World Journal
of Microbiology
and Biotechnology
7, 110-l 14
Peptides as viral vaccines: lessons from experiments with foot-and-mouth disease virus
F. Brown
The highly variable sequence encompassing amino acids 141-160 of VPl, one of the four capsid protelns of foot-and-mouth disease virus, elicits neutralking antlbodles In a range 61 experlmental animals. The sequence has been synthesized chemically by Merrlfleld’s solld phase method and biochemically as part of different fusion protelns. By Incorporating the peptide Into the core proteln of hepatltls B virus, particles are produced which ellclt levels of neutrallzlng antlbody approaching those obtalned with the Intact virus particle. The author Microbiology, ford. Surrev
is with the Department of University of Surrey, GuildGU2 5XH. UK
This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 244% August 1990. --
@I 7991 Rapid
110
Communications
of Oxford
Ltd.
Vaccination against bacterial and viral diseases has been one of the great success stories in the history of human and veterinary medicine. The eradication of smallpox and the control of diseases such as diphtheria, tetanus, measles, poliomyelitis and yellow fever are well recognized. Equally important in veterinary medicine is the development of vaccines to control leptospirosis, rinderpest and foot-andmouth disease in cattle, clostridial diseases in sheep, cattle and pigs, diarrhoea caused by Escherichia co/i in piglets, and Newcastle and Marek’s disease in poultry. Nevertheless, the empiricism underlying the production of these vaccines has meant that the structural basis for immunogenicity and specificity remains largely unexplored. Following the introduction by _Jenner (1798) of cowpox virus as a vaccine for the prevention of smallpox, early immunization studies were directed towards the use of deliberately weakened organisms for the control of infectious diseases. Pasteur and his colleagues developed live vaccines for chicken cholera, anthrax and rabies in rapid succession in the 1880s (e.g. Pasteur 1885), but it was established shortly afterwards that multiplication of the organism was not a necessary requirement for vaccination because proteins, appropriately treated (toxoided), would afford protection against diphtheria and tetanus. Moreover, the demonstration by Semple in 1919 that vaccination against rabies could be achieved by inoculation of the inactivated virus emphasized the role that non-replicating organisms could play in the control of infectious diseases. This principle was amply underlined at the end of the 1940s and beginning of the 1950s by the large-scale application of killed vaccines against foot-and-mouth disease (Frenkel 1947) and poliomyelitis (Salk 1955). With the recent emergence of recombinant DNA technology and techniques for nucleic acid sequencing, monoclonal antibody production and X-ray crystallography of proteins, it has now become possible to study the structural basis for immunogenicity and the immune response to proteins at the molecular level. Using foot-and-mouth disease virus as an example, the progress that has been made towards such goals is described in this paper.
Peptides as vaccines
The Disease
and its Control
Foot-and-mouth disease is the most economically important viral disease of farm animals. Its control is important, not only because of the loss of productivity which follows infection but also because of the embargoes on trading imposed on a country in which the disease is present. This control in endemic areas is by vaccination, using a classical inactivated vaccine produced by inactivating virus grown in bovine tongue epithelial fragments or in the baby hamster kidney (BHK 21) cell line. These vaccines are powerful immunogens and one inoculation is sufficient to elicit levels of neutralizing antibody sufficient to protect cattle and pigs against a challenge dose of 10,000 ID,, of virus inoculated directly into the tongue (for cattle) or foot (for pigs).
The
Virus
and Virus-related
Particles
Foot-and-mouth disease virus is a spherical particle, 30 nm in diameter and sedimenting at 146S, which consists of one molecule of SSRNA, mol. wt 2.6 X IO6 and 60 copies of each of four proteins, mol. wt 24 x IO3 for VW, VP2 and VP3 and 10 x IO3 for VP4. Also present in harvests of the virus-infected cells are empty particles, of a size similar to that of the infectious virus particle but devoid of RNA. They are composed of the same proteins but VP2 and VP4 are covalently linked. In addition, there are also present a particle comprising five copies of each of the three proteins VP1 to VP3 (the so-called 12s particle) and the inactive form of the virus polymerase (the virus infection associated or VIA antigen). The virus particle and the empty particles are immunogenic but the VIA antigen is devoid of activity. The 12s particle has very low immunogenicity (ca 1% of that of the virus particle), which is significant in view of the location of a sequential epitope of considerable immunogenicity on VPI. This issue is discussed below.
