Analysis of neutralizing antigenic sites on the surface of type A12 foot-and-mouth disease virus.

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0022-538X/89/052143-09$02.00/0

Analysis of

Neutralizing Antigenic

Sites

on

the Surface

of Type A12

Foot-and-Mouth Disease Virus

B. BAXT,l* V. VAKHARIA,2t D. M. MOORE,' A. J. FRANKE,' ANDD. O. MORGAN1

Department ofMolecular Biology, United States DepartmentofAgriculture-Agricultural Research Service, NorthAtlantic

Area, Plum Island AnimalDiseaseCenter, Greenport, New York 11944-0848,1and Department of Microbiology, State

University ofNew YorkatStony Brook, StonyBrook, New York 11794-86212 Received6 July 1988/Accepted 6 February 1989

A series ofseven neutralizing monoclonal antibodies (nMAbs) directed against type A12 foot-and-mouth

diseaseviruswasusedto generateneutralization-resistantvariants. Both plaquereductionneutralization and

microneutralizationassaysshowedthatthe variantswerenolongerneutralized

by.

thenMAbs usedto generate them, althoughsomeofthevariantsstill reacted with the nMAbsathighantibody concentrations. Resultsof

cross-neutralization studiesby both plaquereductionneutralization andmicroneutralizationassayssuggested

thepresenceofatleastoneimmunodominant antigenic siteonthesurface oftypeA12foot-and-mouthdisease

virus, alongwith evidence ofa second antigenic site onthe viral surface. Two ofthe variants had reduced

virulence in tissue culture asevidenced by their inability toinhibit cellular protein synthesis anda marked

reduction in virus-inducedcellular morphological alterations. Nucleotide sequencing ofthevariant genomes placed threeepitopes ofthe major antigenic siteon VP1 and thefourth epitope on VP3 and VP1. Theone

epitopeof the minor siteappearstoreside onlyonVP1.

Foot-and-mouth disease virus (FMDV), the only aphtho-virus in the Picornaviridae, is characterized by antigenic variability in tissue culture and in the field (16, 23, 31, 53,

54). By using protease sensitivity, surface iodination, and antibody binding studies, viral protein 1 (VP1) has been

shown to be the majormacromoleculeon the virus surface

(8, 26, 46), although VP2 and VP3 arealso exposed on the

virion surface (40). It has been shown that isolated VP1 and

small peptides from VP1 can elicit neutralizing antibodies

andprotectanimals fromviruschallenge(3-5, 10, 13, 18, 28, 35, 58).

We have described a series of neutralizing monoclonal

antibodies (nMAbs) against type

A12

FMDV which, based

on reactivityand mechanism ofneutralization, appeared to recognize threeorfourantigenic sites (9, 47). Whilesomeof

these sites have beenmapped (47), otherswhichare

depen-denton conformation have not.

Recently, antigenic sites on a number of picornaviruses

havebeenmapped by isolatingvariants whichwereresistant to monoclonal antibody neutralization. Sequencing the ge-nomes of those variants has allowed the placement of the

antigenicsitesontheprimarysequenceofthe viral structural

proteins (12, 17, 20, 37, 38, 41, 51, 52, 55, 61) and, inalmost

every case,hasconfirmed the existence ofmultipleantigenic

sites onthepicornavirus capsid.

Recent results with FMDV type 01 variants have also

identified threeorfourantigenic sites onthesurface ofthat

virus(43, 57, 62). Most of these sites werelocated onVP1,

butsiteswerealso identifiedonVP2and VP3(43). Recently,

Thomasetal.(59)studied neutralization-resistant variantsof

FMDV type

Alo

and located two major antigenic sites on

VP1and VP3.

Inthisreport,wedescribe theisolation of nMAb-resistant

variants of type

Al,

FMDV. Cross-neutralization studies

*Correspondingauthor.

t Present address: Center forAgricultural Biotechnology, College

ofVeterinary Medicine, University of Maryland at College Park, CollegePark, MD 20742.

detected an immunodominant antigenic site containing at

least four epitopes. We alsodetected a second site

contain-ing a single epitope. Nucleic acid sequence analysis

indi-cated that thesequencechangewhich influences theactivity

ofone ofthese epitopes is located onVP3.

MATERIALS AND METHODS

Cells and virus. FMDV type

A12

strain 119, large-plaque

ab variant(type A12),was propagated inBHK-21 cell roller

bottle cultures. Unlabeled virus was concentrated by poly-ethylene glycol precipitation and purified by CsCl density gradientcentrifugation (7). A continuous bovine kidneycell line (LF-BK)was usedfor plaque assays,

microneutraliza-tion(MN) assays, and thegeneration ofvariants.

Hybridomasandmonoclonalantibodies. Theproduction of

nMAb-secreting hybridomas from mice immunized with

various type

A12-derived

antigens has been previously

de-scribed (9, 25, 47) (Table 1). Thecomplete nMAb

designa-tion is given in Table 1. In the text, a short form of the

designation, usingthe firstfourorfivenumbers and lettersto

the left of the decimal point, will be employed (i.e.,

2PD11.12.8.1 will be referredtoas2PD11).The nMAbswere

purified from cell-free hybridoma supernatants by affinity chromatography onprotein A-Sepharose (Pharmacia Inc.).

