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Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies
Veterinary Immunology and Immunopathology, 30 (1992) 275-292 Elsevier Science Publishers B.V., Amsterdam
275
Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies G. B u t c h a i a h a, J.L. C a r d b a n d D . O . M o r g a n b'* aSouthern Regional Station, Indian Veterinary Research Institute, Bangalore 560 024, India bUnited States Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Molecular Biology Laboratory, P. 0. Box 848, Greenport, NY 11944, USA (Accepted 19 December 1990)
ABSTRACT Butchaiah, G., Card, J.L. and Morgan, D.O., 1992. Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies. Vet. Immunol. Immunopathol., 30: 275-292. A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of footand-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion ( 140S); Class II, restricted to 140S and the virion protein subunit ( 12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-l, A, O1 and C but neutralizing capacity was restricted to serotype Asia- 1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.
INTRODUCTION F o o t - a n d - m o u t h d i s e a s e v i r u s ( F M D V ) , t h e sole m e m b e r o f t h e g e n u s aphthovirus of the family Picornaviridae, consists of seven distinct serotypes. E a c h s e r o t y p e is c o m p o s e d o f a m u l t i t u d e o f s u b t y p e s a m o n g w h i c h t h e r e is e x t r e m e a n t i g e n i c d i v e r s i t y . S e r o t y p e s O, A a n d C o c c u r in E u r o p e , S o u t h A m e r i c a , A f r i c a a n d A s i a ; S A T 1, S A T 2 a n d S A T 3 a r e r e s t r i c t e d t o A f r i c a , a n d Asia-1 is w i d e l y d i s t r i b u t e d t h r o u g h o u t A s i a . T h e i c o s a h e d r a l F M D V c a p s i d c o n t a i n s 60 c o p i e s e a c h o f f o u r s t r u c t u r a l p r o t e i n s t e r m e d V P 1, V P 2 , V P 3 *To whom correspondence should be addressed.
276
G. BUTCHAIAH ET AL.
and VP4 enclosing a single-stranded positive sense RNA molecule of about 8000 nucleotides (Bachrach, 1977). The intact 140S virions (Brown and Newman, 1963; Morgan, 1983), 12S protein subunits (12Sps) (Morgan et al., 1980), VP1 or its fragments (Laporte et al., 1973; Bachrach et al., 1975; Strohmaier et al., 1982 ) and biosynthetic or synthetic peptides (Kleid et al., 1981; Bittle et al., 1982; Morgan and Moore, 1990) all elicit neutralizing antibodies and varying degrees of protective immunity in large animals. Antibodies that neutralize viral infectivity provide an important mechanism of protection against FMD. Polyclonal antiserum is useful for identifying the components of FMDV that elicit an immune response and for comparing the antigenic relationships of FMDV serotypes; however, the limits of its discriminating capacity is exceeded by the variation amongst subtypes. On the other hand, monoclonal antibodies (MAbs) are powerful tools for the precise identification of epitopes and analysis of closely related strains of FMDV. Much of the antigenic diversity of FMDV is due to rapid changes in antigenic makeup that occur in the partially immune host population found in epizootic areas and it is necessary to constantly update the composition of vaccines to maintain their efficacy. Thus, it is important to define those epitopes that elicit the protective immune response and their variation or conservation among variants, subtypes and serotypes. The antigenic structure of FMDV serotypes O, A and C is being elucidated through the use of monoclonal antibodies to detail its complexity (McCullough and Butcher, 1982; Meloen et al., 1983; Baxt et al., 1984; Duchesne et al., 1984; Morgan et al., 1984; Ouldridge et al., 1984; Robertson et al., 1984; Grubman et al., 1985; Grubman and Morgan, 1986/1987; Stave et al., 1986, 1988; Mateu et al., 1987, 1988; Baxt et al., 1989). With the exception of a few anti-Asia-1 virus MAbs (Butchaiah and Rao, 1989 ), no detailed MAb analysis of serotype Asia1 FMDV has been reported. This report describes the production and characterization of a panel of MAbs elicited with three different isolates of serotype Asia-1 FMDV. Their reactivity with 10 serotype Asia-1 virus isolates and representations of FMDV serotypes O, A and C is described. MATERIALS AND METHODS
Viruses and cells Foot-and-mouth disease viruses of serotype Asia-1, India 63/72, 21/80, 4/ 86, 10/86, 66/86, MFB/86 and GDG/87, were isolated from bovine tongue tissue from various regions of India. Other viruses of serotype Asia-1, Pak/ 57, Iran/73 and Israel/68 were from stocks of the Plum Island Animal Disease Center. With the exception of Israel/68, all of these serotype Asia-1 viruses belong to the same subtype. The last two digits in the virus designation
ANTIGENIC RELATIONSHIPS OF FOOT-AND-MOUTH DISEASE VIRUS ASIA-1
277
refer to the year of isolation. The bovine tongue tissue viruses were passaged in BHK-21 monolayer cell cultures and stored at - 7 0 ° C. A continuous bovine kidney cell line LF-BK (Swaney, 1988 ) was used for plaque assay.
Purified viral antigens Virus from infected cultures of BHK-21 cells was precipitated with polyethylene glycol and purified by CsC1 density gradient centrifugation (Wagner et al., 1970). The purified virus ( 140S ) was dialyzed against tris-buffered saline (TBS:0.05 MTris HC1, 0.3 MNaC1, pH 7.5 ) and quantitated by optical density. The 10 virus isolates of serotype Asia- 1 and preparations of serotypes O, A and C were purified and quantitated in this manner. The 12Sps was prepared by overnight dialysis of purified 140S virions against citrate-phosphate buffer, pH 4.5, followed by dialysis against TBS and clarification by centrifugation (3000 rpm, 30 min, 4 °C ) (Cowan, 1968 ). Purified VP 1 Was prepared from urea-disrupted 140S virions by DEAE anion-exchange chromatography (Bernard et al., 1974) and checked for purity by SDS-urea-PAGE electrophoresis (Matheka and Bachrach, 1975 ).
Monoclonal antibodies Adult BALB/c mice were infected with purified live virus (103-105 suckling mouse LDs0) and immune cells from these mice were used to generate hybridomas secreting anti-FMDV MAbs. Fusions were made at 7 and 14 postinoculation days (PID) as described by Stave et al. (1986, 1988). Fusion series 26, 30 and 31 were produced at the PIADC and the series 2 at The Indian Veterinary Research Institute (Table 1 ). Hybridomas were grown in minimal essential medium (MEM; Gibco) supplemented with 20% fetal bovine serum (FBS). The culture supernatants were screened by a liquid-phase radioimmunoassay (RIA) for the presence of virus and virus subunit reactive antibodies. Hybridomas producing antibodies of interest were cloned at least twice by limiting dilution and stored in liquid nitrogen. Isotype of the MAbs was determined from hybridoma culture supernatants by Ouchterlony precipitation with anti-mouse isotype specific antisera (Litton Bionetics, Kensington, MD, USA) in 1% agar, pH 7.9 buffered with 1 Mglycine, 0.0125 M sodium barbital, 0.15 M NaC1. The complete MAb designation (i.e. decimals indicate cloning cycles) is given in Table 1, but elsewhere in the report a short form of the designation is used (e.g., 31HC2 replaces 31HC2.1.1 ).
Radioimmunoassay Labelled virions were prepared by reacting 100-200/tg of purified 140S in 100/tl TBS with 0.25-0.5 mCi 1251(Amersham.Searle) in the presence of 25 pg iodogen as described previously (Fraker and Speck, 1978 ). The unincorporated 1251was removed by gel filtration through Sephadex G-25 (Pharmacia). Aliquots of the labelled virus were made to 3% bovine serum albumin
278
G. B U T C H A I A H ET AL.
(BSA) and stored at - 7 0 ° C . The labelled virus was diluted in RIA-BSA buffer (0.12 M boric acid, 0.02 M sodium borate, 0.15 M NaCI, 0.1% BSA, pH 8.5) for initial RIA screening of hybridoma culture supernatants. The 12~I-labelled virus was prepared by centrifugation on a 20-50% discontinuous sucrose gradient (45 000 rpm, 1.5 h, 4°C; Beckman SW 55 Ti rotor) immediately prior to use in determining the specific reactivity of the MAbs to intact 140S virions. A liquid-phase radioimmuno-precipitation (LP-RIA) assay was used to test the MAbs in culture supernatants for their reactivity with labelled virus. Briefly, the MAb culture supernatant (50 #1) was mixed with labelled virus (50/~1; 20 000-30 000 cpm) and incubated (37°C, 1 h). The antigen-antibody complexes were precipitated by adding 50 #1 of 1% protein-A bearing Staphylococcus aureus (armed with rabbit anti-mouse immunoglobulins) followed by incubation (37 ° C, 1 h). The precipitates were pelleted (2 min, Eppendorf Centrifuge Model 5413 ) and washed twice in RIA-BSA buffer containing 0.05% Tween-20 (RIA/BSA/Tween). The amount of radioactivity associated with the pellet was determined and the 30% antibody binding endpoint titer was calculated (Trautman and Harris, 1977). A solid-phase RIA (SP-RIA) was used to determine the reactivity of the MAbs with 12Sps and isolated VP 1 of the virus. Solutions of 12Sps (0.6/~g/ 50 #1 RIA buffer) and VP 1 (0.8 #g/50 #1 0.2 M ethylmorpholine, 6 M urea, 10 m3/2-mercaptoethanol, pH 8.5 ) were adsorbed directly to the surface of Immulon No. 2 plastic wells (Dynatech) overnight at 4°C. The remaining protein binding capacity of the wells was blocked with RIA buffer containing 10% FBS (250 #1; 37°C, 2 h). The MAbs (50/tl) were added, incubated (37°C, 1 h), the wells washed thrice with RIA/Tween buffer and reacted with 125I-labelled goat anti-mouse immunoglobulin (50/tl; 30 000 cpm) at 37°C, 1 h. After a final washing, the radioactivity associated with each well was measured in a gamma counter.
Ouchterlony reaction The capacity of the MAbs to precipitate purified 140S virions and 12Sps was determined by Ouchterlony reaction using 0.7% agarose in 0.014 M TrisHC1, 0.15 M NaC1, pH 8.0, as described by Stave et al. (1988). Plaque reduction neutralization test (PRT) Dilutions of MAbs were incubated with equal volumes of FMDV suspension ( 100 PFU/0.05 ml) for 1 h at 37°C. This reaction mixture (0.1 ml) was adsorbed to monolayers of LF-BK cells (37 ° C, 5% CO2, 1 h) and overlayed with 6% gum tragacanth (37°C, 5% CO> 42 h) (Stave et al., 1986); 70% plaque reduction neutralization (PRT7o) titers were determined using the logit-log transformation (Finney, 1964).