Antigenic
Variability
of the Virus
The virus occurs as seven major serotypes, A, 0, C, Southern African Territories SATI, SAT2, SAT3 and Asia 1. The occurrence of the virus as a multiplicity of serotypes complicates the problem of control because the immunity induced by vaccination with a product of one serotype fails to protect against infection with viruses of the other serotypes. Moreover, there is sufficient antigenic variation within a serotype to necessitate the production of vaccines from viruses which are closely related antigenically to those occurring in the field. Although antigenic variability is a major problem for vaccine manufacturers, it has provided important clues for the identification of immunogenic sites (see below). The sequencing data have shown that VP4 is a highly conserved molecule (2 98%), VP2 and VP3 have ca 90% homology, whereas VP1 is only about 80% homologous. The variable regions of VP1 occur in three main blocks, encompassing amino acids 42-61, 138-160 and 194-204. The 13fG-160 region is particularly variable although the sequence arggb asp which occurs at positions 145, 146 and 147 is present almost without exception in the 20 or more viruses for which sequences are available. The 145147 tripeptide is intimately connected with the attachment of the virus to susceptible cells (Fox et al. 1989).
Location
of Immunogenic
Treatment of the virus particle with with some viruses the immunogenicity 0-BFS, a virus of serotype 0, the the immunogenicity of the treated virus particles. This provided an
Sites on the Virus
Particle
trypsin lowers its infectivity considerably and is also seriously impaired. Thus with strain infectivity is reduced by more than 99% and particles is approximately 1% of the parent important clue regarding the position of the
111
F. Brorvn immunodominant site because, as judged by polyacrylamide gel electrophoresis, only VP1 appeared to be affected by the enzyme treatment. Moreover, Laporte et al. (1973) showed that when the virus particle was disrupted and the proteins separated, only VP1 possessed immunogenic activity. However, it is possible that immunogenic sites on the other proteins of the virus or which encompass more than one protein are also destroyed when the virus is disrupted or even when VP1 alone is cleaved by trypsin. Nevertheless the apparent dominance of VP1 as the immunogenic protein of the virus has led to a focusing of attention on this protein, The immunogenic activity of the protein itself is very low; compared with the virus particle itself it is more than four orders of magnitude less active. However, immunogenic sites on the protein were identified by cleaving it with proteolytic enzymes or cyanogen bromide (Strohmaier et al. 1982). These sites coincided with two of the variable regions, 138160 and 194-204 referred to previously. On the basis of these observations, peptides corresponding to these regions were synthesized. Sequences corresponding to the 138160 region of all the serotypes have been found to be highly active, but the 194-204 region has comparatively low activity and no activity was found in the remainder of the molecule (Bittle et al. 1982).
Presentation
of the Peptide
to the Host
The initial experiments showed that one inoculation of approximately 100 pg of the 141-160 region, conjugated to a carrier protein, would elicit levels of neutralizing antibody in guinea pigs sufficient to protect them against a severe challenge (10,000 ID,,) of homologous virus directly into the foot pad. However, as little as 0.02 pg of the same amino acid sequence, present as 60 copies on the intact virus particle, will elicit the same level of neutralizing antibody. Consequently a variety of methods for improving the presentation of the peptide has been investigated. These studies have led to the conclusion that multiple copies of the sequence presented on a particle will elicit high levels of neutralizing antibody. For example, a fusion protein consisting of the 141-160 sequence at the N terminus of the hepatitis B virus core protein assembles into particles which elicit levels of neutralizing antibody in guinea pigs approaching those obtained with the equivalent amount of the peptide sequence on the parent virus particle (Clarke et a/. 1987). Experiments in progress indicate that even better antibody responses are obtained by insertion of the 141-160 region into the loop region of the core protein predicted by Argos & Fuller (1988). Clearly the relevant response, in this case neutralizing antibody, is dictated by the conformation of the peptide sequence in the engineered construction. It will be recalled that the level of neutralizing antibody elicited by isolated VP1 or the 12s particle containing five
Table 1. Amino A, subtype 12 Virus A 0
acid sequence
01 the 141-160
region
of VP1 01 four
c D
112
of FMDV,
141 GUY
Ser
GUY
Val
Arg
‘JY
Asp
C D
A 0
variants
Ser Leu
serotype
GIY
150 Ser
Leu
160 Pro
Ser Phe 151 Leu
Ala
Leu Pro Ser Pro
Arc!
