Immunology 1989 68 265-271

Foot-and-mouth disease virus-neutralizing antibodies induced in

mice by anti-idiotypic antibodies

A. E. GARMENDIA, D. 0. MORGAN & B. BAXT

Molecular Biology Laboratory, USDA, ARS, Plum Island Disease Center, Greenport, New York, U.S.A.

Acceptedfor publication 28 June 1989

SUMMARY
A neutralizing monoclonal antibody (nmAb) to foot-and-mouth disease virus (FMDV) was used as
antibody-I (ABI) to induce anti-idiotypic antibodies (a-IdAb) in rabbits. The rabbit a-IdAb (AB2)
were isolated on protein A-Sepharose, followed by cycles of separation on idiotype and isotype
affinity columns. The specificity of the AB2 for the paratope of AB I was determined by direct binding
to AB I in solid-phase radioimmunoassay (SP-RIA), and by competition RIA (C-RIA) with virus for
binding to the AB 1. The AB2, termed a-2PDI 1, was utilized to immunize six groups of female Swiss
mice at weekly intervals with either one of three formulations, in doses of 50 ug or 5 yg, given in single
subcutaneous (s.c.) spots. Anti-viral antibody (AB3) was first detected by RIA at the fifth week in the
50 pg/dose groups, and maximum levels were reached at the sixth week in the 50 and 5 pg/dose
groups. The AB3 levels were at least three times higher for mice given 50 pg doses. In addition, the
AB3 were also shown to neutralize FMDV infectivity in tissue culture and in a suckling mouse
protection assay. Overall, mice exhibited variable responses to immunization with AB2. In a
subsequent trial, mice received multispot s.c. and footpad injections of 50 pg of a-2PD 1I coupled to
keyhole limpet haemocyanin (KLH) on a weekly basis. In these mice, AB3 was detected earlier than
in mice immunized with single s.c. injections. These results support the use of a-IdAb as potential
surrogates of critical determinants for FMD vaccines.

