JOURNAL OF VIROLOGY, OCt. 1992, p. 5744-57510022-538X/92/105744-08$02.00/0Copyright © 1992, American Society for Microbiology

Vol. 66, No. 10

Monoclonal Anti-Idiotypes Induce Neutralizing Antibodiesto Enterovirus 70 Conformational EpitopesJAMES 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 ofMicrobiology 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 weregenerated and characterized in order to produce anti-idiotypic MAbs (MAb2s) for use as surrogateimmunogens. Western immunoblot and radioimmunoprecipitation assays suggested that all the MAbsrecognize 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 theinteraction of MAb/ev-12 with EV-70 in dot immunobinding inhibition and immunofluorescence assays. Inaddition, these five MAb2s inhibited virus neutralization mediated by MAb/ev-12, suggesting that theyrecognize paratope-associated idiotopes. In competition enzyme immunosorbent assays, none of the five MAb2srecognized other neutralizing and nonneutralizing EV-70-specific MAbs, demonstrating that the MAb2s werespecific for private idiotopes. Immunization with each of the MAb2s was carried out for the production ofanti-anti-idiotypic antibodies (Ab3). All five MAb2s induced an immune response. Moreover, results suggestedthat they share idiotopes, since MAb2-MAb/ev-12 binding could be inhibited by homologous as well asheterologous Ab3s. Ab3 sera were shown to possess antibodies capable ofimmunoprecipitating 5S-labeled viralproteins in the same manner as MAb/ev-12. Nine of 15 mice immunized with MAb2s demonstrated Ab3neutralizing activity specific for the prototype EV-70 strain, J670/71. The potential application of MAb2s toserve as surrogate immunogens for conformational epitopes is substantiated by the results presented in thisreport.

Enterovirus 70 (EV-70) belongs to the family Picornavir-idae, genus Enterovirus, and is the causative agent of acutehemorrhagic conjunctivitis (AHC). In the past 20 years,EV-70 has caused two major pandemics of AHC. Both ofthese pandemics have been confined to crowded coastalregions lying within what has been referred to as the "AHCbelt" of the world (32). AHC, a self-limiting disease, ischaracterized by such symptoms as an acute onset of edemaand hemorrhaging of the subconjunctiva and eyelids as wellas the sensation of a foreign body in the eye (23, 38).However, outbreaks of EV-70 AHC have also been linked toneurological complications involving the central nervoussystem. Symptoms of these sequelae have included a polio-myelitislike paralysis of the limbs and acute isolated cranial-nerve palsy (17, 39, 40).

Like those of all picornaviruses, the EV-70 virion iscomposed of a positive-sense single strand of genomic RNAencapsulated by a naked protein shell derived from theinteraction of four structural proteins. These four proteinsare VP1 (35,000-molecular-weight protein [35K]), VP2(28K), VP3 (27K), and VP4 (9K) (12). In other picornavi-ruses, VP4 is concealed on the inner surface of the virion andappears not to be detected by the immune system. Theremaining three proteins are of immunological significance.Their interactions are responsible for the configurations thatform the epitopes on the virion surface which are recognizedby the immune system and for the formation of cellularattachment sites. Since the configuration of such epitopes isconformationally dependent on the interaction of peptidechains, these epitopes are referred to as discontinuous (28).

* Corresponding author.

Discontinuous or conformational epitopes associated withvirus neutralization have already been mapped on the sur-face of several picornaviruses (22, 26, 35, 41). The objectiveof this study was to elicit a protective immune responseagainst EV-70 by using anti-idiotypic antibodies (Ab2s)which mimic conformational neutralizing epitopes on thevirus. Such epitopes have been shown to be of importance inthe pathogenesis of other viral infections (19).Ab2s are antibodies which are directed against the idio-

typic determinants of another antibody. Idiotypic determi-nants are confined to the variable region of the immunoglob-ulin molecule. Ab2s have been categorized according to thelocations of the specific idiotypic determinants they recog-nize (16). Ab2s which recognize a determinant distal to theparatope of the original antibody (Abl) are referred to asAb2a. The binding of Ab2a does not inhibit the bindingbetween the Abl and its antigen. If the target idiotope islocated proximal to or overlaps with the paratope of the Ablsuch that the binding of Abl to its antigen is inhibited, thenthe Ab2 is referred to as Ab2y (10). Ab2s which bind directlyto the paratope of Abl are categorized as Ab2I. Not only doAb2Is inhibit antigen-Abl binding, but they are also said topossess the internal image of the original antigen, since bothantigen and Ab2I bind to the same Abl paratope. SinceAb2,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 re-sponse are referred to as anti-anti-idiotypic antibodies, orAb3s.

