Antigenic sites of foot-and-mouth disease virus (FMDV): an analysis of the specificities of anti-FMDV antibodies after vaccination of naturally susceptible host species

N. Aggarwal1 and P. V. Barnett1

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey GU24 0NF, UK1

Author for correspondence: Neeraj Aggarwal. Fax +44 1483 236430. e-mail neeraj.aggarwal{at}bbsrc.ac.uk


   Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Of the known neutralizing antigenic sites of foot-and-mouth disease virus (FMDV), site 1 or A, formed in part by the G–H loop of VP1, has historically been considered immunodominant because of evidence implicating its importance in the induction of a protective immune response. However, no systematic study has been done to determine the relative importance of the various specificities of antibodies against the known neutralizing antigenic sites of FMDV in the polyclonal immune response of a natural host after vaccination. In this report, we have adopted a monoclonal antibody-based competition ELISA and used antibodies specific to sites 1, 2 and 3 to provide some insight into this issue. Following vaccination of the three main target species, cattle, pigs and sheep, with an O1 serotype strain, results indicate that none of these three antigenic sites can be considered immunodominant in a polyclonal serum. Interestingly, pigs did not respond to epitopes on the carboxy terminus end of VP1 as efficiently as the ruminant species. In addition to the known sites, other as yet undefined sites might also be important in the induction of a protective immune response. Possible implications for the design of new vaccine strategies for foot-and-mouth disease are discussed.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Foot-and mouth-disease (FMD) is economically the most important and highly contagious disease of cloven-hoofed livestock; it is present in all continents except Australia and North America. Indeed, the recent outbreak of FMD in the UK and mainland Europe has been a savage reminder of its devastating consequences. Foot-and mouth-disease virus (FMDV) is a single-stranded RNA virus belonging to the genus Aphthovirus in the family Picornaviridae. It causes high morbidity and some mortality in young animals. Control of the disease encompasses an exclusion and slaughter policy, particularly for the FMD-free countries, or vaccination in the endemic areas. There are seven distinct serotypes of FMD, and immunization with inactivated whole virus of one vaccine strain does not confer protection against the other serotypes, or sometimes even between subtypes. Therefore the choice of vaccine strain is of fundamental importance.

It is generally accepted by FMD researchers that the specific humoral immune response is the most important factor in conferring protection against disease. In this respect, there is strong correlation between virus neutralizing antibody and protection for at least one of the main target species, bovines (Pay & Hingley, 1987 ). Numerous studies have been undertaken to identify these neutralizing antigenic sites in more detail, with the aim of developing more effective vaccines (reviewed in Mateu, 1995 ). Such studies have mainly involved the sequencing of escape mutants produced after selection with neutralizing monoclonal antibodies (mAbs). This approach was used successfully in delineating the neutralizing antigenic sites of viruses representing the O serotype (Barnett et al., 1989 ; Kitson et al., 1990 ; Crowther et al., 1993 ), A serotype (Thomas et al., 1988a ; Baxt et al., 1989 ) and C serotype (Mateu et al., 1990 ). Most of these studies relied on the use of murine hybridomas, and it was recently shown that the mouse recognizes similar antigenic features to those seen by bovines (Barnett et al., 1998 ).

FMDV has an icosahedral symmetry with a viral capsid that is non-enveloped and composed of 60 copies of each of the four structural proteins, VP1, VP2, VP3 and VP4 (for a review see Sobrino et al., 2001 ). Three of these proteins, VP1, VP2 and VP3, contribute to the formation of five known antigenic sites of type O1 FMDV (Kitson et al., 1990 ; Crowther et al., 1993 ). The {beta}G–{beta}H loop and carboxy terminus of VP1 contribute to site 1, the critical residues being 144, 148 and 154 and 208. Amino acids at positions 31, 70–73, 75 and 77 of VP2 contribute to site 2, and site 3 is formed in part by residues 43 and 44 of the {beta}B–{beta}C loop of VP1. Only one critical residue, at position 58 of VP3, has so far been identified for site 4. The fifth site, characterized by an amino acid at position 149 of VP1, is probably formed by interaction of the VP1 loop region with other surface amino acids. Site 1 is linear and trypsin sensitive, where as all the other identified sites are conformational and trypsin resistant.

