Download Foot-and-mouth disease virus epitope dominance in the antibody

Journal of General Virology (2012), 93, 488–493
Short
Communication
DOI 10.1099/vir.0.037952-0
Foot-and-mouth disease virus epitope dominance
in the antibody response of vaccinated animals
M. Mahapatra, P. Hamblin and D. J. Paton
Correspondence
Institute for Animal Health, Pirbright Laboratory, Ash Road, Woking, Surrey GU24 0NF, UK
M. Mahapatra
[email protected]
Received 26 September 2011
Accepted 1 December 2011
Five neutralizing antigenic sites have been identified on the surface of serotype O foot-and-mouth
disease virus (FMDV). A set of mAb neutralization-escape mutant viruses was used for the first
time to evaluate the relative use of known binding sites by polyclonal antibodies from three target
species: cattle, sheep and pigs. Antibodies to all five neutralizing antigenic sites were detected in
all three species, with most antibodies directed against antigenic site 2, followed by antigenic
site 1. In 76 % of cattle, 65 % of sheep and 58 % of pigs, most antibodies were directed against
site 2. Antibodies specific to antigenic sites 3, 4 and 5 were found to be minor constituents in the
sera of each of the target species. This implies that antigenic site 2 is a dominant neutralization
immunogenic site in serotype O FMDV and may therefore be a good candidate for designing
novel vaccines.
Foot-and-mouth disease (FMD) is a severe, clinically
acute vesicular disease of cloven-hoofed domestic and wild
animals and is present in all continents except Australasia
and North America. The causative agent of FMD is Footand-mouth disease virus (FMDV), the prototype member of
the genus Aphthovirus in the family Picornaviridae. There
are seven distinct serotypes of FMDV (O, A, C, Asia-1,
SAT-1, SAT-2 and SAT-3) and multiple strains, indicating
significant antigenic variability. Currently, immunization
with conventional inactivated whole-virus vaccine is the
main method of FMD control, but strain composition of
vaccines must be selected with care. The humoral immune
response has been generally accepted as the most important
factor in conferring vaccine-induced protection against the
disease, as a strong correlation has been reported between
the levels of virus-neutralizing antibody produced after
vaccination and subsequent protection of cattle, one of the
main target species (Pay & Hingley, 1987). The mechanism
by which antibodies protect against FMDV is poorly
understood, and different factors may be involved in vivo
compared with the situation measured in vitro (Dunn et al.,
1998; McCullough et al., 1992). Several studies have been
carried out in the last two decades to identify the targets on
the virus surface for the binding of neutralizing antibodies,
and mAbs have been powerful tools for identifying the
amino acid footprint of different antigenic sites, mainly by
sequencing mAb-resistant (mar) mutants. This approach
has been used successfully to delineate the neutralizing
antigenic sites of viruses representing serotypes O (Aktas &
Samuel, 2000; Barnett et al., 1998; Crowther et al., 1993b),
A (Baxt et al., 1989; Bolwell et al., 1989; Mahapatra et al.,
2011; Thomas et al., 1988a), C (Mateu et al., 1990), Asia-1
(Grazioli et al., 2003) and SAT (Crowther et al., 1993a;
Grazioli et al., 2006).
488
FMDV serotype O is the most prevalent globally and has
been studied most extensively. Five neutralizing antigenic
sites (1–5), involving three of the capsid proteins (VP1–3),
have been identified on the surface of serotype O FMDV
(Aktas & Samuel, 2000; Barnett et al., 1998; Crowther et al.,
1993b; Kitson et al., 1990; McCahon et al., 1989; Xie et al.,
1987). Site 1 is linear and trypsin-sensitive, whereas the rest
of the identified antigenic sites have been reported to be
conformation-dependent and trypsin-resistant. The most
prominent surface projection is formed by the bG–bH (G–
H) loop of VP1, which, together with the C terminus of
VP1, contributes to antigenic site 1 with critical residues at
positions 144, 148, 154 and 208. Amino acid residues at
positions 70–73, 75, 77 and 131 of VP2 contribute to
antigenic site 2, whereas residues 43 and 44 of the bB–bC
(B–C) loop of VP1 constitute site 3. Amino acid residues at
positions 56 and 58 of VP3 have been reported to be
critical for antigenic site 4, whereas site 5, characterized by
an amino acid at position 149 of VP1, is probably formed
by interaction of the VP1 G–H loop region with other
surface-located amino acids.
Historically, antigenic site 1 has been considered to be
immunodominant and its linear structure has made it an
obvious target for the development of peptide vaccines.