Val
Ala
Arg
Gln
Peptides as vaccines copies of WI, VP2 and VP3 is several obtained with the intact virus particle.
The
Need
for a Helper
T Cell
orders
of magnitude
lower
than that
Epitope
The ideal vaccine would contain only those regions of the virus that are required to elicit a protective immune response. In our initial experiments we showed that the 141-160 sequence alone, without carrier protein, would elicit good levels of neutralizing antibody in guinea pigs, provided it contained an added CJJ residue at the C terminus. The CJS residue presumably allows the formation of dimers because, when it is blocked or absent, the response is very low. The response is improved considerably by presenting the sequence as a tetramer or octamer, using the method of multiple antigenic peptide synthesis described by Tam (1988). The response of pigs and cattle to the 141-160 9s peptide alone was poor. This has led us to investigate whether the genetic restriction could be overcome by the addition of appropriate T cell epitopes. Preliminary experiments in mice have shown that genetic restriction can be overcome (Francis et al. 1987). Using mice belonging to the H-2d haplotype, which do not respond to the 141-160 sequence, peptides consisting of residues 141-160 and T cell epitopes from either ovalbumin or sperm whale myoglobin will elicit neutralizing antibody responses as high as are obtained in mice belonging to the H-2k haplotype, which do respond to the 141-160 sequence alone. Extension of this approach to cattle and pigs will show whether the restricted response to the 141-160 sequence can be overcome by the inclusion of either (a) foreign T cell epitopes such as those described for the mouse experiments or those present on the hepatitis B virus core, or (b) epitopes from other regions of the virus particle.
Structural
Studies
The specific immune response of the host to an epitope depends on its shape. In an attempt to define the structural basis for antigenic variation, which is so crucial in foot-and-mouth disease, the structure of the virus has been analysed by X-ray crystallography. Although most of the structure of the virus has yielded to this analysis, that of the immunogenic region of the virus in the 141-160 sequence of VP1 has not yet been solved. However, progress has been made with peptides corresponding to this region. The response of guinea pigs to peptides differing only in positions 148 and 153 of the 141-160 sequence of a virus of serotype A can be distinguished by neutralization and immunoprecipitation tests with the corresponding virus and anti-peptide antisera. Four peptides, with the sequences shown in Table I, could be grouped into two pairs, one consisting of peptides with a pro residue at 153 and the other with a residue of leu or ser (Rowlands et al. 1983). Examination of the peptides by nuclear magnetic resonance and circular dichroism showed that the salt bridge between residues asp 147 and arg 154 in A and C was not present in B and D because of the breakage of the helix by the presence of the pro residue at 153 (Mascagni et al. submitted). Extension of this approach should allow the design of peptides with predetermined specificity and may lead to an understanding of the structural basis of antigenic differences between the serotypes.
Conclusions The results described in this paper show that it is possible vaccine comprising the immunogenic B cell epitope in the virus, linked to a T cell epitope appropriate for the be crucial to identify appropriate T cell epitopes for the
to construct an effective the 141-160 sequence of recipient species. It will relevant species and then
113
F. Brown to present the hybrid peptide as a multimer. With the octamer approach described by Tam (1988) this has become a feasible prospect which has the additional advantage that no carrier protein is required. Moreover, our structural studies at the level of atomic resolution have revealed that the shapes of peptides of antigenic variants of the virus are distinguishable and it is to this area that much attention will be focused in the immediate future. Aside from any practical application of this information it will lead to a better understanding of the immune response at the molecular level.