Early FMDV studies using trypsin-treated virus or proteins isolated by chemical or enzymic treatment of intact FMDV highlighted the importance of the VP1 protein in the antigenicity and immunogenicity of the virus (Wild et al., 1969 ; Bachrach et al., 1975 ; Kleid et al., 1981 ; Strohmaier et al., 1982 ). This was further supported by the observation that both neutralizing antibodies and protection were conferred in guinea pigs (Bittle et al., 1982 ; Pfaff et al., 1982 ) and cattle (DiMarchi et al., 1986 ) by immunizing them with peptides corresponding to parts (141–160 or 141–158 and 200–213) of the sequence of VP1. This led to the perception that this site was immunodominant. However, the systemic response following disease or FMD vaccination has never been scrutinized in enough detail to confirm this.

A preliminary study of the serum of O1-vaccinated cattle by Samuel (1997) , using a mAb-based competition ELISA, showed that, for some animals at least, there was a relatively higher titre of site 2 specific antibodies as compared to the other four neutralizing antigenic sites. Indirect evidence from other studies using serotype C (Feigelstock et al., 1992 ; Mateu et al., 1995 ) or serotype A (Thomas et al., 1988b ) FMDV also indicates the important participation of other sites beside site 1 in the generation of an immune response following either natural infection or vaccination. Garmendia et al. (1989) demonstrated the immune response in convalescent bovine and swine sera to be directed to different epitopes but within the same antigenic site. However, no systematic study has been undertaken to quantify the relative amounts of these antibodies against the known neutralizing antigenic sites of FMDV in the polyclonal responses of the three main target hosts.

To estimate the relative proportion of anti-FMDV antibodies with different antigenic site specificities present in the antiserum from cattle, swine and sheep, conventionally immunized with O1 serotype vaccine, we have used a capture competition ELISA (Barnett et al., 1998 ). This test is based on the competition between the site-specific anti-FMDV antibodies present in a hyperimmune polyclonal antiserum and the virus-specific neutralizing mAbs representing the same independent sites. Five anti-O1 Manisa mAbs used in this study have been shown to be directed against sites 1 and 2 (Aktas & Samuel, 2000 ); mAbs representing sites 1 and 3, raised against the O1 Swiss 1965 strain of FMDV (Brocchi et al., 1983 ), but also reactive with O1 Manisa, were also included. The aim was to substantiate the existence of a so-called immunodominant site, or in the absence of this, the relative importance of some of the known antigenic sites in the anti-FMDV polyclonal response of each of the three main target species.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Virus.
FMDV O1 Manisa was prepared from infected baby hamster kidney 21 cells and purified using a 5–30% sucrose density gradient following inactivation of the virus with bromoethyleneimine (Brown & Cartwright, 1963 ). Purified virus was stored at -70 °C.

{blacksquare} Animal sera.
Polyclonal sera from sheep, cattle and pigs vaccinated with inactivated O1 Manisa FMD vaccine were collected during various vaccine trials done at the Institute for Animal Health, Pirbright, UK. Animals were vaccinated either intramuscularly (if an oil adjuvanted vaccine was used) or subcutaneously (for the aluminium hydroxide/saponin vaccine). Sheep and pig sera were collected 28 days post-vaccination and the cattle sera were collected 21 days post-vaccination. In some trials pigs and cattle were challenged with live virus, in the latter case in accordance with European Pharmacopoeia (Monograph for FMD vaccine potency testing).