Isolation of nMAb-resistant variants. Two independent

isolationsof nMAb-resistant variantswereperformed

essen-tially as described previously (57). These isolations are

designated by either a B or a D as the first symbol in the

variant designation. Additional variants were isolated from

the B variant stocksby selecting plaquesgrowingunder1%

agarose, which contained purified nMAb at aconcentration

of 10 ,ug/ml. These variants were plaquepurified two

addi-tional times in the presenceofnMAb and designatedwitha

numberfollowingadecimalpoint (i.e., B2PD.1).Inallcases,

the nMAb usedtoselectthevariantisincludedin the variant

designation (i.e., B2PD.1 was selectedwith nMAb 2PD11).

The parental strain from which these variantswere isolated will be referred toas wild type (wt).

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2144 BAXT ET AL.

TABLE 1. nMAbs used forvirus variantgeneration

Inducing

_antigeng

nMAbreactivity"

nMAb demonstrated Isotype

antigen with:

Virus 2PD11.12.8.1 Virus IgG-h

2PE4.12.1 Virus IgM

2FF11.11.4 Virus, 12S" IgG3 13Kd' 7SF3.1.H3 Virus, 12S. VP1 IgG3

VP1 6FFS.1.3 Virus, 12S, VP1 IgG2b

6HC4.1.3 Virus, 12S, VP1 IgG2b 6EE2.1.2 Virus,12S. VP1 IgG2,1

"Reactivity with various antigens was determined by either liquid- or

solid-phase radioimmunoassayasdescribed previously(9,24.47). "12S, 12Sprotein subunits.

'*Apeptidegeneratedbytreating isolatedVP1with CNBr andspanningthe

regionbetween methionines atresidues54and 179.

Neutralization assays. Plaque reduction neutralization (PRN) assays were performed in LF-BK cells asdescribed previously (9). MN assays wereperformed in 96-well tissue culture dishes seeded with LF-BK cells. Serial twofold

dilutions of nMAbs (initialconcentration, 1 mg/ml) in

mini-mumessential medium containing 25 mM

N-2-hydroxyeth-ylpiperazine-N'-2-ethanesulfonic acid (HEPES) buffer (pH

7.5) were mixed with the variants (final concentration, 104 50% tissue culture infectious doses permilliliter) and incu-bated for 1 h at room temperature. A 25-li sample of each antibody-virus mixture was inoculated into duplicate wells

and incubated for 1 h at 37°C. An additional 25 lI of

minimum essential medium was addedtoeach well, and the cellswereincubatedfor 24 hat37°C. The wellswerestained with crystal violet-Formalin. The number of intact cell sheets that remained were used to calculatea50%protective dose ofantibody

(PD5,)

bythe methodofSpearman-Karber

(21).

Labeling and immunoprecipitation ofproteins in infected cells. Monolayers of LF-BK cells in 35-mm tissue culture

dishes were infected with the variants at 10 PFU per cell

(with certain exceptions as described in the text) in the presence of the nMAbs and labeled with

[t3S]methionine

at 4 hafterinfection as previously described (6). The

prepara-tion of cytoplasmic extracts and analysis of the proteins

synthesized by immunoprecipitation and sodium dodecyl

sulfate-polyacrylamidegelelectrophoresiswasperformedas

described previously (6).

Sequencing ofviral RNA. RNA from purified virions was

extracted with phenol-chloroform-isoamyl alcohol (24) and stored in ethanol. Oligonucleotide primers (17- or 18-mers) were synthesized by using

3-cyanoethyl

phosphoramidites (American BioNuclear) and a Microsyn 1450 DNA synthe-sizer(Systec, Inc.). The oligonucleotides werecleavedfrom

theglass support with NH40Hat50°Covernightandpurified through Sep-Pak C18 columns (Waters Associates, Inc.) by themethod of Lo et al. (29). Atotal of 20 primers, covering theentire P1 region, were used. Sequencing reactions were performed by reverse transcriptase-catalyzed synthesis of

cDNA fragments directly from the RNA template in the

presence ofdeoxynucleotides and dideoxynucleotides and of r(-35S]dATP (51). Labeled fragments were run on 45- or 60-cm8%polyacrylamide-urea gels for sequence determina-tion. Insomereactions, dITP and ddITP were substituted for dGTP and ddGTP, respectively, to alleviate sequencing problems due to compressions caused by G-C-rich regions

(36).

RESULTS

Selection of variants. Viral variantswere selectedbytheir

abilitytogrow in the presence of nMAbs. Seven antibodies

which have been previously characterized (9, 25, 47) were

usedtoselect the variants. Table 1 showsabrief

character-izationof the nMAbs. Three of the nMAbs(2PD11, 2FF11,

2PE4) represent conformationalepitopesasdefined bytheir reactionwith either intact virus (2PD11, 2PE4) or both intact virus and 12S protein subunits (2FF11). The other four antibodies, in addition toreacting with viral structures, also react with isolated VP1 and VP1 fragments and probably define linear epitopes of the molecule.

Characterization ofnMAb-resistantvariants. The variants

were characterized by their ability to be neutralized by each of theseven nMAbs,by usingtwomethods. The PRN assay was used on selected variants and is shown in Table 2. The numbers represent a log dilution of antibody needed to reduce the number of plaques by 70% as calculated by a

logit-log transformation method (60). Thus, the lower the number, the more resistant the virus is toantibody-mediated neutralization.

