Journal of General Virology (1990), 71, 629-637. Printed in Great Britain 629

A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus of serotype C

M. G. Mateu, ~ M. A. Martinez, 2 L. Capucci, 4 D. Andreu, 3 E. Giralt, 3 F. Sobrino, 2 E. Brocchi 4 and E. Domingo 1.

1 Centro de Biolog& Molecular, Universidad Aut6noma de Madrid, Canto Blanco, 28049 Madrid, 2Departamento de Sanidad Animal lnstituto Nacional de Investigaciones Agrarias, c/Embajadores, 68, 28012 Madrid, 3 Departament de Qulmica Orghnica, Universitat de Barcelona, 08028 Barcelona, Spain and 4 Istituto Zooprofilattico Sperimentale della Lombardia e dell'Emilia, 25125 Brescia, Italy

Neutralizing monoclonal antibodies (nMAbs) elicited against foot-and-mouth disease virus (FMDV) of serotype C were assayed with field isolates and variant FMDVs using several immunoassays. Of a total of 36 nMAbs tested, 23 recognized capsid protein VP1 and distinguished at least 13 virion conformation- independent epitopes involved in neutralization of FMDV C. Eleven epitopes of FMDV C-S8cl have been located in segments 138-156 or 192-209 of VP1 by quantifying the reactivity of nMAbs with synthetic peptides and with nMAb-resistant mutants of FMDV C-S8cl carrying defined amino acid substitutions. The

main antigenic site of FMDV C-S8cl (VP1 residues 138 to 150) consists of multiple (at least 10), dis- tinguishable, overlapping epitopes. Some amino acid replacements abolished one of the epitopes, whereas other replacements affected several epitopes in this region. The conservative substitution His(146) --, Arg, found in many nMAb-resistant mutants analysed, abolished the reactivity of the virus with all nMAbs that recognized epitopes in the main antigenic site of FMDV C-S8cl. This indicates that a minimum genetic change can result in a highly amplified phenotypic effect, as regards the antigenicity of FMDV.

Introduction

Foot-and-mouth disease virus (FMDV) is a picornavirus that causes an economically important disease of cattle and other cloven-hoofed animals (reviews in Bachrach, 1968; Brown, 1979; Pereira, 1981 ; Domingo et al., 1990). The region around amino acids 140 to 160 of the structural protein VP1 contains a major antigenic determinant for the neutralization of FMDV (Stroh- maier et al., 1982; Pfaff et al., 1982; Bittle et al., 1982; Rowlands et al., 1983; Geysen et al., 1984, 1985). This region includes several epitopes (Pfaff et al., 1988; Stave et al., 1988; Thomas et al., 1988; Bolwell et al., 1989; Mateu et al., 1989) and for FMDV of serotype O some of the epitopes also contain residues of VP1 segment 200- 213 (Parry et al., 1985, 1989; Xie et al., 1987), thus indicating a complex antigenic structure. Peptides representing this antigenic site may constitute synthetic vaccines for FMD (reviews in Brown, 1988, 1989), but a major drawback for their efficacy is the remarkable potential for antigenic variation of FMDV (Mateu et al., 1987, 1988, 1989; Gebauer et al., 1988; reviewed by Domingo et al., 1990). Analysis of the reactivity of monoclonal antibodies (MAbs) with field isolates of

FMDV type C has shown that very few of the epitopes involved in the neutralization of viral infectivity are conserved (Mateu etal., 1988). This observation prompt- ed us to suggest that each FMDV isolate is antigenically unique in its fine epitopic composition (Mateu et al., 1988). This would follow from the quasispecies (extreme genetic heterogeneity) nature of FMDV populations (Domingo et al., 1985, 1990; Steinhauer & Holland, 1987; Domingo & Holland, 1988; Eigen & Biebricher, 1988; Domingo, 1989). In this view, each isolate would be characterized by an average antigenic composition and individual virions of that isolate may deviate from the average antigenic behaviour (Rowlands et al., 1983; Mateu et al., 1989; Diez et al., 1989). It is not possible to predict to what extent any given amino acid substitution fixed at an antigenic domain will affect the interaction of the antigen with different antibodies and the nature of this interaction has only recently begun to be clarified at the atomic level (reviews in Mariuzza et al., 1987; Getzoff et al., 1988; Colman, 1988).

In this report we show that the 138-156 VP1 region of FMDV of serotype C consists of multiple, overlapping epitopes involved in neutralization of viral infectivity. A number of amino: acid substitutions fixed at this

0000-9198 © 1990 SGM

630 M. G. M a t e u and others

ant igenic site in the field or in cell cul ture affected one or a few such epi topes . In contrast , F M D V mutan t s wi th the amino ac id r ep l acemen t His(146) -* Arg in VP1 d id not react wi th any of the ava i lab le neu t ra l iz ing ( n ) M A b s tha t define the different ep i topes on tha t an t igen ic domain . This result p rovides d i rec t ev idence tha t ce r ta in amino acid subst i tut ions , should they be fixed dur ing virus evolut ion, can lead to r emarkab l e an t igenic change in the major d e t e r m i n a n t o f F M D V .