This paper describes the development and characteriza-tion of three generations of antibodies synthesized inBALB/c mice by using EV-70 as the original antigen. These

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antibodies include (i) monoclonal antibodies (MAbs) specif-ically directed against EV-70, (ii) anti-idiotypic MAbs(MAb2s) specifically directed against an EV-70-reactiveMAb, and (iii) Ab3s. We demonstrate that it is possible toelicit an Ab3 immune response which will specifically neu-tralize the original antigen, EV-70.

MATERIALS AND METHODS

Cells and viruses. Rhesus monkey kidney cells, LLC-MK2derivative (ATCC CCL 7.1), were used throughout thisstudy for propagation of EV-70 and in the assays pertainingto the development of the antibodies produced. All strains ofEV-70 used in this study were obtained from the Centers forDisease Control (Atlanta, Ga.). All other picornaviruseswere obtained from the Viral Products Division of theBureau of Biologics, Health and Welfare Canada, Ottawa,Ontario, Canada. EV-70 was purified from cell lysate bycesium chloride equilibrium density gradient centrifugationfor 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 fluidproduction were obtained from the Animal Resources Divi-sion, Health Protection Branch, Health and Welfare Canada,Ottawa, Ontario, Canada.Immunization of mice with EV-70. Preimmune serum sam-

ples were taken from each mouse prior to immunization withpurified EV-70. One hundred microliters of Freund's com-plete adjuvant-virus solution was injected intraperitoneally.Subsequent injections with Freund's incomplete adjuvantwere given every 3 weeks. Trial bleeds were taken 1 weekafter each boost, and samples were tested by immunofluo-rescence (IF) and in plaque reduction assays to determinethe EV-70-specific antibody titer. When the neutralizationtiter reached 1:4,000, the mouse in question was given a finalboost and exsanguinated 4 days later. The spleen wasremoved for use in a fusion experiment to produce MAbs.

Fusion procedure and MAb production. Spleen cells fromimmunized mice were fused with nonsecreting plasmacy-toma SP2/0 as previously described (6). The hybridomaswere cultured in modified Dulbecco's minimal essentialmedia (GIBCO, Burlington, Ontario, Canada) supplementedwith 15% bovine calf serum, 2 mM L-glutamine (SigmaChemical Co., St. Louis, Mo.), and 50 p,g of gentamicin(Sigma) per ml in the presence of hypoxanthine, aminop-terin, and thymidine (Sigma) as selective agents. Fourteendays after the fusion, aminopterin was removed from theculture medium. Class and subclass determinations of theMAbs were carried out with the Fisher Biotech class-subclass determination kit in accordance with the manufac-turer's instructions (Fisher Scientific, Orangeburg, N.Y.).Selected hybridomas were cloned twice by limiting dilutionand then used for ascites production according to the methodof Brodeur and Tsang (7). In this study, we also used twoEV-70-specific neutralizing MAbs, 72-SE and 73-2F, whichwere kindly donated by L. J. Anderson (3).

Microneutralization assay. Initially, the hybridoma cloneswere screened for the production of neutralizing antibodiesby using a microneutralization assay. Ninety-six-well tissueculture plates were seeded with LLC-MK2 cells and allowedto approach 100% confluence. Duplicate samples of 125 pl ofhybridoma culture supernatant were removed from the fu-sion plates and incubated with 5,000 PFU of EV-70 for 60min at 37°C. Culture supernatant from a hybridoma produc-

ing antibody specific for cytomegalovirus was tested as acontrol. LLC-MK2 cell culture fluid was replaced with thevirus-hybridoma culture supernatant mixture. Plates wereincubated for 5 days at 33°C. On days 3, 4, and 5, each wellwas examined for cytopathic effect (CPE). The hybridomascorresponding to those wells which did not display any CPErelative to controls were retained for further testing.

Plaque reduction assay. For all further characterizations ofthe antibody neutralizing activity, plaque reduction assayswere carried out as previously described (42). A modificationof this assay was used to show inhibition of neutralization byMAb2. A MAb dilution giving 90% neutralization was incu-bated with MAb2 for 60 min at 37°C prior to the addition ofthe virus (approximately 200 PFU). Following this, the assaywas performed as described above. All plaque reduction andinhibition-of-neutralization assays were done in duplicateand repeated several times. Neutralization activity wasconsidered present if 50% or more of the input PFU wereneutralized.