Toanalyze all of the variants, an MN assay was done,as

described in Materials and Methods, and the results are

shown inFig. 1.

In order to determine areas of changein the viral polypep-tides which might correspond to the loss of MAb-neutral-izing activity of the variants, viral RNA wasextracted and sequenced by using reverse transcriptase and dideoxynucle-otides (50). The genome region corresponding to VP1 was sequenced for those variants which were generated from nMAbs defining linear epitopes (7SF3, 6FF5, 6HC4, 6EE2). The entire P1 region of RNA was sequenced for variants generated from conformation-dependent nMAbs (2PD11,

2PE4,2FF11). Results are shown inTable 3. Onlynucleotide changes leading to amino acid changes are shown. In only one instance was there a nucleotide change which did not result in a new amino acid. Amino acids are identified by a four-digit numbering system in which the first digit repre-sents the

polypeptide (i.e., VP1, VP2,

or

VP3)

and the remaining three digits represent the amino acidnumber (e.g., aminoacid 1152 denotes the 152nd amino acid on VP1) (11, 41).

(i) 2FFIl variants. All the variants inthis group were able to be neutralized by all the nMAbs with the exception of 2FF11(Fig. 1). The only exception was variantB2FF, which was partially resistant to neutralization by antibody 6EE2 (Fig. 1). The PRN assay indicated, however,thatthis variant could be fully neutralized by 6EE2 (Table 2). Sequence

TABLE 2. Cross-neutralization by PRNassay"

PRNassay values"ofmonoclonal antibodies: Variants

2FF11 2PE4 2PD11 6EE2 6HC4 6FF5 7SF3

WT 5.59 3.77 4.79 2.78 2.99 3.18 4.09

B2FF <1.50 3.31 3.50 3.62 4.00 >3.50 3.50 B2PE 3.90 < 1.40 3.93 3.22 3.58 3.05 2.53

B2PD 3.05 < 1.40 < 1.40 >2.30 3.91 3.20 2.25 B6EE 2.83 4.99 >4.10 1.72 <1.40 3.83 3.10 B6HC 2.75 3.38 5.34 <1.40 <1.40 <1.40 2.15

B6FF 3.33 3.33 5.0)4 3.10 3.59 1.61 2.16

B7SF 5.00 3.71 >4.10 2.79 3.02 <1.40 1.75

"Individual vairiants were assayed with the panel of nMAbs by the PRN

assay(9).

"Numbers representlog,(709 endpoint titers. Titers inboldface represent assay ofthe variant and themonoclonal antibodyused to generate it.

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B2FF S B2FF.1 e B2FF.2 * B2FF.3 *

B2PE 0 D2PE 0 B2PD 5

B2PD.1 *

B2PD.2 e B2PD.3 5 D2PD ( B6EE 0 B6EE.1 0 B6EE.20 B6EE.3 0 BB6HC 0 B6HC.1 0 B6HC.20 B6HC.3e D6HC 0 B6FF e B6FF.1 0 B6FF.2 0 B6FF.3 0 B7SF e B7SF.1 0 B7SF.2 e B7SF.3 0 D7SF 0

WT 0

It cli It in C.,

wJ C W 0 L

tL a. w U.

cli cli (D t-D e' c' e O~ O 0

ooeoo

0

0 0 0 0 0 0

* O O O e 0

*o e0o

* 0000O

*.

*O O

*e

0 *O*e00O

e~0 0 0 0

0 0 e 0

0 0 0 0

0 0 0 0 0

0 0 0 0

e 0 0 0

00 0

0 0 0 0

0 0 0 0 0

O O e O0

e

O

e

0

*

0 0 0 0 0

O O O

*O

0 0 0 0 0 0

FIG. 1. Results of MN assaysoftype

A12

nMAb-resistant

vari-ants. Variantswereassayed byMN assayasdescribed inMaterials and Methods. The log1o PD50 titers were arbitrarily divided into

resistant (0 [log PD50 = <0.6 to 0.9]), partially resistant (E [log

PD50 = 0.91 to 1.5]), orsensitive (O[log PD50 = 1.51 to2.11) to

rTMAb-inducedneutralization.

analysis of the P1 region of the variants showed only amino

acidchanges in VP1(Table3). Allfourvariants hadachange atresidue 1173 from HistoTyr. Oneof the variants (B2FF)

hadasecond mutationat residue 1147. It is clearhowever,

that only a single amino acid change at residue 1173 is

sufficient to confer resistance tothis nMAb.

(ii) 2PE4 variants. This nMAb is a

conformation-depen-dent immunoglobulin M (IgM), and we were only able to

isolatetwostable variants. Variant B2PEwasfully resistant

to2PE4byPRNandpartially resistant toantibody7SF3 and

2FF11(Table 2). By MN assay, however, both variantswere

fully resistantto 2PE4while B2PEwaspartially resistantto

antibody6FF5 and D2PE waspartially resistant to

antibod-ies6EE2 and7SF3(Fig. 1). Bothvariants hadamutation in

amino acidresidue 1201 from His toTyr, but variant B2PE

was adouble mutant, havinganadditional changeatresidue

1152from Pro to Leu.