Methods

Viruses. The origin and preparation of the majority of the FMDV field isolates used in the present study have already been described (Mateu et al., 1987, 1988). In addition, FMDV C1 Vosges (isolated in France, 1960), C, Corneze (France, 1966), C1 Haute Loire (France, 1969) and C~ Bouches du Rh6ne (France, 1978) were also analysed. They were amplified in BHK-21 cells from the original stocks, kindly provided by M. Lombard (Rh6ne M6rieux, Lyon, France). MAb- resistant mutants were selected with nMAb 4G3, either from FMDV C-S8cl (C~ Santa Pau; Spain, 1970) or from an nMAb SD6-resistant mutant of this virus, using described procedures (Mateu et al., 1989). For the selection of nMAb 4G3-resistant mutants, 104 to 105 p.f.u, were incubated for 1 h at 4 °C with a 1:10 dilution of ascitic fluid. Rabbit anti-mouse IgG (Cappel) was then added to a 1:40 dilution and the mixture was further incubated for 1 h at 4 °C and plated with a 1:200 dilution of nMAb 4G3 included in the agar overlay. Unless stated otherwise each MAb-resistant mutant analysed in the present study was selected from virus prepared from a different single plaque, each derived from the three-times plaque-purified FMDV C-S8cl (Mateu et al., 1989).

Synthetic peptides. Peptides were synthesized manually by solid phase procedures (Merrifield, 1963) on either 4-methylbenzhydryl- amine or N-phenylacetyl-p-aminobenzoic acid-type solid supports (Giralt et al., 1981 ; Mateu et al., 1987, 1989). Synthetic protocols were based on the classical butoxycarbonyl/benzoic acid protection strategy and included systematic double coupling for each residue. Following HF cleavage and deprotection, the crude peptides were purified by gel filtration, followed by reverse-phase liquid chromatography to give HPLC-homogeneous (>95~ pure) products, which were further characterized by amino acid analysis and, in most cases, 1H-nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. After conjugation to keyhole limpet haemocyanin (KLH) the ratio of peptide to KLH was determined by amino acid analysis.

Monoclonal antibodies. MAbs were prepared against FMDV C1 Brescia (Italy, 1964) (Capucci et al., 1984), FMDV C-S8cl (Mateu et al., 1987), FMDV C1 Vosges (MAb 8E8, kindly supplied by M. Lombard, Rh6ne M6rieux, Lyon, France) or FMDV C3 Indaial (Brazil, 1971) (Mateu et al., 1988). Their relevant characteristics are sum- marized in Table 1.

Immunological assays. Enzyme-linked immunodot (EID) and en- zyme-linked immunoelectrotransfer blot (EITB) assays were as described (Mateu et al., 1987), except that goat anti-mouse IgG coupled to peroxidase (Bio-Rad, 1:2000 dilution, 1 h at room temperature) was used instead of rabbit anti-mouse IgG and Protein A-peroxidase. Two types of virus neutralization (VN) assays were used. For titration of MAbs against FMDV C~ Brescia, mixtures of 100 TCIDs0 of FMDV C~ Brescia and serial dilutions of supernatant of hybridoma culture were incubated for 1 h at 37 °C and titrated on IBRS-2 cell monolayers

grown on multiwell plates (four wells/dilution). At 48 h post-infection titres were calculated using the method of Reed & Muench (1938). Any other neutralization tests and titres were by a plaque reduction assay, as described in Mateu et al. (1987).

Nucleotide sequencing. FMDV RNA was sequenced by primer extension and dideoxynucleotide chain termination, as described (Sobrino et al., 1986; Martinez et al., 1988).

Results

Mult iple epitopes in the 140-160 VP1 antigenic site o f F M D V o f serotype C

Seven out o f 17 n M A b s p roduced aga ins t F M D V C1 Brescia reac ted specif ical ly wi th VP1 in E ITB assays (Table 1). These VPl - spec i f i c n M A b s revealed four d is t inct pa t t e rns o f reac t iv i ty in immunoassays wi th a set of 24 field isolates o f F M D V of sero type C (Fig. 1) thus defining, on this basis, at least four d is t inguishable epi topes. These results, a long wi th conclusions of a previous analysis ca r r i ed out wi th 16 VPl - spec i f i c n M A b s p r e p a r e d aga ins t F M D V C-S8cl and F M D V C3 Inda ia l (Mateu et al., 1987, 1988 and da t a not shown), indica te tha t 23 out o f 36 n M A b s el ic i ted wi th type C viruses are VPl - spec i f i c (Table 1) and d is t inguish at least 13 vi r ion c on fo rma t ion - inde pe nde n t ep i topes involved in neu t ra l iza t ion o f F M D V s of sero type C. Eleven o f these epi topes are represen ted in F M D V C-S8cl (see groups in Table 1).