IF assay. Indirect IF assays were carried out as previouslydescribed (42). A modification of this assay was used toshow inhibition of fluorescence by MAb2. A minimumamount of any EV-70 MAb giving a strong fluorescencereaction was preincubated with dilutions of MAb2 for 60 minat 37°C. The mixture was then applied to the wells, and theassay was completed as described above.RIP assay. All radioimmunoprecipitation (RIP) assays

were performed as follows. LLC-MK2 cells were infected ata 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 wereincubated in methionine-free medium (ICN BiochemicalsCanada, Mississauga, Ontario, Canada) for 90 min. Follow-ing methionine starvation, 200 WCi of Tran (35S]methionine(ICN Biochemicals Canada) was added to each culture. Themock-infected control and one infected culture were thenallowed to incubate for 3 h. A remaining infected culture wasallowed to proceed to +4 CPE. Following the 3-h incuba-tion, cells were scraped from the mock- and EV-70-infectedcultures. Cells were washed three times in RIP buffer (50mM Tris-HCl, 150 mM NaCl [pH 7.2]) at 4°C and then lysedin lysis buffer (50mM Tris-HCl, 150mM NaCl, 0.1% sodiumdodecyl sulfate [SDS], 0.5% sodium deoxycholate, 2% Tri-ton X-100) at 4°C. The nuclei were pelleted at 16,000 x g for60 s (Eppendorf centrifuge model 5415C), and supernatantswere frozen at -20°C for future use. The culture, which wasallowed to proceed to +4 CPE, was frozen and thawed threetimes, and the virus was purified by cesium chloride gradientcentrifugation.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 con-tained equal amounts of [35Slmethionine. These mixtureswere allowed to incubate for 1 h at 37°C or overnight at 4°Cwith constant agitation. Protein A-Sepharose beads (Phar-macia, Montreal, Quebec, Canada) were hydrated in phos-phate-buffered saline (PBS) and incubated in 0.05% bovineserum albumin (BSA) for 1 h at 37°C to block nonspecificbinding sites. Beads were washed three times in RIP bufferand suspended in serum-free medium to a final concentrationof 30% (vol/vol). The suspension was then aliquoted in equalvolumes to each of the samples. Samples were incubated for1 h at 37°C or overnight at 4°C with constant agitation. Thebeads were then pelleted and washed three times in RIPbuffer. Following the final wash, dissociation buffer (0.3 mMTris [pH 6.81, 5% [wt/vol] SDS, 50% [vol/vol] glycerol,

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5746 WILEY ET AL.

0.05% bromophenol blue, 10% [vol/vol] j-mercaptoethanol)was added, and the samples were heated for 5 min in boilingwater and centrifuged at 16,000 x g (Eppendorf centrifugemodel 5415C) for 45 s. Supernatants were analyzed byelectrophoresis on an SDS-polyacrylamide gel (18). The gelwas 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 (PierceChemicals, Rockford, Ill.), depending on the isotype of theMAb to be purified, and eluted with 0.1 M glycine (pH 3.0).Purity of the preparation was assessed by SDS polyacryl-amide gel electrophoresis (PAGE), and the biological activ-ity was tested by IF and neutralization assays.The F(ab)'2 preparations were made by pepsin digestion of

the purified immunoglobulin fractions according to themethod described by Parham (25). Briefly, 25 mg of antibodywas 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 Sepharosebeads (Pierce Chemicals). The digestion was stopped by theaddition of 500 ,ul of 1.0 M Tris (pH 7.5). Beads were

removed by centrifugation, and the supernatant was dia-lyzed against PBS (pH 7.2) for 16 h. The undigested immu-noglobulin fraction was separated from the F(ab)'2 fractionby passage through a protein A-Sepharose column. Thepurity of the F(ab)'2 fraction was assessed by SDS-PAGE,and the biological activity was confirmed by IF. The F(ab)'2preparation was titrated for use in enzyme immunoassay(EIA).Immunization with MAbs for production ofAb2s and Ab3s.