ANTIGENIC RELATIONSHIPS OF FOOT-AND-MOUTH DISEASE VIRUS ASIA-I
279
Mouse protection test (MPT) Dilutions of MAbs were mixed with equal volumes of FMDV suspension (200 suckling mouse LDs0/0.03 ml) and incubated (37°C, 1 h), and were each inoculated intraperitoneally (0.03 ml/dose) into two litters of suckling mice (eight, 7-9 days old) (Stave et al., 1986). The number of mice alive at PID 5 was used to calculate the 50% mouse protective test (MPTso) of the antibody preparation by the Spearman-Karber method (Finney, 1964). RESULTS
MAb production, characterization and epitope/location classification A panel of 26 MAbs was generated against three different FMD serotype Asia-1 viruses isolated in India (66/86, 10/86, 63/72) (Table 1). Monoclonal antibodies developed with immune cells from mice at 7 PID were isotype IgM, whereas, MAbs derived at 14 PID and later were mainly IgG. Hybridomas were selected for cloning on the basis that they react in the LP-RIA with their eliciting virus. The MAbs were classified by the location of their epitope relative to the intact 140S virion, the 12Sps and VP 1 (Table 2 ). Class I MAbs reacted with the freshly centrifuged labelled 140S virus in the LPRIA but failed to react with either the 12Sps or VP 1 in the SP-RIA. Class II MAbs reacted with 14OS virus in LP-RIA and 12SpS in SP-RIA but did not react with VP 1. Class III MAbs reacted positively with labelled 140S virus in the LP-RIA and in both the 12Sps and VP1 SP-RIAs. The Class IV 12Sps specific MAbs reacted only in the 12Sps SP-RIA. Class V MAbs are restricted to reaction in either the virus protein SP-RIA or reducing PAGE dot blots.
MAb cross-reactivity amongst FMD VAsia-1 subtypes: binding, neutralization and precipitation in agar gel Binding. Eight MAbs (Class I ) bound specifically with 140S in RIA but only four of them neutralized viral infectivity (Tables 3 and 4 ). Two of these Class I MAbs derived from mice at 14 PID were isotype IgA (Table 1 ). Twelve MAbs (Class II) bound to both the 140S and 12Sps particles. Four MAbs (Class III) reacted with 140S/12S/VP 1 in RIA, but only two MAbs (Class IV) bound specifically to 12Sps. These MAbs, as hybridoma supernatants, had relatively high RIA titers with 12Sps but neither precipitated the antigen in the Ouchterlony reaction nor neutralized infectivity (Tables 3 and 5 ) The binding pattern of each of the MAbs to 10 different viruses of Asia-1 serotype is shown in Table 3. The MAbs elicited with viral isolate India 10/ 86 (fusion 26 ) bound to most of the Indian isolates and to Pak/57 and Iran/ 73. A large portion of the non-neutralizing MAbs were RIA reactive with the majority of the Asia-1 viruses tested while greater variation was observed in the binding pattern of the neutralizing MAbs. Conformation-independent
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G. BUTCHAIAH ET AL.
TABLE 1 Characterization o f MAbs elicited with F M D V Asia-1 isolates MAb
Isotype
Class a
Neutralization
Antigen source b
2C2.1.1 c 2C5.1.1
IgG3 IgG2b
II III
+d +
Asia-1 India 6 3 / 7 2 Killed 90-PID
26IH7.1.1 26JH6.1.1 26KA8.1.1.1.1 26KE6 r 26LA10.1.1
IgG2b IgG2a IgM IgG2b IgG2b
II1 II II 1 II
+ _ e +
Asia-1 India 10/86 Infect 14-PID
30EF9.1.1 30GG7.1.1 30HA7.1.1 30JD5.1.1
IgM
I
-
Asia-1 India 66/86 Infect 7-PID
IgM
I
-
IgM
II
-
lgM
IV
-
31AH6.1.1 31AHll.I.1 31DAI0.1.1 31DD8.1.1 31DF6 31EB3.1.1 31GAI0.1.1 31HB8 31HC2.1.1 31HD8.1.1 31HE8.1.1 31IE6.1.1 31IH7.1.1 31KC3.1.1.1 31KE2.1.1
IgG
I
+
IgA IgG2a IgG3 IgG2a
I I II II II II I IIl II I II IV III I1
+ + -
IgM
IgG 1 IgG2a IgG2a IgG2a IgG3 lgG2b IgG2a IgM
IgG2b
Asia-I India 6 6 / 8 6 Infect 14-PID
+
+ + +
+
aClass indicates location o f MAb reactive epitope in relation to intact virion, 12Sps and VP1 (see Table 2 ). bKilled=inactivated F M D V ; I n f e c t = i n f e c t i o u s F M D V ; P I D = p o s t - i n f e c t i o n / v a c c i n a t i o n day on which i m m u n e spleen cells were harvested. CHybridoma identification, the n u m b e r s after the points indicate the n u m b e r o f cloning cycles (i.e. • 1.1 has been cloned twice ); d + , Neutralizes eliciting virus. e _ , N o neutralization o f infectivity detected. flnitial isolate with no further cloning.
MAb 31HC2 (Class III) bound to only 50% of the Asia-1 virus isolates, whereas, the other three Class III MAbs bound 70% or more (Table 3 ). The Class IV MAbs 30JD5 and 311H7 uniformly bound, with one exception, to all FMDV 12Sps preparations tested regardless of serotype (Tables 3 and 6 ). The majority of Indian isolates and Pak/57, Iran/73 and Israel/68 were recognized by MAbs 2C2 and 2C5 (elicited by India 63/72 ) but the neutraliza-
ANTIGENIC RELATIONSHIPS OF FOOT-AND-MOUTH DISEASEVIRUS ASIA-I
281
TABLE 2 Classification of MAbs elicited with FMDV Class
Location of epitope
I
Restricted to the intact virion (140S)
1I
Available on both 140S and the 12S protein subunit (12Sps)
III
Available on 140S, 12Sps and a virus protein (VP)
IV
Restricted to the 12Sps
V
Restricted to a VP"
aPreviously reported by Robertson et al. (1984). 0
tion spectrum of 2C2 was larger than that of 2C5. Twelve of the panel of 26 MAbs recognized FMDV Asia-1 Israel/68, a subtype of Asia-1 usually considered distinct from the Indian subtypes.
Neutralization. Viral infectivity was neutralized to varying degrees by 12 of the MAbs. The Class I, IgA MAbs 31AH 11 and 31AH6 bound the virus with a low RIA titer but neutralized viral infectivity efficiently (Tables 3 and 4). In contrast, the MAbs 31HE8 (Class I), 31KE2 (Class II) and 26IH7 (Class III), reacted strongly with the virus in RIA but exhibited weak neutralization activity. In the PRT assay, the MAbs 2C5, 31AH11 and 31HC2 exhibited strong neutralizing activity against their eliciting viruses (Table 4). The Class I, isotype IgA MAb 31AH11 neutralized the infectivity of all Asia-1 viruses tested. On the other hand, the Class III MAbs 2C5 and 31HC2 neutralized only 50% of viruses tested. The majority of the neutralizing MAbs neutralized more than one virus with the exception of 31KE2 (Class II) which was restricted to its eliciting virus. Similar results were obtained against the 10 FMDV serotype Asia-1 isolates in both the PRT and MPT assays with the exception of MAb 31AH6 Class I that showed moderate titers in PRT but protected mice poorly (Table 4). At least seven of these 12 MAbs appear to recognize different epitopes (neutralizing) based on cross-reactivity patterns within each epitope class (Table 7 ). Precipitation in the Ouchterlony reaction. Fourteen MAbs precipitated purified preparations of 140S virus in the Ouchterlony reaction (Table 5) and this included all of the neutralizing MAbs (Table 4). Two of the precipitating MAbs (31DD8 and 31HD8 Class II) precipitated both 140S virions and 12Sps in the Ouchterlony reaction (Table 5 ). The analysis of the MAbs capacity to precipitate Asia- 1 viruses showed that
282
G. BUTCHAIAH ET AL.
TABLE 3 M o n o c l o n a l a n t i b o d y b i n d i n g a n a l y s i s o f F M D V s e r o t y p e Asia-1 i s o l a t e s MAb
F M D V Asia-1 i s o l a t e s a India 63/72
India 21/80
India 4/86
India 10/86
India 66/86
India MFB/86
India GDG/87
Pak 57
Iran 73
Israel 68
Class I 26KE6
0.8 b
0.8
1.2
0.8 ~
0.8
_d
1.2
0.6
-
--
30EF9
-
0.6
-
0.8
0.8
-
0.6
-
-
-
30GG7
0.4
0.5
0.6
0.8
0.6
-
0.3
0.6
-
-
-
0.4
1.1
0.5
1.4
0.5
0.3
l.l
-
-
31AHll(+)
0.5
0.8
0.8
0.8
1.1
1.1
1.1
1.1
0,6
0.8
31DAI0(+)
0.8
0.6
1.6
0.6
1.4
0.4
0.6
0.6
0.3
0.3
31HB8
-
0.5
1.1
0.3
0.8
-
0.1
0.3
-
-
31HE8(+)
1.2
0.5
2.7
3.1
3.3
3.5
0.3
0.4
3.1
3.1
MAb Class lI 2C2(+) 26JH6 26KA8 26LA10(+) 30HA7 31DD8
2.7 3.1 1.2 2.8 2.5
2.2 ND f 1.1 0.8 2.1
2.5 2.7 1.6 3.1 1.1 3.2
4.1 1.4 3.2 0.8 4.1
2.5 4.2 1.5 3.4 1.1 4.1
2.7 1.8 3.8 1.5 4.1
0.3 ND 1.6 3.2 0.8 3.4
2.7 3.1 1.1 1.1 3.8
2.3 4.0 1.4 3.6
2.8 3.7 4.1
31DF6 31EB3(+) 31GA10 31HD8 31IE6 31KE2(+)
1.4 0.6 2.7 . .
0.7 0.4 2.5 0.6 .
0.8 0.3 3.1 0.8
0.8 0.3 0.5 4.2 0.3
2.7 0.8 2.3 4.2 0.8 2.1
1.2 1.7 4.5 .
1.2 0.7 1.2 3.1 0.8 .
0.4 0.8 0.5 3.3 .
0.8 0.5 4.2 -
0.3 2.5 4.2 -
C l a s s II1 2C5(+) 26IH7 ( + ) 31HC2(+) 31KC3(+)
3.6 1.7 ND
2.5 0.4 0.3 0.5
2.5 2.8 0.3
2.7 2.8 0.3
2.8 3.1 0.3
1.4 3.4 2.5 0.3
2.7 3.2 0.3
2.4 0.3
4.1 3.1 2.7 0.1
4.1 -
ClassIV 30JD5 311H7
2.2 3.1
1.6 2.2
1.8 3.5
2.5 4.6
2.7 4.6
2.1 4.6
2.1 3.7
1.6 3.8
2.5 4.6
2.4 4.1
31AH6(+)
e
.
.
.
3.1
a P u r i f i e d F M D V Asia-1 i s o l a t e s l a b e l l e d w i t h ~25I. b 0 . 8 = t h e 30% a n t i b o d y b i n d i n g c a p a c i t y e n d - p o i n t t i t e r ( T r a u t m a n a n d H a r r i s , 1977 ). c U n d e r l i n e d v a l u e s i n d i c a t e r e a c t i o n w i t h t h e e l i c i t i n g v i r u s , d-, N o r e a c t i o n w a s d e t e c t e d . e( + ), N e u t r a l i z e s v i r a l i n f e c t i v i t y . fND = test not done.