References ARGOS, P. & FULLER, SD. 1988 A model for hepatitis B virus core protein: prediction of antigenic sites and relationship to RNA virus capsid proteins. EMBO Journal 7, 81’+824. BITTLE, J.E., 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 synthesised peptide predicted from the viral nucleotide sequence. Nature 298, 30-33. CLARKE, B.E., NEWTON, SE., CARROLL, A.R., FRANCIS, M. J., APPLEYARD, G., SYRED, A.D., HIGHFIELD, P.E., ROWLANDS, D.J. & BROWN, F. 1987 Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 330, 381-384. Fox, G., PARRY, N.R., BARNETT, P.V., MCGINN, B., ROWLANDS, D. J. & BROWN, F. 1989 The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD. Journal of General Virology 70, 625632. FRANCIS, M. J., HASTINGS, G.Z., SYRED, A.D., MCGINN, B., BROWN, F. & ROWLANDS, D.J. 1987 Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T cell determinants. Nature 330, 168170. FRENKEL, H.S. 1947 La culture du virus de la fitvre aphteuse sur l’epithelium de la langue des bovides. Bulletin de I’Oflce International des Epixooties 28, 155162. JENNER, E. 1798 An Enquiry into the Causes and Effects of the Variohe Vaccinae. London: Sampson Low. LAPORTE, J., GROSCLAUDE, J., WANTYGHEM, J., BERNARD, S. & ROUZE, P. 1973 Neutralisation en culture cellulaire du pouvoir infectieux du virus de la fitvre aphteuse par des serums provenant de ports immunises h l’aide d’une promine virale purifiee. Comptes
rendus de I’Acadimie
des Sciences Shie
D 276,
3399-3401.
MASCAGNI, P., SILIGARDI, G., VAN DER HOEVEN, S.J., BROWN, F., DRAKE, A.F., NICCOLAI, N., JAMES, C.H., ROWLANDS, D. & GIBBONS, W.A. Structural basis for the antigenic specificity of foot-and-mouth disease virus peptides as deduced from solution conformation studies. Proceedings oftbe NationalAcademy ofsciences ojthe USA Submitted. PASTEUR, L. 1885 Methode pour prevenir la rage apres morsure. Comptes rendus des Shnces de I’Acadimie
des Sciences 101, 765-772.
ROWLANDS, D. J., CLARKE, B.E., CARROLL, A.R., BROWN, F., NICHOLSON, B.H., BITTLE, J.L., HOUGHTEN, R.A. & LERNER, R.A. 1983 Chemical basis of antigenic variation in foot-and-mouth disease virus. Nature 306, 694-697. SALK, J.E. 1955 A concept of the mechanism of immunity for preventing paralysis in poliomyelitis. Annals of the New York Academy of Sciences 61, 10231036. SEMPLE, D. 1919 On the nature of rabies and antirabic treatment. British Medical Journa/ 2, 333371. STROHMAIER, K., FRANZE, R. & ADAM, K.H. 1982 Localization and characterisation of the antigenic portion of the foot-and-mouth disease virus immunizing protein. JournuC of General
Viroloa
59, 295-306.
TAM, J.P. 1988 Synthetic peptide vaccine design: synthesis and properties of a high density multiple antigenic peptide system. Proceedings of the National Academy of Sciences of the USA
114
85, 540’%5413.
of Microbiology
and Biotechnology
7, 110-l 14
Peptides as viral vaccines: lessons from experiments with foot-and-mouth disease virus
F. Brown
The highly variable sequence encompassing amino acids 141-160 of VPl, one of the four capsid protelns of foot-and-mouth disease virus, elicits neutralking antlbodles In a range 61 experlmental animals. The sequence has been synthesized chemically by Merrlfleld’s solld phase method and biochemically as part of different fusion protelns. By Incorporating the peptide Into the core proteln of hepatltls B virus, particles are produced which ellclt levels of neutrallzlng antlbody approaching those obtalned with the Intact virus particle. The author Microbiology, ford. Surrev
is with the Department of University of Surrey, GuildGU2 5XH. UK
This paper was presented at the IUMS Symposium on New Developments in Diagnosis and Control of Infectious Diseases held in conjunction with the Eighth International Congress of Virology, Berlin, Germany, 244% August 1990. --
@I 7991 Rapid
110
Communications
of Oxford
Ltd.