W e have used synthe t ic pep t ides represen t ing VP1 segments for ep i tope locat ion. In E I D assays 20 VP1- specific n M A b s reac ted quan t i t a t ive ly wi th pep t ide A 19 (residues 138 to 156 o f VP1) and on ly one n M A b , 7JA1, reac ted wi th pep t ide C18 (residues 192 to 209 of VP1); none o f the c on fo rma t ion -de pe nde n t n M A b s reac ted wi th any of the pep t ides (Table 1 and Fig. 2a). The epi topes recognized by n M A b s 7AB5 and 7CH1 are not represented in F M D V C-S8cl and have not been located.

A more precise m a p p i n g o f the different ep i topes located in the 130-160 VP1 region was achieved wi th over l app ing pep t ides (Fig. 2b). n M A b s 5A2, SD6, 7 A H I , 7FC12 and 7EE6 reac ted wi th pep t ides A24 (residues 133 to 156) and A19 (138 to 156), but not wi th A13 (144 to 156), A9 (148 to 156) or A7 (150 to 156). Thus, residues f rom segment 138-143 cont r ibu te to the five ep i topes recognized by those M A b s . The absence of reac t iv i ty wi th pep t ide A '9 (136 to 144) suggests tha t residues f rom segment 145-156 also pa r t i c ipa t e in these five epi topes, a l though these residues are not sufficient to p roduce posi t ive b ind ing wi th pep t ide A13. The conclu- sions of the reac t iv i ty of those n M A b s wi th synthet ic pep t ides are cons is ten t wi th the obse rva t ion tha t n M A b SD6-res is tant mu tan t s included rep lacements at amino ac id pos i t ions 138, 139 or 146 of VP 1 (Mateu et al., 1989).

Overlapping epitopes in F M D V type C 631

T a b l e 1. nMAbs raised against F M D V o f serotype C

Reactivity with homologous F M D V

Reactivity with F M D V C-S8cl

Whole virus Viral Whole Viral Synthetic Eliciting protein virus protein peptide

MAb F M D V Isotype ELISA VN* EITB t EID EITB EID:~ Group§

3E5 C1 Brescia (140S) IgG2a + +(2.6) VP1 + VP1 A19 IV 4B8 IgG3 + + (2-7) VP1 + VP1 A 19 IV 4C4 ND[[ + + (2-0) - + VP 1 A 19 IV 4F 1 IgG 3 + + (2.9) VP 1 + VP 1 A 19 I 4F11 IgG3 + + (2.7) VP1 + VP1 A19 IV 5A2 IgG2a + + (1.5) VP1 + VP1 A 19 II 6 D l l IgG1 + +(2-1) VP1 + VP1 A19 III IG5 IgG2b + + (2.0) - + - - 2A12 IgG3 + +(2.1) - + - - 2E5 IgG 1 + + (2.4) - + - - 3C2 IgG2b + + (1.8) + - 3E9 IgG2b + + (2.0) - + - - 3 G 11 IgG 1 + + (0.9) - + - - 4 D 7 IgG1 + + (1.8) - + - - 4F4 IgG 1 + + (0.6) - + - - 5C4 IgG2b + + (2.0) - + - - 5H10 ND + +(2-1) - + - -

SA3 C-S8cl (140S) IgG1 + + VP1 + VP1 AI9 I SA4 IgG1 + + VP1 + VPI A19 I SB3 lgG 1 + + VP 1 + VP 1 A 19 I SB10 ND + + VPI + VPI A t9 I SC9 ND + + VPI + VP1 A19 I SD6 IgG 1 + + (3"0) VP 1 + VP 1 A 19 I 4G3 IgG1 + +(1-0) VP1 + VP1 A19 IX

8E8 CtVosges (140S) ND + +(1.0) - + - -

7AH1 C31ndaial (140S) ND + +(1.4) VPI + VP1 A19 V 7FC12 IgG1 + + (3.5) VP1 + VP1 A19 VI 7CA 11 IgG 1 + + (3"6) VP 1 + VP 1 A 19 VII 7CA8 IgG3 + + (3-6) VP1 + VP1 A 19 VIII 7JDI IgG1 + + (3.3) VP1 + VP1 A19 VIII 7EE6 IgG3 + + (3'3) VP 1 + - A 19 X 7JA1 IgG2b + + (0.4) VP1 + VP1 C18 XI 7AB5 IgG3 + +(3 '5) VPl - - 7CH 1 IgG 1 + + (2.0) VP 1 - - 7LA5 IgG3 + + (1.4) - + -- 7DF10 IgG 1 + + (1.5) - + - -

* VN, virus neutralization assays. Average neutralizing titres (in parentheses) are given. For MAbs against F M D V C 1 Brescia, titres were expressed as the log10 reciprocal dilution of supernatant of hybridoma culture (SHC) that inhibited virus-specific c.p.e, development (100 TCIDso) in 5 0 ~ of the wells in a classic microneutralization assay. For the remaining MAbs, titres were given as the log10 reciprocal dilution of SHC that causes neutralization of 7 0 ~ of p.f.u, in a plaque reduction assay (Mateu et al., 1987).

t The reacting viral protein is indicated. - indicates no protein reacted with the MAb. :~ The peptide reacting with the M Ab in an EID assay is indicated. A19, peptide spanning residues 138 to 156 of VP1 ; C18, peptide spanning

residues 192 to 209 of VP1. - , no peptide reacted. § The MAbs that recognize VP1 segments 138-156 or 192-209 of F M D V C-$8 were assigned to different groups (I to XI, see also Fig. 4) when a

distinct pattern of reactivity with a set of F M DV variants was observed (data from Fig. 1 and Mateu et al., 1988). Minor differences in the pattern of reactivity were not taken into account.