(iii) 2PD11 variants. We have analyzed five variants

gen-erated against thisconformation-dependent nMAb. Variant

B2PD was notonly resistant to2PD11byPRNbut wasalso

resistant to 2PE4 and partially resistant to7SF3 and2FF11

(Table 2). When all of the variants were analyzed by MN

assay (Fig. 1), the resistance to both 2PD11 and 2PE4 was

seen, as was complete or partial resistance to 2FF11. In addition, four of the five variants were also resistant to

[image:3.612.102.249.66.392.2]

antibodies

6FF5

and7SF3.

All

ofthevariants had mutations in VP3 (Table 3). Variant D2PD had a single mutation at

TABLE 3. Amino acid changes in typeA12 nMab-resistant variants"

Amino acid resultingfrom change at amino acidresidues":

Variant

1083 1147 1151 1152 1173 1201 1209 3175 3178

WT Asp Phe Ala Pro His His Gly Thr Thr

B2FF Leu Tyr

B2FF. 1 Tyr

B2FF.2 Tyr

B2FF.3 Tyr

B2PE Leu Tyr

D2PE Tyr

B2PD Asn Leu Ala Ala

B2PD. 1 Leu Ala Ala

B2PD.2 Leu Ala Ala

B2PD.3 Leu Ala Ala

D2PD Ala

B6EE Arg

B6EE.1 Arg

B6EE.2 Arg

B6EE.3 Arg

B6HC Ser Arg

B6HC.1 Leu Val

B6HC.2 Ser Arg

B6HC.3 Ser Arg

D6HC Arg

B6FF Ser

B6FF. 1 Ser

B6FF.2 Ser

B6FF.3 Ser

B7SF Gln

B7SF.1 Gln

B7SF.2 Gln

B7SF.3 Gln

D7SF Val

"Amino acids were deduced from the nucleotide sequence of wt and variantgenomes.

"Amino acidsresiduesaredenotedbyafour-digit numberingsystem(11,

41) as described in Results.

residue 3178 from Thr to Ala. The other four variants had identical amino acid changes at residues 3175 and 3178. In

addition, eachof the variants B2PD, B2PD.1, B2PD.2,and

B2PD.3 hadamutationatresidue 1152fromProtoLeu. Itis interestingto notethatvariant D2PDwhichhadonlyasingle

amino acid change in VP3 was only partially resistant to

2PD11 andsensitiveto6FF5and

7SF3,

indicatingthatwhile

changesatresidue 3175, residue3178, orbothare sufficient

toconfer some resistance to 2PD11, residue 1152 seems to

be involved in theepitope. Inaddition, antibodies 6FF5 and 7SF3 appear to have a requirementfor residue 1152, since

D2PDis still sensitiveby neutralization bythoseantibodies.

(iv) 6EE2 variants. All the variantsgenerated against this monoclonalantibodywereresistantonlyto6EE2and 6HC4

by MN assay (Fig. 1). Variant B6EE also appeared to be

partiallyresistantto2FF11by PRNassay (Table 1). Onlya

single amino acid change was detected in VP1, at residue

1209, fromGlytoArg(Table 3).Thisresidue is fourresidues fromtheC terminusofVP1.

(v)

6U4C4

variants. Variants to this nMAb presented a mixed patternofreactivity.Allfive variantswereresistantto both 6HC4 and6EE2byMNassay,

indicating

that thesetwo antibodies reactwith the same

epitope (Fig.

1). Fourof the variants, however, were also resistant to 6FF5

(Fig.

1).

Variant B6HC, when tested by PRN assay (Table 2), was also resistant to6HC4, 6EE2, and6FF5 and

partially

resis-tantto7SF3 and2FF11.Thevariants whichwereresistantto 6FF5 were double mutants with

changes

at residue 1209

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2146 BAXT ET AL.

I.

0

a

a.

a b c

7SF3

Antibody Dilution

FIG. 2. Liquid-phase radioimmunoassay analysis of nMAb-re-sistantvariants. Variants B2PD (a), B2FF (b), B7SF (c), B6FF (d), B6EE(e), andB6HC (f)werebiosynthetically labeled by growing in

the presence of[3H]uridine. Purified variants along with purified

[3H]Nridine-labeled wtwere reacted with nMAbs inaliquid-phase radioimmunoassay as described previously (26). The nMAbswere

purified and used at a starting concentration of 1 mg/ml. The antibodies usedareintheupperleftcornerof eachpanel. Symbols:

0,wt;0, nMAb-resistant variant.

from Glyto either Arg or Val and changesat residue 1152 from Pro to either Ser or Leu (Table 3). Variant D6HC,

which was only resistant to 6HC4 and 6EE2, was a single

mutant atresidue 1209 from GlytoArg(Table 3).

(vi) 6FF5 and 7SF3 variants. The pattern ofreactivity of

variants generated against these two nMAbs indicates that they react with the same epitope. By PRN assay, variants

B6FF and B7SF were both resistant to 6FF5 and 7SF3 (Table 2). Analysis of neutralization by MN assay showed

that all the variants were resistant to 6FF5, however, only

the7SF3 variants were resistant7SF3. The only exception wasD7SF, whichwas sensitiveto7SF3 byMN assay even

though itwas generated by 7SF3. Sequence analysis of the

variant genomes showed that all the variants had a single

mutation in VP1. The 6FF5 variants all had a change at

residue 1152 from Pro to Ser, while four of the five 7SF3

variantshadachangeat1152 from ProtoGlnand D7SF had

achangeto 1151 fromAlato Val.