{blacksquare} MAbs.
Anti-O1 Manisa mAbs used in this study have been described previously (Aktas & Samuel, 2000 ) and the epitope specificities are detailed in Table 1. In the absence of a complete panel of mAbs covering the epitopes of the five known neutralizing sites of FMDV O1 Manisa, it was decided to also include the three well-characterized mAbs B2, D9 and C8, raised against O1 Swiss 1965 strain of FMDV (Kitson et al., 1990 ). The epitope specificities of these three mAbs are also detailed in Table 1.


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Table 1. Epitope specificity and critical amino acid residues of the panel ofvirus-neutralizing anti-FMDV O1 mAbs used in this study

 
{blacksquare} Virus neutralization test.
Neutralizing antibody titres in the hyperimmune serum to FMDV type O1 were determined in a micro-neutralization test essentially according to the method of Golding et al. (1976) . End-point titres were calculated as the reciprocal of the last serum dilution to neutralize 100 TCID50 of homologous FMDV in 50% of the wells.

{blacksquare} ELISA techniques
(i) Liquid phase blocking ELISA (LPB-ELISA).
Anti-virus antibodies were determined by LPBE according to the protocol described by Kitching et al. (1988) , and routinely used at the OIE/FAO World ReferenceLaboratory (WRL) for FMD, Pirbright, UK.

(ii) Competition ELISA.
A capture competition ELISA (Barnett et al., 1998 ) was adopted to determine the relative response of antibodies to antigenic sites 1, 2 and 3 in anti-FMDV polyclonal hyperimmune serum. Initially, mAbs were titrated to determine the dilution that would give 70% of maximal binding in a capture ELISA. For this, Maxisorb plates (Nunc) were coated overnight at 4 °C with rabbit polyclonal anti-FMDV serum diluted 1:5000 in carbonate–bicarbonate buffer (pH 9·6, Sigma). The bound antibody was used to capture virus from a 1 µg/ml suspension of inactivated virus stock. This was followed by addition of mAbs at twofold dilutions. Specific binding was detected by addition of HRP-conjugated anti-mouse antibodies (Dakopatts) followed by developing the reaction with O-phenylenediamine. Absorbance was read at 492 nm after stopping the colour development with 1·25 M sulphuric acid.

For the competition assay, twofold serial dilutions of polyclonal anti FMDV serum from vaccinated animals were mixed with the pre-determined mAb dilution. The residual binding of mAbs was detected as in the capture ELISA. Polyclonal antibodies directed to equivalent epitopes on the virus or epitopes in close proximity to those defined by the mouse mAbs would be highlighted by reduced absorbance values compared to those observed in the absence of the ‘competitor’. The percentage inhibition was calculated as [1-(c/t)]x100, where c and t represent the absorbance in the presence and absence of polyclonal serum respectively.

To establish the specificity of this approach, 14 or 21 day post-vaccinal sera from three pigs and three cattle were examined initially. These animals were immunized with 20 µg of a novel hepatitis B core particle construct incorporating the VP1 140–160 amino acid sequence of FMDV O1 Kaufbeuren (Brown et al., 1991 ) and thus represented a site 1 specific response alone. The virus neutralizing antibody titres of these antisera ranged from 1·5–2·4 log10 SN50/100 TCID50.


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Analysis of anti-FMDV antibodies in the polyclonal serum
Antisera from the FMD vaccinated animals, representing the three main target species, were initially quantified for anti-virus antibodies against O1 Manisa using the LPB-ELISA and virus neutralization test. As expected most of the animals selected for this study had high virus-specific antibody titres following a single vaccination (see Table 2).


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Table 2. LPB-ELISA and VNT titres in anti FMDV polyclonal sera from vaccinated animals representing the three main target species

 
Epitope-specific responses in the polyclonal serum from FMDV-vaccinated animals
By using the competition-based ELISA and mAbs specific to epitopes within antigenic sites 1, 2 and 3, the relative responses to these individual epitopes were measured in the anti-FMD polyclonal sera. Serum titres from individual vaccinates were quantified by determining the dilution that would provide an arbitrary minimum inhibition of 50% mAb binding (see Figs 2, 3 and 4). The relative responses to each antigenic site could then be compared directly from individual responses.