[I ND, Not determined.

S u c h c o n c l u s i o n s a r e a l s o i n a c c o r d w i t h t h e r e a c t i v i t i e s

o f t h e M A b - r e s i s t a n t m u t a n t s w i t h n M A b s 5 A 2 , 7 A H 1

a n d 7 F C 1 2 ( F i g . 3 ; s e e b e l o w ) .

I n c o n t r a s t t o t h e s e a n t i b o d i e s , n M A b s 4 G 3 , 7 C A 1 1

a n d 7 J D 1 a l s o r e a c t e d w i t h p e p t i d e A 1 3 , i n d i c a t i n g t h a t

p o s i t i o n s 144 t o 147 c o n t r i b u t e t o t h e t h r e e e p i t o p e s

d e f i n e d b y t h e l a t t e r M A b s . B e c a u s e p e p t i d e s A 1 3 a n d

A 1 9 b o u n d t o a s i m i l a r e x t e n t t o M A b s 4 G 3 , 7 C A 1 1 a n d

7 J D 1 , r e s i d u e s w i t h i n s e g m e n t 1 3 8 - 1 4 3 d o n o t a p p e a r t o

c o n t r i b u t e s i g n i f i c a n t l y t o t h e e p i t o p e s d e f i n e d b y t h e s e

n M A b s . C o n s i s t e n t w i t h t h e s e r e s u l t s , i n M A b 4 G 3 -

r e s i s t a n t m u t a n t s t h e p o s i t i o n s i n v o l v e d w e r e 146, 148,

149 a n d 150, a n d s u b s t i t u t i o n s i n 138 a n d 139 d i d n o t

a f f e c t t h e r e a c t i v i t y o f n M A b s 4 G 3 , 7 C A 1 1 a n d 7 J D 1

632 M.G. Mateu and others

Ct GC-Ger/2(

C2997-U.K./-~ i Ct Loupoigne

C~ Vosges-Fr/

C~ Corneze-F

C~ Haute Loil

C~ Bouches d

C~ Santa Pau-

C~ Vail de Bi~

C~ Lleida-Sp/l

C~ Serra Dar(

C~ Barcelona-

C2 Pando-Ur/

C3 Resende-13

C4 Tierra de ]

C~ Arg/69

C3 Leticia-Cc

C3 Indaial-Br

C3 Santa Fe-~

C3 Arg/83 )

CPFA43652-

C3 Arg/84

: C3 Arg/85 I CPFA45913-

Fig. 1. Reactivity in EITB assays of FMDV C field variants with VP1- specific nMAbs elicited with FMDV C~ Brescia. After the designation of the isolate the country and year of isolation are abbreviated. Abbreviations and symbols: Ger, Germany; U.K., United Kingdom; Bel, Belgium; Fr, France; Sp, Spain; Ur, Uruguay; Br, Brazil; Arg, Argentina and Col, Columbia. For each MAb the reactivity of each isolate is referred to that of FMDV C-S8c 1 and is given as: II, positive (signal above 50~ of that of the control); k~, a reduced signal is detected (signal under 50 ~ of control); IS], no signal detected; ND, not determined.

(Table 2 and Fig. 3). nMAbs 4C4 and 6Dll bound significantly less strongly to peptide A13 than to A19 or A24 (Fig. 2b), suggesting that residues in segment 138- 143 may somehow contribute to the epitopes recognized by these two nMAbs. This was confirmed by results of reactivity of MAb-resistant mutants with nMAbs 4C4 and 6D11 (Fig. 3). These results, summarized in Fig. 4 (see also below), permit grouping of the epitopes at the 138-156 site of VP1 in two antigenic subregions.