INTRODUCTION greatly increases their potential as vaccines or immunothera-

Foot-and-mouth disease virus (FMDV) is an aphthovirus of the
Picornaviridae family, and the aetiological agent of one of the
most devastating diseases of livestock. The control of FMD is
based upon quarantine and extensive worldwide vaccination
programme. While there are several vaccine formulations used
to control FMD, the high genetic mutation rates of the virus
(Domingo, Davila & Ortin 1980), inappropriate inactivation
and low stability of the vaccines (Kleid et al., 1981; Bittle et al.,
1982) are still major drawbacks. Consequently, a new genera-
tion of antigens being tested for use as vaccines for FMDV
include genetically engineered and expressed proteins and
peptides (Kleid et al., 1981) and synthetic peptides (Bittle et al.,
1982) representing neutralizing epitopes, both of which have
been shown to induce protective immunity to FMDV. More
recently anti-idiotypic antibodies (a-IdAb) have been evaluated
as surrogates for FMDV neutralization epitopes (Baxt et al.,
1989a).
It has been shown that a-IdAb may enhance or suppress
specific immune responses (reviewed by Dalgleish & Kennedy,
1988; Hiernaux, 1988; Norton et al., 1985), a feature which
Correspondence: Dr B. Baxt, Molecular Biology Laboratory,
USDA, ARS, Plum Island Animal Disease Center, P.O. Box 848,
Greenport, NY 11944, U.S.A.
265
peutic agents. Sacks, Esser & Sher (1982) first reported the
protection of experimental animals immunized with a-IdAb
against a protozoan agent; however, a genetic restriction was
evident in this system. Subsequently, immunization with inter-
nal image a-IdAb, termed AB2-beta by Jerne (1974), was shown
to induce immune responses against some viral (Sharpe et al.,
1984; Marriot et al., 1987; Zhou et al., 1987; Uytdehaag &
Osterhaus, 1985; Paque & Miller, 1987), bacterial (McNamara,
Ward & Kohler, 1984) and parasitic agents (reviewed by
Murray, 1987). It has also been shown that some non-internal
image AB2 can induce immune responses against viral antigens
(Schick, Dressman & Kennedy, 1987). In addition, AB2-beta
has been used to induce immunoresistance to tumours in
humans and experimental animals (Herlyn et al., 1987; Viale et
al., 1987), and as probes for identification of cellular receptors in
at least two viral systems (Mariott et al., 1987; Noseworthy et
al., 1983).
In a previous study, a series of seven anti-FMDV type A12
neutralizing monoclonal antibodies (nmAb) were utilized to
generate a-IdAb in rabbits (Baxt et al., 1989a). Two of these
a-IdAb induced, in mice, antibodies capable of binding labelled
FMDV; however, neutralizing antibodies could not be demon-
strated (Baxt et al., 1989a). To address further whether a-IdAbs
could be used as surrogate antigens capable of inducing
266 A. E. Garmendia, D. 0. Morgan & B. Baxt
neutralizing anti-FMDV responses, experimental immuniza-
tions of mice with a-IdAb were conducted. For this purpose, a
nmAb that defines a conformational viral epitope located on the
viral capsid proteins VP3 and VP1 (Baxt et al., 1984; Grubman
& Morgan, 1986; Baxt et al., 1989b) was selected. Polyclonal
a-IdAb generated against this mAb in rabbits induced a
neutralizing anti-FMDV response in immunized mice.
MATERIALS AND METHODS
Viruses and cells
The large plaque ab variant of FMDV type A12 was grown in
BHK-21 cells, and utilized for induction of monoclonal anti-
bodies, immunoassays and radiolabelling essentially as des-
cribed previously (Baxt et al., 1984). Virus was radiolabelled
with [3H]uridine, and 140 S viral particles were purified as
described elsewhere (Baxt et al., 1984). Additional [3H]uridine-
labelled FMDV types, and mAb neutralization escape variants,
have also been described (Grubman & Morgan, 1986; Baxt et
al., 1989b).
Induction and separation of anti-idiotypic antibodies (AB2)
To induce a-IdAb, 200 pg of mAb 2PD1 1, purified on protein
A-Sepharose and emulsified in an equal volume of Freund's
complete adjuvant (CFA), were injected s.c. into rabbits,
followed by bi-weekly booster injections of 2PD 11 in Freund's
incomplete adjuvant (IFA). To separate the a-IdAb, the rabbit
serum was adsorbed onto protein A-Sepharose equilibrated
with borate-buffered saline (BBS; 0-1 M boric acid, 001 M
sodium borate, 0-5 M NaCI, pH 8-5). For equilibration and
elution from affinity columns, the same buffers were used in all
separation procedures. The IgG fraction, eluted with glycine-
1LCI (0- 1 M glycine, 0-2 M NaCI, 0-1 N HCI, pH 3 0) from protein
A-Sepharose, contained anti-idiotypic and anti-isotypic anti-
bodies. These antibodies were adsorbed further on an idiotype
matrix (Idm), which was 2PDl 1 coupled to Affi Gel-1O (Bio-
Rad, Richmond, CA), followed by several cycles of separation
on a isotype matrix (Ism), which was 6HC4 coupled to Affi Gel-
10 (6HC4 is a murine anti-FMDV nmAb of the same isotype as
but different specificity to 2PD1 1). The fractions eluted from the
Idm that passed through the Ism contained predominantly a-