MAbs were coupled to keyhole limpet hemocyanin (Sigma)at a 1:1 (wt/wt) ratio for 90 min at room temperature withconstant agitation in the presence of 0.05% (final concentra-tion) glutaraldehyde. The sample was then dialyzed againstPBS (pH 7.2) for 24 h. Prior to the first injection, a sample ofpreimmune serum was taken from each mouse. Injections(80 ,ug of conjugate) were given intraperitoneally with Fre-und's adjuvant or subcutaneously with Quil A (CedarlaneLaboratories, Toronto, Ontario, Canada) (25 p,g per mouse)at 4-week intervals. Serum samples were taken 10 days aftereach injection and assessed for the presence of Ab2 by EIAand inhibition of neutralization. In the case of MAb2 pro-duction, if a sufficient Ab2 serum titer was detected, a finalintravenous boost without Quil A was given. Four dayslater, the mouse was exsanguinated and the spleen wasremoved for hybridoma production.EIA screening for MAb2. The screening of MAb2 hybrid-

oma supernatant was performed as described elsewhere (5,15). In brief, EIAs were standardized so that each well wascoated with 0.13 jig of the F(ab)'2 fraction of MAb/ev-12 in100 ,ul of PBS (pH 7.2) overnight at room temperature. Theplates were washed once in PBS-0.02% Tween 20 (PBS-Tween). Any remaining binding sites were blocked with 200,ul of 1% BSA in PBS (pH 7.2) for 1 h at 37°C followed bythree washes with PBS-Tween. One hundred microliters ofhybridoma supernatant was added to each well, and plateswere incubated for 1 h at 37°C. Following three PBS-Tweenwashes, 100 ,ul of alkaline phosphatase-conjugated anti-mouse immunoglobulin G (IgG), Fc specific (Cappel-Orga-non Teknika, West Chester, Pa.), in 3% BSA-PBS (pH 7.2)was added to each well. The plates were incubated for 1 h at37°C and then washed three times with PBS-Tween. Seven-ty-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

TiteiA gMAb

IF Neutralization isotype Specifici

MAb/ev-1 8,000 0 3(K) All strainsMAb/ev-6 8,000 50 3(K) All strainsMAb/ev-12 200 16,000 2a(K) Prototype J670/7172-5EC 500 >8,000 3 All strainsd73-2Fc 500 >8,000 2b All strainsdMAb/P2-4e 0 0 2a(K) No strains

a Reciprocal endpoint dilution of ascites fluid giving + 1 fluorescence (IF) or50% 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 wasread with a Dynatech MR7000 Microplate Reader.A modification of this procedure was performed as a

competition assay with Ab3 antisera. Inhibition of MAb2-MAbW binding was assessed by prior incubation of MAb2hybridoma supernatant with Ab3 antiserum for 1 h at 37°C.The mixture was then added to the F(ab)'2-coated plates for1 h at 37°C. Following this, the assay was completed asdescribed above.

Inhibition of binding of MAb/ev-12 to EV-70 by MAb2. Thedot immunobinding inhibition assay used in this study wasmodified from that described by Brodeur et al. (4). Virus washarvested, partially purified by removal of cellular debris,and pelleted. The virus suspension was diluted 1:10 inblotting buffer (10 mM Tris-HCl [pH 7.6], 150mM NaCl) andaliquoted at 50 ,ul per well, and the plates were incubated ateither 37°C for 1 h or 4°C overnight. The wells were blockedwith 3% nonfat dry milk for 1 h at 37°C and then washedthree times with PBS-Tween. One-hundred microliters ofMAb/ev-12 hybridoma supernatant and 100 pu1 of an appro-priate dilution of MAb2 hybridoma supernatant were incu-bated for 1 h at 37°C and then applied to the membrane.After a 60-min incubation at 37°C, the membrane wasremoved from the dot blot apparatus and blocked again with3% nonfat dry milk for 30 min. "I-labeled anti-mouse IgG(0.1 ,uCi/ml; ICN Biochemicals Canada) was added, and theincubation was continued for 1 h at 37°C. The membrane wasthen 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 fusionexperiments, a panel consisting of 12 EV-70-specific MAbswas made. MAb/ev-1 through MAb/ev-11 displayed eitherlow or nonexistent neutralization titers against EV-70,whereas MAb/ev-12 showed a high EV-70 neutralizationtiter when tested by both microneutralization and plaquereduction assays (Table 1). The former group all carried theIgG3 isotype, and MAb/ev-12 was of the IgG2a isotype. Allof the MAbs possessed kappa light chains. MAb/ev-1 andMAb/ev-6 were further characterized for this study. Both ofthese MAbs demonstrated strong IF reactivity with theprototype EV-70 strain J670/71 as well as with a selection ofother EV-70 strains (KW97, R6, RU3875, 1604, and V1205)as previously described (42). However, MAb/ev-12 showedIF activity and neutralizing activity only against the proto-type strain. Further evidence that target epitopes for MAb/