Characterization of variants by direct antibody binding.

Variants were biosynthetically labeled with [3H]uridine and

liquid-phase radioimmunoassays were performed as

de-scribed previously (26). Figure2 shows the results ofeach

assaycomparing the variant withwtA12. Antibodies 2PD11

(Fig. 2a),2FF11 (Fig. 2b), and 6EE2 (Fig. 2e)gave little or noreactionwiththevariant,whilethey reactedwell withthe wt.ThenMAb 2PE4, which isanIgM, waspoorly bound to protein-A-containing Staphylococcus aureus, with the

countsboundrepresenting less than 1% of the inputcounts

used in this assay.

Arming

the S. auireus with rabbit

anti-mouse IgM did not seem to enhance the

binding

of the

antigen-2PE4 complex

(data

not

shown).

Antibodies 7SF3

(Fig. 2c) and 6FF5

(Fig. 2d)

reacted with variants at

high

antibody

concentration, however,

duetothe lowtiter ofthe

nMAb

against

the

variant,

the

reactivity

was

rapidly

diluted out. It is

possible

thatthis

binding

at

high

antibody

concen-tration may be duetothenMAb

having

alowered

affinity

for

the variant. Wecannotrule out,

however,

some wt

contam-inationofthevariant seed.

Antibody 6HC4,

however,reacts

with the variant to the same extent as with wt. The

signifi-cance of

antibody

reactivity

with a viral variant which

cannot be neutralized

by

that

antibody

isnot clearbut has

been noted for FMDV type 01-resistant variants

(62)

and

also for type A1, nMAbs which bind but do not neutralize

other type A

subtypes

aswell asother serotypes

(25).

Biological activityof nMAb-resistant variants. Toexamine whether the variants werealtered in theirabilitytogrow in cells or to affect cellular processes, LF-BK cells were infected with selected variants and

pulse-labeled

with

[35S]methionine

when

cytopathic

effect

appeared.

When a

multiplicity

of 10 PFU per cell was

used,

allofthevariants

withtheexceptions ofB7SF and B2FF causedcell

rounding

within 4 h after infection(data not shown). Variants B7SF

and B2FF

required

very

high

multiplicities (about

500 PFU per

cell)

tocausecell

rounding

within 4 to6 hafter infection

(data

not shown).

Labeling

ofinfected cells with

[35S]methionine

at4hafter

infectionis shown in

Fig.

3A. Cellswereinfected with B7SF

and B2FFatabout 500 PFU per

cell,

whilethe

multiplicity

of

infection of the othervariantswas10PFUpercell. With the

exception

ofvariants B7SF and B2FF

(lanes

2and

6),

viral

protein

synthesis

occurredin cellsinfected with all the other

variants. Cellular

protein

synthesis

was

markedly

inhibited

in wt-,

B6HC-,

and B6EE-infected cells

(lanes 1, 7,

and

8)

andto alesserextentin

B6FF-,

B2PD-, and B2PE-infected

cells

(lanes

3

through

5). In B7SF- andB2FF-infected cells

(lanes 2 and 6), however, there

appeared

to be little or no

inhibition ofcellular

protein

synthesis

evenafterinfectionat

sucha

high

multiplicity.

Thelack ofinhibition

by

thesetwo variantscorrelateswell with thediminution of virus-induced

morphological

alterations.

Inallvariant-infected

cells, however,

the

synthesis

of viral

proteins,

could be demonstrated

by

immunoprecipitation

withguinea

pig hyperimmune

serum

(Fig.

3B). Cellsinfected

with B6FF, B2PE, B6HC, and B6EE

(lanes

3, 5, 7 and

8)

showedagreateramount of

synthesis

ofviral

proteins

than othervariant-infected cells. Extracts of the infected labeled cells were also

immunoprecipitated

with the individual monoclonal antibodies.

Figure

4 shows that

only

viral pro-teins in cells infected with B6HC and B6EEwere

immuno-precipitated

with the monoclonalantibodiesusedtogenerate them. The results shown in

Fig.

4 are in agreement with those shown in

Fig.

2. Purified B6EEwas not

immunopre-cipitated by

6EE2

(Fig. 2),

and it is not clear

why

viral

proteins

from infected cells reacted with the

antibody.

Antibody

2PE4 gavepoor

reactivity

with wtviral

proteins,

andwe could notdetect

immunoprecipitated products (data

not

shown).

Changesin FMDVtypeA12wtsequence. The

sequencing

of

thevariants

through

the P1

region

wasdone with

sequencing

of wt RNA for

comparison. During

the course of the

sequencing,

it was noticed that there were differences in sequencefrom that

published

for type

A12 (45).

The results of the

sequencing directly

from RNAareshown in Table4.

Therewere atotal of 18 nucleotide

changes

in 16codons. In J. VIROL.

