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J. gen. Virol. (1985), 66, 2347-2354. Printedin Great Britain
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Key words: FMDV/vaccine/oligopeptide/immunologicalpriming
Immunological Priming with Synthetic Peptides of Foot-and-Mouth Disease
Virus
By
M. J. F R A N C I S , *
C. M. F R Y ,
D.J.'ROWLANDS,
F. B R O W N ,
J. L. B I T T L E , 1 R. A. H O U G H T E N 1 AND R. A. L E R N E R 1
Wellcome Biotechnology Ltd, F M D Division, Ash Road, Pirbright, Woking, Surrey GU24 ONQ,
U.K. and 1Research Institute of Scripps Clinic, La Jolla, California 92037, U.S.A.
(Accepted 12 August 1985)
SUMMARY
A sub-immunizing dose of a synthetic peptide corresponding to the amino acids 141
to 160 region of protein V P 1 from foot-and-mouth disease virus (FMDV), serotype O1,
coupled to keyhole limpet haemocyanin (141-160KLH) has been shown to prime the
immune system of guinea-pigs for an FMDV serotype-specific neutralizing antibody
response to a second sub-immunizing dose of the same peptide. Optimal priming
required an interval of 42 days between the priming dose and the booster dose. No
priming was observed in the absence of adjuvant. The secondary response was not
restricted by the carrier since animals primed with 141-160KLH could be boosted with
uncoupled 141-160 or 141-160 coupled to tetanus toxoid. It has also been shown that
uncoupled peptide 141-160 will prime for a neutralizing antibody response when it is
incorporated into a relatively non-immunogenic carrier such as small unilamellar
liposomes. These results indicate that the 141-160 peptide of FMDV, as well as
containing an important neutralizing antibody site, can initiate its own T-helper cell
response.
INTRODUCTION
The foot-and-mouth disease virus (FMDV) particle is composed of one molecule of singlestranded RNA (mol. wt. 2.6 × 106) and 60 copies of each of four structural proteins, VP1, VP2,
VP3 (mol. wt. 24 x 103) and VP4 (mol. wt. 10 x 103). Of these structural proteins VP1 appears
to play a key role in the antigenic and immunogenic activity of the virus particle (Wild et al.,
1969; Laporte et al., 1973; Bachrach et al., 1975). Detailed study of both enzymically and
chemically cleaved fragments of VP1 from virus of serotype O has identified two regions,
between amino acids 138 to 154 and 200 to 213, which are found on the surface of the virus and
fragments containing these regions are able to induce neutralizing antibodies against the
homologous virus (Strohmaier et al., 1982). Furthermore, studies using chemically synthesized
peptides corresponding to several regions of VP1 have led to the identification of similar sites on
the molecule (141 to 160 and 200 to 213) which elicit neutralizing antibodies that can protect
guinea-pigs against experimental infection (Bittle et al., 1982; Pfaffet al., 1982). Although such
peptides have an immunizing activity of only 1 ~o or less of that of the inactivated virus particle
on an equal weight basis, the level of neutralizing antibody produced is several orders of
magnitude greater than that obtained with the whole VP1 molecule (Bittle et al., 1982).
Moreover, anti-peptide antibodies mimic the subtype specificity of the intact virus particle
(Clarke et al., 1983; Rowlands et al., 1983).
Peptides corresponding to the immunogenic regions of poliovirus VP1 have been used to
prime the immune system of rabbits to produce high neutralizing antibody titres after a second
injection of a sub-immunizing dose of the intact virus particle (Emini et al., 1983). Peptides of
the haemagglutinin molecule of influenza virus will also prime for a secondary response to virus
particles (R. Arnon & F. Melchers, personal communications). A similar priming effect has also
been observed with anti-idiotype antibodies to hepatitis B which can be used to enhance the
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M . J . FRANCIS AND OTHERS
i m m u n e response to a s u b s e q u e n t injection o f the s a m e anti-idiotype a n t i b o d y or hepatitis B
surface antigen ( K e n n e d y & D r e e s m a n , 1984). A l t h o u g h it has not yet b e e n s h o w n w h e t h e r
p e p t i d e - p r i m e d animals will be p r o t e c t e d against s u b s e q u e n t infection, it has b e e n p o i n t e d out
that the strategy o f p r i m i n g could be used to identify neutralizing e p i t o p e s on the virion ( E m i n i
et al., 1983).
In a p r e l i m i n a r y study we h a v e d e s c r i b e d a p r i m i n g effect o f p e p t i d e s o f the VP1 protein o f
F M D V ( F r a n c i s et al., 1985). T h e s e o b s e r v a t i o n s h a v e led us to investigate p r i m i n g w i t h F M D V
p e p t i d e s in m o r e detail w i t h a v i e w to studying the role of carriers and a d j u v a n t s in the antip e p t i d e response.