(a) 1 I I I I I I 1 I I I I I I I I I I I

(~

F

C /

t

t

I I I I I I I I I I I !

t

K

Fig. 2. (a) Reactivity in EID assays of FMDV C-S8cl andofsynthetic peptides with nMAbs raised against FMDV C~ Brescia. Antigens applied were: PBS, phosphate-buffered saline alone (no antigen added); FMDV C-S8c 1 (containing 25 pmol of VP 1 per dot); peptides A24, A19 or C18 (30 pmol) coupled to KLH (0.5 ktg) [these peptides represent VP1 amino acid residues 133 to 156, 138 to 156 and 192 to 209, respectively]; KLH, 0-5ktg carrier protein alone. They were reacted with the indicated nMAbs. (b) Reactivity of overlapping peptides with nMAbs that recognize distinct epitopes in the 138-156 VP1 region. Antigens were applied to nitrocellulose: PBS, no antigen added; C-$8, FMDV C-S8cl (containing 25 pmol of VPI); synthetic peptides (80 pmol each) representing segments of VP1 of FMDV C- $8cl, coupled to KLH (0.5 ~tg) [A24 (amino acids 133 to 156), A19 (138 to 156, A13 (144 to 156), A9 (148 to 156), A7 (150 to 156), A'9 (136 to 144)]; KLH, 0-5 p,g carrier protein alone. They were reacted with PBS, (no antibody added), anti-KLH serum (ctC-KLH) or the indicated MAbsl which represent each of the groups defined in Table 2. A mouse serum against peptide A7 coupled to BSA gave a positive reaction with peptides A24, A19, A13, A9 and A7 coupled to KLH, but not with KLH alone.

A single amino acid substitution affects multiple overlapping epitopes in the 140-160 VP1 antigenic site of FMDV of serotype C

W e have quan t i f i ed the r eac t iv i ty in i m m u n o a s s a y s o f F M D V type C v a r i a n t s w i t h subs t i t u t i ons in the VP1 reg ion a r o u n d a m i n o ac ids 140 to 160, u s ing VP l - spec i f i c n M A b s tha t def ine d i f fe rent epi topes. The resul ts o f r eac t iv i ty of M A b - r e s i s t a n t m u t a n t s wi th n M A b s in E I T B assays (Fig. 3) show tha t the subs t i t u t i ons f o u n d c a n be classified in to th ree groups , a cco rd ing to the i r effect o n the a n t i g e n - a n t i b o d y in te rac t ion . T h e first

Overlapping epitopes in F M D V type C 633

Selec FMDV M/

Parent C-SScl MAR SD6-1 : MAR SD6-2 : MAR SD6-7 MAR SD6-6 MAR SD6-11 MAR SD6-18 MAR SD6-19 MAR SD6-28 MAR SD6-40 SD6 MAR 4G3-2 4G3 MAR 4G3-11 4G3 MAR4G3-10 4G3 MAR 4G3-9 4G3 MARSD64G3-1 ,~# MAR SD64G3-3 ]~] MAR SD64G3-2 ]~

Fig. 3. Reactivity in EITB assays of VP1 of MAb-resistant (MAR) mutants with nMAbs that recognize different epitopes in the 138-156 VP1 region of FMDV C-S8cl. Each of mutants SD6-1, -7, -6, -11, -28, -40 and 4G3-2 was selected from virus prepared from a different plaque. Mutants resistant to both SD6 and 4G3 (MAR SD6-4G3) were selected in the presence of MAb 4G3 from mutant SD6-6. For each MAb the reactivity of any mutant is referred to that of FMDV C-S8cl and is given as: II, positive (signal above 50% of that of FMDV C- $8cl); k~, a reduced signal is detected (signal under 50%); D, no signal detected.

133 138 144 150 156 I I 1 I ]

TTTYTA S A S H LTTTH ARH LP Epitope , , , , , S D 6 - I ~ ~

5A2-I I ~ l , ~ . ~ k :

6D 11- I 11 ) i ~ ' q ~ ' ~ = ~ _ 4C4-I V ~ ' ~ , ~ ~

7AH I-V ] ~ \ \ ~ X ~ . \ \ ~ " ~ 7FCI2-VI l :~

7CAI I-VII

7JDI-VIII 1 ~'~'~'N~ ~

4G3-1X I -~'~i~,,",~'~k

Fig. 4. An outline of the antigenic structure of the 138-156 VP1 determinant of FMDV C-S8c 1. nMAbs that recognize distinguishable epitopes (assigned to different groups in Table 1) are listed (left column). The amino acid sequence (single-letter code) between residues 133 and 156 of VP 1 of FMDV C-S8cl is shown. Residues which upon alignment are equivalent to the 137-156 loop of FMDV OIBFS 1860 (Acharya et al., 1989) are underlined. Dots under the sequence indicate positions with substituted amino acids in the variants analysed. The conserved sequence R-G-D-L-A is boxed. The information obtained for each epitope is summarized. A box delimits the shortest peptide found to be reactive with the corresponding MAb. Filled triangles indicate positions where one or more amino acid substitutions affected the binding of the MAb in both EID and EITB assays. Positions at which substitutions had an effect in EITB but not in EID assays (empty triangles), or in EID but not tested in EITB assays (empty squares) are also indicated. Substitutions at 140, 147 and/or 149 affect the reactivity with MAbs 5A2, 7AHI and 7FC12, but the substitution(s) responsible are unknown. Hatched regions indicate the minimal location of each epitope (assumed to be completely linear) as defined by the available data (compare Fig. 2b and 3).

