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New approaches to vaccination against foot-and-mouth disease
New approaches to vaccination against foot-and-mouth disease Fred Brown
The economically important foot-and-mouth disease has been successfully controlled in Western Europe by comprehensive immunization using killed vaccine. The author discusses the wisdom of abandoning this policy, and outlines research into alternative vaccines using recombinant DNA technology, in particular using synthetic peptides.
Keywords:Foot-and-mouth disease : immunization policy; recombinant DNA technology; peptide HISTORICAL
INTRODUCTION
Vaccination against infectious diseases has been one of the more successful stories in the history of human and veterinary medicine. Not only has it contributed in large measure to the eradication of the devastating disease of smallpox but it has also been instrumental in the control of other human diseases such as diphtheria, tetanus, measles, poliomyelitis, rubella and yellow fever. Equally important has been the development in veterinary medicine of vaccines to control leptospirosis, rinderpest and foot-and-mouth disease in cattle, clostridial diseases in sheep, cattle and pigs, diarrhoea caused by Escherichia coil in piglets, and Newcastle and M a r e k ' s disease in poultry. Nevertheless, the empiricism underlying these vaccines leaves much to be desired. Although variolation was practised in many Far Eastern countries as long ago as the tenth century, the introduction by Jenner ~ of cowpox virus as a vaccine for the prevention of smallpox is generally regarded as the start of vaccination. Almost a century later, Pasteur and his colleagues developed live vaccines for chicken cholera, anthrax and rabies in rapid succession (see, for example, Pasteur-'). All these vaccines consisted either of a naturally occurring variant or relative {in the case of smallpox t of the virulent organism or one which had been 'weakened" by serendipitous laboratory manipulations. The products, which infected the host for the most part without causing clinical disease, elicited protective immune responses. It was established shortly afterwards, however, that United States Department of Agriculture, PO Box 848, Greenport, New York, 11944-0848, USA 0264410)(,.'92/1410224:)5 ~3 1992 Butterworth-HeinemannLtd 1022 Vaccine, Vol. 10, Issue 14, 1992
multiplication of the organism was not a necessary requirement for vaccination because secreted proteins, appropriately treated (toxoided) would afford protection against diphtheria and tetanus. Moreover, the demonstration by Scruple 3 that protection 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 + and poliomyelitis s respectively. The application of comprehensive vaccination policies against foot-and-mouth disease ( F M D ) in Western Europe, starting with Holland in 1952. has been so successful in controlling the disease, with tlo outbreaks since 1989, that the European Community countries, perhaps misguidedly, have now ceased to wtccinate against the disease. The information in Figures I and 2 shows how devastating the disease can be and emphasizes the impact of vaccination in three of the countries in Western Europe. Since the whole purpose of vaccination is to prevent the occurrence of a diseasc, rather than merely to control it, the effect of abandoning vaccination in Western Europe will be observed keenly throughout the veterinary and farming communities. The reasons for the abandonment of the vaccination programmes against foot-and-mouth disease appear to have been based on first, cost and second, the indisputable fact that most of the outbreaks in recent years have been caused either by escape of virus front vaccine production units or the use of improperly inactivated vaccines ~'. With modern methods of containment and the proven efl'ectiveness of imines for inactivating the virus ++s, this failure to ensure safety is indefensible. It ix regrettable, therefore, that alternative vaccines emerging from the new recombinant D N A technology are not yet available, first, to obviate the need to produce the quantities of virus that are needed for the production of conventional inactivated vaccines, and second, to eliminate entirely the need to use virus in the vaccines. Nevertheless, research on alternative vaccines continues becausc it is perceived that such products may be required in the less developed countries. In considering such products, it is worthwhile at the outset to list the commercial criteria to be met by any new wtccine. The new product must be innocuous and it must also be as
Vaccination against foot-and-mouth disease: F. Brown 100000
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Figure 1 Farms affected by foot-and-mouth disease in (a) Holland and (b) Germany between 1880 and 1930
a