METHODS
Syntheticpeptides. 01 Kaufbeuren peptides were synthesized by the solid-phase method using a Beckman model
990B peptide synthesizer at the Scripps Clinic, U.S.A. (Bittle et al., 1982) based on the 213 amino acid sequence of
VP1 published by Kurz et al. (1981). Peptides were coupled to keyhole limpet haemocyanin (KLH) through an
additional cysteine on the carboxy-terminus using N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as a
coupling agent and to tetanus toxoid (TT) using glutaraldehyde.
Animals. Female Dunkin-Hartley guinea-pigs, approximately 12 weeks old and weighing between 450 and
500 g, maintained as a closed randomly bred colony at the Animal Virus Research Institute, Pirbright, U.K., were
used.
Challenge test. 01 Kaufbeuren virus suspension in a 0.02 ml dose, containing 500 guinea-pig IDs0, was injected
intradermally into the left hind footpad and the animals were examined daily for 7 days. Guinea-pigs with no
lesions or lesions_only at the injection site were regarded as protected and those with more extensive lesions as
unprotected.
Neutralization assay. The neutralizing activity of serum samples against 100 TCID5o of FMDV was determined
using a microneutralization test (Francis & Black, 1983). Each test was performed in triplicate and the results were
recorded as the mean log~0 reciprocal of the serum dilution that gave confluent cell sheets in 50 ~ of the microplate
wells (SNso).
Enzyme-linked immunosorbent assay (EL1SA). A modification of the indirect ELISA technique described by
Voller & Bidwell (1976) was used to assay anti-virus particle and anti-peptide IgG responses. Briefly, microplates
were coated overnight at room temperature with purified FMDV (Brown & Cartwright, 1963) or uncoupled
synthetic peptide at a concentration of 2 ~tg/ml. The plates were washed and test serum samples, either at a
standard 1:50 dilution or a range of doubling dilutions from 1:10, were added. After incubation for 1 h at 37 °C,
plates were washed and anti-guinea-pig IgG-peroxidase conjugate was added. After a further hour at 37 °C the
plates were washed and an enzyme substrate (0.04% o-phenylenediamine + 0.004% hydrogen peroxide in
phosphate/citrate buffer) was added. After 5 to 7 min, colour development was stopped with 12.5~ sulphuric acid
and the absorbance at 492 nm was measured in a Titertek Multiskan (Flow Laboratories).
For the fixed 1 : 50 dilutions, A492 readings of sera taken at intervals after inoculation of the guinea-pigs were
corrected by subtracting from them the corresponding 0 day values (mean 0 day A492value for peptide was 0-07 +
0.02 and for virus was 0-19 _+ 0.03) obtained in the same test. Alternatively, the A,,92 values obtained from
doubling dilutions of post-inoculation samples were plotted against the log~0 reciprocal antiserum dilution and the
antibody titres were calculated by reference to a negative standard (a 1 : 10 dilution of pre-inoculation serum). The
results reported are the means of two tests, using duplicate wells for each serum dilution in each test.
Preparation ofliposomes. Liposomes were prepared according to the method described by Souhami et al. (1981).
Negatively charged liposomes were made using the molar ratio of phosphatidylcholine (7), cholesterol (2) and
dicetylphosphate (1); neutral liposomes contained phosphatidylcholine and cholesterol, in the ratio 4:1. Briefly,
dried lipid films obtained by rotary evaporation were shaken off the inner surface of a round-bottom flask with a
30 vtg/ml solution of peptide 141-160 prepared in 0.04 M-phosphate buffer pH 7-6, to give a final suspension of 3
(w/v). This preparation was referred to as large multilamellar liposomes. In order to produce small unilamellar
liposomes, the large multilamellar liposomes were sonicated with 20 bursts of 30 s in an ice-bath, using a 'Rapids'
sonicator (Ultrasonics, U.K.).
RESULTS
Timing o f the secondary inoculation
Sixteen guinea-pigs were inoculated i n t r a m u s c u l a r l y w i t h 30 ttg o f p e p t i d e 141-160 coupled to
K L H and formulated in 0.5 ~ A1 (OH)3, as an adjuvant. T h e y were subsequently d i v i d e d into
four groups o f four animals and r e i n o c u l a t e d intramuscularly w i t h the s a m e p r e p a r a t i o n either
14, 28, 42 or 84 days later. S e r u m samples were collected at the start of the e x p e r i m e n t , at the
t i m e of reinoculation and then weekly for 5 weeks.