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Fig. 2. Log serum concentration required for 50% inhibition of mAb binding.

 


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Fig. 3. Log serum concentration required for 50% inhibition of mAb binding.

 


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Fig. 4. Log serum concentration required for 50% inhibition of mAb binding.

 
The animals immunized with the novel hepatitis B core construct, representing a site 1 specific response only, competed strongly with site 1 mAb but not with a mAb specific to site 3. However, some animals competed with site 2 specific mAb C6. A representative profile of the competitive responses from two of these animals is shown in Fig. 1.



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Fig. 1. Competition ELISA using polyclonal antisera from cow SS58 (A) and pig SS61 (B). Twofold dilutions of polyclonal antiserum from hyperimmunized animals were mixed with a dilution of mAb that gave 70% of maximal binding; the mAb binding was detected and percentage inhibition calculated as described in text.

 
For cattle (Fig. 2) and sheep (Fig. 3), antibodies were detected against all the epitopes examined with no prominent response observed to any one individual site. Indeed, quantitatively the relative responses to the different antigenic sites were similar, dismissing the idea of immunodominance to any one antigenic region. In some animals only weak reactivity was observed against the epitope represented by mAb D9. Whilst the basis for this is unclear, it does indicate that individual variation to some antigenic sites does occur. It is worth noting that for four cattle, TD44, TD45, TD46 and TD48, such weak responses to the D9 epitope had no bearing on their ability to be protected from live virus challenge (Table 2). Notably, sera from all the vaccinated cattle which were not protected from virus challenge (TD50, UE48, UE49, UE50, UE51 and UE52) were either incapable of inhibiting the panel of mAbs and/or had poor virus neutralization titres.

Immunization of pigs with FMD vaccine also showed no indication of a single antigenic site being immunodominant in the polyclonal response (Fig. 4). Aside from the two non-competitors, TY13 and UE40, all the pigs developed antibodies directed to epitopes within sites 1 or 2. Three pigs, TY10, TY12 and TY14, showed little inhibition against the site 3 specific mAb C8. Again, such weak responses to this epitope had no bearing on their ability to protect against live virus challenge. Surprisingly, no inhibition was observed against the site 1 epitope defined by mAb SA 176 in 9 of the 10 pigs examined, the one exception, UE37, being barely detectable. The specificity of this mAb differs from the other representative site 1 mAbs in that it is directed toward the carboxy terminus end of VP1 and includes residue 198.


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
To date, five independent antigenic sites involving the three capsid proteins, VP1, VP2 and VP3, have been described for FMDV serotype O1 (Kitson et al., 1990 ; Crowther et al., 1993 ). Site 1, involving the {beta}G–{beta}H loop and the carboxy terminus of the VP1 protein, has generally been perceived as an immunodominant site. The aim of this work was to obtain a better perspective on the relevance of this site, and some of the other known antigenic sites, in a polyclonal response following FMD vaccination of three target livestock species.

The competition that was observed using site 2 mAb C6 in cattle and pigs immunized with a novel site 1 specific construct may not be that surprising given that C6 is a monovalent antibody (McCullough et al., 1987 ) and thus might not have competed well enough because of poor affinity. However, in subsequent analyses different site 2 mAbs (raised against O1 Manisa) were used that may or may not have behaved similarly. Sometimes competition might be seen because of stearic interference due to the large size of the antibody molecule and the relative proximity between antigenic sites on the FMDV capsid. Nevertheless, a competition ELISA-based approach has been used successfully to define the epitopes of FMDV (Barnett et al., 1998 ; McCullough et al., 1987 ; Thomas et al., 1988b ). For confirmatory purposes, refinements to this approach such as the use of Fab fragments of antibodies or profiling by the use of site-specific mutant viruses should be considered.