Guinea pigs and cattle immunized with peptides corresponding to parts of VP1 (aa 141–160 or 141–158 and 200–
213) conferred neutralizing antibody and protection
(Bittle et al., 1982; DiMarchi et al., 1986; Pfaff et al.,
1982). However, these peptide vaccines have not been
fully effective in cattle (Taboga et al., 1997). Using a mAbbased competition ELISA (cELISA), Samuel (1997)
reported the presence of relatively higher titres of site 2specific antibodies in cattle. In contrast, using a similar
assay, Aggarwal & Barnett (2002) reported that none of
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FMD virus epitope dominance
antigenic sites 1–3 can be considered as immunodominant
in bovine polyclonal sera. However, a potential problem
with this approach is that antibody bound to one site may
interfere with a competitive ligand targeted elsewhere on
the virus surface, due to the small size of the virion and
close vicinity of different antigenic sites. Other studies
involving serotype C (Mateu et al., 1995) and serotype A
(Thomas et al., 1988b) also indicated the participation of
other antigenic sites in addition to site 1 in generation of
an immune response following either natural infection or
vaccination.
The relative importance of the different neutralizing
antigenic sites in FMDV vaccine-induced protection has
been poorly studied. We have addressed this question by
using site-specific mar-mutant viruses to quantify the
relative amount of antibodies against the known neutralizing antigenic sites of FMDV in the polyclonal sera of
three different target species: cattle, pigs and sheep. Most of
the animals exhibited significantly higher levels of antibodies to antigenic site 2. This knowledge may help in
vaccine strain selection, as well as the design of novel
vaccines.
Mar mutants of the O1 Kaufbeuren (O1K) strain of FMDV,
representing each of five antigenic sites, were used in this
study (Crowther et al., 1993b; Table 1). Prior to the
commencement of binding studies, the presence of the
expected mutations was confirmed by capsid sequencing of
all mutant viruses. The inactivated FMD vaccines used to
vaccinate animals were either made in house or obtained
from a commercial source (Merial), and were either O1K, O1
Lausanne or O1 BFS. All of these virus strains belong to the
European–South American topotype. The capsid sequences
of O1 Lausanne and O1 BFS are 99.6 and 99.2 % identical to
that of O1K, respectively, at the amino acid level and have
residues identical to those in O1K at the positions of change
in the mar mutants. All of the animals received a comparable
amount of antigen and the polyclonal antisera were collected
either 21 or 28 days post-vaccination. The polyclonal sera
were initially tested for antibodies against FMDV O1K using
a virus-neutralization (VN) test and samples with a titre of
.2 log10 were selected for further study. In total, 110 serum
samples from vaccinated cattle (n557), pigs (n533) and
sheep (n520) were used in this study. The bovine serum
Table 1. Critical residues of FMDV serotype O mAbs
Numbers in parentheses indicate mar-mutant number (Crowther
et al., 1993a; McCahon et al., 1989; Kitson et al., 1990).
mAb
D9 (480)
C6 (599)
C8 (597)
14EH9 (63)
C3 (O1KC3)
Virus strain
O1
O1
O1
O1
O1
Swiss 1965
Swiss 1965
Swiss 1965
Brugge
Caseros
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Site
Critical residue
1
2
3
4
5
VP1–148
VP2–73
VP1–44
VP3–58
VP1–149
samples were from cattle vaccinated with O1K (n55), O1
Lausanne (n518) or O1 BFS (n534), whereas the samples
from pigs and sheep were from animals vaccinated with the
O1 BFS vaccine.
Titres of neutralizing antibodies against the O1K virus and
various mar-mutant viruses were measured by a microneutralization assay essentially as described by Rweyemamu
(1984). The VN titres were calculated as log10 of the
reciprocal antibody dilution required for 50 % neutralization of 100 TCID50 virus. All tests were carried out in
duplicate and at least twice, and the mean of the results was
used for further analysis. Pre-immune and several control
sera from each species were obtained and analysed, and no
anti-FMDV antibodies were detected. Statistical analyses
were performed on serum titres obtained from the neutralization test using Stata/SE 11.2 (StataCorp LP). Pairwise
comparisons of individual groups (parent and various mar
mutants) were carried out using the Bonferroni-adjusted ttest. For the analysis, the parent group was considered as the
reference, and the variability in the VNT log10 response for
each of the mar mutants was calculated for each species.
Furthermore, differences between each pair of means for
each group were also assessed. Graphs were constructed
using the ggplot2 package (Wickham, 2009) for R 2.13.0
(http://www.R-project.org).
The mar-mutant viruses used in this study are single
mutants, in which the critical residue(s) of one of the
neutralizing epitopes has been mutated, thereby abrogating
the binding to the corresponding mAb (Kitson et al., 1990).