IdAb. These were dialysed against phosphate-buffered saline
(PBS), concentrated to approximately 1 mg/ml on Centricon
filters (Amicon Inc., Danvers, MA), and stored at - 20° in
aliquots for further use.
Radioimmunoassays for anti-idiotypic antibody specificity
For use in a solid-phase radioimmunoassay (SP-RIA), serial
two-fold dilutions of a-IdAb, at a starting concentration of
40 Mg/ml were made in carbonate-bicarbonate buffer (CBB;
13 mm sodium carbonate, 35 mm sodium bicarbonate, pH 9 5),
and adsorbed onto polystyrene wells (Immulon Wells, Dyna-
tech Labs) overnight at 4°. The wells were blocked with RIA
buffer (0- 12 M boric acid, 0-02 M sodium borate, 0-07 M NaCl,
pH 8-2) containing 10% fetal calf serum (RIA-FCS) for 2 hr at
370, and washed with RIA buffer containing 0 02% BSA and
0-05% Tween-20 (RIAT). For binding, 5 x 104 c.p.m./well of
['25I]2PDl 1, labelled as described elsewhere (Baxt et al., 1989a),
were added to the wells and incubated at 370 for 30 min,
followed by 40 for 90 min. The wells were washed with RIAT
and counted directly in a gamma-counter. Labelled, isotype-
matched controls were tested under the same conditions. For
competition radioimmunoassay (C-RIA), 2PD11 (ABI) was
adsorbed overnight at 40 onto Immulon wells, and the wells
blocked as described above. The optimum 2PD1 1 concentration
per well and the amount of [3H]FMDV A12 to be used in the C-
RIA were determined by checkerboard titration in a direct
binding RIA. Increasing concentrations of a-IdAb and constant
amounts of [3H]FMDV type A12, diluted in PBS with 0 1% BSA
(PBS-BSA), were simultaneously added into the ABI wells and
incubated for 90 min at 250. The wells were washed six times
with RIAT, eluted with 0 5 N NaOH for 10 min at 37°, and
counted in a scintillation counter. Normal rabbit IgG, and a
rabbit a-IdAb made against another anti-FMDV nmAb, termed
6EE2, were tested under the same conditions.
Generation of AB3
In Trial 1, three formulations of a-2PDl 1, at two concentrations
each, were tested in six groups of three 60-g female Swiss mice.
Groups 1 and 2 received 50 Mg or 5 Mg, respectively, of purified
rabbit a-2PD1 1 diluted in PBS. Groups 3 and 4 received 50 g or
5 Mg, respectively, of a 2PD 11 coupled to KLH (Viale et al.,
1987) (a-2PDl 1KLH), and Groups 5 and 6 received 50 Mg or
5 Mg, respectively, of a-2PD1 1 that was self-polymerized with
glutaraldehyde (Avrameas, Ternynck & Guesdon, 1978)
(a-2PD I lGlu). For priming, the immunogens were emulsified in
equal volumes of CFA and given in a single s.c. spot, followed
by four weekly booster injections in IFA, and a final i.p.
injection given in IFA at the fifth week. An additional group of
three mice was immunized with 50Mg/dose ofnormal rabbit IgG
(NRIgG) with the same adjuvants under the same conditions.
For antibody analysis, blood was collected from the mice on a
weekly basis, and ascites induced with 180/TG sarcoma cells
aided with an i.p. injection of pristane (Baxt et al., 1989a).
In Trial 2, a group of nine female Swiss mice each received
50 Mg of a-2PDllKLH in CFA, injected in three to four
separate s.c. spots and in one foot pad. Weekly multi-spot
booster s.c. injections were given in IFA, and blood was
collected weekly.
Radioimmunoassay for detection of AB3
Antibody to radiolabelled FMDV type A12 was assayed in
serum and ascitic fluid from immunized mice by a direct
radioimmunoassay in fluid-phase (FP-RIA), aided with Staphy-
lococcus aureus protein A (SAPA) as described previously (Baxt
et al., 1984). Briefly, the serum samples, diluted in PBS
containing 0-1 % BSA, were reacted against a constant amount
of [3H]FMDV for 90 min at 250. Ten microlitres of 10% SAPA,
armed with rabbit anti-mouse IgG, were added to the reaction
mixtures, and incubated for 30 min at 250. After centrifugation,
the pellets were washed three times with RIAT buffer, resus-
pended, eluted with 150 mm Tris-HCl, pH 7 5, containing 2%
SDS, for 15 min at 370, and then counted in a scintillation
counter.
Neutralizing and protection assays
Neutralizing antibodies in serum were determined by virus
neutralization (VN), and in ascitic fluids by plaque-reduction
neutralization assays (PRNA) on confluent monolayers of a
continuous line of bovine kidney cells (BK-LF) grown in 96-well
or 6-well tissue culture dishes, respectively. In VN assays,
100 TCID50 of virus were incubated with two-fold dilutions of
serum for I hr at 250 prior to infection of the cells. Dilutions of
 Anti-idiotype-induced anti-FMD V antibody
I00
0 a-2PDIIGlu
0
0
0 3 6 9 12
a-Id (pg/assay)
Figure 1. Competition radioimmunoassay in solid-phase. Two-fold
dilutions of either a-2PDl 1(0), a-6EE2(@) or normal rabbit IgG (-)
were mixed with a constant amount of [3H]A12 and reacted with 2PDl1
bound onto Immulon wells, as described in the Materials and Methods.
virus and serum were made in minimal essential media (MEM)
containing 25 mM HEPES buffer, pH 7-5. The VN test was