Table 2. Monoclonal antibody 4G3-resistant mutants of F M D V C-$8c1"

Parent Substituted Number of FMDV amino acid'~ mutants

C-S8cl His(146) --, Arg 4 Thr(149) ~ Pro 3 Thr(150) ~ Met 2 Thr(150) ~ Lys 1

SD6-6:~ Thr(148) --* Pro 1 Thr(149) ~ Pro 1 Thr(150) ~ Lys 1 Thr(149) --, Ala; Thr(150) ~ Met 1

* Each MAb-resistant mutant, except those derived from MAb- resistant SD6-6, was selected from virus prepared from a different single plaque.

Number gives amino acid position in VP1. ;~ SD6-6 is a MAb SD6-resistant mutant of FMDV C-S8cl with the

amino acid substitution Ser(139)-~ lie.

group is amino acid replacements that affected only one epitope [e.g. Thr(150) ~ Met or Thr(150) ~ Lys, which decreased the b ind ing of n M A b 4G3 only]. The second group includes subst i tut ions that affected several, but not

all, of the epitopes wi th in the ant igenic site. For example Ala(138) ~ Asp significantly decreased the in terac t ion of the virus with n M A b s SD6, 5A2, 6D11 and 4C4, which recognized four dis t inguishable epitopes, but did not decrease the interact ion with n M A b s 7AH1, 7FC12, 7 C A l l , 7JD1 and 4G3, which recognized five other epitopes (Fig. 3 and Table 1). In the third group one substi tut ion, H i s (146)~ Arg, affected all the epitopes defined by our panel of n M A b s in this ant igenic region. A high proport ion of MAb-res i s tan t mutan t s indepen- dently selected with n M A b SD6 or 4G3 included replacement H i s ( 1 4 6 ) ~ A r g (Mateu et al., 1989 and Table 2). They did not react with any of the n M A b s that defined n ine epitopes in the 138-156 VP 1 region (Fig. 3). Thus, a single amino acid subst i tu t ion was sufficient to affect the b ind ing of all the ant ibodies that recognized the ma in ant igenic region of this F M D V , even though it consists of mult iple epitopes (see Discussion). n M A b 7JA1 recognized an epitope in the C te rminus of VP1

(Fig. 2a) and its reactivi ty was not affected by any of the tested subsi tut ions in the 138-150 region (Fig. 3).

634 M. G. Mateu and others

When comparing reactivities of MAb-resistant mutants in EITB assays (Fig. 3) with those in EID assays (data not shown) we generally found a good correlation between the two techniques. However, a few exceptions were noted with nMAbs 4C4, 7AH1, 7CA11 and 7EE6. In particular, variants with the substitution His(146) -~ Arg reacted with nMAbs 7AH1 and 7CA11 in the EID assay. Also, nMAb 7EE6 reacted in the same assay with FMDV C-S8cl and all MAb-resistant mutants tested, except those carrying the substitution His(146)-~ Arg. Results of neutralization assays with these viruses and nMAbs paralleled the results of EITB, but not of EID assays. Absence of correlation between binding of some antibodies to virions and neutralization of infectivity has previously been observed for FMDV (Grubman & Morgan, 1986; Mateu et al., 1988; McCahon et al., 1989).

Substitutions fixed in the course of a disease episode [Ala(138) ~ Thr, Leu(147) --, Ile and Leu(147) ~ Val in VP1] greatly affect the epitope defined by nMAb SD6 (Mateu et al., 1989). These substitutions, or others found at positions 140(Ala ~ Thr) and 149(Thr ~ Ala), also affect the epitopes recognized by nMAbs 5A2, 7AH1, 7FC12 and 7CAll , but not those recognized by nMAbs 4C4, 6D11, 4G3 and 7JD1 (Fig. 1, Mateu et al., 1988 and unpublished observations). Thus, the genetic diversity that originated during the spread of the virus in the course of one disease episode affected several epitopes at the main antigenic determinant of FMDV.

Complex antigenic structure o f the 138-156 segment o f VP1 of F M D V C-$8cl

The above data permit the outlining of an antigenic structure for the main antigenic site of FMDV C-S8cl (Fig. 4). The determinant involves at least the region of VP1 between amino acids 138 and 150. Upon alignment these residues equate with positions 139 to 155 of FMDV O1 BFS 1860, matching very closely a disordered loop (amino acids 137 to 156) on the virion surface (Acharya et al., 1989). One group of nMAbs (7CAll, 7JD1 and 4G3) interacts with the hypervariable segment located immediately to the carboxy-terminal side of the conserved Arg(141)-Gty-Asp-Leu-Ala(145) (R-G-D-L- A) sequence (Fig. 4). For example, nMAb 4G3 defines an epitope that lies between amino acids 144 and 156, involving at least residues 146, 148, 149 and 150 (compare Table 1 and Fig. 2b and 3). Another group includes the majority of VPl-specific nMAbs elicited against FMDV C-S8cl, C1 Brescia or C3 Indaial, including SD6, 5A2, 6Dll , 4C4 and 7AH1. These nMAbs define different epitopes, which have as a common feature the participation of residues at both sides of the conserved R-G-D-L-A stretch (Fig. 4). A

pivotal role for the amino acid at position 146 on the antigenic structure of the major determinant of FMDV C-S8cl is suggested by the results (Fig. 3 and 4, see also Discussion).