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7SF3 6FF5 2PD1I 2FF11 6HC4 6EE2

wt

vwt vI

H

HFt7

Y

B L 0 w E U e

_ r, cm eVNf.4.oc

t X : m m so m m =

PI-P3- _ _t

P1

pi '*

*

s@

a a a a

AD m 4

VP3-3t a

L-e^ *.t4

2 B- _ -_

[image:5.612.349.507.63.386.2]

12 3 4 5 6 7 9

FIG. 3. Analysis of proteins in variant-infected cells.

Monolay-ersofLF-BKcellswereinfected with the variantsat10PFUpercell

(with the exception of variants B7SFand B2FF, whichwereusedat a multiplicity of about 500 PFU per cell) and labeled with [35S]methionine, and cytoplasmic extracts were prepared as

de-scribed inMaterials and Methods. (A) Extracts wereanalyzed by

10%sodium dodecyl sulfate-polyacrylamide gel electrophoresis.(B)

Extracts were immunoprecipitated with guinea pig hyperimmune

anti-type A12sera, asdescribed in Materials and Methods, followed

by analysis with sodium dodecyl sulfate-polyacrylamide gel electro-phoresis. The locations of viral proteinsareindicatedonthe left side

ofeachpanel.

all, 10 amino acids were different from the published

se-quence. Most ofthenucleotideandamino acid changeswere

inVP2, buttherewerethree amino acid changes in VP3 and

onenucleotide changein VP4. Thenumbering of the amino

acids in VP4 represents a change in the cleavage point

between the Lproteinand VP4toreflect thepresence of the

consensusmyristylation sequenceatthe Nterminus of VP4

recently reported for picornaviruses (15). Thenewcleavage

pointisset atLys-Gly at nucleotide 601. It isinteresting to

note that there were no changes in the wt sequence in the VP1 region of the genome. All the reported changes were

confirmed by dideoxy sequencing of cloned cDNA

frag-ments prepared from RNA which was isolated from virus

grown at about the same time as the virus used for the

originalsequencing.

DISCUSSION

By comparing neutralization mechanisms and by using antigen binding assays, we had previously reported that

there were three antigenic sites in type

A12

(9, 47). In this

study, we have shown that there is one immunodominant

antigenic site on the virion surface containing at least four

epitopes. There alsoappearstobeasecondantigenicsiteon

the virion. Recently, we have also shown that type A12

nMAbswhich definecommonepitopesalso containcommon

idiotypes (B. Baxt, A. E. Garmindia, and D. 0. Morgan,

Viral Immunol., in press). Theuse of nMAb-resistant

vari-FIG. 4. Immunoprecipitation by nMAbs of proteins from vari-ant-infected cells. Cytoplasmic extractsfrom variant (V)- and

wt-infected cells described in the Fig. 3 legend were immunoprecipi-tated with the appropriate nMAb, and the precipitates were

analyzed with 10% sodium dodecyl sulfate-polyacrylamide gel elec-trophoresis asdescribed in Materials andMethods.

ants has shown at least three orfour neutralizing antigenic

sitesontype0FMDV(43, 57, 62)andtwomajorsitesonthe

surface oftype

A1l

(59).

The generation of 29 neutralization-resistant variants

against seven nMAbs and the sequencing of the variant

genomes have enabledustolocatethe neutralizing epitopes

on VP1. We have also shown that one epitope involves

resides on VP3. Antigenic sites on types 01 and

A1o

have

also been mappedto VP2 and VP3 (43, 59). These are the

first.direct demonstrations of neutralization epitopeson an

aphthovirus structural polypeptide other than VP1.

Re-cently, it has been shown that VP2 and VP3 have exposed

residueson thevirion surface (40).

Variants were generated by two independent isolations.

We feel thatthe variants from both oftheseisolations were

present in the original virus stocks and were merely

en-hanced in the presence of the nMAbs. Results reported by

Xie et al. (62) indicate that, even after extensive plaque

purification, neutralization-resistant variants could still be

found in FMDV type 01 seeds. The rapid variability of

FMDV both in tissue culture and in nature has been well

documented (16, 23, 31, 53, 54).

In order to determine which of the variants define the

sameantigenic site, cross-neutralization studies weredone.

A PRN assay (Table 2) was done on a limited number of

variants,while an MN assay (Fig. 1) was performed on all

P3-

P1- 3BCD-compex-j

IABC-3D_

1CD- 3D-

P2-YPO- _m _

2cp-

_

If

VP3-VP1- _l 1, _1

3C- 46

1 2 3 4 5 6 7 9

VP3_

VP1-

VP2-

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[image:6.612.67.308.91.295.2]

2148 BAXT ET AL.

TABLE 4. Changes in nucleotideandamino acid residues in type A12frompublishedsequence (45)

Amino acid Nucleotide Nucleotide Amino acid

residue residue' change change

4015b 645 C--T Nochange

2071 1066 C-> A Pro-Thr

2078 1088 G-* U Arg-*Leu

2079 1090 A-.G Thr-*Glu

1091 CA

2102 1161 G-*A Nochange

2130 1245 G--A Nochange

2131 1246 A--G Thr-sGlu

1247 C -A

2134 1256 C- A Thr--Lys

2136 1263 A-*G Nochange

2137 1264 G--A Glu--Lys

2185 1410 U-G Nochange

2217 1506 G-*A Nochange

2218 1508 U--*A Val-,Glu

3029 1595 A- U Glu-*Val

3034 1610 A- G Lys-sArg

3039 1624 A-*G Arg--Gly

"Nucleotide residue numbers are identical to the published type A1,2

sequence (45).

bThelocation oftheL/P1 cleavagehasbeenchangedsothatamino acid

residue 4001 islocated atnucleotide residue601.Thischangewasmade to

accommodatethe consensusmyristylationsequence(15)atthatlocation.

the variants. The results of both assays and nucleotide

sequence analysis (Table 3) showed that there is a single

immunodominant siteonthe FMDV type A12 surface.