scored by examination of cytopathic effect (CPE) of individual
wells, and/or determination of monolayer destruction by crystal
violet-formalin staining of the plates. The VN titres were the
log1o of the dilution which protected 50% of the monolayers,
determined by the method of Spearman and Karber (Baxt et al.,
1989b). The PRNA was done as described previously (Baxt et
al., 1984). Analysis of neutralizing antibodies in vivo was done
by a standard suckling mouse protection test, as described
previously (Grubman & Morgan, 1986).
RESULTS
Reactivity of the rabbit anti-2PD11 antibody
From a series of seven anti-FMD type A12 nmAbs used to
generate a-IdAB in rabbits (Baxt et al., 1989a), the a-IdAb
chosen as AB2 for the present study was made against 2PDl 1, a
mAb of the IgG2b isotype specific for 140 S viral particles (Baxt
et al., 1984; Grubman & Morgan, 1986). 2PDl defines a
conformational FMDV epitope (Baxt et al., 1989b), and has
high titres both in primary binding RIA and neutralization
assays with FMDV A12 (Baxt et al., 1984; Grubman & Morgan,
1986). The anti-2PDl antibodies (a-2PDl 1) generated in
rabbits against 2PDl were affinity purified as described above
and characterized as follows: (i) a-2PDl was shown to react
with ['25I]2PD1 in SP-RIA in a dose-dependant manner, while
six other a-FMDV nmAb, including isotype-matched controls,
reacted poorly or failed to react (Baxt et al., 1989a); (ii) a-2PD 1I
was able to compete with labelled type A12 FMDV, in a dose-
dependant manner, for binding to 2PDl adsorbed onto wells
(Fig. 1). Neither control NRIgG nor an a-IdAb generated
against nmAb 6EE2 (a-6EE2). which has a different deter-
minant specificity than 2PDI 1, inhibited the binding ofthe virus
to 2PDl (Fig. 1); and (iii) there was no direct binding of
a-2PD1 to the virus (data not shown). Together, these
characteristics indicated that the rabbit a-2PDl reacted with or
near the antigen-binding site of the ABI.
Binding of FMDV by mouse AB3 serum
In Trial 1, mice were divided into groups and immunized with
a-2PD1 as described in the Materials and Methods. Sera was
collected weekly and assayed for AB3 by FP-RIA. The results in
267
a a-2PDII
Ma-2PDIIKLH
- NRIgG
18 - (a)
(b)
15-
to 12
2-
3_
00
3 4 5 6 7 8 3 4 5 6 7 8
Weeks post -immunization
Figure 2. Mouse AB3 binding to [3H]FMDV. Mice were immunized
with either 50 pg (a) or 5 pg (b) of AB2 as described for Trial 1 in the
Materials and Methods. The AB2 was used either unmodified (0), self-
polymerized with glutaraldehyde (-), or coupled to KLH (s). One
group of mice was also immunized with 50 yg of NRIgG (U) (b).
Individual sera were collected and the histograms represent the binding
average of each group in a FP-RIA as described in the Materials and
Methods. The error bars represent one standard deviation of individual
samples from the mean. All sera were diluted 1:2 for this assay.
Figure 2 show that a-FMDV antibody could be demonstrated in
sera of some of the mice of the high-dose (50 pg/dose) groups
five weeks after the first immunization (Fig. 2a). At this time all
mice received an i.p. booster injection with their respective
formulations and doses. At the sixth week, a significant increase
(P < 0-05) in binding response was observed, as were differences
in both the high- and the low-dose groups (Fig. 2a, b). Thus, the
average binding to pH]FMDV A12 at the sixth week was at least
three- to nine-fold greater in mice given 50 pg doses of a-2PD1
(Fig. 2a) compared to mice receiving 5 pg doses (Fig. 2b). The
antigen formulation did not have an apparent effect on the
responses of the high-dose groups, since by the sixth week no
significant differences (P < 0-05) in binding were evident. In the
low-dose groups (Fig. 2b) the group receiving a-2PDlIKLH
appeared to have a better antibody response than the other
groups; however, statistical analysis indicated no significant
difference in binding (P < 0-05). Within an antigen formulation
however, the dose of AB2 given appeared to influence the
amount of AB3 detected. The amount of [3H1A12 bound to
serum of mice receiving 50 pg of a-2PDI lGlu at Week 6 was
greater than mice receiving 5 pg of the same formulation
(P < 0-05), and a similar result was seen for uncoupled a-2PDl
(P < 0-01) at Weeks 6 and 7. The dose of a-2PDl KLH did not
have a significant effect on the AB3 response of mice (P < 0-05).
Figure 2 also shows that there was a high degree of variation of
AB3 responses of individual mice, as evidenced by the high
standard deviations in some of the groups. Sera from mice
immunized with 50 pg of NRIgG were unable to bind labelled
FMDV (Fig. 2b).
Two mouse AB3 sera, collected at the sixth week and
exhibiting the highest binding to wild type virus, were reacted
with a monoclonal antibody-resistant variant of type Al2 that no
longer reacted with 2PD1 (Baxt et al., 1989b). Table 1 shows
that as with the original ABI, both AB3 exhibited little or no
268 A. E. Garmendia, D. 0. Morgan & B. Baxt
Table 1. Reactivity of mouse-AB3 with an 6