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ANTI-IDIOTYPE-INDUCED EV-70-NEUTRALIZING ANTIBODIES 5747

a b c d e f 9 h i j_oo

-68-43

- 29

-18

-14

FIG. 1. Autoradiogram of EV-70 proteins immunoprecipitatedby anti-EV-70 MAbs. 35S-labeled mock-infected cell lysate, EV-70-infected cell lysate, or purified EV-70 virus was immunoprecipitatedwith MAbs and then analyzed by SDS-PAGE as described inMaterials and Methods. Lanes show MAb/ev-1 with mock-infectedcell lysate (lane a), EV-70-infected cell lysate (lane b), or purifiedEV-70 virus (lane c); mock-infected cells alone (lane d); labeledmolecular mass markers (in kilodaltons; lane e); purified EV-70virus alone (lane f); MAb/ev-12 with purified EV-70 (lane g),EV-70-infected cell lysate (lane h), or mock-infected cell lysate (lanei); 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 wasprovided by experiments showing that preincubation of theprototype strain with either of the former MAbs did notblock neutralization by MAb/ev-12. None of the anti-EV-70-specific MAbs showed IF reactivity against a panel ofpicornaviruses exclusive of EV-70. This panel consisted ofcoxsackievirus serotypes Bi and B3; echovirus serotypes 1,9, and 23; poliovirus Sabin serotypes 1, 2, and 3; and humanrhinovirus serotype 14.Western immunoblot and RIP assays were carried out

with each of the MAbs. In Western immunoblot assays, noreactivity 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. Noreaction by the MAbs was observed with the mock-infectedcell lysate. The reactivity of the MAbs for EV-70 wasdemonstrated by this lack of activity against the cellularcomponents and by the corresponding lack of activity of anunrelated MAb against the purified virus.

Characterization of MAb2s. The production of an Ab3-neutralizing viral response by using MAb2s relies on mim-icry of the virion neutralization epitope by the paratope ofthe MAb2. Since MAb/ev-12 possessed a high neutralizationtiter, 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, allIgGl, were chosen for further study on the basis of theirstability and levels of antibody secretion. In order to deter-mine whether MAb2s were directed against paratope-asso-ciated idiotopes of MAb/ev-12, various blocking assays wereperformed. All of the MAb2s were capable of inhibiting boththe IF and the neutralizing activity of MAb/ev-12, suggestingthe paratope specificity of the MAb2s. Table 2 presents datarepresentative of the ability of each of the MAb2s to inhibitthe EV-70-specific neutralization capacity of MAb/ev-12. Inthe absence of a MAb2, MAb/ev-12 neutralized 90% of thePFU challenge. Incubation of MAb/ev-12 with any of the

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

Antibodiesa added PFU/wellb % Inhibition ofto EV-70 PUwl" neutralization

MAb/ev-12 + medium 15, 16 0MAb/ev-12 + MAb2-2 150, 130 100MAb/ev-12 + MAb2-7 160, 150 100MAb/ev-12 + MAb2-9 120, 140 93MAb/ev-12 + MAb2-13 126, 123 89MAb/ev-12 + MAb2-15 150, 150 100MAb/ev-12 + MAb2/3C5C 16, 16 0Medium + MAb2/3C5 146, 141 100

a MAb2 and MAb/ev-12 were preincubated for 1 h at 37°C. They were thenchallenged with virus, and the mixture was incubated for 1 h at 37°C. Theplaque assay procedure was then followed.

b Residual PFU per well following a challenge of 140 PFU per well. Testedin duplicate.

c MAb2 against anti-cytomegalovirus Bi MAbl (29).

MAb2s blocked the neutralization of EV-70. An unrelatedMAb2 had no effect on the ability of MAb/ev-12 to neutralizeEV-70 or on the viral challenge itself. In addition, none ofthe MAb2s alone neutralized EV-70 to any extent, nor didthey react with noninfected LLC-MK2 cells in IF tests (datanot shown). It is noteworthy that none of the MAb2s wereable 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 blockMAb/ev-12 binding to EV-70 in dot immunobinding inhibi-tion assays (Fig. 2). Twofold titration of the MAb2s resultedin a dose-responsive decrease in this inhibition.

In order to further investigate the specificities of theMAb2s for MAb/ev-12, two EV-70-specific neutralizingMAbs from another laboratory (3) plus MAb/ev-1 and MAb/ev-6 were tested for recognition by each of the MAb2s. Norecognition of these MAbs by any of the MAb2s wasobserved in IF inhibition and/or competition EIAs. Thissuggested that the idiotype of each of these four MAbsdiffered from that of MAb/ev-12.