Multiple neutralizing antigenic sites have been found on

type 0 FMDV (32,56-58,62), aswell as onhuman rhinovi-rus 14 (HRV14) (51, 52) andpoliovirus (12, 17, 37, 41, 61). Recentevidence suggeststhat hepatitis Avirus may have a

single immunodominant antigenic site (55). The analysis of

the nature and location ofthe antigenic sites in poliovirus

and rhinovirus has been augmented by theability to locate

the mutations on the three-dimensional (3-D) structural

modelofthose viruses (27, 41, 48).

Recently, Luo et al. (30) have predicted the 3-D surface

structureoftypeA12 FMDVon thebasis oftheknown 3-D

structures ofother picornaviruses and computer-generated sequencealignments.Theactual3-D structure of FMDV has not been reported, although thevirus has beencrystallized

and preliminary X-ray diffraction data have been obtained

(22). The sequence alignments have allowed us to assign

putative locationstotheepitopesand relate theseepitopesto

theknown locationsofepitopesonotherpicornaviruses(20,

41, 48).

The majorityof the variantssequenced (18 of 29or

62%)

had amino acid changes ateither residues 1151 or 1152. The

analogous residues in HRV14 are located in alarge surface

protruding loop region inVP1between,BGand,Hknownas

theFMDVloop (30).Wehadpreviouslymappedepitopes to thisregionby using nMAb reactivity to shortVP1fragments

and fusion-VP1 proteins (47). Variability in this region has

also been shown in naturally occurring type A12 variants (49; D. M. Moore, V. Vakharia, and D. 0.Morgan, Virus Res., in press). In FMDV

A1o,

changes in this areaof VP1 appear

tocombine with changes inVP2forasite analogous to Nim II in HRV14 (59). While we have not detected any VP2

changes in our variants, we have found changes in VP3

associated with changes in residue 1152.

Variants which have been generated with nMAb 2PD11 all havechanges in VP3 in either residue 3178 or both 3175 and 3178. These residues appearto lie inanao-helical portion of

VP3 between G and H,in a surface location close to residue 1152 (30). Four of the five 2PD11 variants also had mutations in 1152 and as a result were also resistanttoantibodies 6FF5

and 7SF3, which seem to react with only 1152. The fifth

variantin thisgroup(D2PD) hadasingle mutationatresidue 3178 and was only partially resistant to 2PD11. Thus, it

appears that the 2PD11 epitope consists of both residues

1152 and3175, 3178,orallthree; however,only mutations in the VP3 residues confer resistanceto 2PD11. Residue 1152 mightaid in binding the antibody, since the variant without this change was only partially resistantto2PD11.

The 2PD11 variantswerealso resistanttoantibodies 2PE4 and 2FF11. Both of these antibodies arealso conformation dependent. Variants generated with antibody 2PE4,

how-ever, were still neutralized by 2PD11. One variant (D2PE)

had a single amino acid change at residue 1201, while the otherwas adoublemutant at 1152 and 1201. The location of theC-terminal residues of VP1 relativetothe other

picorna-virusesisnot known, buttheymayhave surfacelocation by

analogy with HRV14 (30). Given that 2PD11 variants were

resistant to 2PE4, it would indicate that the C-terminal residues of VP1 do lie on the surface and are in the same

antigenic site with residues 3175, 3178, and 1152. Mutations in VP3 may possibly alter the surface in this region sothat

2PE4cannot neutralize, even though it may not react with

those residues directly. A single change at residue 1201 is sufficient to confer resistance to 2PE4-induced neutraliza-tion.

Variants to the third conformation-dependent antibody, 2FF11, all had mutations at residue 1173. This residue may

lie nearthe fivefold axis in the

PI

of VP1 (30). Thisis close

to the Nim 1B antigenicsite inHRV14. One variant (B2FF) was a double mutant with a second mutation at 1147. This residueappears tolie in the FMDV loopnearthelocation of the mutations in the other variants. This residue has been previouslyidentified as being subject to variationinnaturally occurringantigenic variants fromtypeA12 (49). This variant appeared to be partially resistant to 6EE2 (Fig. 1) but was fullyneutralized by that antibody by the more sensitivePRN

assay (Table 2). Thus, 2FF11 appears to define a second antigenic site on surface of type A12. This conclusion is basedonthelocation of the mutation and the fact that 2FF11 variants were onlyresistant to 2FF11. The only data arguing against this conclusion is the fact that 2PD11 variants were

all either fully or partially resistant to 2FF11. This result might indicate that mutations in VP3 could prevent 2FF11

reactivity byinducing a change insurface conformationeven

though the antibody could react with a different antigenic site.