FMDV ABI-resistant variant* 5

to4
'4

X3 V

% binding
02 *,, a

Test antibody Wild type Variant

- t n
f n
2PDl 1 (IOpg/ml) 100 16 0 1 2 3 4 5 6
AB3. I t 34 1 Weeks post- immunization
AB3.51 65 09
Figure 3. Mouse AB3 binding to [3H]FMDV in FP-RIA. Sera were
* The test antibodies were reacted in FP- tested weekly during immunization with a-2PDI IKLH as described in
RIA with labelled wild type or a labelled 2PD 1I Trial 2 in the Material and Methods. The average binding each week is
neutralization-resistant variant of FMDV type represented (*), and the other symbols represent binding by individual
A12 (Baxt et al., 1989b). The values are mice. All sera were diluted 1:2 for this assay.
expressed as a percentage of the binding labelled
virus input.
t Mouse AB3 serum from Group 1 (see the
Materials and Methods) that exhibited the 2 5-
highest-binding to wild-type virus in its group
assayed at a 1:2 dilution.
T Mouse AB3 serum from Group 5 (see the 0

cr 1-5- L
Materials and Methods) that exhibited the 0

highest binding to wild-type virus in its group

assayed at a 1:2 dilution.
05II
reactivity with the variants, suggesting that the AB3 reacted vn _n n,

with the same, or a closely related, epitope defined by 2PD1 1. NJ °0

Nf
CJ -° _ =0 =)
Additional testing, in FP-RIA, of the mouse AB3 sera demon- O a aL cQ-
CL
N CLjNJ
Q.