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A N T I G E N I C R E L A T I O N S H I P S O F F O O T - A N D - M O U T H DISEASE V I R U S ASIA-I
TABLE 4 FMDV
serotype Asia-I infectivity neutralization
c a p a c i t y o f M A b s in t h e p l a q u e r e d u c t i o n
(PRT)
and
mouse protection tests (MPT) MAb a
Class I 31AH6
31AHI 1
31DAI0
31HE8
FMDV
Asia- 1 isolates
India
India
India
India
India
India
India
Pak
Iran
Israel
63/72
21/80
4/86
10/86
66/86
MFB/86
GDG/87
57
73
68
PRT
_b
_
1.5 c
1.5 d
0.6
-
-
MPT
-
-
0.4
0.6
0.4
-
-
-
PRT
1.4
2.4
2.4
2.1
2.4
2.1
1.4
2.4
2.1
1.6
MPT
0.6
1.8
1.6
1.6
1.8
1.4
0.6
1.4
1.4
1.1
PRT
-
1.2
0.7
0.4
1.6
0.7
1.1
1.4
0.9
MPT
1.4
1.6
0.6
1.6
0.8
1.1
1.1
0.7
0.4 0.4
-
PRT
0.5
0.9
0.8
1.2
0.4
-
1.1
0.7
MPT
0.4
0.8
0.8
1.1
0.6
-
0.8
0.6
PRT
1.7
1.6
1.1
0.8
-
-
1.1
0.9
0.8
MPT
1.6
1.4
0.8
0.8
-
-
0.8
0.8
0.6
PRT
-
MPT
.
-
Class II 2C2
26LAI0
31EB3
31KE2
-
0.6
.
PRT
0.4
MPT
0.6
-
PRT
-
MPT
-
. 0.4
-
. 0.7
-
0.4
0.7
0.4
0.6
0.4
0.6
0.8
-
-
0.7
-
-
0.8
Class III 2C5
26IH7
31HC2
31KC3
PRT
2.1
-
-
0.7
0.7
1.1
-
MPT
2.1
-
-
0.6
-
0.8
0.8
-
PRT
-
-
-
0.6
-
-
-
MPT
-
-
-
0.4
-
-
-
PRT
-
0.4
0.9
2.6
0.4
-
-
MPT
-
0.4
0.8
2.1
0.8
-
-
PRT
-
-
-
0.5
0.4
0.4
0.4
0.4
MPT
-
-
-
0.4
0.5
-
0.4
0.4
aMAbs from Table 1 which gave no detectable reaction were excluded from this table. b , N o s i g n i f i c a n t (i.e. > 3 0 ) r e a c t i o n d e t e c t e d . CTiter ( P R T = m a x i m u m dilution (Log base 10) which produces 70% reduction (MPT=maximum d i l u t i o n { L o g b a s e 10} w h i c h p r o t e c t e d 5 0 % o f t h e t e s t m i c e . OUnderlined value indicates reaction with the eliciting virus.
in plaque
count)
or
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G. B U T C H A I A H ET AL.
TABLE 5
Precipitation of FMDV serotype Asia- 1 isolates by anti-Asia- 1 MAbs in the Ouchterlony reaction MAb a
Isotype FMDVAsia-I isolates India 63/72
India 2t/80
India 4/86
India 10/86
India 66/86
India MFB/86
India GDG/87
Pak 57
Iran 73
Israel 68
_c PPT
_ PPT PPT PPT
PPT d PPT PPT PPT
PPT PPT -
PPT ~ PPT PPT PPT
PPT PPT
PPT PPT -
PPT PPT PPT PPT
PPT PPT
PPT PPT PPT PPT .
PPT PPT PPT PPT PPT PPT
PPT PPT PPT -
PPT PPT PPT PPT PPT PPT PPT
PPT PPT PPT -
PPT PPT PPT PPT -
PPT PPT PPT PPT -
PPT PPT
PPT
PPT
PPT
PPT PPT
PPT
PPT PPT
PPT
PPT PPT
PPT
PPT PPT
PPT
PPT
-
PPT
-
PPT
PPT
-
-
Class I 31AH6(+)
b
IgA
31AHII(+) 31HB8 31HE8(+)
IgA IgG2a IgG3
Mab Class II 2C2( + ) 30HA7 31DD8 31EB3(+) 31HD8 311E6 31KE2 ( + )
IgG3 IgM IgG3 IgM lgG2a IgG2b IgG2b
PPT
Mab Class III 2C5(+) 26IH7(+)
IgG2b IgG2b
PPT PPT
31HC ( + )
IgG2a
PPT PPT PPT .
.
.
PPT
PPT
aMAbs from T a b l e 3 ( h y b r i d o m a supernatants) which gave no detectable reaction were excluded from this table. b( + ), Neutralizes viral infectivity. c ( _ ), N o reaction detected.
°PPT, Precipitation in the Ouchterlony reaction. *Underlined value indicates reaction with the eliciting virus.
four non-neutralizing MAbs (31DD8, 31HB8, 31HD8, 31IE6) and two neutralizing MAbs (31AH11, 31HE8) precipitated the majority of the Asia-1 viruses tested. On the other hand, neutralizing MAbs (e.g., 2C2, 2C5, 31AH6, 31EB3, 31HC2, 31KE2 ) exhibited extensive variation in precipitating different Asia- 1 viruses (Table 5 ). Cross-reactivity amongst FMD V serotypes Analysis of the RIA cross-reactivity of the MAbs with eight different viruses from F M D V serotypes O, A and C identified seven MAbs ( 7 / 2 6 ) which reacted with the heterologous F M D V serotypes. Only one Class II MAb ( 31DD8 ) reacted with the heterologous serotypes (i.e., O 1 Brugge). The four Class III VP 1-reactive neutralizing MAbs ( 2C5, 26JH7, 31HC2, 31KC3 ) exhibited cross-reactivity with heterologous F M D V serotypes A and/or C in RIA; however, none of them neutralized the heterologous serotypes (data not
ANTIGENIC
RELATIONSHIPS
OF FOOT-AND-MOUTH
DISEASE
VIRUS
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ASIA-1
TABLE 6 Monoclonal antibodies elicited with F M D V Asia-1 which react with other F M D V serotypes MAb"
F M D V serotype O1 Bru
A5 Spain
Ai2 LP ab
C1
Class II 31DD8
Bind b
___c
Class III 2C5 26IH7 31HC2 31KC3
-----
-----
-Bind Bind Bind
Bind . . .
Class IV 30JD5 31 IH7
Bind Bind
Bind Bind
Bind Bind
-Bind
C2
C3 Indail
C3 Resende
C4
Bind . . .
Bind
B
. . .
Bind Bind
Bind Bind
Bind Bind
-. . .
m
m
Bind Bind
aMAbs which gave no detectable reaction with serotypes other than Asia-1 were excluded from this Table. b B i n d = t h e MAb reacts with the indicated virus serotype in the radioimmunoassay. c , No reaction was detected. TABLE7 Foot-and-mouth disease virus Asia- 1 distinct epitopes related to the neutralization of viral infectivity Epitope
MAb
Class Distinguishing characteristics (within the MAb class)
1"
31AHI 1
I
Binds and neutralizes all serotype Asia-1 viruses tested
2
31HE8
I
Binds to all serotype Asia-1 viruses tested and neutralizes all except India 10/86 and India G D G / 8 7
3
31AH6
I
Binds to all serotype Asia- 1 viruses tested and neutralizes all except Pak/57, Iran/73 or Israel/68
4
2C2
II
Neutralizes serotype Asia-I isolates India 21/80, Iran/73 and Israel/ 68
5
31EB3
II
Does not neutralize serotype Asia- 1 isolates India 21/80, Iran/73 and Israel/68
6
2C5
III
Neutralizes serotype Asia-1 isolate India 63/72 but does not neutralize India 66/88
7
31HC2
III
Neutralizes serotype Asia-I isolate India 66/88 but does not neutralize India 63/72
"The MAbs listed here represent the minimal n u m b e r of neutralization related epitopes on F M D V serotype Asia- 1.
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G. BUTCHAIAH ETAL.