good or better than the current vaccines. If it is also cheaper, then the only remaining concern would be to make it attractive to the market. Experience in Western Europe has demonstrated that the current inactivated F M D vaccines are very efficient when properly administered. Moreover, a single inoculation-will elicit a protective immune response against as severe a challenge as 10 000 IDso of virus inoculated directly into the tongue, or close contact with an infected animal. This is an important feature of the current vaccines, which is a credit to the manufacturers who produce them; this feature is often overlooked by those who make inactivated vaccines against other diseases where multiple inoculations are used to elicit the level of response which will protect. Consequently, the scientific and technical challenges are great. However, the scientific rewards, as well as the commercial rewards, are also great. Our approaches to vaccination have lacked adventure for far too long and the new technology affords many opportunities to remedy that situation. The emergence of recombinant DNA technology and techniques for nucleic acid sequencing, monoclonal antibody production and the more rapid solving of protein structures by X-ray crystallography, allied to the major advances which are being made in our understanding of the immune response at the molecular level, have provided those involved in vaccination with the opportunity to design and develop entirely new products for foot-and-mouth disease bearing in mind that protection is directly related to the level of neutralizing antibody in the serum. Several avenues are being explored which are based on this new technology. The basic information on the virus on which these approaches are being made is next described. VIRUS AND VIRUS-SPECIFIED ANTIGENS
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Four virus-specified antigens are found in harvests of virus grown in tissue culture cells: (a) the infectious virus particle; (b) the so-called 'empty' particle; (c) the 12S protein sub-unit, and (d) the virus infection-associated (VIA) antigen. All four particles have been purified and analysed. Their properties are given in Table 1. The four antigens can readily be separated and these fractionation studies have shown that most of the neutralizing activity is elicited by the intact virus particles. However, the empty particles also elicit neutralizing antibody, provided they are stabilized by a cross-linking agent ~. In contrast, the 12S protein sub-unit elicits only very low levels of neutralizing antibody and the VIA antigen is devoid of this activity.
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Year Figure 2 Effect of mass vaccination on the number of outbreaks of foot-and-mouth disease in (a) Holland, (b) France and (c) the former Federal Republic of Germany. Arrows indicate the start of mass vaccination
With some strains of the virus the immunogenic activity is greatly reduced by treatment with trypsin 1°. Since only VP1 is affected by this treatment the immunodominant region has been assumed to be located on this protein. Moreover, VPI isolated from virus particles possesses low but significant immunogenic activity whereas the other proteins are devoid of activity1 Although these results do not provide decisive evidence that there are no immunogenic sites on VP2, VP3 or VP4, the marked loss of activity which occurs when virus
Vaccine, Vol. 10, Issue 14, 1992
1023
Vaccination against foot-and-mouth disease. F. Brown Table 1
Antigenic components of foot-and-mouth disease virus
Components
Sedimentation constant (S)
Virus particle
146
Empty particle
75
Protein subunit
12
Virus infection-associated antigen
3.8
Composition
Immunogenicity
1 molecule ssRNA ( M 2.6 x 106). 60 copies each of VPI-3 (M 24 x 103) and VP4 (M,: 8 x 103)
High
60 copies each of VPO", VP1 and VP3
High
Pentamer ofVP1 3
Low
RNA polymerase
Nil
( M r : - 5 6 x 103)
aVPO comprises VP2 and VP4 covalently linked
of serotype O is treated with trypsin, when these proteins are apparently unaffected, provides good evidence that the major activity is associated with VPI. These observations led to considerable effort by Biogen and Genentech about a decade ago to express this protein in Escherichia coli cells with the objective of producing a sub-unit vaccine 12. However, the low immunogenic activity of the expressed protein, compared with that of the virus particle, led to its abandonment as a commercial proposition.
Empty particles Although early experiments had indicated that the empty particles had very low immunogenicity compared with the intact virus particles, it was found that 'empties' were as efficient as the full particles in absorbing neutralizing antibody from immune serum 9. The problem was overcome by stabilizing the empties with formaldehyde 9. Subsequent experiments showed that with some strains of the virus the untreated empty particles were as immunogenic as the full particles ~3. Recent experiments by Grubman and his colleagues 14 have shown that empty particles produced by expression of the DNA corresponding to that part of the viral RNA coding for the capsid protein region will elicit neutralizing antibody in guinea-pigs and in pigs. Although the levels of neutralizing activity are low, it is anticipated that stabilization of the product will lead to enhanced activity.