These were used for the first time to quantify the relative
amount of antibodies produced against each epitope
in response to FMDV vaccination. Our hypothesis is that
antibodies produced against an intact or native epitope
will not recognize the mutated epitope, as mutations on
antigenic sites have been shown to completely destroy
binding with relevant virus-specific antibodies in FMDV
(Dunn et al., 1998) and in poliovirus (Rezapkin et al.,
2010). Indeed, Crowther et al. (1993a) reported a 15 %
drop in neutralizing-antibody titre in post-vaccination
cattle sera following a single mutation (G67 to G67+C3
mutant). Therefore, the reduction in the neutralizingantibody titre for a particular mar mutant will indicate the
presence of the relative amount of the antibodies against
that particular epitope in the polyclonal serum, which in
turn reflects the immundominance of that epitope.
The titre reductions of neutralizing antibodies measured
against various mar-mutant viruses compared with the
parental virus O1K are shown in Fig. 1(a, b). Reduced
neutralization was observed with mutants for all five
antigenic sites in all three target species studied, albeit to
various degrees (Fig. 1a) with significantly greater reductions (P,0.01) against antigenic site 2. Similarly significant reductions against antigenic site 1 (P,0.001) were
observed only in cattle and pigs, and the BFS vaccine
group. Differences between species were found to be
statistically not significant (data not shown).
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489
M. Mahapatra, P. Hamblin and D. J. Paton
Fig. 1. Difference in the mean (log10) neutralizing-antibody titre as measured by microneutralization assay between parent
(reference virus) and mar-mutant viruses by species (a) and by vaccination (b). Error bars represent 95 % confidence intervals.
The x-axis represents the mar mutants to various serotype O antigenic sites as shown in Table 1. Reference mean antibody
titres for parent virus are 2.58 log10 for cattle, 2.62 log10 for pig and 2.89 log10 for sheep; 2.74 log10 for O1 BFS, 2.38 log10
for O1 Lausanne and 2.29 log10 for O1 Kaufbeuren. Asterisks indicate that the neutralizing-antibody titre is significantly lower
than that of the parent virus; *P-values between ,0.05 and ,0.01; **P-values between ,0.001 and ,0.0001.
Sera from 57 vaccinated cattle were used in this study and
the results are shown in Fig. 1(a). Antibodies to all five
antigenic sites, although to various degrees, were detected
in these animals. Significantly higher (P,0.001) levels of
antibodies were found to be directed against antigenic site
2, followed by site 1. The majority of the animals used in
this study (76 %) had more antibodies directed against
antigenic site 2, whereas about 15 % of the animals had
more antibodies against antigenic site 1 (Fig. 2a). There
were 6 % of animals that had approximately equal levels of
antibodies against sites 1 and 2 (Fig. 2a). The 57 serotype O
sera tested in this study comprised samples from animals
vaccinated with O1 BFS, O1 Lausanne or O1K vaccine.
When the results were analysed for each vaccine, a consistent picture emerged, with more antibodies directed
against antigenic site 2 followed by site 1 in most cattle
(BFS, 62 %; Lausanne, 78 %; Kaufbeuren, 100 %) (Fig. 2b).
The amount of antibodies produced against other sites
(sites 1, 3, 4, and 5) was variable among the various vaccine
490
strains. Interestingly, the lowest amount of antibody
against site 1 was detected in O1K-vaccinated cattle; this
could be due to the small sample size for this vaccine or
because these five cattle were the only animals that received
a booster dose on day 21 following vaccination. Herremans
et al. (2000) also observed a change in site-specific
dominance associated with booster vaccination, finding a
relative rise in the level of antibody to site 3 in poliovirus.
In the case of the O1 BFS group, 9 % of animals had
approximately equal levels of antibodies against sites 1 and
2 (Fig. 2b).
Sera from 20 vaccinated sheep were used for this study and
the results are depicted in Fig. 1(a). As in cattle, a varied
immune response was observed in these animals; however,
overall, significantly more antibodies (P,0.001) were
directed against site 2. The levels of neutralizing antibodies
against antigenic sites 1, 4, 5 and 3 were not statistically
significant. About 65 % of animals had more antibodies
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Journal of General Virology 93
FMD virus epitope dominance
Fig. 2. Percentage of animals showing highest antibody titres against various neutralizing antigenic sites by species (a) and by
vaccination (b). Error bars represent 95 % confidence intervals.
against site 2, followed by site 1 (20 %) and site 4 (15 %)
(Fig. 2b).