shown). The Class IV 12Sps specific MAbs 311H7 and 30JD5 as reported in Table 6 reacted with all but one of the heterologous serotypes (i.e. 30JD5 did not react with FMDV CI). DISCUSSION
Each MAb was classified according to the location of its reactive site in relation to the intact virion, 12Sps and VP 1. The infectivity neutralization based classification scheme of Stave et al. ( 1988 ) was modified to utilize antibody binding as the sole parameter. This protocol recognizes the fact that the spectrum of FMDV isolates bound frequently exceeds the spectrum of those neutralized as well as the fact that certain FMDV reactive antibodies have no effect on neutralization of viral infectivity. The proposed classes of anti-FMDV antibodies based upon epitope location are presented in Table 2. In effect this scheme simultaneously classifies the epitope and the MAb which defines it. The development of two neutralization MAbs (31AH6, 31AH11 ) of the IgA isotype shows for the first time that circulating IgA may play a role in the immune response to FMDV infection. The isolation of two Class IV MAbs ( 30JDS, 311H7 ) from mice infected with purified live virus demonstrates the in vivo degradation of the virus into 12Sps. Such in vivo degradation of purified, inactivated virus into 12Sps was also described previously after repeated immunization of mice (McCullough and Butcher, 1982 ). One of the Class IV MAbs (30JDS) isolated from mice at 7 PID was IgM, confirming the observation of Stave et al. (1986) that IgM binds to the 12Sps, in contrast to earlier reports using polyclonal antisera (Brown and Smale, 1970). In this study no Class III antibodies were developed from the 7 PID fusion. The Class IV MAbs do not bind the infectious particle and, therefore, do not neutralize viral infectivity. These MAbs bound the 12Sps of all FMDVs with the exception that MAb 30JD5 did not bind to C 1 virus. It seems that such universal epitopes likely involve a fundamental highly conserved architectural feature of the virus; thereby, the absence of such epitopes predicts a basic structural difference with other FMDVs. These 12Sps-specific epitopes are exposed only when the intact virus is degraded into 12Sps. Antibodies to them have also been generated with FMDV serotype SO and A (Morgan et al., 1984; Stave et al., 1986; Saiz et al., 1989). Class IV MAbs have been used to develop an inhibition ELISA for the differential diagnosis of FMD from other vesicular diseases (Smitsaart et al., 1990). Certain non-neutralizing Class II MAbs (e.g. 26JH6, 31GAI0, 31HDS) appear to be serotype specific for Asia- 1 and, as a panel, react with all Asia- 1 viruses tested. These MAbs should be useful for diagnostic serotyping of Asia1 virus isolates. With the single exception of MAb 31DD8 binding to serotype
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O 1 Brugge, the non-neutralizing Class I and II MAbs did not bind to heterologous serotypes of FMDV. The type of assay and the conditions under which it is performed affect the reaction or lack of reaction of a particular MAb: therefore, in this investigation, MAbs have been characterized and classified, in so far as possible, on the basis of positive results. The majority of the neutralizing MAbs (8/12) derived from mice immunized with whole Asia-I virus were conformation dependent. There appears, however, to be a site on VP 1 defined by the conformation-independent, highly efficient neutralizing MAbs 2C5 and 31HC2. Further, it has been shown that VP 1 isolated from Asia- 1 virus, when used as a vaccine, protects cattle against infection with the homologous virus (D.O. Morgan and G. Butchaiah, unpublished data). These results show that both conformation-dependent and conformation-independent epitopes are important in the protection of animals from serotype Asia-1 infection. Conformation-dependent epitopes were also found more frequently than sequential (conformation-independent) epitopes for FMDV serotype O (Stave et al., 1988) and in nearly equal amounts for serotype A virus (McCullough and Butcher, 1982; Meloen et al., 1983; Baxt et al., 1984; Morgan et al., 1984; Ouldridge et al., 1984). Conformation-dependent epitopes are also of major importance in other picornaviruses (Ferguson et al., 1984; Diamond et al., 1985; Blondel et al., 1986; Sherry et al., 1986). Summation of the effect of antibodies on the conformation-dependent epitopes may account for the greater efficacy, on a weight basis, of whole virus vaccines when compared to subunit vaccines. Conformation-dependent neutralization epitopes recognized by Class I MAbs 31AH 11, 31DA 10, 31 HE8 are functionally conserved across several of the isolates of FMDV serotype Asia-1 tested here. Similarly, certain conformation-dependent anti-FMDV serotype A12 MAbs neutralized several different subtypes of FMDV serotype A as described previously (Grubman and Morgan, 1986). In contrast, conformation-independent Class III MAbs such as 2C5, 31HC2 exhibit extensive variation in their reactivity with different Asia-1 virus isolates of the same subtype. In general, the infectivity neutralizing capacity of the Class III MAbs was more restricted than that of the Class I and II MAbs (Table 4). This finding suggests that the epitopes recognized by Class III MAbs are located on the highly variable region of VP1 (amino acids 140-160) as established in other serotypes of FMDV. The conformation-independent neutralizing Class III MAbs (2C5, 26IH7, 31HC2, 31KC3 ) cross-reacted in RIA with other FMDV serotypes A and/or C; however, no neutralization of viral infectivity of the heterologous serotypes was found (Table 6 ). The lack of correlation between the spectrum of viruses bound and the viruses neutralized within a homologous serotype has been well demonstrated. For example, MAb 2C5 elicited by Asia-1 India 63/ 72 virus binds to other Asia-1 viruses, India 21/80, Iran/73 and Israel/68,
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but it does not neutralize their infectivity. Similar results showing binding to heterologous serotypes without neutralizing their infectivity have been reported previously with MAbs raised against FMDV serotypes A12 (Grubman and Morgan, 1986), O1 Brugge (Stave et al., 1988) and poliovirus type 1 (Crainic et al., 1983; Diamond et al., 1985; Blondel et al., 1986). The mechanics of this process remain a paradox. One can logically postulate that a change of a single amino acid residue in the area of an epitope while not altering antibody binding might eliminate the infectivity neutralizing effect of a MAb reaction. An alternative could be that the orientation of the bound MAb on one virus sterically interferes with the virus binding to its cell receptor while on another it does not. Large variations in the magnitude of infectivity neutralization within a group of neutralized viruses such as demonstrated by MAbs 31HC2 and 2C5 (Table 4) is a situation in which a single epitope has a positive role in neutralization but with variable degrees of effectiveness. The scheme of anti-FMDV MAb classification presented in Table 2 is based on the simple surmise that epitopes located on structurally distinct parts of the virus are distinct. Further, the neutralization patterns of the MAbs with 10 different Asia-1 virus isolates suggest that several of the MAbs are recognizing different epitopes. The Class I MAbs 31AH 11, 31DA 10, 31 HE8 bind to all Asia-1 virus tested (Table 3 ) but 31 HE8 has a restricted neutralization pattern (Table 4); whereas MAb 31AH6, also Class I, has a more restricted binding pattern and neutralized only India 4/86, India 66/86 and India MFB/ 86. Similarly, the Class II MAb 31EB3 neutralized India 63/72, India 4/86, India 66/86, India GDG/87, India MFB/86 and Pak/57 and is distinct from another Class II MAb 31KE2 which neutralized only the eliciting virus. The divergent Asia- 1 isolate neutralization patterns of Class III MAbs 31KC3 and 31HC2 indicate that they may well recognize different epitopes and are certainly distinct from 2C5 (Table 4). With foregoing arguments as evidence, it is proposed that a minimum of seven distinct neutralization epitopes exist on the surface of Asia-1 virus India 66/86 (Table 7 ). There is weaker evidence for additional neutralization epitopes. This does not prove that all of these seven neutralization epitopes exist or function in all Asia- 1 viruses. However, given the structural similarities of the Asia- 1 viruses, it strongly suggests that these or their functional surrogates likely exist on all Asia-1 viruses. Five of the seven Indian Asia-1 viruses were isolated recently, i.e., 19861987, and the remaining two were isolated 1972-1980. Remarkably, viruses isolated even a few months apart in different regions in India (Asia-I India 4/86, 10/86, 66/86, MFB/86, G D G / 8 7 ) exhibited extensive variation particularly in conformation-independent neutralization epitopes recognized by Class III MAbs 2C5 and 31HC2. Further, the Asia- 1 viruses of 1980 or earlier (India 63/72, 21/80, Iran/73, Israel/68) possessed both the epitopes recognized by the neutralizing MAbs 2C5 (Class III) and 2C2 (Class II) as demonstrated by binding in RIA. In contrast, Asia-1 virus isolates of 1986-1987
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and the classical Asia-1 virus P a k / 5 7 exhibited the presence o f one of these two epitopes but not both. The extensive antigenic heterogenicity observed among Indian Asia-1 virus field isolates could result from partially i m m u n e (vaccinated) host animals providing a strong selection force for the generation of antigenic variants. Antigenic heterogeneity, as reported here, is not unique to F M D V serotype Asia-1. Such heterogeneity in natural populations of other F M D V serotypes O, A and C ( D o m i n g o et al., 1980; King et al., 1981; Rowlands et al., 1983; Sobrino et al., 1986; Beck and Strohmaier, 1987; Mateu et al., 1988 ); Dengue virus isolates ( M o n a t h et al., 1986) and other R N A viruses (Domingo and Holland, 1987 ) has been well documented. Despite the extensive variation detected by certain neutralizing MAbs, conformation-dependent epitopes recognized by neutralizing Class I MAbs 31AH 11, 31DA 10, 31 HE8 are uniformly conserved in the Asia- 1 viruses tested irrespective of time or geographical location o f their isolation. Furthermore, all o f the isolates were neutralized by polyclonal cattle and mouse anti-Asia- 1 Indian 66/86 virus sera although to varying degrees (data not shown). This indicates that future emphasis should be placed on the construction of conformational epitopes rather than sequential epitopes in order to increase the spectrum o f cross-subtype protection o f synthetic F M D vaccines. ACKNOWLEDGEMENTS G.B. was supported by a Biotechnology Overseas Associateship, Government o f India. We wish to thank Adriene Ciupryk for typing and Sue B. Morgan for editing the manuscript.
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ertson, B.H. and Bachrach, H.L., 1981. Cloned viral protein vaccine for foot-and-mouth disease: responses in cattle and swine. Science, 214:1125-1129. Laporte, J., Grosclaude, J., Wantyghem, J., Bernard, S. and Rouze, P., 1973. Neutralization en culture cellulaire de pouvoir infectieux du virus de la fi6vre aphteuse par des s6rums provenant de pores immunis6s ~t l'aide d'une prot6ine virale purifi6e. C. R. Acad. Sci. (Paris), Ser. D, 276: 3399-3402. Mateu, M.G., Rocha, E., Vicente, O., Vayreda, F., Navalpotro, C., Andreu, D., Pedroso, E., Giralt, E., Eujuanes, L. and Domingo, E., 1987. Reactivity with monoclonal antibodies of viruses from an episode of foot-and-mouth disease. Virus Res., 8:261-274. Mateu, M.G., Da Silva, J.L., Rocha, E., De Brum, D.L., Alonso, A., Enjuanes, L., Domingo, E. and Barahona, H., 1988. Extensive antigenic heterogeneity of foot-and-mouth disease virus ofserotype C. Virology, 167:113-124. Matheka, H.D. and Bachrach, H.L., 1975. N-Terminal amino acid sequences in the major capsid proteins of foot-and-mouth disease virus types A, O and C. J. Virol., 16: 1248-1253. McCullough, K.C. and Butcher, R., 1982. Monoclonal antibodies against foot-and-mouth disease virus 146S and 12S particles. Arch. Virol., 74: 1-9. Meloen, R.H., Briaire, J., Woortmeyer, R.J. and Van Zaane, D., 1983. The main antigenic determinant detected by neutralizing monoclonal antibodies on the intact foot-and-mouth disease virus particle is absent from isolated VP 1. J. Gen. Virol., 64:1193-1198. Monath, T.P., Wands, J.R., Hill, L.J., Brown, N.V., Marciniak, R.A., Wong, M.A., Gentry, M.K., Burke, D.S., Grant, J.A. and Trent, D.W., 1986. Geographic classification of Dengue-2 virus strains by antigen signature analysis. Virology, 154:313-324. Morgan, D.O., 1983. Biological synthesis of subunit vaccines. In: R.W.Loan (Editor), Bovine Respiratory Disease, A Symposium. Texas A&M University Press, College Station, TX, pp. 387-399. Morgan, D.O. and Moore, D.M., 1990. Protection of cattle and swine against foot-and-mouth disease (FMD) with biosynthetic peptide vaccines. Am. J. Vet. Res., 51: 40-45. Morgan, D.O., Moore, D.M. and McKercher, P.D., 1980. Vaccination against foot-and-mouth disease. In: A. Mizrahi, I. Hertma, M. A. Klingberg and A. Kohn (Editors), New Developments with Human and Veterinary Vaccines. Progress in Clinical and Biological Research Series. Alan R. Liss Inc. Pub., New York, NY, pp. 169-178. Morgan, D.O., Robertson, B.H., Moore, D.M., Timpone, C.A. and McKercher, P.D., 1984. Aphthoviruses: control of foot-and-mouth disease with genetic engineering vaccines. In: E. Kurstak (Editor), Control of Virus Diseases. Marcel Dekker, Inc., New York, NY, pp. 135145. Ouldridge, E.J., Barnett, P.V., Parry, N.R., Syred, A., Head, M. and Rweyemamu, M.M., 1984. Demonstration of neutralizing and non-neutralizing epitopes on the trypsin-sensitive site of foot-and-mouth disease virus. J. Gen. Virol., 765: 203-207. Robertson, B.H., Morgan, D.O. and Moore, D.M., 1984. Location of neutralizing epitopes defined by monoclonal antibodies generated against the outer capsid polypeptide, VP 1, of footand-mouth disease virus A 12. Virus Res., 1: 489-500. Rowlands, D.J., Clarke, B.E., Carroll, A.R., Brown, F., Nicholson, B.H.,Bittle, J.L., Houghten, R.A. and Lerner, R.A., 1983. Chemical basis of antigenic variation in foot-and-mouth disease virus. Nature (London), 306: 694-697. Saiz, J.C., Gonzalez, M.J., Morgan, D.O., Card, J.L., Sobrino, F. and Moore, D.M., 1989. Antigenic comparison of different foot-and-mouth disease virus types using monoclonal antibodies defining multiple neutralizing epitopes on FMDV A5 subtypes. Virus Res., 13: 4560. Sherry, B., Mosser, A.G., Colonno, R.J. and Rueckert, R.R., 1986. Use of monoclonal antibodies to identify four neutralization immunogens on a Comcon cold picornavirus, human rhinovirus 4. J. Virol., 57: 246-257.