strated reactivity against two other FMDV types, A24 and A27, C\l C~l
0
0

also recognized by the AB 1; however, there was no reaction with 0

type A76, which did not react with the ABl (Table 2). In contrast,
mouse AB3 failed to react with types A5 and A79, which do react
with nmAb 2PD 1. The induction, by a-2PDI 1, of a-FMDV
responses in mice confirmed that the polyclonal AB2 contained
internal image AB2 (AB2-beta) (Ertl & Bona, 1988; Kennedy,
Henkel & Dreesman, 1986).
Immunization groups
Figure 4. AB3 neutralization of FMDV. Sera collected at Week 6 from
mice used in Fig. 2 were tested for viral neutralizing activity by VN assay
as described in the Materials and Methods. The histogram represents
the average VN50 titre as described in Table 3, and the error bars
represent one standard deviation of individual samples from the mean.

Table 2. Reactivity of mouse AB3

with subtypes of type A FMDV in In Trial 2, in which mice received multispot immunizations
FP-RIA
with 50 yg of a-2PDllKLH, AB3 was detected within 15-21
days after the initial immunization in the serum of at least one
Antibody mouse, and maximum binding occurred by the fifth week
(Fig. 3). Thus, the period from the first immunization to the
Subtype 2PDI1* Mouse AB3t detection of antibody responses was shorter in Trial 2 than in
Trial 1, suggesting that multispot s.c. and footpad injections
A12 + + possibly aided in priming of mice, since no other variables were
A5 + - introduced in the immunization. Although the assays were not
A24 + +
done at the same time, and therefore are not directly compar-
A27 + +
able, it appears that the responses were not different from those
A76 -
A79 + - of the group which received the same a-2PDl 1 formulation and
dose in Trial 1. Figure 3 also shows that there were wide degrees
* Affinity-purified nmAb at a
of individual variation in antibody responses among mice.
concentration of 10 yg/ml.
t The test antibody is a pool of Neutralization of FMDV by mouse AB3
ascitic fluids from Group 5, (see the The neutralizing ability of the serum AB3 collected at the sixth
Materials and Methods), collected 4
and 5 weeks after the last immuniza- week of immunization was demonstrated in tissue culture VN
tion, and assayed at a 1:2 dilution. assays (Fig. 4). As with the RIA results, it is apparent that the
t + indicates binding to greater serum from the high-dose groups receiving a-2PDlI and
than 25% of labelled virus input. a-2PD I I Glu had higher average neutralizing titres than those of
 Anti-idiotype-induced anti-FMD V antibody 269
Table 3. Protection of suckling the low-dose groups while, in contrast, both the high- and low-
mice to lethal doses of FMDV by dose groups of mice receiving a-2PD 1 KLH showed little or no
mouse AB3 and FMDV mAb differences in neutralizing titre. Viral neutralization was also
demonstrated in ascitic fluids collected 4 or 5 weeks after the last
Antibody Log PDso* immunization, by PRNA (data not shown).
Analysis of a selected ascitic fluid from Group 5 (see the
AB3t 1-44 Materials and Methods), demonstrated the presence of anti-
2PDllT 2-66 bodies that protected suckling mice against lethal doses of
2PE4$ 1-87 FMDV (Table 3). The titre of this serum was approximately
2FFI 1$ 1-62 1 log below that obtained with the purified ABI (2PDl 1),
NMAF§ < 0 30 although it had a neutralizing titre in a range similar to that
other purified monoclonal antibodies to FMDV A12 which were
*
Loglo of the dilution of anti- tested simultaneously (Table 3).
body that protected 50% of eight In Trial 2, the sera collected from mice at the fifth week were
suckling mice per dilution given tested for binding to virus by RIA and viral neutralization. The
100 LD50 of virus. results (Table 4) show that the AB3 levels in Trial 2 did not differ
t The test antibody was an asci- significantly from those of Trial 1. Four of seven mice that were
tic fluid from Group 5 (see the positive in FP-RIA had log VN50 titres ranging from 0-6 to 1-5
Materials and Methods) that exhi- 5 weeks after the first immunization, and sera of two of those
bited the highest binding to virus.
Serial 10-fold dilutions were used mice protected suckling mice against a lethal dose of FMDV
for the assay. (Table 4). Two of the mice tested negative in all three assays
I Affinity-purified nmAb anti- throughout the 6 weeks of testing.
bodies to FMDV A12 at a starting
concentration 1 mg/ml.
§ Ascitic fluid induced in unim- DISCUSSION
munized mice. This is the first report of the induction of neutralizing antibodies
to FMDV by immunization of mice with anti-idiotypic anti-
Table 4. Serum AB3 anti-FMDV titres of individual bodies. It has previously been shown that non-neutralizing anti-
mice in in vitro and in vivo assays* FMDV antibodies were found in mice immunized with some
AB2 preparations (Baxt et al., 1989a). Two previous studies
Assay system with other picornaviruses, poliovirus (Uytdehaag & Osterhaus,
1985) and Coxackie virus (Paque & Miller, 1987), have also
Mouse no. FP-RIAt Log VNOT Log PDso§ demonstrated the ability ofAB2 to induce neutralizing antibody
and cell-mediated responses, respectively. We have generated an
AB2 that appears to react with the paratopic determinants of an
1 1-6 15 095 anti-FMDV nmAb (2PDl 1), and thus appears to mimic the
2 1.0 <0 6¶ <0-60 corresponding viral neutralizing epitope. Following this ap-
3 0-85 0-6 <0-60 proach, we expected to obtain an antibody that shared con-
4 0-85 0-6 <0-60 formational similarity (Williams et al., 1988; Hwei-Ling Chen et