We conclude that the 138-156 VP1 region of FMDV C-S8cl contains multiple overlapping epitopes. One conservative amino acid substitution, His(146) -~ Arg, is sufficient to eliminate all the epitopes probed.

Discussion

The three-dimensional structure of FMDV of serotype O (strain O1 BFS 1860; Acharya et al., 1989) has revealed that the region of VP1 between residues 137 and 156 is a disordered loop on the virion surface; this segment constitutes the major antigenic site of FMDV (Stroh- maier et al., 1982; Pfaff et al., 1982; Bittle et al., 1982). The loop includes the conserved sequence Arg-Gly-Asp, probably part of the cell attachment site (Fox et al., 1989), flanked by two hypervariable regions, nMAbs SD6 (Mateu et al., 1989), 5A2, 6Dll and 7AH1 were elicited by different FMDVs of serotype C and each recognizes a different epitope that involves the two hypervariable regions surrounding the Arg-Gly-Asp sequence. In addition nMAbs SA3, SA4, SB10, SC9 and 4F1 appear to recognize the same epitope as MAb SD6 (compare Table 1 and Fig. 4). These results suggest immunodominance of this group of related epitopes in FMDV of serotype C. Of a total of 36 nMAbs, 23 were VPl-specific and 20 recognized synthetic peptides representing the VP1 sequence 138-156 of FMDV C- $8cl. The epitopes recognized by those 20nMAbs involved at least some residues from segments 138-140 and 146-150. These observations suggest that segment 138-150 of VP1, which matches very closely the disordered loop of FMDV-O~ BFS (Fig. 5), is the major antigenic determinant of FMDV of serotype C.

Bolwell et al. (1989) have defined three epitopes in the 138-154 VP1 segment of FMDV A22 Iraq 24/64. Despite little homology between the predicted loop residues of FMDV C-S8cl and A22 Iraq 24/64 (Fig. 5), the two viruses share a number of antigenic properties. Most nMAbs raised against these viruses recognize virion conformation-independent epitopes located in the FMDV loop. In both viruses epitopes are found that include residues from the two hypervariable regions around the Arg-Gly-Asp segment; other epitopes involve only the region at the carboxy-terminal side of the determinant. Several of the epitopes overlap. Finally, substitutions at an equivalent position, residue 146 in FMDV C-S8cl or 150 in A22 Iraq 24/64 (Fig. 5), were the most frequent among MAb-resistant mutants isolated with these viruses. Thus, the general antigenic features of

Overlapping epitopes in F M D V type C 635

O~ BFS 1860

A22 Iraq 24/64

Ct-S8cl

FMDV loop 161 131 ~ ~ I

yN G G C R y S R N AV PN L'~I{G_-_D]LQVL AQ KVA R T L P 131 160

YNGTSKY~AOGTG" RiR GD!L6P L AARIVAAQ LP 130 156

YTGTTTYT[A . . . . $ AIRGD',L AH LTTT]HAR H L P

Fig. 5. A comparison between the amino acid sequences of the major antigenic site of FMDV O~ BFS 1860 (Acharya et al., 1989), Az2 Iraq 24/64 (Bolwell et al., 1989) and C-S8cl (Sobrino et al., 1986). The VP1 residues (single-letter code) have been aligned to provide the maximum nucleotide sequence homology among 15 different FMDVs of serotypes A, O and C (Dopazo et al., 1988). A dot in the sequence indicates deletion of one amino acid. A line above the sequence of FMDV O1 BFS indicates the VP1 G-H loop of this virus (Acharya et aL, 1989). The conserved RGD (Arg-Gly-Asp) sequence is boxed with a discontinuous line. The major antigenic site of FMDV A22 Iraq and that of FMDV C-S8cl are boxed with a solid line. Symbols for amino acids found to be involved in epitopes within these antigenic sites are enhanced.

the main determinant of VP1 appear to be maintained in FMDVs of serotypes C and A. This was not the case when viruses of serotypes A and O were compared (Bolwell et al., 1989).

Data in Fig. 1 and 3 and in Mateu et al. (1988) suggest the presence of at least 10 distinguishable epitopes in the antigenic loop of FMDV C-S8cl. It is likely that, as more nMAbs are tested, additional epitopes will be found within this short amino acid stretch. Harrison (1989) has suggested that, as with a peptide in solution, the FMDV loop has no unique conformation; it can adopt, with little cost in free energy, the conformation required to interact with any antibody elicited against a peptide represented in the loop sequence. In this view, the overlapping epitopes defined by our anti-virus nMAbs would correspond to multiple immunogenic conformations of the flexible loop on the virion. Each of them will induce the synthesis of antibodies that will recognize the loop when present in the appropriate conformation.