Characterization ofAb3 response. Each MAb2 was used as

MAb2-2 0 * ,

MAb2-7 *

MAb2-9 *

MAb2-13 9

MAb2-15 * Sa

MAb2/3C5 * * 9 9 9

1 2 3 4 5 6 7

FIG. 2. Autoradiogram of dot immunobinding assay of inhibitionof MAb/ev-12 binding to EV-70 by MAb2s. MAb/ev-12 hybridomasupernatant was incubated with twofold dilutions of hybridomasupernatant of each MAb2 or an unrelated MAb2 (MAb2/3C5). Theantibody preparations were then reacted with the semipurifiedEV-70 preparation bound to the nitrocellulose membrane, andMAb/ev-12 binding was revealed with '25I-labeled anti-mouse IgG.Viral material was omitted when control MAb2/3C5 was diluted1:32. Lanes show MAb/ev-12 alone (lane 1); MAb/ev-12 preincu-bated 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 antiserumbMAb2

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

MAb2-2 94 57 71 3 33MAb2-7 95 92 87 69 86MAb2-9 88 51 91 66 18MAb2-13 93 65 82 93 61MAb2-15 93 87 86 58 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 MAb2binding 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 productionof Ab3 antisera. In competition EIAs, serum obtained fol-lowing the third injection demonstrated that each mouse hadmade an Ab3 response to its MAb2 challenge. As shown inTable 3, each homologous MAb2-MAb interaction was in-hibited by more than 90% by the corresponding Ab3 antise-rum at a 1:500 dilution of Ab3. This level of inhibitiongradually subsided to 50% or less at 1:10,000 (data notshown). The inhibition levels observed in the heterologousMAb2-MAb competitions varied over a wide range, whichimplied the cross-reactivity of each Ab3 serum with most ofthe other MAb2s. Two exceptions to this were that Ab3serum raised to MAb2-13 did not recognize MAb2-2, andAb3 serum raised to MAb2-15 did not recognize MAb2-2 andMAb2-9.

Since MAb2s had clearly stimulated immune responses inthese mice, we next wanted to determine whether the Ab3antisera could neutralize the infectivity of EV-70. As illus-trated in Table 4, the immunization of syngeneic mice withMAb2s elicited a neutralizing immune response specific forEV-70. Mice which received MAb2-2 produced the bestneutralizing response. Sera from the three mice in this groupshowed more than 50% neutralization activity at an Ab3serum dilution of 1:25. Of the mice which received MAb2-9and MAb2-15, two of the three in each group displayed a50% or greater neutralization activity at this serum dilution.Of the mice which received MAb2-7 and MAb2-13, only onemouse from each group displayed a 50% neutralizationactivity at an Ab3 dilution of 1:25.

In an effort to assess whether higher neutralization titerscould be generated, a group of mice were immunized withMAb2-2 or MAb2-15 and either Freund's adjuvant or Quil A.However, no differences in the neutralization titers wereobserved following four injections in a 12-week immuniza-tion regimen. The highest 50% neutralization titers were

TABLE 4. Neutralizing activity of Ab3 antiserum against EV-70

% Neutralization' at Ab3Ab3 antiserum serum dilution of:

1:25 1:50

Preimmune 0 NDAnti-MAb2-2 86, 77, 67 54, 45, 14Anti-MAb2-7 59, 34, 18 12, 12, 0Anti-MAb2-9 70, 61, 27 14, 0, 0Anti-MAb2-13 54, 40, 12 15, 0, 0Anti-MAb2-15 82, 54, 33 35, 0, 0

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

from a mouse which received MAb2-15 with Freund's adju-vant (1:200) and from a mouse which received MAb2-2 withQuil A (1:100) (data not shown).Serum from the mouse which produced the highest Ab3

neutralization titer was tested against the same heterologousEV-70 strains as MAb/ev-12 had been tested against. Neitherthe Ab3 serum nor MAb/ev-12 demonstrated any neutraliz-ing activity against these heterologous EV-70 strains. Thissuggested that the specificities of the two preparations wereidentical. The MAb/ev-12-like specificity of the Ab3s forEV-70 proteins was further assessed by RIP. Serum from amouse immunized with MAb2-15 in Freund's adjuvant andserum from a mouse immunized with MAb2-2 in Quil A weretested. As seen in Fig. 3, the same viral bands wereprecipitated with anti-MAb2-15 antiserum (lane d) and anti-MAb2-2 antiserum (lane g) as were observed with MAb/ev-12 (lane a). The reactivity of the Ab3 antiserum for EV-70was supported by the absence of these viral bands when anunrelated Ab3 antiserum was used (lane b) and by the factthat no cellular material reacted with either of the twoantisera (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