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Smitsaart, E.N., Saiz, J.C., Yedloutschnig, R.J. and Morgan, D.O., 1990. Detection of foot-andmouth disease virus by competitive ELISA using a monoclonal antibody specific for the 12S protein subunit from six of the seven serotypes. Vet. Immunol. Immunopathol., 26" 251265. Sobrino, F., Palma, E.L., Beck, E., Davila, M., De la Torre, J.C., Negro, P., Villanueva, N., Ortin, J. and Domingo, E., 1986. Fixation of mutations in the viral genome during an outbreak of foot-and-mouth disease: Heterogeneity and rate variation. Gene, 50" 149-159. Stave, J.W., Card, J.L. and Morgan, D.O., 1986. Analysis of foot-and-mouth disease virus type 01 Brugge neutralization epitopes using monoclonal antibodies. J. Gen. Virol., 67: 20832092. Stave, J.W., Card, J.L., Morgan, D.O. and Vakharia, V.N., 1988. Neutralization sites of type O1 foot-and-mouth disease virus defined by monoclonal antibodies and neutralization-escape virus variants. Virology, 162:21-29. Strohmaier, K., Franze, R. and Adam, K.-H., 1982. Location and characterization of the antigenic portion of the FMDV immunizing protein. J. Gen. Virol., 59: 295-306. Swaney, L.M., 1988. A continuous bovine kidney cell line for routine assays of foot-and-mouth disease virus. Vet. Microbiol., 18" 1-14. Trautman, R. and Harris, W.F., 1977. Modeling and computer simulation approach to the mechanism of foot-and-mouth disease virus neutralization assays. Scan. J. Immunol., 6:831841. Wagner, G.G., Card, J.L. and Cowan, K.M., 1970. Immunochemical studies of foot-and-mouth disease. VII Characterization of foot-and-mouth disease virus concentrated by polyethylene glycol precipitation. Arch. Virusforsch., 30: 343-352.
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Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies G. B u t c h a i a h a, J.L. C a r d b a n d D . O . M o r g a n b'* aSouthern Regional Station, Indian Veterinary Research Institute, Bangalore 560 024, India bUnited States Department of Agriculture, Agricultural Research Service, Plum Island Animal Disease Center, Molecular Biology Laboratory, P. 0. Box 848, Greenport, NY 11944, USA (Accepted 19 December 1990)
ABSTRACT Butchaiah, G., Card, J.L. and Morgan, D.O., 1992. Antigenic relationships of foot-and-mouth disease virus serotype Asia-1 isolates demonstrated by monoclonal antibodies. Vet. Immunol. Immunopathol., 30: 275-292. A panel (26) of monoclonal antibodies (MAbs) was elicited against three distinct isolates of footand-mouth disease virus (FMDV) serotype Asia-1. Each MAb was characterized according to the location of its epitope: Class I, restricted to the intact virion ( 140S); Class II, restricted to 140S and the virion protein subunit ( 12Sps); Class III, available on 140S, 12Sps and virus protein 1; Class IV, restricted to 12Sps. In addition, the MAbs were further categorized by isotype, neutralization of viral infectivity, capacity to bind in radioimmunoassay and precipitation in the Ouchterlony reaction. Neutralization of FMDV infectivity by a MAb of the IgA isotype is reported for the first time. A minimum of seven distinct neutralization epitopes were described on FMDV Asia-1. Some of the neutralizing MAbs bound FMDVs in addition to those that they neutralized. The MAbs defined epitopes common to FMDV serotypes Asia-l, A, O1 and C but neutralizing capacity was restricted to serotype Asia- 1. Class IV MAbs defined epitopes highly conserved throughout the FMDV serotypes. Identification of FMDV neutralization epitopes makes possible the direct selection of optimal FMDV strains for vaccine fabrication. In addition, these data are crucial to the design of future synthetic vaccines.
INTRODUCTION F o o t - a n d - m o u t h d i s e a s e v i r u s ( F M D V ) , t h e sole m e m b e r o f t h e g e n u s aphthovirus of the family Picornaviridae, consists of seven distinct serotypes. E a c h s e r o t y p e is c o m p o s e d o f a m u l t i t u d e o f s u b t y p e s a m o n g w h i c h t h e r e is e x t r e m e a n t i g e n i c d i v e r s i t y . S e r o t y p e s O, A a n d C o c c u r in E u r o p e , S o u t h A m e r i c a , A f r i c a a n d A s i a ; S A T 1, S A T 2 a n d S A T 3 a r e r e s t r i c t e d t o A f r i c a , a n d Asia-1 is w i d e l y d i s t r i b u t e d t h r o u g h o u t A s i a . T h e i c o s a h e d r a l F M D V c a p s i d c o n t a i n s 60 c o p i e s e a c h o f f o u r s t r u c t u r a l p r o t e i n s t e r m e d V P 1, V P 2 , V P 3 *To whom correspondence should be addressed.
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G. BUTCHAIAH ET AL.
and VP4 enclosing a single-stranded positive sense RNA molecule of about 8000 nucleotides (Bachrach, 1977). The intact 140S virions (Brown and Newman, 1963; Morgan, 1983), 12S protein subunits (12Sps) (Morgan et al., 1980), VP1 or its fragments (Laporte et al., 1973; Bachrach et al., 1975; Strohmaier et al., 1982 ) and biosynthetic or synthetic peptides (Kleid et al., 1981; Bittle et al., 1982; Morgan and Moore, 1990) all elicit neutralizing antibodies and varying degrees of protective immunity in large animals. Antibodies that neutralize viral infectivity provide an important mechanism of protection against FMD. Polyclonal antiserum is useful for identifying the components of FMDV that elicit an immune response and for comparing the antigenic relationships of FMDV serotypes; however, the limits of its discriminating capacity is exceeded by the variation amongst subtypes. On the other hand, monoclonal antibodies (MAbs) are powerful tools for the precise identification of epitopes and analysis of closely related strains of FMDV. Much of the antigenic diversity of FMDV is due to rapid changes in antigenic makeup that occur in the partially immune host population found in epizootic areas and it is necessary to constantly update the composition of vaccines to maintain their efficacy. Thus, it is important to define those epitopes that elicit the protective immune response and their variation or conservation among variants, subtypes and serotypes. The antigenic structure of FMDV serotypes O, A and C is being elucidated through the use of monoclonal antibodies to detail its complexity (McCullough and Butcher, 1982; Meloen et al., 1983; Baxt et al., 1984; Duchesne et al., 1984; Morgan et al., 1984; Ouldridge et al., 1984; Robertson et al., 1984; Grubman et al., 1985; Grubman and Morgan, 1986/1987; Stave et al., 1986, 1988; Mateu et al., 1987, 1988; Baxt et al., 1989). With the exception of a few anti-Asia-1 virus MAbs (Butchaiah and Rao, 1989 ), no detailed MAb analysis of serotype Asia1 FMDV has been reported. This report describes the production and characterization of a panel of MAbs elicited with three different isolates of serotype Asia-1 FMDV. Their reactivity with 10 serotype Asia-1 virus isolates and representations of FMDV serotypes O, A and C is described. MATERIALS AND METHODS
Viruses and cells Foot-and-mouth disease viruses of serotype Asia-1, India 63/72, 21/80, 4/ 86, 10/86, 66/86, MFB/86 and GDG/87, were isolated from bovine tongue tissue from various regions of India. Other viruses of serotype Asia-1, Pak/ 57, Iran/73 and Israel/68 were from stocks of the Plum Island Animal Disease Center. With the exception of Israel/68, all of these serotype Asia-1 viruses belong to the same subtype. The last two digits in the virus designation
ANTIGENIC RELATIONSHIPS OF FOOT-AND-MOUTH DISEASE VIRUS ASIA-1
277
refer to the year of isolation. The bovine tongue tissue viruses were passaged in BHK-21 monolayer cell cultures and stored at - 7 0 ° C. A continuous bovine kidney cell line LF-BK (Swaney, 1988 ) was used for plaque assay.
Purified viral antigens Virus from infected cultures of BHK-21 cells was precipitated with polyethylene glycol and purified by CsC1 density gradient centrifugation (Wagner et al., 1970). The purified virus ( 140S ) was dialyzed against tris-buffered saline (TBS:0.05 MTris HC1, 0.3 MNaC1, pH 7.5 ) and quantitated by optical density. The 10 virus isolates of serotype Asia- 1 and preparations of serotypes O, A and C were purified and quantitated in this manner. The 12Sps was prepared by overnight dialysis of purified 140S virions against citrate-phosphate buffer, pH 4.5, followed by dialysis against TBS and clarification by centrifugation (3000 rpm, 30 min, 4 °C ) (Cowan, 1968 ). Purified VP 1 Was prepared from urea-disrupted 140S virions by DEAE anion-exchange chromatography (Bernard et al., 1974) and checked for purity by SDS-urea-PAGE electrophoresis (Matheka and Bachrach, 1975 ).
Monoclonal antibodies Adult BALB/c mice were infected with purified live virus (103-105 suckling mouse LDs0) and immune cells from these mice were used to generate hybridomas secreting anti-FMDV MAbs. Fusions were made at 7 and 14 postinoculation days (PID) as described by Stave et al. (1986, 1988). Fusion series 26, 30 and 31 were produced at the PIADC and the series 2 at The Indian Veterinary Research Institute (Table 1 ). Hybridomas were grown in minimal essential medium (MEM; Gibco) supplemented with 20% fetal bovine serum (FBS). The culture supernatants were screened by a liquid-phase radioimmunoassay (RIA) for the presence of virus and virus subunit reactive antibodies. Hybridomas producing antibodies of interest were cloned at least twice by limiting dilution and stored in liquid nitrogen. Isotype of the MAbs was determined from hybridoma culture supernatants by Ouchterlony precipitation with anti-mouse isotype specific antisera (Litton Bionetics, Kensington, MD, USA) in 1% agar, pH 7.9 buffered with 1 Mglycine, 0.0125 M sodium barbital, 0.15 M NaC1. The complete MAb designation (i.e. decimals indicate cloning cycles) is given in Table 1, but elsewhere in the report a short form of the designation is used (e.g., 31HC2 replaces 31HC2.1.1 ).