5 <0-85** <0-6 <0 60
6 <0-85 <0-6 <0-60 al., 1988; Kennedy et al., 1986; Smith, Bost & Blalock, 1977)
7 0-85 <0-6 <0-60 with the corresponding viral epitope that is recognized by the
8 0-85 09 1-13 AB 1, and therefore could be used as a substitute antigen for
9 0-85 <0-6 <0-60 FMDV. This assumption was supported by the immune
responses to FMDV that a-2PDl 1 generated in mice (i.e. AB3),
* Mice were immunized in multiple s.c. spots and which were evidenced by binding and neutralization of the virus.
one footpad with 50 pg of 2PDlIKLH weekly for Furthermore, there was no reactivity of mouse AB3 with a
4 weeks. Sera was collected 5 weeks after the first FMDV type A12 variant, induced by selective pressure with
injection of AB2. 2PD 11 (Baxt et al., 1989b), indicating that these AB3 were able
t Loglo of the reciprocal of the serum dilution to discriminate subtle but critical sequence changes on the
that precipitated 30% or more oflabelled virus input. epitope in a fashion similar to the AB I (Baxt et al., 1989b). The
I Virus neutralizing titres determined by VN AB3 were also able to react with the same, or similar, epitopes
assay as described in the Materials and Methods. identified by the AB I on types A24 and A27 (Grubman & Morgan
¶ No virus neutralization under the conditions of 1986), although under the experimental conditions used here,
assay. the AB3 failed to react with types A5 and A79 which do react with
§ Virus neutralization titres as determined by a the AB 1. Both these characteristics indicated a specificity of the
suckling mouse protection assay as described in the
Materials and Methods. Titres are represented as AB3 similar to that of the AB I (Baxt et al., 1989b; Grubman &
described in Table 3. Morgan, 1986).
** The highest sera concentration used. This Under our experimental conditions, the AB3 induced in
value indicates that at this dilution less than 30% of mice appeared to exhibit lower a-FMDV reactivity than ABI.
the input virus was bound and is considered a The differences may be explained, in part, by the relative
negative result. concentrations of ABI and AB3 tested in the assays since the
270 A. E. Garmendia, D. 0. Morgan & B. Baxt
AB I was purified nmAb 2PD1 1 assayed at a starting concentra-
tion of 1 mg/ml. The nature of the inducing immunogens used,
however, must also be considered. While the ABI was generated
against the original epitope on viral particles, AB3 was induced
by a polyclonal AB2 in which the stimulus depended on antigen
mimicry, and the actual concentration of internal image, on a
weight basis, was undetermined, although obviously present. As
with other polyclonal a-IdAb systems (Uytdehaag & Osterhaus,
1985), it appears that the limiting factor here was the actual dose
ofinternal image given, as shown by the more effective induction
of a-FMDV responses when higher doses of a-IdAb were given
to mice (Fig. 2). Conversely, insufficient amounts of internal
image per dose may explain the weaker responses exhibited by
the low-dose groups.
Results in Figs 2 and 3 show that there was a wide range of
individual variation among animals, and relatively low AB3
titres were achieved. It was possible, however, to demonstrate
the induction of neutralizing a-FMDV responses using a
polyclonal a-IdAb that mimics a critical neutralizing site on
FMDV (Baxt et al., 1989b). Therefore, the results presented
here support the use of a-IdAb as surrogates of critical
determinants on FMDV for use in experimental FMD vaccines.
Thus, it is conceivable that, using monoclonal AB2 (mAb2)
specific for 2PD I1, the dosage of internal image can be
controlled and thus the induction of AB3 optimized. Further-
more, it may also be possible, with mAb2, to minimize the
variations in the responses derived from the immunogen itself,
and evaluate other factors associated with variability.
In summary, the a-2PDl 1 described here had a predominant
reactivity with the binding site of AB I molecules. This feature is
supported by the capacity of a-2PD 1 both to compete for AB I
with the virus, and to induce neutralizing a-FMDV responses in
mice that had no prior exposure to the virus. In addition, we
have recently shown that a-2PDl 1 can serve as a probe to
identify an idiotype associated with neutralization of FMDV in
at least two other species, which had either been infected with
FMD or had received FMD vaccine (Garmendia et al., 1989).
These features of a-2PD 11 were consistent with the proposed
functional criteria of an AB2-beta-bearing internal image
(Jerne, 1974; Kennedy et al., 1986; Ertl & Bona, 1988).
However, while we were able to induce specific FMDV-
neutralizing AB3 responses by immunization with this a-IdAb,
a direct extrapolation of these experimental results, suggesting
whether similar epitopes may be critical in susceptible species,
cannot be made. Therefore, testing of the immunogenic proper-
ties of the AB2 in these species is indicated. Consequently, to
extend this investigation our laboratory is attempting to
produce monoclonal AB2 for the FMDV system.
ACKNOWLEDGMENTS
The author's wish to thank Dr M.J. Grubman for supplying the labelled
FMDV subtypes, A. J. Franke, R. Goldsmith, D. J. Gerstner and
F. Lyburt for excellent technical assistance, and M. A. Wigmore and
A. Ciupryk for help in manuscript preparation.
REFERENCES
AvRAMEAs S., TERNYNCK T. & GUESDON J.L. (1978) Coupling of
enzymes to antibodies and antigens. Scand. J. Immunol. 8, 7.
BAxr B., GARMENDIA A.E. & MORGAN D.O. (1989a) Characterization of