FIG. 3. Autoradiogram of EV-70 proteins immunoprecipitatedby Ab3 antiserum. 35S-labeled mock-infected cell lysate or purifiedEV-70 virus was immunoprecipitated with Ab3 antiserum and thenanalyzed by SDS-PAGE as described in Materials and Methods.Lanes show MAb/ev-12 with purified EV-70 virus (lane a); unrelatedAb3 antiserum with purified EV-70 (lane b); anti-MAb2-2 antiserumwith mock-infected cell lysate (lane c) or purified EV-70 virus (laned); labeled molecular mass markers (in kilodaltons; lane e); purifiedEV-70 virus alone (lane f); and anti-MAb2-15 antiserum with puri-fied EV-70 (lane g) or with mock-infected cell lysate (lane h).

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TABLE 5. Inhibition of Ab3 neutralizing activity by homologous MAb2

Residual PFU/well at MAb2 inhibitor dilution of°:Ab3 antiserum

No inhibitor 1:50,000 1:5,000 1:1,000 1:500 1:100 1:10

Anti-MAb2-2 0, 8 0, 0 0, 0 0, 0 0, 1 >100, >100 102, 87Anti-MAb2-9 0, 2 1, 2 2, 4 ND 40, 68 ND NDAnti-MAb2-15 0, 1 0, 0 1, 3 > 100, > 100 > 100, > 100 > 100, > 100 > 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 testswith a 140-PFU challenge of EV-70. ND, not done.

(Table 2). In a similar manner, the ability to inhibit Ab3neutralization activity by using the homologous MAb2 wasdemonstrated (Table 5). The degree of inhibition reflectedthe titers of both the Ab3 antiserum and MAb2. Completeinhibition of neutralization was observed at low dilutions ofthe 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 effective-ness as surrogate immunogens, we first made EV-70-specificMAbs. Of the EV-70 MAbs described in this study, we foundthat those which possess the highest IF titers exhibit little orno neutralization activity, whereas MAb/ev-12, which has ahigh neutralizing titer, has a comparatively lower IF titer. Inthis regard, our MAbs are similar to other previously de-scribed EV-70-specific MAbs (3). In addition, those of ourMAbs which shared a higher IF titer were reactive with allEV-70 strains tested, whereas MAb/ev-12 was specific forthe J670/71 strain only. This implies the existence of at leasttwo epitopes on the prototype strain, with one neutralizingand the other nonneutralizing. The nonneutralizing epitopeseems to be conserved in all of the EV-70 strains used in thisstudy.The reactivity of the EV-70-specific MAbs in Western

immunoblot and RIP assays suggests that epitopes areconformationally dependent. Dissociation of the virionsprior to exposure to the MAbs, as in Western immunoblotassays, precluded antibody binding. This is analogous to thefailure of picornaviruses to recognize cellular receptorsfollowing modification of their viral capsids (9). Conversely,the MAbs were able to recognize virions that had not beendenatured, as depicted in the RIP assays (Fig. 1), suggestingthe recognition by the MAbs of conformation-dependentdiscontinuous epitopes.The selection of MAb/ev-12 for the production of MAb2s

is based on the potential biological importance of MAb/ev-12-recognized epitopes in the pathogenesis of EV-70. MAb/ev-12 recognition of EV-70 was inhibited by each of theMAb2s produced (Fig. 2 and Table 3). No inhibition ofanti-EV-70 activity was recorded when four other EV-70-specific MAbs were tested with each of the MAb2s. Inaddition, MAb/ev-12 is the only antibody capable of inhibit-ing MAb2-MAb1 binding, suggesting that the idiotope rec-ognized on MAb/ev-12 is a private idiotope and is not sharedwith these other MAbs. The ability of each of the MAb2s tospecifically inhibit the anti-EV-70 activity of MAb/ev-12suggests that the idiotope they recognize on MAb/ev-12 isjuxtaposed to, overlaps with, or lies within the MAb/ev-12paratope that recognizes the viral epitope associated withneutralization. Therefore, by definition, these MAb2s areeither Ab2y or Ab2p (10, 16).