All of the variants to 6HC4 and 6EE2 had an aminoacid

substitution at residue 1209, which is four residues away from the C terminus of VP1. It had been previously sug-gested that the epitope which reacts with these antibodies was located between residues 1169 and 1179 (47). This identification was based on the fact that VP1 blocked

anti-body binding to 12S as did a CNBr fragment spanning residues 1055 to 1179, while peptides covering 1001 to 1144 and 1137 to 1168 did not. Our sequencing studies did not reveal any amino acid change in the region of 1169 to 1179. As with the 2PE4 variants, which had a change at the C terminus of VP1 and residue 1152, four of the nine variants generated with 6HC4 and 6EE2 were double mutants at residues 1152 and 1209, thus strengthening the conclusion thattheC-terminal VP1 residues must lie close to the FMDV loopregion on the viral surface. While there is evidence that C-terminal amino acids define antigenic sites on type 0 J. VIROL.

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FMDV (18, 33, 58) and there is some suggestion that C-terminal VP1residues areimportant in type Avirus (34), resultsfor types A12 and

A1o

(59) directly demonstrate that C-terminal amino acids of VP1 are part of type A-neutral-izing antigenic sites.

Finally,variantsto6FF5 and7SF3areallcharacterized by

asingle amino acidchangeat either 1151 or1152. Antibody

7SF3 had beenpreviously mapped to thisregion of VP1 by

using reactivity to short VP1 fragments and VP1-fusion

proteins (47). Others have also shown the variabilityof this

residue in naturally occurring type A12 variants (49; Moore

et al., in press).

Thus, FMDV type A12 seems tohave two antigenic sites

onthe surface of the virion. Six of the seven nMAbsdefine

amajor siteanalogous to theNim II site on HRV14(48) or

site 2 on poliovirus type 1 (41). Site 2, defined by nMAb

2FF11, appears tobe similarto the Nim IB site on HRV14

(48). There are similarities and differences between our

results for type A12 and the recently reported results for type

A1o

(59). Both subtypes of type A lack an antigenic site

similar to the Nim IA of HRV14. This site is the major

antigenic site ofpoliovirus type 3 (37) but is not found in

poliovirus type 1 (41). In contrast, none of our nMAbs

appeartodefineanantigenicsiteanalogoustothe Nim III of HRV14 (48, 51) or site 3 in poliovirustype 1 (41), which is oneof the major sites found ontype

A1o.

The involvement of the C terminus of VP1 appears to

differ between A12 and

A1o.

In type

A1lo

the C terminus formsa separate antigenic site, whereas in A12, ourresults

indicate that this area ofVP1 appears toforma partof the

major

antigenic site. This is similar to HRV14 (51, 52) and FMDV type 01(62).

Viral proteins in cells infected with the 6EE2 and 6HC4

variantsreactedwith theirrespective monoclonal antibodies

(Fig.

4),and purified, biosyntheticallylabeled B6HC reacted with6HC4 inanRIA(Fig. 2). Thus,it ispossiblethatresidue 1209

might

not be necessaryforattachment of6HC4.There has beenasuggestion ofasimilar situationinpoliovirus (12).

Alternatively,

it is possiblethat a mutationat residue 1209 mayallow the antibodyto bind but not neutralize, perhaps

by changing the orientation of antibody binding. Variants

representing the other monoclonal antibodies clearly have

littleor no

reactivity

withtheirrespectivenMAbs(Fig.2and

4). This would imply that, while other residues might be involved in theantibody binding sites,thechanged residues

clearly

havea

major

influence on

antibody binding.

While three of the variants appeared to have a reduced

ability

to inhibit cellular protein synthesis (Fig. 3A), two

variants, B7SF and B2FF, showed a markedly impaired

ability to both inhibit cellular protein synthesis and cause

virus-induced morphological alterations to cells. The basis

for reduced in vitro virulenceofthe variants isnot known. The genome ofB7SFwasonly sequencedinthe VP1region,

and the only mutationwas at amino acid residue 1152. The entire P1

region

of variant B2FF was sequenced and, in

additiontothechangesataminoacid residues1147 and1173,

the

only

other change was a silent nucleotide change in amino acid residue2063.Thesevariants havenotbeen tested in animals for attenuation. Recently, Prabhakar et al. (44)

reportedthatcoxsackievirus B4variantswereattenuated in

suckling

mice. In picornaviruses, the molecular basis of attenuation has been studiedmost extensivelywith

poliovi-ruses. Analysis ofneurovirulent and attenuated strains of

poliovirus

type 3 suggests thatanucleotidechangein the 5'

noncoding regionof the genome might be relatedto

attenu-ation (14, 19, 39), and in poliovirus type 1, areas in the 5'

half of thegenomehave been implicated in virulence (1, 2). InFMDV,attenuation has been relatedto ashortening ofthe poly(C)tract, adeletion inprotein 3ABCD,orboth (42,50). Moreextensiveanalysis ofourpoorly growing variants will be necessarytodeterminethemolecular basis of attenuation of the variants.

ACKNOWLEDGMENTS

WethankJohn Dunn andWilliamCrockett, Brookhaven National

Laboratory, for helpwithpreparation of the oligonucleotide prim-ers.Wealsoexpress ourappreciationtoMichael Rossmann,Purdue

University, for sharing data on the three-dimensional structural

predictions ofFMDV priortopublication. Finally, wethankMary

Wigmore for helpinpreparation ofthe manuscript.

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