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Immunological priming with FMD V peptides
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Fig. 1. (a, b) Neutralizing, (c, d) anti-virus particle, (e,J) anti-peptide 141 160 and (g, h) anti-peptide
200-213 antibody responses of guinea-pigs primed with 141 160KLH(O, O), 200-213KLH (A, A) or
KLH alone (ai, D) and boosted (T) 42 days later with 141 160KLH (a, c, e, g) or 200-213KLH (b, d,f,
h).
No anti-peptide or neutralizing antibody was detected in any of the groups at the time of
reinoculation. Low levels of anti-peptide 141-160 antibody were produced after reinoculation at
either 14 or 28 days after the primary injection. These were associated with neutralizing and
anti-virus particle antibodies which were slow to develop and did not reach peak activity for 21
to 28 days. Reinoculation at 42 days, however, produced significant anti-peptide 141-160, antivirus particle and neutralizing antibody responses (see Fig. 1) which reached peak activity
within 14 days. A similar booster response was observed when the second inoculation was given
at 84 days. Therefore, a 42 day time interval between primary and secondary inoculations was
used in all subsequent experiments.
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M.J.
FRANCIS AND OTHERS
Specificity of priming with peptide
Three groups of eight guinea-pigs were inoculated intramuscularly with 30 ktg of peptide 141160 coupled to KLH (141-160KLH), 30 ~tg of peptide 200-213 coupled to KLH (200-213KLH)
or 20 ktg of untreated KLH alone, each formulated with 0.5 ~o AI(OH)3. After 42 days they were
subdivided into groups of four and reinoculated intramuscularly with either 30 p.g of peptide
141-160KLH or 30 ~tg of peptide 200-213KLH, each formulated with 0.5~ AI(OH)3. Serum
samples were collected at regular intervals after both primary and secondary inoculations.
No neutralizing activity, anti-virus particle or anti-peptide 141-160 was detected in any of the
groups after primary inoculation (Fig. 1). However, after a secondary inoculation the group
that had been primed and boosted with peptide 141-160KLH (Fig. 1a) produced significant
levels of neutralizing antibodies within 7 days and a peak titre of 2-3 log~0 SNs0 was reached
within 14 days. This neutralizing antibody production was associated with the appearance of
anti-virus particle (Fig. I c) and anti-peptide 141-160 (Fig. l e) activity. No such neutralizing
activity could be detected against the heterotypic viruses A24 Cruzeiro or C Noville. The
priming for peptide 141-160KLH did not occur when guinea-pigs were given a primary
inoculation of untreated KLH alone. However, a low level of neutralizing activity was observed
in the group primed with peptide 200-213KLH and boosted with peptide 141-160KLH (Fig.
la).
Groups of guinea-pigs primed with peptide 141 160KLH, peptide 200-213KLH or KLH
alone which received peptide 200-213KLH as the second injection Produced no detectable
neutralizing, anti-virus particle or anti-peptide 141-160 antibody (Fig. 1). However, there was a
primary anti-peptide 200-213 antibody response in the group primed with peptide 200-213KLH
(Fig. 1g, h). There was also an anti-peptide 200-213 response following the second inoculation
with 200-213KLH, although this was less marked in the groups which had received 141160KLH or KLH alone as the primary inoculation (Fig. 1 h).
Effect of adjuvant
Three groups of four guinea-pigs were primed and boosted after 42 days with 30 ~tg of peptide
141-160KLH, either alone or formulated with 0-5~o AI(OH)3 or 1:1 with incomplete Freud's
adjuvant (IFA). Serum samples were collected at weekly intervals.
No neutralizing antibody was detected in any of the groups after primary inoculation.
Furthermore, no detectable neutralizing antibody was produced following a second injection of
peptide 141-160KLH in the absence of adjuvant (Fig. 2). There was also no detectable anti-virus
particle (peak A49z of <0.1) or anti-peptide 141-160 (peak A492 of <0"1) antibody in these
animals. However, the groups inoculated with either AI(OH)3- or IFA-adjuvanted preparations
produced significant levels of neutralizing, anti-virus particle (peak A492 of 0"6 to 1'3) and antipeptide 141-160 (peak A492 of 0"8 to l'0) antibodies following the second injection (Fig. 2).
Effect of carrier on secondary response
Three groups of four guinea-pigs were inoculated with 30 ~tg of peptide 141-160KLH plus
0.5~ AI(OH)3, and reinoculated with either 30 ~g of peptide 141-160KLH, 30 ~g of peptide
141-160 coupled to TT (141-160TT) or 30 ~g of uncoupled peptide 141-160, each formulated
with 0"5~o AI(OH)3. One additional group was given 30 p.g of 141-160TT with 0-5~ AI(OH)3 as
both the primary and secondary inoculation. Serum samples were collected at weekly intervals.