Radioimmunoassay Labelled virions were prepared by reacting 100-200/tg of purified 140S in 100/tl TBS with 0.25-0.5 mCi 1251(Amersham.Searle) in the presence of 25 pg iodogen as described previously (Fraker and Speck, 1978 ). The unincorporated 1251was removed by gel filtration through Sephadex G-25 (Pharmacia). Aliquots of the labelled virus were made to 3% bovine serum albumin
278
G. B U T C H A I A H ET AL.
(BSA) and stored at - 7 0 ° C . The labelled virus was diluted in RIA-BSA buffer (0.12 M boric acid, 0.02 M sodium borate, 0.15 M NaCI, 0.1% BSA, pH 8.5) for initial RIA screening of hybridoma culture supernatants. The 12~I-labelled virus was prepared by centrifugation on a 20-50% discontinuous sucrose gradient (45 000 rpm, 1.5 h, 4°C; Beckman SW 55 Ti rotor) immediately prior to use in determining the specific reactivity of the MAbs to intact 140S virions. A liquid-phase radioimmuno-precipitation (LP-RIA) assay was used to test the MAbs in culture supernatants for their reactivity with labelled virus. Briefly, the MAb culture supernatant (50 #1) was mixed with labelled virus (50/~1; 20 000-30 000 cpm) and incubated (37°C, 1 h). The antigen-antibody complexes were precipitated by adding 50 #1 of 1% protein-A bearing Staphylococcus aureus (armed with rabbit anti-mouse immunoglobulins) followed by incubation (37 ° C, 1 h). The precipitates were pelleted (2 min, Eppendorf Centrifuge Model 5413 ) and washed twice in RIA-BSA buffer containing 0.05% Tween-20 (RIA/BSA/Tween). The amount of radioactivity associated with the pellet was determined and the 30% antibody binding endpoint titer was calculated (Trautman and Harris, 1977). A solid-phase RIA (SP-RIA) was used to determine the reactivity of the MAbs with 12Sps and isolated VP 1 of the virus. Solutions of 12Sps (0.6/~g/ 50 #1 RIA buffer) and VP 1 (0.8 #g/50 #1 0.2 M ethylmorpholine, 6 M urea, 10 m3/2-mercaptoethanol, pH 8.5 ) were adsorbed directly to the surface of Immulon No. 2 plastic wells (Dynatech) overnight at 4°C. The remaining protein binding capacity of the wells was blocked with RIA buffer containing 10% FBS (250 #1; 37°C, 2 h). The MAbs (50/tl) were added, incubated (37°C, 1 h), the wells washed thrice with RIA/Tween buffer and reacted with 125I-labelled goat anti-mouse immunoglobulin (50/tl; 30 000 cpm) at 37°C, 1 h. After a final washing, the radioactivity associated with each well was measured in a gamma counter.
Ouchterlony reaction The capacity of the MAbs to precipitate purified 140S virions and 12Sps was determined by Ouchterlony reaction using 0.7% agarose in 0.014 M TrisHC1, 0.15 M NaC1, pH 8.0, as described by Stave et al. (1988). Plaque reduction neutralization test (PRT) Dilutions of MAbs were incubated with equal volumes of FMDV suspension ( 100 PFU/0.05 ml) for 1 h at 37°C. This reaction mixture (0.1 ml) was adsorbed to monolayers of LF-BK cells (37 ° C, 5% CO2, 1 h) and overlayed with 6% gum tragacanth (37°C, 5% CO> 42 h) (Stave et al., 1986); 70% plaque reduction neutralization (PRT7o) titers were determined using the logit-log transformation (Finney, 1964).
ANTIGENIC RELATIONSHIPS OF FOOT-AND-MOUTH DISEASE VIRUS ASIA-I
279
Mouse protection test (MPT) Dilutions of MAbs were mixed with equal volumes of FMDV suspension (200 suckling mouse LDs0/0.03 ml) and incubated (37°C, 1 h), and were each inoculated intraperitoneally (0.03 ml/dose) into two litters of suckling mice (eight, 7-9 days old) (Stave et al., 1986). The number of mice alive at PID 5 was used to calculate the 50% mouse protective test (MPTso) of the antibody preparation by the Spearman-Karber method (Finney, 1964). RESULTS
MAb production, characterization and epitope/location classification A panel of 26 MAbs was generated against three different FMD serotype Asia-1 viruses isolated in India (66/86, 10/86, 63/72) (Table 1). Monoclonal antibodies developed with immune cells from mice at 7 PID were isotype IgM, whereas, MAbs derived at 14 PID and later were mainly IgG. Hybridomas were selected for cloning on the basis that they react in the LP-RIA with their eliciting virus. The MAbs were classified by the location of their epitope relative to the intact 140S virion, the 12Sps and VP 1 (Table 2 ). Class I MAbs reacted with the freshly centrifuged labelled 140S virus in the LPRIA but failed to react with either the 12Sps or VP 1 in the SP-RIA. Class II MAbs reacted with 14OS virus in LP-RIA and 12SpS in SP-RIA but did not react with VP 1. Class III MAbs reacted positively with labelled 140S virus in the LP-RIA and in both the 12Sps and VP1 SP-RIAs. The Class IV 12Sps specific MAbs reacted only in the 12Sps SP-RIA. Class V MAbs are restricted to reaction in either the virus protein SP-RIA or reducing PAGE dot blots.
MAb cross-reactivity amongst FMD VAsia-1 subtypes: binding, neutralization and precipitation in agar gel Binding. Eight MAbs (Class I ) bound specifically with 140S in RIA but only four of them neutralized viral infectivity (Tables 3 and 4 ). Two of these Class I MAbs derived from mice at 14 PID were isotype IgA (Table 1 ). Twelve MAbs (Class II) bound to both the 140S and 12Sps particles. Four MAbs (Class III) reacted with 140S/12S/VP 1 in RIA, but only two MAbs (Class IV) bound specifically to 12Sps. These MAbs, as hybridoma supernatants, had relatively high RIA titers with 12Sps but neither precipitated the antigen in the Ouchterlony reaction nor neutralized infectivity (Tables 3 and 5 ) The binding pattern of each of the MAbs to 10 different viruses of Asia-1 serotype is shown in Table 3. The MAbs elicited with viral isolate India 10/ 86 (fusion 26 ) bound to most of the Indian isolates and to Pak/57 and Iran/ 73. A large portion of the non-neutralizing MAbs were RIA reactive with the majority of the Asia-1 viruses tested while greater variation was observed in the binding pattern of the neutralizing MAbs. Conformation-independent
280
G. BUTCHAIAH ET AL.
TABLE 1 Characterization o f MAbs elicited with F M D V Asia-1 isolates MAb
Isotype
Class a
Neutralization
Antigen source b
2C2.1.1 c 2C5.1.1
IgG3 IgG2b
II III
+d +
Asia-1 India 6 3 / 7 2 Killed 90-PID
26IH7.1.1 26JH6.1.1 26KA8.1.1.1.1 26KE6 r 26LA10.1.1
IgG2b IgG2a IgM IgG2b IgG2b
II1 II II 1 II
+ _ e +
Asia-1 India 10/86 Infect 14-PID
30EF9.1.1 30GG7.1.1 30HA7.1.1 30JD5.1.1
IgM
I
-
Asia-1 India 66/86 Infect 7-PID
IgM
I
-
IgM
II
-
lgM
IV
-
31AH6.1.1 31AHll.I.1 31DAI0.1.1 31DD8.1.1 31DF6 31EB3.1.1 31GAI0.1.1 31HB8 31HC2.1.1 31HD8.1.1 31HE8.1.1 31IE6.1.1 31IH7.1.1 31KC3.1.1.1 31KE2.1.1
IgG
I
+
IgA IgG2a IgG3 IgG2a
I I II II II II I IIl II I II IV III I1
+ + -
IgM
IgG 1 IgG2a IgG2a IgG2a IgG3 lgG2b IgG2a IgM
IgG2b
Asia-I India 6 6 / 8 6 Infect 14-PID
+
+ + +
+
aClass indicates location o f MAb reactive epitope in relation to intact virion, 12Sps and VP1 (see Table 2 ). bKilled=inactivated F M D V ; I n f e c t = i n f e c t i o u s F M D V ; P I D = p o s t - i n f e c t i o n / v a c c i n a t i o n day on which i m m u n e spleen cells were harvested. CHybridoma identification, the n u m b e r s after the points indicate the n u m b e r o f cloning cycles (i.e. • 1.1 has been cloned twice ); d + , Neutralizes eliciting virus. e _ , N o neutralization o f infectivity detected. flnitial isolate with no further cloning.
MAb 31HC2 (Class III) bound to only 50% of the Asia-1 virus isolates, whereas, the other three Class III MAbs bound 70% or more (Table 3 ). The Class IV MAbs 30JD5 and 311H7 uniformly bound, with one exception, to all FMDV 12Sps preparations tested regardless of serotype (Tables 3 and 6 ). The majority of Indian isolates and Pak/57, Iran/73 and Israel/68 were recognized by MAbs 2C2 and 2C5 (elicited by India 63/72 ) but the neutraliza-
ANTIGENIC RELATIONSHIPS OF FOOT-AND-MOUTH DISEASEVIRUS ASIA-I
281
TABLE 2 Classification of MAbs elicited with FMDV Class
Location of epitope
I
Restricted to the intact virion (140S)
1I
Available on both 140S and the 12S protein subunit (12Sps)
III
Available on 140S, 12Sps and a virus protein (VP)
IV
Restricted to the 12Sps
V
Restricted to a VP"
aPreviously reported by Robertson et al. (1984). 0
tion spectrum of 2C2 was larger than that of 2C5. Twelve of the panel of 26 MAbs recognized FMDV Asia-1 Israel/68, a subtype of Asia-1 usually considered distinct from the Indian subtypes.