Rowlands et al. (1983) and Geysen et al. (1984, 1985) identified several positions in the VP1 region around amino acids 140 to 160 of FMDV that, when changed, resulted in a decrease in reactivity of the antigen with guinea-pig or rabbit antiviral sera. Use of MAbs has permitted further probing of the effect of substitutions on the interaction between FMDV and a specific antibody molecule. Conservative, frequently occurring amino acid replacements, which were fixed in VP1 of FMDV type C during a disease episode, greatly diminished the binding of nMAb SD6 to VP1 or to virions (Mateu et al., 1989). Since the relevant substi- tutions were Ala(138) --, Thr and Leu(147) --, lie or Leu(147) ~ V al, we suggested that the decreased interac- tion could result from restriction of local mobility imposed by the bulky radicals on the/%carbon of Thr, lie

and Val, thus impairing the display of the conformation recognized by nMAb SD6. The free energy expended in distorting the peptide chain to fit the antibody-combin- ing site would probably result in reduced binding affinity (Dyson et al., 1988). In the present report we have extended to other nMAbs the previous analysis with SD6 and shown that whereas some replacements affected only one epitope, most of them affected several epitopes (Fig. 4) and the substitution His(146) ~ Arg greatly decreased the reactivity with all nMAbs tested. This is a chemically conservative substitution (Dayhoff et al., 1972), display- ing a high degree of acceptance during protein evolution (Feng et al., 1985). It is also noteworthy that out of seven possible amino acid replacements at this position, all with a similar degree of acceptability (Feng et al., 1985), only His ~ Arg was repeatedly selected for in SD6 and 4G3 MAb-resistant mutants (Mateu et al., 1989 and Table 3). Remarkably, when the replacement His ~ Arg was introduced at the relevant position of a synthetic hexapeptide that exactly represented positions 143 to 148 of FMDV C-S8cl VP1, it was not scored as antigenicaUy significant regarding the reaction of the peptides with rabbit anti-FMDV serum (Geysen et al., 1985). Among other possibilities, our nMAbs may not reflect the antibody composition of the serum used by Geysen and colleagues, or the hexapeptide used may have been shorter than required to represent in their entirety most of the epitopes that we have defined in the 138-156 VP1 region.

A pivotal antigenicity role for the amino acid at VP1 position 146 is also supported by a previous study of virus variants recovered from cattle persistently infected with FMDV C3 (Gebauer et al., 1988). At the equivalent position in VP1 of FMDV C3 (residue 148 in Fig. 5 of Gebauer et al., 1988), the parental C3 clones and viruses 783/63 and 788/63 recovered from cattle had Asp or His. They all reacted with nMAbs whose corresponding epitopes have now been positioned at the 138-156 site (compare Table 2 in Gebauer et al., 1988 and Fig. 4). In contrast, variant 785/63 from carrier cattle included Asp --, Gin at the relevant position as the only replace- ment in this VP1 segment and did not react with most of the above MAbs (Table 2 in Gebauer et al., 1988). Thus, VP1 substitution Asp(148)~ Gin in FMDV C3 paral- leled the effect of His(146)--* Arg in the equivalent position of FMDV C-S8cl, as regards loss of reactive epitopes.

The above results indicate that a single critical replacement fixed in the FMDV loop may have a strong effect on its antigenicity by affecting many epitopes within that site. This leads to an important phenotypic amplification of a modest genetic modification. Thus, a low level of genetic heterogeneity, such as the one found among viruses of a single disease episode that differed in

636 M. G. Mateu and others

about 0.7~ to 2.2~ of nucleotides (Domingo et al., 1980; Sobrino et al., 1986), may cause relevant antigenic diversity (Martinez et al., 1988; Mateu et al., 1988). This reinforces our view that the designing of synthetic vaccines against FMD should include consideration of these variations and their formulation should include variant sequences of critical determinants, and/or be composed of several independent antigenic sites (Domingo, 1989; Domingo et al., 1990; Mateu et al., 1989).

We are indebted to M. Lombard (Rh6ne M~rieux, Lyon, France) and H. Barahona (Pan American Foot-and-Mouth Disease Center, Rio de Janeiro, Brazil) for supplying some viral isolates and MAbs. We thank E. Rocha and B. Borrego for the preparation of some viruses and M. D~vila for assistance. Work at CBM was supported by Comisi6n Asesora para la Investigaci6n Cientifica y T&nica, Fondo de Investigaciones Sanitarias, Consejo Superior de Investigaciones Cient- ificas (Spain) and Fundaci6n Ram6n Areces. Work at INIA was supported by foundations of this Institute and the U.S.-Spanish Joint Committee for Scientific and Technological Cooperation. Work at the University of Barcelona was supported by Comisi6n Interministerial de Ciencia y Tecnologia and by the U.S.-Spanish Joint Committee for Scientific and Technological Cooperation.

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(Received 1 August 1989; Accepted 23 October 1989)