice following challenge of 110 PFU per well. ND, not done. Data for individual animals are given. Tests were done in duplicate.

FIG. 3. Autoradiogram of EV-70 proteins immunoprecipitated by Ab3 antiserum. 35S-labeled mock-infected cell lysate or purified EV-70 virus was immunoprecipitated with Ab3 antiserum and then analyzed by SDS-PAGE as described in Materials and Methods. Lanes show MAb/ev-12 with purified EV-70 virus (lane a); unrelated Ab3 antiserum with purified EV-70 (lane b); anti-MAb2-2 antiserum with mock-infected cell lysate (lane c) or purified EV-70 virus (lane d); labeled molecular mass markers (in kilodaltons; lane e); purified EV-70 virus alone (lane f); and anti-MAb2-15 antiserum with purified EV-70 (lane g) or with mock-infected cell lysate (lane h).

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ANTI-IDIOTYPE-INDUCED EV-70-NEUTRALIZING ANTIBODIES

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TABLE 5. Inhibition of Ab3 neutralizing activity by homologous MAb2 Residual PFU/well at MAb2 inhibitor dilution of°:

Ab3 antiserum

Anti-MAb2-2 Anti-MAb2-9 Anti-MAb2-15

No inhibitor

1:50,000

1:5,000

0, 8 0, 2 0, 1

0, 0 1, 2 0, 0

0, 0 2, 4 1, 3

1:1,000

0, 0 ND > 100, > 100

1:500

1:100

1:10

0, 1 40, 68 > 100, > 100

>100, >100 ND > 100, > 100

102, 87 ND > 100, > 100

a Ab3 antisera diluted 1:25 were preincubated with various dilutions of MAb2 ascites fluid prior to virus neutralization assay. Results are from duplicate tests with a 140-PFU challenge of EV-70. ND, not done.

(Table 2). In a similar manner, the ability to inhibit Ab3 neutralization activity by using the homologous MAb2 was demonstrated (Table 5). The degree of inhibition reflected the titers of both the Ab3 antiserum and MAb2. Complete inhibition of neutralization was observed at low dilutions of the homologous MAb2. As the MAb2 was further diluted, the neutralizing activity of the Ab3 serum became evident. DISCUSSION In order to produce MAb2s and investigate their effectiveness as surrogate immunogens, we first made EV-70-specific MAbs. Of the EV-70 MAbs described in this study, we found that those which possess the highest IF titers exhibit little or no neutralization activity, whereas MAb/ev-12, which has a high neutralizing titer, has a comparatively lower IF titer. In this regard, our MAbs are similar to other previously described EV-70-specific MAbs (3). In addition, those of our MAbs which shared a higher IF titer were reactive with all EV-70 strains tested, whereas MAb/ev-12 was specific for the J670/71 strain only. This implies the existence of at least two epitopes on the prototype strain, with one neutralizing and the other nonneutralizing. The nonneutralizing epitope seems to be conserved in all of the EV-70 strains used in this

study. The reactivity of the EV-70-specific MAbs in Western immunoblot and RIP assays suggests that epitopes are conformationally dependent. Dissociation of the virions prior to exposure to the MAbs, as in Western immunoblot assays, precluded antibody binding. This is analogous to the failure of picornaviruses to recognize cellular receptors following modification of their viral capsids (9). Conversely, the MAbs were able to recognize virions that had not been denatured, as depicted in the RIP assays (Fig. 1), suggesting the recognition by the MAbs of conformation-dependent discontinuous epitopes. The selection of MAb/ev-12 for the production of MAb2s is based on the potential biological importance of MAb/ev12-recognized epitopes in the pathogenesis of EV-70. MAb/ ev-12 recognition of EV-70 was inhibited by each of the MAb2s produced (Fig. 2 and Table 3). No inhibition of anti-EV-70 activity was recorded when four other EV-70specific MAbs were tested with each of the MAb2s. In addition, MAb/ev-12 is the only antibody capable of inhibiting MAb2-MAb1 binding, suggesting that the idiotope recognized on MAb/ev-12 is a private idiotope and is not shared with these other MAbs. The ability of each of the MAb2s to specifically inhibit the anti-EV-70 activity of MAb/ev-12 suggests that the idiotope they recognize on MAb/ev-12 is juxtaposed to, overlaps with, or lies within the MAb/ev-12 paratope that recognizes the viral epitope associated with neutralization. Therefore, by definition, these MAb2s are either Ab2y or Ab2p (10, 16).