Vaccination against bacterial and viral diseases has been one of the great success stories in the history of human and veterinary medicine. The eradication of smallpox and the control of diseases such as diphtheria, tetanus, measles, poliomyelitis and yellow fever are well recognized. Equally important in veterinary medicine is the development of vaccines to control leptospirosis, rinderpest and foot-andmouth disease in cattle, clostridial diseases in sheep, cattle and pigs, diarrhoea caused by Escherichia co/i in piglets, and Newcastle and Marek’s disease in poultry. Nevertheless, the empiricism underlying the production of these vaccines has meant that the structural basis for immunogenicity and specificity remains largely unexplored. Following the introduction by _Jenner (1798) of cowpox virus as a vaccine for the prevention of smallpox, early immunization studies were directed towards the use of deliberately weakened organisms for the control of infectious diseases. Pasteur and his colleagues developed live vaccines for chicken cholera, anthrax and rabies in rapid succession in the 1880s (e.g. Pasteur 1885), but it was established shortly afterwards that multiplication of the organism was not a necessary requirement for vaccination because proteins, appropriately treated (toxoided), would afford protection against diphtheria and tetanus. Moreover, the demonstration by Semple in 1919 that vaccination against rabies could be achieved by inoculation of the inactivated virus emphasized the role that non-replicating organisms could play in the control of infectious diseases. This principle was amply underlined at the end of the 1940s and beginning of the 1950s by the large-scale application of killed vaccines against foot-and-mouth disease (Frenkel 1947) and poliomyelitis (Salk 1955). With the recent emergence of recombinant DNA technology and techniques for nucleic acid sequencing, monoclonal antibody production and X-ray crystallography of proteins, it has now become possible to study the structural basis for immunogenicity and the immune response to proteins at the molecular level. Using foot-and-mouth disease virus as an example, the progress that has been made towards such goals is described in this paper.
Peptides as vaccines
The Disease
and its Control
Foot-and-mouth disease is the most economically important viral disease of farm animals. Its control is important, not only because of the loss of productivity which follows infection but also because of the embargoes on trading imposed on a country in which the disease is present. This control in endemic areas is by vaccination, using a classical inactivated vaccine produced by inactivating virus grown in bovine tongue epithelial fragments or in the baby hamster kidney (BHK 21) cell line. These vaccines are powerful immunogens and one inoculation is sufficient to elicit levels of neutralizing antibody sufficient to protect cattle and pigs against a challenge dose of 10,000 ID,, of virus inoculated directly into the tongue (for cattle) or foot (for pigs).
The
Virus
and Virus-related
Particles
Foot-and-mouth disease virus is a spherical particle, 30 nm in diameter and sedimenting at 146S, which consists of one molecule of SSRNA, mol. wt 2.6 X IO6 and 60 copies of each of four proteins, mol. wt 24 x IO3 for VW, VP2 and VP3 and 10 x IO3 for VP4. Also present in harvests of the virus-infected cells are empty particles, of a size similar to that of the infectious virus particle but devoid of RNA. They are composed of the same proteins but VP2 and VP4 are covalently linked. In addition, there are also present a particle comprising five copies of each of the three proteins VP1 to VP3 (the so-called 12s particle) and the inactive form of the virus polymerase (the virus infection associated or VIA antigen). The virus particle and the empty particles are immunogenic but the VIA antigen is devoid of activity. The 12s particle has very low immunogenicity (ca 1% of that of the virus particle), which is significant in view of the location of a sequential epitope of considerable immunogenicity on VPI. This issue is discussed below.
Antigenic
Variability
of the Virus
The virus occurs as seven major serotypes, A, 0, C, Southern African Territories SATI, SAT2, SAT3 and Asia 1. The occurrence of the virus as a multiplicity of serotypes complicates the problem of control because the immunity induced by vaccination with a product of one serotype fails to protect against infection with viruses of the other serotypes. Moreover, there is sufficient antigenic variation within a serotype to necessitate the production of vaccines from viruses which are closely related antigenically to those occurring in the field. Although antigenic variability is a major problem for vaccine manufacturers, it has provided important clues for the identification of immunogenic sites (see below). The sequencing data have shown that VP4 is a highly conserved molecule (2 98%), VP2 and VP3 have ca 90% homology, whereas VP1 is only about 80% homologous. The variable regions of VP1 occur in three main blocks, encompassing amino acids 42-61, 138-160 and 194-204. The 13fG-160 region is particularly variable although the sequence arggb asp which occurs at positions 145, 146 and 147 is present almost without exception in the 20 or more viruses for which sequences are available. The 145147 tripeptide is intimately connected with the attachment of the virus to susceptible cells (Fox et al. 1989).