Peptides A third approach has been provided by the observation that a portion of VPI will elicit neutralizing antibodylS 17. About 30 years ago Andercr 18 showed that fragments of the coat protein of tobacco mosaic virus would elicit the production of antibodies which neutralized virus infectivity. Synthesis of the fragment in an active form using peptide chemistry led to the expectation that totally synthetic vaccines could be produced and the work of Sela and his colleagues with the bacteriophage MS2 a9 and a variety of microorganisms reinforced this view. Not only would such an approach be aesthetically pleasing but it would also have considerable practical advantages (Table 2), not least of which would be the stability of a peptide, so that it could be stored indefinitely at ambient temperatures. Moreover, provided adequate delayed release mechanisms can be designed, booster doses of the peptide could be delivered from implants. This approach depends on being able to identify the active fragment or fragments on the virus
1024
Vaccine, Vol. 10, Issue 14, 1992
Table 2 1 2 3 4 5 6 7
Advantages of a peptide vaccine
Product chemically defined Stable indefinitely No infectious agent present No large-scale production plant required No downstream processing required Can be designed to stimulate appropriate immune responses Provides opportunity to use delayed release mechanisms
particle. Four methods havc bcen used to identify putative immunogenic sequences on the capsid protein VP1 of FMDV. Strohmaier and his colleagues ~5 used the classical method of hydrolysing the protein with cyanogen bromide or various proteolytic enzymes and testing the fragments for tlteir ability to clicit neutralizing antibody. This approach allowed the identification of two regions 146 154 and 200 213 which they predicted would elicit such a response. In an indirect approach which took advantage of the occurrence of the virus as a multiplicity of antigenic variants, Bittle et al. ~ reasoned that such variation would be reflected in sequence differences in the immunogenic protein. Comparison of the sequences of VP1 molecules from viruses belonging to three different serotypes showed that, while much of the sequence is conserved, there is considerable variability at residues 42-61, 131 160 and 193 204. Antibody against the 42 61 region did not react with intact virus particles suggesting that this region was not on the surface. In contrast the 131 160 and 193 204 regions elicited neutralizing antibody although the 131 160 region was considerably more active than the 193 204 sequence. Subsequent studies by X-ray crytallography 2° have shown that both these regions are on the surface of the virus particle whereas 42 61 lies underneath the promincnt GH surface loop. The "Pepscan" method is a more direct serological approach, in which ovcrlapping peptides corresponding to the entire sequence of VP1 were synthesized on polyethylene pins, which were then allowed to react with neutralizing antibody 2~. Those peptides which reacted with the antibody were then detected by screening with anti-species antibody against the virus immune serum. This approach also pinpointed a region within the 141-160 sequence of VPI. The fourth approach ~v was based on the reasoning that a good candidate would possess a strong helical structure with hydrophobic and hydrophilic zones on
Vaccination against foot-and-mouth disease: F. Brown
opposite sides of the helix. Such a structure was found at residues 144-159. The fact that all four approaches highlighted the importance of the 141-160 region resulted in a concentration of effort on this sequence. The recently solved structure of the virus 2° has revealed that the dominant immunogenic site is located within a prominent disordered loop, which is in accord with the contention that immunogenicity is greater in those segments of protein molecules which are flexible. Additionally, the attachment site of the virus is also located within the same loop region so that antibody directed against this sequence will prevent virus attachment to susceptible cells. Moreover, the C-terminus of VP1 is located close to the loop region, providing structural evidence for the enhanced immunogenicity which has been reported for the hybrid peptide comprising residues 141-160 and 200-213 referred to above z2'23. P R E S E N T A T I O N O F T I l E 141-160 P E P T I D E TO T H E H O S T Because of the cost of experiments in the natural hosts, most of the work with experimental vaccines is done in mice and guinea-pigs. These experiments have provided interesting and significant insights into the response of the host animal to the peptide antigen. For example, it is frequently stated that peptides alone are not immunogenic. In our first experiments with the 141 - 160 peptide we found that if it was linked to keyhole limpet haemocyanin, a single inoculation of ~100/~g was sufficient to protect guinea-pigs against a severe challenge infection 16. However, the peptide was also immunogenic in the absence of a carrier protein if it was polymerized either with glutaraldehyde or air-oxidized after adding a cysteine residue at each terminus 17. Moreover, subsequent work has shown that the unconjugated peptide is immunogenic when delivered in liposomes or when allowed to form disulphide dimers via an added non-natural cysteine at the C-terminus. This carrierindependent