Sera from 33 vaccinated pigs were used for this study
and the results are shown in Fig. 1(a). Although a varied
immune response was observed in these animals, as a
whole, significantly higher (P,0.0001) levels of antibodies
were directed against site 2, followed by site 1. The majority
of the animals used in this study (58 %) had more
antibodies against antigenic site 2, whereas about 18 %
had more antibodies against antigenic site 1 (Fig. 2a). In
addition, 15 % of animals had approximately equal levels of
antibodies against sites 1 and 2 (Fig. 2a).
Although a three- or four-site mutant was neutralized
using FMDV post-infection sera (McCahon et al., 1989;
Crowther et al., 1993a), use of post-vaccination sera
showed a different pattern. A neutralization test involving
multiple mutant G67 (a four-site mutant) and FMDV
post-vaccination sera demonstrated the group mean
residual percentage titre to be 15 % (presented as a
percentage of the titre found against the parent O1K
virus). However, when the fifth site was mutated
http://vir.sgmjournals.org
(G67+C3, a five-site mutant), the mean residual percentage titre decreased to an undetectable level, indicating that
the drop was due to the loss of neutralization against the
fifth site (Crowther et al., 1993a). It was only a single
mutation at position 149 of VP1 that resulted in about
15 % drop in neutralizing-antibody titre.
In this study, we have quantified the relative amounts of
antibody produced against each antigenic site in response
to vaccination against serotype O FMDV using a panel
of mar mutants. The results indicate that most of the
antibodies produced in response to FMDV vaccination in
cattle, sheep and pigs are against antigenic site 2, followed
by site 1. Using a serum immunoglobulin-fractionation
study involving serotype C FMDV, Mateu et al. (1995) also
reported that about 27 % of virus-neutralizing activity
from vaccinated pigs corresponded to antigenic site 1,
whereas the majority of the antibodies were directed
against other antigenic sites. Other reports are also
consistent with our findings: (i) mAbs produced following
immunization with serotype O FMDV vaccines are often
found to be against antigenic site 2 (Barnett et al., 1998; M.
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491
M. Mahapatra, P. Hamblin and D. J. Paton
Mahapatra, unpublished data), indicating that this site is
often recognized by antibody-producing cells. (ii) The VP1
G–H loop (site 1) is not essential for protection in natural
hosts against FMDV infection (Fowler et al., 2008, 2010;
Rieder et al., 1994), and vaccination with VP1 G–H loop
peptide vaccines has not been fully successful in eliciting
complete protection (Krebs et al., 1993; Rodriguez et al.,
2003; Taboga et al., 1997). However, our results contradict
those of Aggarwal & Barnett (2002), who used a mAbbased cELISA involving mAbs to antigenic sites 1–3, some
homologous and some heterologous, to determine the
relative importance of various antigenic sites in polyclonal
responses to serotype O FMDV. Their results indicated
that none of these antigenic sites can be considered as
immunodominant. Interestingly, they also reported that
pigs did not respond to epitopes on the C-terminal end of
VP1 as efficiently as ruminants. Although a cELISA-based
approach has been used successfully to define the epitopes
of FMDV (Barnett et al., 1998; McCullough et al., 1987;
Thomas et al., 1988b) and poliovirus (Rezapkin et al., 2005,
2010), competition has been reported to be dependent on
the affinity of the mAbs (Thomas et al., 1988b) and,
sometimes, competition might be observed due to steric
interference because of the large size of the antibody
molecule and the relative proximity of the antigenic sites
on the surface of FMDV.
In summary, it appears that, following vaccination, the
predominant antibody response in the case of serotype O
FMDV is against antigenic site 2, followed by site 1. This
trend was consistent in all of the three different target
species studied (cattle, pigs and sheep), and also with three
different vaccine formulations (BFS, Lausanne and Kaufbeuren). The antibody profile induced in humans following vaccination with inactivated and live-attenuated
oral polio vaccine was found to be significantly different
(Rezapkin et al., 2010). Crowther et al. (1993a) also
reported a significantly higher VN titre in post-infection
sera than in post-vaccination sera using a G67 (four-site
mutant) virus. Hence, it is possible that the antibody
profile following FMDV infection could also be different
from that following vaccination.
Acknowledgements
We would like to thank B. Haas, B. Statham, P. Barnett, Y. Li, N.
Ferris, A. Dekker and A. Samuel for making some of the reagents
available for this study and A. Di-Nardo for help in statistical analysis
of the data. This work was supported financially by DEFRA grants
SE2922 and SE2937. D. J. P. is a Jenner Institute Investigator and
is funded by the Biotechnology and Biological Sciences Research
Council (BBSRC).
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