The results indicated that most of the animals responded to all the epitopes, recognized by our panel of anti-FMDV mAbs, following vaccination. The use of different adjuvants in the vaccine preparations did not affect the recognition of antigenic epitopes. However, some variation was observed between species and individual animals to the specific epitopes examined. In all the animals examined, mAb B2 was a better competitor antibody than mAb D9. A similar observation was made by McCullough et al. (1987) and might be related to differences in affinity.

The ruminants, cattle and sheep, showed a fairly even response to sites 1, 2 and 3 in their anti-FMDV polyclonal response. This was in contrast to swine, which appeared not to recognize some of the epitopes that define site 1, in particular the carboxy terminus sequence of VP1, defined by mAb SA 176. This suggests that, unlike ruminants, pigs do not normally raise a significant response to this region of the virus following vaccination. The mechanism(s) responsible for this apparent difference is not known, but may relate to differing processing events following immunization.

Animals SV85, SV86, SV88, TY13, UE40, TD50 and UE52 seroconverted with detectable and sometimes significant virus neutralizing antibody titres but were unable to compete with the panel of mAbs used in this study. It is possible that antibodies in these animals were directed to site 4, a neutralizing site that was not tested in this study. However, it seems unlikely that all the antibodies would be against this site alone. This is supported by the difficulty in producing site 4 specific mAbs in spite of the recognition of the similar antigenic features of FMDV in both mice and bovines (Barnett et al., 1998 ). The inability of hyperimmune sera to compete with the panel of mAbs used in this study might relate to further unidentified neutralizing antigenic sites (Dunn et al., 1998 ). Another possibility could be that antibodies produced in these animals had a low affinity for the sites tested in this study (Thomas et al., 1988b ).

Overall, we conclude that of the three known antigenic sites examined in this study, none can be considered immunodominant following vaccination with FMDV of serotype O1.

A similarly broad repertoire of epitope specificities following vaccination has been observed in previous studies (Thomas et al., 1988b ; Mateu et al., 1995 ) although only a small number of animals were examined and the studies did not encompass three main target species.

Such a broad antibody response would be advantageous to the host, as specific mutational changes in the virus are less likely to evade the host’s immune defence, compared to one in which the response is narrower and limited to one or two sites, such as that against a peptide construct. This may also partly explain the lower immunogenicity and the limited success of FMD peptide vaccines when applied in the target host (DiMarchi et al., 1986 ; Morgan & Moore, 1990 ; Taboga et al., 1997 ), despite the initial promise from guinea pig experiments (Bittle et al., 1982 ; Pfaff et al., 1982 ). Current research with genetically engineered FMD vaccines that mirror the virus (Mason et al., 1997 ; Ward et al., 1997 ; Mayr et al., 1999 ; Sanz-Parra et al., 1999 ) must be considered to have greater potential because they can present an animal with the intact viral capsid and raise immunity against all the possible epitopes, including those associated with protection.

This study has shown that immunodominance cannot be demonstrated for type O FMDV. However, this may not necessarily be the case for other serotypes of FMDV. The recognition and response to structural features of the virus capsid may be quite different for other serotypes. However, to our knowledge, this is the first study delineating the status of antigenic site-specific anti-FMDV antibodies in a polyclonal response of all three main target species following FMD vaccination.


   Acknowledgments
 
The authors would particularly like to thank Drs F. De Simone and E. Brocchi of the Istituto Zooprofilattico Sperimentale della Lombardia e delle Emilia, Brescia, Italy and Drs A. Samuel and S. Aktas of the Institute for Animal Health, Pirbright, UK, for the generous gift of the mAbs used in this study. We also thank Bayer AG, Cologne, for making available some of the serum samples used in this study, Mrs Teli Rendle of WRL for FMD at Pirbright for undertaking the LPB-ELISAs and VNT and animal attendants for helping in the various vaccine trials. Critical review of the manuscript by Dr A. Samuel is greatly appreciated. This work was funded by the Ministry of Agriculture, Fisheries and Food (now DEFRA), UK, under project number SE2912.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 10 August 2001; accepted 29 November 2001.