Location
of Immunogenic
Treatment of the virus particle with with some viruses the immunogenicity 0-BFS, a virus of serotype 0, the the immunogenicity of the treated virus particles. This provided an
Sites on the Virus
Particle
trypsin lowers its infectivity considerably and is also seriously impaired. Thus with strain infectivity is reduced by more than 99% and particles is approximately 1% of the parent important clue regarding the position of the
111
F. Brorvn immunodominant site because, as judged by polyacrylamide gel electrophoresis, only VP1 appeared to be affected by the enzyme treatment. Moreover, Laporte et al. (1973) showed that when the virus particle was disrupted and the proteins separated, only VP1 possessed immunogenic activity. However, it is possible that immunogenic sites on the other proteins of the virus or which encompass more than one protein are also destroyed when the virus is disrupted or even when VP1 alone is cleaved by trypsin. Nevertheless the apparent dominance of VP1 as the immunogenic protein of the virus has led to a focusing of attention on this protein, The immunogenic activity of the protein itself is very low; compared with the virus particle itself it is more than four orders of magnitude less active. However, immunogenic sites on the protein were identified by cleaving it with proteolytic enzymes or cyanogen bromide (Strohmaier et al. 1982). These sites coincided with two of the variable regions, 138160 and 194-204 referred to previously. On the basis of these observations, peptides corresponding to these regions were synthesized. Sequences corresponding to the 138160 region of all the serotypes have been found to be highly active, but the 194-204 region has comparatively low activity and no activity was found in the remainder of the molecule (Bittle et al. 1982).
Presentation
of the Peptide
to the Host
The initial experiments showed that one inoculation of approximately 100 pg of the 141-160 region, conjugated to a carrier protein, would elicit levels of neutralizing antibody in guinea pigs sufficient to protect them against a severe challenge (10,000 ID,,) of homologous virus directly into the foot pad. However, as little as 0.02 pg of the same amino acid sequence, present as 60 copies on the intact virus particle, will elicit the same level of neutralizing antibody. Consequently a variety of methods for improving the presentation of the peptide has been investigated. These studies have led to the conclusion that multiple copies of the sequence presented on a particle will elicit high levels of neutralizing antibody. For example, a fusion protein consisting of the 141-160 sequence at the N terminus of the hepatitis B virus core protein assembles into particles which elicit levels of neutralizing antibody in guinea pigs approaching those obtained with the equivalent amount of the peptide sequence on the parent virus particle (Clarke et a/. 1987). Experiments in progress indicate that even better antibody responses are obtained by insertion of the 141-160 region into the loop region of the core protein predicted by Argos & Fuller (1988). Clearly the relevant response, in this case neutralizing antibody, is dictated by the conformation of the peptide sequence in the engineered construction. It will be recalled that the level of neutralizing antibody elicited by isolated VP1 or the 12s particle containing five
Table 1. Amino A, subtype 12 Virus A 0
acid sequence
01 the 141-160
region
of VP1 01 four
c D
112
of FMDV,
141 GUY
Ser
GUY
Val
Arg
‘JY
Asp
C D
A 0
variants
Ser Leu
serotype
GIY
150 Ser
Leu
160 Pro
Ser Phe 151 Leu
Ala
Leu Pro Ser Pro
Arc!
Val
Ala
Arg
Gln
Peptides as vaccines copies of WI, VP2 and VP3 is several obtained with the intact virus particle.
The
Need
for a Helper
T Cell
orders
of magnitude
lower
than that
Epitope
The ideal vaccine would contain only those regions of the virus that are required to elicit a protective immune response. In our initial experiments we showed that the 141-160 sequence alone, without carrier protein, would elicit good levels of neutralizing antibody in guinea pigs, provided it contained an added CJJ residue at the C terminus. The CJS residue presumably allows the formation of dimers because, when it is blocked or absent, the response is very low. The response is improved considerably by presenting the sequence as a tetramer or octamer, using the method of multiple antigenic peptide synthesis described by Tam (1988). The response of pigs and cattle to the 141-160 9s peptide alone was poor. This has led us to investigate whether the genetic restriction could be overcome by the addition of appropriate T cell epitopes. Preliminary experiments in mice have shown that genetic restriction can be overcome (Francis et al. 1987). Using mice belonging to the H-2d haplotype, which do not respond to the 141-160 sequence, peptides consisting of residues 141-160 and T cell epitopes from either ovalbumin or sperm whale myoglobin will elicit neutralizing antibody responses as high as are obtained in mice belonging to the H-2k haplotype, which do respond to the 141-160 sequence alone. Extension of this approach to cattle and pigs will show whether the restricted response to the 141-160 sequence can be overcome by the inclusion of either (a) foreign T cell epitopes such as those described for the mouse experiments or those present on the hepatitis B virus core, or (b) epitopes from other regions of the virus particle.