No primary neutralizing antibody response was observed in any of the groups. Each of the
peptide preparations used for secondary inoculation produced significant levels of neutralizing
antibody (Fig. 3) and this was associated with the appearance of comparable levels of anti-virus
particle (peak A492 of 0.2 to 1.0) and anti-peptide 141-160 (peak A492 of 0.5 to 1-0) antibody.
Furthermore, the neutralization titres in the group reinoculated with uncoupled peptide 141-160
were similar to those in the group which was reinoculated with peptide 141-160KLH.
The group primed with peptide 141-160KLH which was reinoculated with peptide 141160TT produced lower levels of neutralizing antibody. However, these levels were not
significantly lower than those present in the group primed and boosted with peptide 141-160TT.
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Immunological priming with FMD V peptides
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Fig. 2. Secondary neutralizing antibody response of guinea-pigs primed and boosted after 42 days with
141 160KLH, using either IFA (A) or AI(OH) 3 ( 0 ) as adjuvant, or no adjuvant ( I ) .
Fig. 3. Secondary neutralizing antibody response of guinea-pigs primed with 141-160KLH (O, A, I )
or 141 160TT (A) and boosted after 42 days with 141-160KLH (O), 141 160TT (A, A) or 141-160
alone ( I ) .
The lower levels obtained with this peptide indicated that it had lower intrinsic activity than the
KLH-coupled peptide.
Effect of incorporating peptide into liposomes
Four groups of four guinea-pigs were primed and boosted with 30 ktg of uncoupled peptide
141-160 incorporated into liposomes. The samples consisted of either negatively charged, or
neutral, large multilamellar liposomes or small unilamellar tiposomes, derived by sonication of
the multilamellar preparations. Two further groups of animals, injected with either 30 ~tg of
peptide 141-160 which had been mixed with preformed negatively charged small unilamellar
liposomes, or 30 p.g of peptide 141-160 alone, were included as controls. Serum samples were
collected at regular intervals after the primary and secondary inoculations.
No neutralizing activity was detectable in any group after the primary inoculation (Fig. 4).
However, following a second inoculation neutralizing activity was produced in the groups which
received small unilamellar liposomes, either neutral or negatively charged, which incorporated
the peptide (Fig. 4a, b). This was associated with the appearance of anti-virus particle (2.8 to 3-0
log10) and anti-peptide 141-160 (3.0 to 3.4 logto) activity. Furthermore, 75 to 100~o of the
animals from these groups were protected against challenge 56 days after secondary inoculation.
No detectable neutralizing activity was observed in the pooled sera from groups which received
large multilamellar liposomes incorporating peptide (Fig. 4a, b) or a small unilamellar
liposome/peptide mixture up to 42 days after secondary inoculation (Fig. 4c). Individual sera
collected at 56 days after the second inoculation had neutralizing titres up to 1.2 loglo SNso and
this resulted in a low degree of protection (0 to 25~o). Peptide alone produced no detectable
neutralizing activity (Fig. 4c) and no protection.
DISCUSSION
It is well established that, following primary immunization, animals will generally produce a
rapid and enhanced immune response to a second stimulus (Glenny & Sudmersen, 1921).
Furthermore, this so-called memory state can be adoptively transferred by transplanting cells
from previously immunized donors into isologous hosts (Makela & Mitchison, 1965). An
analysis of immunological memory demonstrated that animals may be primed without
producing detectable levels of primary antibody formation (Nossal et al., 1965). The present
study has shown that a sub-immunizing dose of FMDV peptide 141-160KLH can prime the
immune system of guinea-pigs for a serotype-specific neutralizing antibody response to a second
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M. J. FRANCIS AND OTHERS
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Time after primary inoculation (days)
Fig. 4. Neutralizing antibody response of guinea-pigs primed and boosted (T) after 42 days with (a)
negatively charged multilamellar (O) or small unilamellar (0) liposomes incorporating peptide 141160, (b) neutral mulfilamellar (A) or small unilamellar (A) liposomes incorporating peptide 141 160,or
(c) small unilamellar liposomes mixed with peptide 141 160 (D) or peptide 141 160 alone (m).