Since Ab2,s mimic the conformation of epitopes, reactivity with viral receptors on host cell surfaces may be a feature of some of them. In fact, this has been the focus of much research regarding these antibodies (1, 8, 20, 21, 43). In this study, none of the MAb2s reacted with uninfected cell monolayers, nor did they protect susceptible cells from viral infection. This suggests that the MAb2s elicited in response to MAb/ev-12 do not recognize the cellular structure used for viral attachment. The recognition by MAb/ev-12 of a neutralizing epitope which is not part of the viral attachment site would explain the inability of the MAb2s to recognize the corresponding cellular structure in a manner similar to that postulated for coxsackievirus B4 (21). This may be due to a canyonlike structure which has been found on certain picornaviruses (30, 31, 36). Functional differentiation of Ab2a, Ab2y, and Ab2p may be achieved by analyzing the Ab3 immune response elicited following immunization with the MAb2s (10, 11, 16). As a result of molecular mimicry, Ab2,B should be able to stimulate antigen-specific immune responses and serve as a vaccine. However, complexity in Ab2 classifications has resulted in some instances in which Ab2aos were also able to elicit an antigen-specific immune response in the absence of the original antigen (13, 33). Although Ab2, is still considered the ideal vaccine candidate, the induction of Abl-like antibodies which possess idiotope markers and antigenbinding specificities identical to those of Abl is a mandatory prerequisite in the selection of a candidate Ab2. In this study, the immunization regimen used for the production of Ab3 antiserum resulted in high Ab3 titers. The higher levels of inhibition seen in the homologous MAb2-MAb/ev-12 interactions relative to that seen for the heterologous MAb2s indicate a greater degree of specificity for the homologous MAb2s. The wide range of inhibition levels in the heterologous MAb2-MAb interactions suggests that despite the presence of shared idiotopes, the MAb2s may be distinct from each other. Thus, in addition to Ab3s specific for a homologous MAb2, an Ab3 antiserum may or may not contain cross-reactive antibodies recognizing heterologous MAb2s. The presence and absence of such cross-reactive antibodies are exemplified by the Ab3 antisera elicited by using MAb2-13 and MAb2-15. Neither of these antisera was able to inhibit the binding of all heterologous MAb2s to MAb. This distinctiveness may apply not only to the physical structure of the MAb2s but also to their regulatory roles in the immune response. Confirmation of the Abl-like properties of our Ab3 antisera was crucial to the legitimacy of using the MAb2s as surrogate immunogens. Of the five MAb2s used, MAb2-2, MAb2-9, and MAb2-15 elicited 50% neutralization activity at an Ab3 serum dilution of 1:25 in all or most of the mice immunized. It is noteworthy that those mice which received MAb2-2 produced the highest EV-70 neutralization activity

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WILEY ET AL.

and also demonstrated a consistently high level of inhibition of the homologous and heterologous MAb2-MAb/ev-12 interaction. On an individual basis, the highest Ab3 50% neutralization titers were obtained from mice which received either MAb2-15 in Freund's adjuvant (1:200) or MAb2-2 in Quil A (1:100). The Ab3 viral neutralization titers reported here and the ranges over which they vary are comparable with those reported for other viral systems (24, 27, 37). It is also possible that injection of xenogeneic polyclonal Ab2s rather than syngeneic MAb2s would induce a significantly higher Ab3 neutralizing response. The fact that the Ab3 antisera possess Abl-like properties is demonstrated by the ability of homologous MAb2s to inhibit both Ab3 and

MAb/ev-12 neutralizing activity. It has been demonstrated in influenza and respiratory syncytial virus models (2, 24) that Abl and antigen-reactive Ab3s may have distinct specificities for the antigen. The level of fidelity of the immune response manifested in our study permitted the restricted neutralization spectrum of MAb/ev-12 for the prototype J670/71 strain to be maintained by the Ab3s. In RIP assays, Ab3s recognized an EV-70 surface epitope which caused the immunoprecipitation of the same EV-70 proteins as MAb/ev-12. Maintenance of such a strain-specific immune response has also been demonstrated previously for an Ab3 to reovirus type 3 hemagglutinin (14, 34). In summary, we have generated MAb2s which elicited a neutralizing response to the prototype strain of EV-70. The facts that the elicited Ab3 response is against a conformational epitope and that both synthetic peptide and recombinant DNA technologies have not yet been able to fully mimic such epitopes reinforce the value of Ab2s as surrogate immunogens in the study of viral pathogenesis. REFERENCES 1. Abdelmagid, 0. Y., D. J. Orten, W. Xue, F. Blecha, and H. C. Minocha. 1992. Anti-idiotypic antibodies to bovine herpes-1 inhibit virus infection in cell cultures. Arch. Virol. 122:163-173. 2. Anders, E. M., G. P. Kapaklis-Deliyannis, and D. 0. White. 1989. Induction of immune response to influenza virus with anti-idiotypic antibodies. 63:2758-2767. 3. Anderson, L. J., M. H. Hatch, M. F. Flemister, and G. E. Marchetti. 1984. Detection of enterovirus 70 with monoclonal antibodies. J. Clin. Microbiol. 20:405-408. 4. Brodeur, B. R., S. Faucher, M. V. O'Shaughnessy, and J. Hamel. 1991. Monoclonal idiotypic and anti-idiotypic antibodies to human immunodeficiency virus type 1 envelope glycoprotein. J. Gen. Virol. 72:51-58. 5. Brodeur, B. R., J. Hamel, D. Martin, and P. Chong. 1992. Synthetic peptides and anti-idiotypic antibodies as immunogens for the induction of antibody response to Haemophilus influenzae type b. J. Infect. Dis. 165(Suppl. 1):106-108. 6. Brodeur, B. R., Y. Larose, P. Tsang, J. Hamel, F. Ashton, and A. Ryan. 1985. Protection against infection with Neisseria meningitidis group B serotype 2b by passive immunization with serotype-specific monoclonal antibody. Infect. Immun. 50:510516. 7. Brodeur, B. R., and P. S. Tsang. 1986. High yield monoclonal antibody production in ascites. J. Immunol. Methods 86:239241. 8. Co, M. S., G. N. Gaulton, B. N. Fields, and M. I. Greene. 1985. Isolation and biochemical characterization of the mammalian reovirus type 3 cell-surface receptor. Proc. Natl. Acad. Sci. USA 82:1494-1498. 9. Colonno, R. J. 1987. Cell surface receptors for picornaviruses. BioEssays 5:270-275. 10. Dalgleish, A. G., and R. C. Kennedy. 1988. Anti-idiotypic antibodies as immunogens: idiotypic-based vaccines. Vaccine 6:215-220.

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