anti-idiotypic antibodies generated against foot-and-mouth disease
virus neutralizing monoclonal antibodies. Viral Immunol. 2, 103.
BAXT B., MORGAN D.O., ROBERTSON B.H. & TIMPONE C.A. (1984)
Epitopes on foot-and-mouth disease virus outer capsid protein VP1
involved in neutralization and cell attachments J. Virol. 51, 298.
BAXT B., VAKHARLA V., MooRE D., FRANKE J.A& MORGAN D.O. (1989b)
Analysis of neutralizing antigenic sites on the surface of type A12
FMDV. J. Virol. 63, 2143.
BITTLE J.L., HOUGHTEN R.A., ALEXANDER H., SHINNICK T.M., SUT-
CLIFFE J.G., LERNER R.A., ROWLANDS D.J. & BROWN F. (1982)
Protection against foot-and-mouth disease virus by immunization
with a chemically synthesized peptide predicted from the viral
nucleotide sequence. Nature (Lond.), 298, 30.
DALGLEIsH A.G. & KENNEDY R.C. (1988) Anti-idiotypic antibodies as
immunogens: idiotype-based vaccines. Vaccine, 6, 215.
DOMINGO E., DAVILA M. & ORTIN J. (1980) Nucleotide sequence
heterogeneity of the RNA from a natural population of foot-and-
mouth disease virus. Gene, 11, 333.
ERTL H.C.J. & BONA C. (1988) Criteria to define anti-idiotypic
antibodies carrying the internal image of an antigen. Vaccine, 6, 80.
GARMENDIA A.E., BORCA M.V., MORGAN D.O. & BAXT B. (1989)
Analysis of foot-and-mouth disease virus neutralizing idiotypes from
immune bovine and swine with anti-murine idiotype antibody probes.
J. Immunol. (in press).
GRUBMAN M.J. & MORGAN, D.O. (1986) Antigenic comparison of foot-
and-mouth disease virus serotypes with monoclonal antibodies. Virus
Research, 6, 33.
HERLYN D., WETTENDORFF M., SCHMOLL E., ILIOPOULOs D., SCHEDEL I.,
DREIKHAUSEN U., RAAB R., Ross A.H., JAKSCHE H., SCRIBA M. &
KoPROWSKI H. (1987) Anti-idiotype immunization of cancer patients:
Modulation of the immune response. Proc. nail. Acad. Sci. U.S.A. 84,
8055.
HIERNAUX J.R. (1988) Idiotype vaccines and infectious diseases. Infect.
Immun. 56, 1407.
HWEi-LINGC., SOOD A.K., WARD R.E., KIEBER-EMMONsT. & KOHLER H.
(1988) Structural basis of stimulatory anti-idiotypic antibodies. Mol.
Immunol. 25, 33.
JERNE N.K. (1974) Towards a network theory of the immune system.
Ann. Immunol. (Inst. Pasteur), 125c, 373.
KENNEDY R.C., HENKEL R.D. & DREESMAN G.R. (1986) Further
characterization immunoglobulin receptors on murine hybridoma
cells secreting antibodies to hepatitis B surface antigen. Scand. J.
Immunol. 23, 481.
KLEID D.G., YANSURA D., SMALL B., DOWBENKO D., MOORE D.M.,
GRUBMAN M.J., McKERCHER P.D., MORGAN D.O., ROBERTSON B.H.
& BACHRACH H.L. (1981) Cloned viral protein vaccine for foot-and-
mouth disease: responses in cattle and swine. Science, 214, 1125.
McNAMARA M.K., WARD R.E. & KOHLER H. (1984) Monoclonal
idiotype vaccine against Streptococcus pneumonia infection. Science,
226, 1325.
MARRIOT S.J., ROEDER D.J. & CONSIGLI R.A. (1987) Anti-idiotypic
antibodies to a polyomavirus monoclonal antibody recognize cell
surface components of mouse kidney cells and prevent polyomavirus
infection. J. Virol., 61, 2747.
MURRAY P.K. (1987) Prospects for molecular vaccines in veterinary
parasitology. Vet. Parasitol. 25, 121.
NORTON F.L., MORROW P.R., LEUNG C.Y. & BENJAMINI E. (1985) The
idiotypic characterization of the immune response to a defined
epitope of a protein antigen and the specific in vivo suppression of the
immune response to this epitope by anti-idiotypic antibodies. J. Im-
munol. 134, 3226.
NOSEWORTHY J.H., FIELDS B.N., DITCHER M.A., SOBOTKA C., PIZER E.,
PERRY L.L., NEPOM J.T. & GREENE M.I. (1983) Cell receptors for the
mammalian reovirus I. Syngeneic monoclonal anti-idiotypic anti-
body identifies a cell surface receptor for reovirus. J. Immunol. 131,
2533.
PAQuE R.E. & MILLER R. (1987) Modulation ofmurine Coxsackie virus-
induced myocarditis utilizing anti-idiotypes. Viral Immunol. 1, 207.
SACKS A.L., ESSER K.M. & SHER A. (1982) Immunization of mice
 Anti-idiotype-induced anti-FMD V antibody
against African trypanosomiasis using anti-idiotypic antibodies. J.
exp. Med. 155, 1108.
SCHICK M.R., DREsSMAN G.R. & KENNEDY R.C. (1987) Induction of an
anti-hepatitis surface antigen response in mice by noninternal image
(AB2a) anti-idiotypic antibodies. J. Immunol. 138, 3419.
SHARPE A.H., GAULTON G.N., McDADE K.K., FIELDS B.N. & GREENE
M.I. (1984) Syngeneic monoclonal antiidiotype can induce immunity
to reovirus. J. exp. Med. 160, 1195.
SMITH L.R., BOST K.L. & BLALOCK, J.E. (1987) Generation of idiotypic
and anti-idiotypic antibodies by immunization with peptides encoded
by complementary RNA: A possible molecular basis for the network
theory. J. Immunol. 137, 7.
UYTDEHAAG F.G.C.M. & OSTERHAUS A.D.M.E. (1985) Induction of
neutralizing antibody in mice against polyovirus type II with
monoclonal anti-idiotypic antibody. J. Immunol. 34, 1225.
271
VIALE G., GRASSI F, PELAGI M., AZANI R., MENARD S., MIOTTI S.,
BUFFA R., GINI A. & SIccARDI A.G. (1987) Anti-human tumor
antibodies induced in mice and rabbits by 'internal image' anti-
idiotypic monoclonal immunoglobulins. J. Immunol. 139, 4250.
WILLIAMS W.V., Guy H.R., RUBIN D.H., ROBEY F., MYERS J.N.,
KIEBER-EMMONS T., WEINER D.B. & GREENE M.I. (1988) Sequences of
the cell-attachment sites of reovirus type 3 and its anti-idiotypic/
antireceptor antibody: Modeling of their three-dimensional struc-
tures. Proc. natl. Acad. Sci. U.S.A. 85, 6488.
ZHOU E.M., CHANH T.C., DRESSMAN R.G., KANDA P. & KENNEDY R.
(1987) Immune response to human immunodeficiency virus. In vivo
administration of anti-idiotype induces an anti-gpl60 response
specific for a synthetic peptide. J. Immunol. 139, 2950.