dose of the same peptide. An interval of at least 42 days between primary and secondary
inoculations is required to produce the optimal boosting effect. This may be explained by the
observation that B-cell memory appears to require several weeks for full development
(Cunningham & Sercarz, 1971)
The presence of a low level of neutralizing activity in the sera of guinea-pigs primed with 200213KLH and boosted with 141 160KLH would indicate some sequence homology between the
two peptides. Examination of the published sequence of VP1 for this virus (Strohmaier et al.,
1982) reveals that the sequences of amino acids 151 to 155 and 209 to 213 are similar but in
reverse order. However, it is difficult to understand this observation because this sequence
relationship is unlikely to be reflected in any structural similarity. Moreover, 141-160KLH did
not appear to prime for 200-213KLH, and whereas 200-213KLH produced a good primary antipeptide 200-213 response no neutralizing activity was detected. This provides further support to
the published observations on the greater neutralizing efficiency of antibodies to the 141-160
region over antibodies to the 200 213 region (Bittle et al., 1982).
The fact that no detectable neutralizing antibody response was produced in the absence of
AI(OH)3 or I F A indicates that an adjuvant is required either for priming or boosting a
neutralizing antibody response to the F M D V peptides. However, adequate priming was
obtained using either AI(OH)3 or I F A as the adjuvant.
In previous studies with a variety of viral peptides a number of different protein carriers and
coupling techniques have been used to enhance their immunizing activity (for review, see
Palfreyman et al., 1984). However, relatively little attention appears to have been directed
towards the role of the carrier in producing an enhanced anti-peptide response. Studies with
hapten-protein conjugates have shown that T-cells recognize, and respond to, carrier
determinants on the protein in order to enable hapten-reactive B-cells to proliferate and
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Immunological priming with F M D V peptides
2353
differentiate into antibody-forming cells (Raft, 1970; Mitchison, 1971). Furthermore, an
optimal boosting effect requires the hapten to be coupled to the same carrier as that used for
priming, or the animals to have been previously primed with the same carrier, non-haptenized
(Ovary & Benacerraf, 1963; Rajewsky et al., 1969).
While these studies have provided valuable information on the induction of humoral
responses the function of the carrier in a peptide response may be different since peptides
containing 10 to 30 amino acids, although they appear to be poor immunogens, cannot be
regarded as true haptens. Furthermore, the results of this study have shown that the role of K L H
in priming for F M D V peptide is fundamentally different from its role in hapten priming since
no carrier was required to boost the response of peptide-KLH-primed animals. The lower boost
observed using TT-coupled peptide may have been due to minor differences in the conformation
of K L H - and TT-coupled peptides resulting in the lower intrinsic activity of the latter coupled
peptide, since if 141-160 coupled to TT is used to prime and boost guinea-pigs a similar reduced
response is observed. This conformational difference could be a result of the different methods
used to couple peptide to the protein carrier (see Methods). Therefore, it seems likely that the
carrier is simply acting as a delivery system for the peptide by increasing its size, the epitope
density and/or by influencing the conformation in which it is presented to the immune system.
The potent protein carriers frequently used in studying the immunogenic activity of viral
peptides may, in any case, not be necessary for stimulating a satisfactory immune response since
polymerized F M D V peptides will induce a neutralizing antibody response in the absence of
carrier (Bittle et al., 1984). In the present study, liposomes which are themselves very poor
antigens have been successfully used as carriers in order to enhance the immunogenicity of the
141-160 peptide. Although no attempt was made to control the epitope density it was clear that
the size of the liposomes was of major importance since sonication to a small unilamellar form
was required to optimize their adjuvanticity. The charge of the liposomes did not appear to have
any marked influence on their immunizing activity. However, it was necessary to incorporate
the peptide within the liposomes, since a similar weight of peptide added to preformed small
unilamellar liposomes was less immunogenic. The low level of antibody activity which was
observed in this control group may have been due to the free peptide associating with the 'empty'
liposomes. It is possible that the small unilamellar liposomes are 'targeting' the peptide directly
to antigen-presenting cells, for example follicular dendritic cells of the germinal centres. These
results emphasize the potential value of small unilamellar liposomes as adjuvants for synthetic
vaccines.
In conclusion, a low-immunizing dose of peptide 141-160 will prime the immune system of
guinea-pigs for a F M D V neutralizing antibody response. Furthermore, the observations that
this priming can be achieved with a relatively non-immunogenic carrier and that the secondary
response is not carrier-restricted indicates that the 14l 160 peptide can initiate its own T-helper
cell activity without the involvement of antigenic carrier molecules. Therefore, the 141-160
peptide contains both B-cell, neutralizing antibody recognition sites, and T-cell determinants.
The authors wish to thank Dr H. M. Patel of the Department of Biochemistry, Charing Cross Hospital Medical
School, London, U.K. for his practical help and guidance on the techniques of liposome preparation.
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