Structural
Studies
The specific immune response of the host to an epitope depends on its shape. In an attempt to define the structural basis for antigenic variation, which is so crucial in foot-and-mouth disease, the structure of the virus has been analysed by X-ray crystallography. Although most of the structure of the virus has yielded to this analysis, that of the immunogenic region of the virus in the 141-160 sequence of VP1 has not yet been solved. However, progress has been made with peptides corresponding to this region. The response of guinea pigs to peptides differing only in positions 148 and 153 of the 141-160 sequence of a virus of serotype A can be distinguished by neutralization and immunoprecipitation tests with the corresponding virus and anti-peptide antisera. Four peptides, with the sequences shown in Table I, could be grouped into two pairs, one consisting of peptides with a pro residue at 153 and the other with a residue of leu or ser (Rowlands et al. 1983). Examination of the peptides by nuclear magnetic resonance and circular dichroism showed that the salt bridge between residues asp 147 and arg 154 in A and C was not present in B and D because of the breakage of the helix by the presence of the pro residue at 153 (Mascagni et al. submitted). Extension of this approach should allow the design of peptides with predetermined specificity and may lead to an understanding of the structural basis of antigenic differences between the serotypes.
Conclusions The results described in this paper show that it is possible vaccine comprising the immunogenic B cell epitope in the virus, linked to a T cell epitope appropriate for the be crucial to identify appropriate T cell epitopes for the
to construct an effective the 141-160 sequence of recipient species. It will relevant species and then
113
F. Brown to present the hybrid peptide as a multimer. With the octamer approach described by Tam (1988) this has become a feasible prospect which has the additional advantage that no carrier protein is required. Moreover, our structural studies at the level of atomic resolution have revealed that the shapes of peptides of antigenic variants of the virus are distinguishable and it is to this area that much attention will be focused in the immediate future. Aside from any practical application of this information it will lead to a better understanding of the immune response at the molecular level.
References ARGOS, P. & FULLER, SD. 1988 A model for hepatitis B virus core protein: prediction of antigenic sites and relationship to RNA virus capsid proteins. EMBO Journal 7, 81’+824. BITTLE, J.E., 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 synthesised peptide predicted from the viral nucleotide sequence. Nature 298, 30-33. CLARKE, B.E., NEWTON, SE., CARROLL, A.R., FRANCIS, M. J., APPLEYARD, G., SYRED, A.D., HIGHFIELD, P.E., ROWLANDS, D.J. & BROWN, F. 1987 Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 330, 381-384. Fox, G., PARRY, N.R., BARNETT, P.V., MCGINN, B., ROWLANDS, D. J. & BROWN, F. 1989 The cell attachment site on foot-and-mouth disease virus includes the amino acid sequence RGD. Journal of General Virology 70, 625632. FRANCIS, M. J., HASTINGS, G.Z., SYRED, A.D., MCGINN, B., BROWN, F. & ROWLANDS, D.J. 1987 Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T cell determinants. Nature 330, 168170. FRENKEL, H.S. 1947 La culture du virus de la fitvre aphteuse sur l’epithelium de la langue des bovides. Bulletin de I’Oflce International des Epixooties 28, 155162. JENNER, E. 1798 An Enquiry into the Causes and Effects of the Variohe Vaccinae. London: Sampson Low. LAPORTE, J., GROSCLAUDE, J., WANTYGHEM, J., BERNARD, S. & ROUZE, P. 1973 Neutralisation en culture cellulaire du pouvoir infectieux du virus de la fitvre aphteuse par des serums provenant de ports immunises h l’aide d’une promine virale purifiee. Comptes
rendus de I’Acadimie
des Sciences Shie
D 276,
3399-3401.
MASCAGNI, P., SILIGARDI, G., VAN DER HOEVEN, S.J., BROWN, F., DRAKE, A.F., NICCOLAI, N., JAMES, C.H., ROWLANDS, D. & GIBBONS, W.A. Structural basis for the antigenic specificity of foot-and-mouth disease virus peptides as deduced from solution conformation studies. Proceedings oftbe NationalAcademy ofsciences ojthe USA Submitted. PASTEUR, L. 1885 Methode pour prevenir la rage apres morsure. Comptes rendus des Shnces de I’Acadimie
des Sciences 101, 765-772.
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