Neutralization. Viral infectivity was neutralized to varying degrees by 12 of the MAbs. The Class I, IgA MAbs 31AH 11 and 31AH6 bound the virus with a low RIA titer but neutralized viral infectivity efficiently (Tables 3 and 4). In contrast, the MAbs 31HE8 (Class I), 31KE2 (Class II) and 26IH7 (Class III), reacted strongly with the virus in RIA but exhibited weak neutralization activity. In the PRT assay, the MAbs 2C5, 31AH11 and 31HC2 exhibited strong neutralizing activity against their eliciting viruses (Table 4). The Class I, isotype IgA MAb 31AH11 neutralized the infectivity of all Asia-1 viruses tested. On the other hand, the Class III MAbs 2C5 and 31HC2 neutralized only 50% of viruses tested. The majority of the neutralizing MAbs neutralized more than one virus with the exception of 31KE2 (Class II) which was restricted to its eliciting virus. Similar results were obtained against the 10 FMDV serotype Asia-1 isolates in both the PRT and MPT assays with the exception of MAb 31AH6 Class I that showed moderate titers in PRT but protected mice poorly (Table 4). At least seven of these 12 MAbs appear to recognize different epitopes (neutralizing) based on cross-reactivity patterns within each epitope class (Table 7 ). Precipitation in the Ouchterlony reaction. Fourteen MAbs precipitated purified preparations of 140S virus in the Ouchterlony reaction (Table 5) and this included all of the neutralizing MAbs (Table 4). Two of the precipitating MAbs (31DD8 and 31HD8 Class II) precipitated both 140S virions and 12Sps in the Ouchterlony reaction (Table 5 ). The analysis of the MAbs capacity to precipitate Asia- 1 viruses showed that
282
G. BUTCHAIAH ET AL.
TABLE 3 M o n o c l o n a l a n t i b o d y b i n d i n g a n a l y s i s o f F M D V s e r o t y p e Asia-1 i s o l a t e s MAb
F M D V Asia-1 i s o l a t e s a India 63/72
India 21/80
India 4/86
India 10/86
India 66/86
India MFB/86
India GDG/87
Pak 57
Iran 73
Israel 68
Class I 26KE6
0.8 b
0.8
1.2
0.8 ~
0.8
_d
1.2
0.6
-
--
30EF9
-
0.6
-
0.8
0.8
-
0.6
-
-
-
30GG7
0.4
0.5
0.6
0.8
0.6
-
0.3
0.6
-
-
-
0.4
1.1
0.5
1.4
0.5
0.3
l.l
-
-
31AHll(+)
0.5
0.8
0.8
0.8
1.1
1.1
1.1
1.1
0,6
0.8
31DAI0(+)
0.8
0.6
1.6
0.6
1.4
0.4
0.6
0.6
0.3
0.3
31HB8
-
0.5
1.1
0.3
0.8
-
0.1
0.3
-
-
31HE8(+)
1.2
0.5
2.7
3.1
3.3
3.5
0.3
0.4
3.1
3.1
MAb Class lI 2C2(+) 26JH6 26KA8 26LA10(+) 30HA7 31DD8
2.7 3.1 1.2 2.8 2.5
2.2 ND f 1.1 0.8 2.1
2.5 2.7 1.6 3.1 1.1 3.2
4.1 1.4 3.2 0.8 4.1
2.5 4.2 1.5 3.4 1.1 4.1
2.7 1.8 3.8 1.5 4.1
0.3 ND 1.6 3.2 0.8 3.4
2.7 3.1 1.1 1.1 3.8
2.3 4.0 1.4 3.6
2.8 3.7 4.1
31DF6 31EB3(+) 31GA10 31HD8 31IE6 31KE2(+)
1.4 0.6 2.7 . .
0.7 0.4 2.5 0.6 .
0.8 0.3 3.1 0.8
0.8 0.3 0.5 4.2 0.3
2.7 0.8 2.3 4.2 0.8 2.1
1.2 1.7 4.5 .
1.2 0.7 1.2 3.1 0.8 .
0.4 0.8 0.5 3.3 .
0.8 0.5 4.2 -
0.3 2.5 4.2 -
C l a s s II1 2C5(+) 26IH7 ( + ) 31HC2(+) 31KC3(+)
3.6 1.7 ND
2.5 0.4 0.3 0.5
2.5 2.8 0.3
2.7 2.8 0.3
2.8 3.1 0.3
1.4 3.4 2.5 0.3
2.7 3.2 0.3
2.4 0.3
4.1 3.1 2.7 0.1
4.1 -
ClassIV 30JD5 311H7
2.2 3.1
1.6 2.2
1.8 3.5
2.5 4.6
2.7 4.6
2.1 4.6
2.1 3.7
1.6 3.8
2.5 4.6
2.4 4.1
31AH6(+)
e
.
.
.
3.1
a P u r i f i e d F M D V Asia-1 i s o l a t e s l a b e l l e d w i t h ~25I. b 0 . 8 = t h e 30% a n t i b o d y b i n d i n g c a p a c i t y e n d - p o i n t t i t e r ( T r a u t m a n a n d H a r r i s , 1977 ). c U n d e r l i n e d v a l u e s i n d i c a t e r e a c t i o n w i t h t h e e l i c i t i n g v i r u s , d-, N o r e a c t i o n w a s d e t e c t e d . e( + ), N e u t r a l i z e s v i r a l i n f e c t i v i t y . fND = test not done.
283
A N T I G E N I C R E L A T I O N S H I P S O F F O O T - A N D - M O U T H DISEASE V I R U S ASIA-I
TABLE 4 FMDV
serotype Asia-I infectivity neutralization
c a p a c i t y o f M A b s in t h e p l a q u e r e d u c t i o n
(PRT)
and
mouse protection tests (MPT) MAb a
Class I 31AH6
31AHI 1
31DAI0
31HE8
FMDV
Asia- 1 isolates
India
India
India
India
India
India
India
Pak
Iran
Israel
63/72
21/80
4/86
10/86
66/86
MFB/86
GDG/87
57
73
68
PRT
_b
_
1.5 c
1.5 d
0.6
-
-
MPT
-
-
0.4
0.6
0.4
-
-
-
PRT
1.4
2.4
2.4
2.1
2.4
2.1
1.4
2.4
2.1
1.6
MPT
0.6
1.8
1.6
1.6
1.8
1.4
0.6
1.4
1.4
1.1
PRT
-
1.2
0.7
0.4
1.6
0.7
1.1
1.4
0.9
MPT
1.4
1.6
0.6
1.6
0.8
1.1
1.1
0.7
0.4 0.4
-
PRT
0.5
0.9
0.8
1.2
0.4
-
1.1
0.7
MPT
0.4
0.8
0.8
1.1
0.6
-
0.8
0.6
PRT
1.7
1.6
1.1
0.8
-
-
1.1
0.9
0.8
MPT
1.6
1.4
0.8
0.8
-
-
0.8
0.8
0.6
PRT
-
MPT
.
-
Class II 2C2
26LAI0
31EB3
31KE2
-
0.6
.
PRT
0.4
MPT
0.6
-
PRT
-
MPT
-
. 0.4
-
. 0.7
-
0.4
0.7
0.4
0.6
0.4
0.6
0.8
-
-
0.7
-
-
0.8
Class III 2C5
26IH7
31HC2
31KC3
PRT
2.1
-
-
0.7
0.7
1.1
-
MPT
2.1
-
-
0.6
-
0.8
0.8
-
PRT
-
-
-
0.6
-
-
-
MPT
-
-
-
0.4
-
-
-
PRT
-
0.4
0.9
2.6
0.4
-
-
MPT
-
0.4
0.8
2.1
0.8
-
-
PRT
-
-
-
0.5
0.4
0.4
0.4
0.4
MPT
-
-
-
0.4
0.5
-
0.4
0.4
aMAbs from Table 1 which gave no detectable reaction were excluded from this table. b , N o s i g n i f i c a n t (i.e. > 3 0 ) r e a c t i o n d e t e c t e d . CTiter ( P R T = m a x i m u m dilution (Log base 10) which produces 70% reduction (MPT=maximum d i l u t i o n { L o g b a s e 10} w h i c h p r o t e c t e d 5 0 % o f t h e t e s t m i c e . OUnderlined value indicates reaction with the eliciting virus.
in plaque
count)
or
284
G. B U T C H A I A H ET AL.
TABLE 5
Precipitation of FMDV serotype Asia- 1 isolates by anti-Asia- 1 MAbs in the Ouchterlony reaction MAb a
Isotype FMDVAsia-I isolates India 63/72
India 2t/80
India 4/86
India 10/86
India 66/86
India MFB/86
India GDG/87
Pak 57
Iran 73
Israel 68
_c PPT
_ PPT PPT PPT
PPT d PPT PPT PPT
PPT PPT -
PPT ~ PPT PPT PPT
PPT PPT
PPT PPT -
PPT PPT PPT PPT
PPT PPT
PPT PPT PPT PPT .
PPT PPT PPT PPT PPT PPT
PPT PPT PPT -
PPT PPT PPT PPT PPT PPT PPT
PPT PPT PPT -
PPT PPT PPT PPT -
PPT PPT PPT PPT -
PPT PPT
PPT
PPT
PPT
PPT PPT
PPT
PPT PPT
PPT
PPT PPT
PPT
PPT PPT
PPT
PPT
-
PPT
-
PPT
PPT
-
-
Class I 31AH6(+)
b
IgA
31AHII(+) 31HB8 31HE8(+)
IgA IgG2a IgG3
Mab Class II 2C2( + ) 30HA7 31DD8 31EB3(+) 31HD8 311E6 31KE2 ( + )
IgG3 IgM IgG3 IgM lgG2a IgG2b IgG2b
PPT
Mab Class III 2C5(+) 26IH7(+)
IgG2b IgG2b
PPT PPT
31HC ( + )
IgG2a
PPT PPT PPT .
.
.
PPT
PPT
aMAbs from T a b l e 3 ( h y b r i d o m a supernatants) which gave no detectable reaction were excluded from this table. b( + ), Neutralizes viral infectivity. c ( _ ), N o reaction detected.
°PPT, Precipitation in the Ouchterlony reaction. *Underlined value indicates reaction with the eliciting virus.
four non-neutralizing MAbs (31DD8, 31HB8, 31HD8, 31IE6) and two neutralizing MAbs (31AH11, 31HE8) precipitated the majority of the Asia-1 viruses tested. On the other hand, neutralizing MAbs (e.g., 2C2, 2C5, 31AH6, 31EB3, 31HC2, 31KE2 ) exhibited extensive variation in precipitating different Asia- 1 viruses (Table 5 ). Cross-reactivity amongst FMD V serotypes Analysis of the RIA cross-reactivity of the MAbs with eight different viruses from F M D V serotypes O, A and C identified seven MAbs ( 7 / 2 6 ) which reacted with the heterologous F M D V serotypes. Only one Class II MAb ( 31DD8 ) reacted with the heterologous serotypes (i.e., O 1 Brugge). The four Class III VP 1-reactive neutralizing MAbs ( 2C5, 26JH7, 31HC2, 31KC3 ) exhibited cross-reactivity with heterologous F M D V serotypes A and/or C in RIA; however, none of them neutralized the heterologous serotypes (data not
ANTIGENIC
RELATIONSHIPS
OF FOOT-AND-MOUTH
DISEASE
VIRUS
285
ASIA-1
TABLE 6 Monoclonal antibodies elicited with F M D V Asia-1 which react with other F M D V serotypes MAb"
F M D V serotype O1 Bru
A5 Spain
Ai2 LP ab
C1
Class II 31DD8
Bind b
___c
Class III 2C5 26IH7 31HC2 31KC3
-----
-----
-Bind Bind Bind
Bind . . .
Class IV 30JD5 31 IH7
Bind Bind
Bind Bind
Bind Bind
-Bind
C2
C3 Indail
C3 Resende
C4
Bind . . .
Bind
B
. . .
