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
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Abstract |
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Introduction |
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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
G
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, 7073, 75 and 77 of VP2 contribute to site 2, and site 3 is formed in part by residues 43 and 44 of the
B
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 (141160 or 141158 and 200213) 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.
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Methods |
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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).
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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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 carbonatebicarbonate 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 140160 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 1·52·4 log10 SN50/100 TCID50.
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Results |
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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.
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Discussion |
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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 hosts 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.
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Acknowledgments |
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References |
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Bachrach, H. L., Moore, D. M., McKercher, P. D. & Polatnick, J. (1975). Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus. Journal of Immunology 115, 1636-1641.[Abstract]
Barnett, P. V., Samuel, A. R., Pullen, L., Ansell, D., Butcher, R. N. & Parkhouse, R. M. E. (1998). Monoclonal antibodies, against O1 serotype foot-and-mouth disease virus, from a natural bovine host, recognize similar antigenic features to those defined by the mouse. Journal of General Virology 79, 1687-1697.[Abstract]
Barnett, P. V., Ouldridge, E. J., Rowlands, D. J., Brown, F. & Parry, N. R. (1989). Neutralization epitopes of type O foot-and-mouth disease virus. I. Identification and characterization of three functionally independent, conformational sites. Journal of General Virology 70, 1483-1491.[Abstract]
Baxt, B., Vakharia, V., Moore, D. M., Franke, A. J. & Morgan, D. O. (1989). Analysis of neutralising antigenic sites on the surface of type A12 foot-and-mouth disease virus. Journal of Virology 63, 2143-2151.[Medline]
Bittle, J. L., Houghten, R. A., Alexander, H., Shinnick, T. M., Sutcliffe, J. G., Lerner, R. A., Rowlands, D. J. & Brown, F. (1982). Protection against foot-and-mouth disease by immunization with a chemically synthesized peptide predicted from the viral nucleotide sequence. Nature 298, 30-33.[Medline]
Brocchi, E., Civardi, A., De Dimone, F. & Panina, G. F. (1983). Characterisation of foot-and-mouth disease virus antibodies. 20th Congress of the Italian Society of Microbiology, Gardone, Italy. Atti della Societa Italiana delle Scienze Veterinarie 36, 576-578.
Brown, F. & Cartwright, B. (1963). Purification of radioactive foot-and-mouth disease virus. Nature 199, 1168-1170.[Medline]
Brown, A. L., Francis, M. J., Hastings, G. Z., Parry, N. R., Barnett, P. V., Rowlands, D. J. & Clarke, B. E. (1991). Foreign epitopes in immunodominant regions of hepatitis B core particles are highly immunogenic and conformationally restricted. Vaccine 9, 595-601.[Medline]
Crowther, J. R., Farias, S., Carpenter, W. C. & Samuel, A. R. (1993). Identification of a fifth neutralizable site on type O foot-and-mouth disease virus following characterization of single and quintuple monoclonal antibody escape mutants. Journal of General Virology 74, 1547-1553.[Abstract]
DiMarchi, R., Brooke, G., Gale, C., Cracknell, V., Doel, T. & Mowat, N. (1986). Protection of cattle against foot-and-mouth disease by a synthetic peptide. Science 232, 639-641.[Medline]
Dunn, C. S., Samuel, A. R., Pullen, L. A. & Anderson, J. (1998). The biological relevance of virus neutralisation sites for virulence and vaccine protection in the guinea pig model of foot-and-mouth disease. Virology 247, 51-61.[Medline]
Feigelstock, D., Mateu, M. G., Piccone, M. E., De Simone, F., Brocchi, E., Domingo, E. & Palma, E. L. (1992). Extensive antigenic diversification of foot-and-mouth disease virus by amino acid substitutions outside the major antigenic site. Journal of General Virology 73, 3307-3311.[Abstract]
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. Journal of Immunology 143, 3015-3019.