Since Ab2,s mimic the conformation of epitopes, reactiv-ity with viral receptors on host cell surfaces may be a featureof some of them. In fact, this has been the focus of muchresearch regarding these antibodies (1, 8, 20, 21, 43). In thisstudy, none of the MAb2s reacted with uninfected cellmonolayers, nor did they protect susceptible cells from viralinfection. This suggests that the MAb2s elicited in responseto MAb/ev-12 do not recognize the cellular structure used forviral attachment. The recognition by MAb/ev-12 of a neu-tralizing epitope which is not part of the viral attachment sitewould explain the inability of the MAb2s to recognize thecorresponding cellular structure in a manner similar to thatpostulated for coxsackievirus B4 (21). This may be due to acanyonlike structure which has been found on certain picor-naviruses (30, 31, 36).

Functional differentiation of Ab2a, Ab2y, and Ab2p maybe achieved by analyzing the Ab3 immune response elicitedfollowing immunization with the MAb2s (10, 11, 16). As aresult of molecular mimicry, Ab2,B should be able to stimu-late antigen-specific immune responses and serve as a vac-cine. However, complexity in Ab2 classifications has re-sulted in some instances in which Ab2aos were also able toelicit an antigen-specific immune response in the absence ofthe original antigen (13, 33). Although Ab2, is still consid-ered the ideal vaccine candidate, the induction of Abl-likeantibodies which possess idiotope markers and antigen-binding specificities identical to those of Abl is a mandatoryprerequisite in the selection of a candidate Ab2. In thisstudy, the immunization regimen used for the production ofAb3 antiserum resulted in high Ab3 titers. The higher levelsof inhibition seen in the homologous MAb2-MAb/ev-12interactions relative to that seen for the heterologous MAb2sindicate a greater degree of specificity for the homologousMAb2s. The wide range of inhibition levels in the heterolo-gous MAb2-MAb interactions suggests that despite the pres-ence of shared idiotopes, the MAb2s may be distinct fromeach other. Thus, in addition to Ab3s specific for a homol-ogous MAb2, an Ab3 antiserum may or may not containcross-reactive antibodies recognizing heterologous MAb2s.The presence and absence of such cross-reactive antibodiesare exemplified by the Ab3 antisera elicited by usingMAb2-13 and MAb2-15. Neither of these antisera was ableto inhibit the binding of all heterologous MAb2s to MAb.This distinctiveness may apply not only to the physicalstructure of the MAb2s but also to their regulatory roles inthe immune response.

Confirmation of the Abl-like properties of our Ab3 anti-sera was crucial to the legitimacy of using the MAb2s assurrogate immunogens. Of the five MAb2s used, MAb2-2,MAb2-9, and MAb2-15 elicited 50% neutralization activity atan Ab3 serum dilution of 1:25 in all or most of the miceimmunized. It is noteworthy that those mice which receivedMAb2-2 produced the highest EV-70 neutralization activity

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5750 WILEY ET AL.

and also demonstrated a consistently high level of inhibitionof the homologous and heterologous MAb2-MAb/ev-12 in-teraction. On an individual basis, the highest Ab3 50%neutralization titers were obtained from mice which receivedeither MAb2-15 in Freund's adjuvant (1:200) or MAb2-2 inQuil A (1:100). The Ab3 viral neutralization titers reportedhere and the ranges over which they vary are comparablewith those reported for other viral systems (24, 27, 37). It isalso possible that injection of xenogeneic polyclonal Ab2srather than syngeneic MAb2s would induce a significantlyhigher Ab3 neutralizing response. The fact that the Ab3antisera possess Abl-like properties is demonstrated by theability of homologous MAb2s to inhibit both Ab3 andMAb/ev-12 neutralizing activity.

It has been demonstrated in influenza and respiratorysyncytial virus models (2, 24) that Abl and antigen-reactiveAb3s may have distinct specificities for the antigen. Thelevel of fidelity of the immune response manifested in ourstudy permitted the restricted neutralization spectrum ofMAb/ev-12 for the prototype J670/71 strain to be maintainedby the Ab3s. In RIP assays, Ab3s recognized an EV-70surface epitope which caused the immunoprecipitation of thesame EV-70 proteins as MAb/ev-12. Maintenance of such astrain-specific immune response has also been demonstratedpreviously for an Ab3 to reovirus type 3 hemagglutinin (14,34).

In summary, we have generated MAb2s which elicited aneutralizing response to the prototype strain of EV-70. Thefacts that the elicited Ab3 response is against a conforma-tional epitope and that both synthetic peptide and recombi-nant DNA technologies have not yet been able to fully mimicsuch epitopes reinforce the value of Ab2s as surrogateimmunogens in the study of viral pathogenesis.

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