Bind Bind
Bind Bind
Bind Bind
-. . .
m
m
Bind Bind
aMAbs which gave no detectable reaction with serotypes other than Asia-1 were excluded from this Table. b B i n d = t h e MAb reacts with the indicated virus serotype in the radioimmunoassay. c , No reaction was detected. TABLE7 Foot-and-mouth disease virus Asia- 1 distinct epitopes related to the neutralization of viral infectivity Epitope
MAb
Class Distinguishing characteristics (within the MAb class)
1"
31AHI 1
I
Binds and neutralizes all serotype Asia-1 viruses tested
2
31HE8
I
Binds to all serotype Asia-1 viruses tested and neutralizes all except India 10/86 and India G D G / 8 7
3
31AH6
I
Binds to all serotype Asia- 1 viruses tested and neutralizes all except Pak/57, Iran/73 or Israel/68
4
2C2
II
Neutralizes serotype Asia-I isolates India 21/80, Iran/73 and Israel/ 68
5
31EB3
II
Does not neutralize serotype Asia- 1 isolates India 21/80, Iran/73 and Israel/68
6
2C5
III
Neutralizes serotype Asia-1 isolate India 63/72 but does not neutralize India 66/88
7
31HC2
III
Neutralizes serotype Asia-I isolate India 66/88 but does not neutralize India 63/72
"The MAbs listed here represent the minimal n u m b e r of neutralization related epitopes on F M D V serotype Asia- 1.
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G. BUTCHAIAH ETAL.
shown). The Class IV 12Sps specific MAbs 311H7 and 30JD5 as reported in Table 6 reacted with all but one of the heterologous serotypes (i.e. 30JD5 did not react with FMDV CI). DISCUSSION
Each MAb was classified according to the location of its reactive site in relation to the intact virion, 12Sps and VP 1. The infectivity neutralization based classification scheme of Stave et al. ( 1988 ) was modified to utilize antibody binding as the sole parameter. This protocol recognizes the fact that the spectrum of FMDV isolates bound frequently exceeds the spectrum of those neutralized as well as the fact that certain FMDV reactive antibodies have no effect on neutralization of viral infectivity. The proposed classes of anti-FMDV antibodies based upon epitope location are presented in Table 2. In effect this scheme simultaneously classifies the epitope and the MAb which defines it. The development of two neutralization MAbs (31AH6, 31AH11 ) of the IgA isotype shows for the first time that circulating IgA may play a role in the immune response to FMDV infection. The isolation of two Class IV MAbs ( 30JDS, 311H7 ) from mice infected with purified live virus demonstrates the in vivo degradation of the virus into 12Sps. Such in vivo degradation of purified, inactivated virus into 12Sps was also described previously after repeated immunization of mice (McCullough and Butcher, 1982 ). One of the Class IV MAbs (30JDS) isolated from mice at 7 PID was IgM, confirming the observation of Stave et al. (1986) that IgM binds to the 12Sps, in contrast to earlier reports using polyclonal antisera (Brown and Smale, 1970). In this study no Class III antibodies were developed from the 7 PID fusion. The Class IV MAbs do not bind the infectious particle and, therefore, do not neutralize viral infectivity. These MAbs bound the 12Sps of all FMDVs with the exception that MAb 30JD5 did not bind to C 1 virus. It seems that such universal epitopes likely involve a fundamental highly conserved architectural feature of the virus; thereby, the absence of such epitopes predicts a basic structural difference with other FMDVs. These 12Sps-specific epitopes are exposed only when the intact virus is degraded into 12Sps. Antibodies to them have also been generated with FMDV serotype SO and A (Morgan et al., 1984; Stave et al., 1986; Saiz et al., 1989). Class IV MAbs have been used to develop an inhibition ELISA for the differential diagnosis of FMD from other vesicular diseases (Smitsaart et al., 1990). Certain non-neutralizing Class II MAbs (e.g. 26JH6, 31GAI0, 31HDS) appear to be serotype specific for Asia- 1 and, as a panel, react with all Asia- 1 viruses tested. These MAbs should be useful for diagnostic serotyping of Asia1 virus isolates. With the single exception of MAb 31DD8 binding to serotype
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O 1 Brugge, the non-neutralizing Class I and II MAbs did not bind to heterologous serotypes of FMDV. The type of assay and the conditions under which it is performed affect the reaction or lack of reaction of a particular MAb: therefore, in this investigation, MAbs have been characterized and classified, in so far as possible, on the basis of positive results. The majority of the neutralizing MAbs (8/12) derived from mice immunized with whole Asia-I virus were conformation dependent. There appears, however, to be a site on VP 1 defined by the conformation-independent, highly efficient neutralizing MAbs 2C5 and 31HC2. Further, it has been shown that VP 1 isolated from Asia- 1 virus, when used as a vaccine, protects cattle against infection with the homologous virus (D.O. Morgan and G. Butchaiah, unpublished data). These results show that both conformation-dependent and conformation-independent epitopes are important in the protection of animals from serotype Asia-1 infection. Conformation-dependent epitopes were also found more frequently than sequential (conformation-independent) epitopes for FMDV serotype O (Stave et al., 1988) and in nearly equal amounts for serotype A virus (McCullough and Butcher, 1982; Meloen et al., 1983; Baxt et al., 1984; Morgan et al., 1984; Ouldridge et al., 1984). Conformation-dependent epitopes are also of major importance in other picornaviruses (Ferguson et al., 1984; Diamond et al., 1985; Blondel et al., 1986; Sherry et al., 1986). Summation of the effect of antibodies on the conformation-dependent epitopes may account for the greater efficacy, on a weight basis, of whole virus vaccines when compared to subunit vaccines. Conformation-dependent neutralization epitopes recognized by Class I MAbs 31AH 11, 31DA 10, 31 HE8 are functionally conserved across several of the isolates of FMDV serotype Asia-1 tested here. Similarly, certain conformation-dependent anti-FMDV serotype A12 MAbs neutralized several different subtypes of FMDV serotype A as described previously (Grubman and Morgan, 1986). In contrast, conformation-independent Class III MAbs such as 2C5, 31HC2 exhibit extensive variation in their reactivity with different Asia-1 virus isolates of the same subtype. In general, the infectivity neutralizing capacity of the Class III MAbs was more restricted than that of the Class I and II MAbs (Table 4). This finding suggests that the epitopes recognized by Class III MAbs are located on the highly variable region of VP1 (amino acids 140-160) as established in other serotypes of FMDV. The conformation-independent neutralizing Class III MAbs (2C5, 26IH7, 31HC2, 31KC3 ) cross-reacted in RIA with other FMDV serotypes A and/or C; however, no neutralization of viral infectivity of the heterologous serotypes was found (Table 6 ). The lack of correlation between the spectrum of viruses bound and the viruses neutralized within a homologous serotype has been well demonstrated. For example, MAb 2C5 elicited by Asia-1 India 63/ 72 virus binds to other Asia-1 viruses, India 21/80, Iran/73 and Israel/68,
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but it does not neutralize their infectivity. Similar results showing binding to heterologous serotypes without neutralizing their infectivity have been reported previously with MAbs raised against FMDV serotypes A12 (Grubman and Morgan, 1986), O1 Brugge (Stave et al., 1988) and poliovirus type 1 (Crainic et al., 1983; Diamond et al., 1985; Blondel et al., 1986). The mechanics of this process remain a paradox. One can logically postulate that a change of a single amino acid residue in the area of an epitope while not altering antibody binding might eliminate the infectivity neutralizing effect of a MAb reaction. An alternative could be that the orientation of the bound MAb on one virus sterically interferes with the virus binding to its cell receptor while on another it does not. Large variations in the magnitude of infectivity neutralization within a group of neutralized viruses such as demonstrated by MAbs 31HC2 and 2C5 (Table 4) is a situation in which a single epitope has a positive role in neutralization but with variable degrees of effectiveness. The scheme of anti-FMDV MAb classification presented in Table 2 is based on the simple surmise that epitopes located on structurally distinct parts of the virus are distinct. Further, the neutralization patterns of the MAbs with 10 different Asia-1 virus isolates suggest that several of the MAbs are recognizing different epitopes. The Class I MAbs 31AH 11, 31DA 10, 31 HE8 bind to all Asia-1 virus tested (Table 3 ) but 31 HE8 has a restricted neutralization pattern (Table 4); whereas MAb 31AH6, also Class I, has a more restricted binding pattern and neutralized only India 4/86, India 66/86 and India MFB/ 86. Similarly, the Class II MAb 31EB3 neutralized India 63/72, India 4/86, India 66/86, India GDG/87, India MFB/86 and Pak/57 and is distinct from another Class II MAb 31KE2 which neutralized only the eliciting virus. The divergent Asia- 1 isolate neutralization patterns of Class III MAbs 31KC3 and 31HC2 indicate that they may well recognize different epitopes and are certainly distinct from 2C5 (Table 4). With foregoing arguments as evidence, it is proposed that a minimum of seven distinct neutralization epitopes exist on the surface of Asia-1 virus India 66/86 (Table 7 ). There is weaker evidence for additional neutralization epitopes. This does not prove that all of these seven neutralization epitopes exist or function in all Asia- 1 viruses. However, given the structural similarities of the Asia- 1 viruses, it strongly suggests that these or their functional surrogates likely exist on all Asia-1 viruses. Five of the seven Indian Asia-1 viruses were isolated recently, i.e., 19861987, and the remaining two were isolated 1972-1980. Remarkably, viruses isolated even a few months apart in different regions in India (Asia-I India 4/86, 10/86, 66/86, MFB/86, G D G / 8 7 ) exhibited extensive variation particularly in conformation-independent neutralization epitopes recognized by Class III MAbs 2C5 and 31HC2. Further, the Asia- 1 viruses of 1980 or earlier (India 63/72, 21/80, Iran/73, Israel/68) possessed both the epitopes recognized by the neutralizing MAbs 2C5 (Class III) and 2C2 (Class II) as demonstrated by binding in RIA. In contrast, Asia-1 virus isolates of 1986-1987
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and the classical Asia-1 virus P a k / 5 7 exhibited the presence o f one of these two epitopes but not both. The extensive antigenic heterogenicity observed among Indian Asia-1 virus field isolates could result from partially i m m u n e (vaccinated) host animals providing a strong selection force for the generation of antigenic variants. Antigenic heterogeneity, as reported here, is not unique to F M D V serotype Asia-1. Such heterogeneity in natural populations of other F M D V serotypes O, A and C ( D o m i n g o et al., 1980; King et al., 1981; Rowlands et al., 1983; Sobrino et al., 1986; Beck and Strohmaier, 1987; Mateu et al., 1988 ); Dengue virus isolates ( M o n a t h et al., 1986) and other R N A viruses (Domingo and Holland, 1987 ) has been well documented. Despite the extensive variation detected by certain neutralizing MAbs, conformation-dependent epitopes recognized by neutralizing Class I MAbs 31AH 11, 31DA 10, 31 HE8 are uniformly conserved in the Asia- 1 viruses tested irrespective of time or geographical location o f their isolation. Furthermore, all o f the isolates were neutralized by polyclonal cattle and mouse anti-Asia- 1 Indian 66/86 virus sera although to varying degrees (data not shown). This indicates that future emphasis should be placed on the construction of conformational epitopes rather than sequential epitopes in order to increase the spectrum o f cross-subtype protection o f synthetic F M D vaccines. ACKNOWLEDGEMENTS G.B. was supported by a Biotechnology Overseas Associateship, Government o f India. We wish to thank Adriene Ciupryk for typing and Sue B. Morgan for editing the manuscript.
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