Golding, S. M., Hedger, R. S. & Talbot, P. (1976). Radial immuno-diffusion and serum neutralisation techniques for the assay of antibodies to swine vesicular disease. Research in Veterinary Science 20, 142-147.[Medline]
Kitching, R. P., Rendle, R. & Ferris, N. P. (1988). Rapid correlation between field isolates and vaccine strains of foot-and-mouth disease virus. Vaccine 6, 403-408.[Medline]
Kitson, J. D. A., McCahon, D. & Belsham, G. J. (1990). Sequence analysis of monoclonal antibody resistant mutants of type O foot-and-mouth disease virus: evidence for the involvement of the three surface exposed capsid proteins in four antigenic sites. Virology 179, 26-34.[Medline]
Kleid, D. G., Yansura, D., Small, B., Dowbenko, D., Moore, D. M., Grubman, M. J., 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, 1125-1129.[Medline]
McCullough, K. C., Crowther, J. R., Carpenter, W. C., Brocchi, E., Capucci, L., De Simone, F., Xie, Q. & McCahon, D. (1987). Epitopes on foot-and-mouth disease virus particles. I. Topology. Virology 157, 516-525.[Medline]
Mason, P. W., Piccone, M. E., McKenna, T. S. C., Chinsangaram, J. & Grubman, M. J. (1997). Evaluation of a live-attenuated foot-and-mouth disease virus as a vaccine candidate. Virology 227, 96-102.[Medline]
Mateu, M. G. (1995). Antibody recognition of picornaviruses and escape from neutralisation: a structural view. Virus Research 38, 1-24.[Medline]
Mateu, M. G., Martínez, M. A., Capucci, L., Andreu, D., Giralt, E., Sobrino, F., Brocchi, E. & Domingo, E. (1990). A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-and-mouth disease virus of serotype C. Journal of General Virology 71, 629-637.[Abstract]
Mateu, M. G., Camarero, J. A., Giralt, E., Andreu, D. & Domingo, E. (1995). Direct evaluation of the immunodominance of a major antigenic site of foot-and-mouth disease virus in a natural host. Virology 206, 298-306.[Medline]
Mayr, G. A., Chinsangaram, J. & Grubman, M. J. (1999). Development of replication-defective Adenovirus serotype 5 containing the capsid and 3C protease coding regions of foot-and-mouth disease virus as a vaccine candidate. Virology 263, 496-506.[Medline]
Morgan, D. O. & Moore, D. M. (1990). Protection of cattle and swine against foot-and-mouth disease, using biosynthetic peptide vaccines. American Journal of Veterinary Research 51, 40-45.[Medline]
Pay, T. W. F. & Hingley, P. J. (1987). Correlation of 140S antigen dose with the serum neutralising antibody response and the level of protection induced in cattle by foot-and-mouth disease vaccines. Vaccine 5, 60-64.[Medline]
Pfaff, E., Mussgay, M., Bohm, H. O., Schulze, G. E. & Schaller, H. (1982). Antibodies against a preselected peptide recognize and neutralize foot-and-mouth disease virus. EMBO Journal 1, 869-874.[Medline]
Samuel, A. R. (1997). Genetic and antigenic studies on foot-and-mouth disease virus type O. PhD thesis, University of Hertfordshire, UK.
Sanz-Parra, A., Jiménez-Clavero, M. A., Garca-Briones, M. M., Blanco, E., Sobrino, F. & Ley, V. (1999). Recombinant viruses expressing the foot-and-mouth disease virus capsid precursor polypeptide (P1) induce cellular but not humoral antiviral immunity and partial protection in pigs. Virology 259, 129-134.[Medline]
Sobrino, F., Sáiz, M., Jiménez-Clavero, M. A., Núñez, J. I., Rosas, M. F., Baranowski, E. & Ley, V. (2001). Foot-and-mouth disease virus: a long known virus, but a current threat. Veterinary Research 32, 1-30.[Medline]
Strohmaier, K., Franze, R. & Adam, K. H. (1982). Location and characterization of the antigenic portion of the FMDV immunizing protein. Journal of General Virology 59, 295-306.[Abstract]
Taboga, O., Tami, C., Carrillo, E., Núñez, J. I., Rodríguez, A., Sáiz, J. C., Blanco, E., Valero, M., Roig, X., Camarero, J. A., Andreu, D., Mateu, M. G., Giralt, E., Domingo, E., Sobrino, F. & Palma, E. L. (1997). A large-scale evaluation of peptide vaccines against foot-and-mouth disease: Lack of solid protection in cattle and isolation of escape mutants. Journal of Virology 71, 2606-2614.[Abstract]
Thomas, A. A. M., Woortmeijer, R. J., Puijk, W. & Barteling, S. J. (1988a). Antigenic sites on foot-and-mouth disease virus type A10. Journal of Virology 62, 2782-2789.[Medline]
Thomas, A. A. M., Woortmeijer, R. J., Barteling, S. J. & Meloen, R. H. (1988b). Evidence for more than one important, neutralizing site on foot-and-mouth disease virus. Archives of Virology 99, 237-242.[Medline]
Ward, G., Rieder, E. & Mason, P. W. (1997). Plasmid DNA encoding replicating foot-and-mouth disease virus genomes induces antiviral immune responses in swine. Journal of Virology 71, 7442-7447.[Abstract]
Wild, T. F., Burroughs, J. N. & Brown, F. (1969). Surface structure of foot-and-mouth disease virus. Journal of General Virology 4, 313-320.[Medline]
Received 10 August 2001;
accepted 29 November 2001.