activity is due to the presence of sites in the 20 amino acid sequence which not only elicited neutralizing antibody (B-cell epitopes) but also sites which provided T-cell help for antibody production (Th-cell epitopes). The marked immunogenicity of the peptide without the need for carrier protein and the observation that its administration as a polymer enhances its activity made it an ideal candidate for evaluating Tam's multiple antigenic peptide (MAP) system z4. This system, which provides for a direct solid-phase synthesis of a peptide antigen on a lysine backbone, was first used as an octamer. |n an experiment with the 141 160 peptide to compare the responses to monomer, dimer, tetramer and octamer constructs built on the same principle, we have found that neutralizing antibody responses known to protect guinea-pigs against challenge infection were obtained with a single inoculation of 0.8 4.0/~g of peptide, presented as a tetramer or octamer, whereas 20/~g of dimer were required to elicit a similar level of antibody 25. The important message to emerge from the work, therefore, was that presentation of the peptide as multiple copies gives a greatly enhanced response. This conclusion amply confirms the demonstration that presentation of the peptide fused to the N-terminus of hepatitis B core antigen, which spontaneously
assembles into 27 nm particles with approximately 100 copies of the peptide repeated over the surface, is also highly immunogenic, with as little as 0.2 #g of peptide eliciting protective levels of neutralizing antibody 26. THE NEED FOR T-CELL EPITOPES Although the neutralizing antibody response to the uncoupled peptide in guinea-pigs approached that obtained with the virus particle, cattle and pigs gave a very much poorer response. This non-responsiveness has been studied in a genetically defined population of inbred mice 2v. Congenic B10 mice, genetically identical at all loci except those of the H-2 complex, were compared for their neutralizing antibody response to the peptide. Of the strains studied only B10.BR and B10.RIII(7INS) responded, with the first of these strains producing a higher response than that ofoutbred Charles River mice. Among the non-responding congenic mice the B10.D2 strain (which is H-2 d haplotype) was chosen because several H-2d-restricted T-cell epitopes had been characterized previously. It was found that sequences from ovalbumin and sperm whale myoglobin, synthesized as hybrid peptides at the C-terminus of the FMDV peptide, overcame the non-responsiveness of the B10.D2 mice. More recently, synthetic peptides corresponding to regions of VP1 have been identified which stimulate the proliferation of peripheral blood lymphocytes of cattle immunized with an inactivated FMDV vaccine. A peptide corresponding to amino acids 21-40 induced a population of T cells which recognized the native protein and provided help when linked to the 141 160 sequence28. THE PROBLEM OF ANTIGENIC VARIATION The occurrence of FMDV as seven distinct serotypes poses considerable problems in the control of the disease because animals recovered from infection with a virus from one serotype, or vaccinated with a monvalent vaccine, are still susceptible to infection with viruses from the remaining six serotypes. Moreover, antigenic variation within a serotype is so great that a vaccine prepared from one isolate may not afford protection against infection with other isolates from the s a m e serotype. Sequencing the gene coding for the capsid protein region of the virus has shown that the greatest variability occurs within the sequence coding for amino acids 141-160 of VP1. One example will serve to illustrate the importance of this sequence in determining specificity. Four viruses, isolated from a single sample of bovine tongue epithelium from an animal infected with a virus of serotype A, were found to have the same amino acid sequence across the entire capsid protein region except for substitutions at positions 148 and 153 of VP1. These viruses can be distinguished by cross-neutralization and radioimmunoprecipitation tests using antibody against the virus particles and the corresponding peptides. Since antigenic variation is determined by the configuration of the epitope, this sequence variation has provided the opportunity to study this problem with a small molecule possessing immunological activity. Circular dichroism and nuclear magnetic resonance studies have shown that configurational differences in the peptides can be detected 29'3°. This correlation of configuration with
Vaccine, Vol. 10, Issue 14, 1992 1025
V a c c i n a t i o n a g a i n s t f o o t - a n d - m o u t h d i s e a s e . F. B r o w n
serological properties means that this type of structural analysis will be valuable for studying the folding of the peptides and may eventually lead to the design of molecules with defined antigenic properties. CONCLUSIONS There would be clear advantages if it were possible to construct a peptide, with the appropriate T-cell epitope, that not only provided a wide spectrum of protection against the antigenic variants that occur in the field but also presented none of the dangers, perceived or otherwise, associated with the current vaccines. Moreover, such a molecule, which would be free from the variability found with biological products, would offer the opportunity to investigate the basis of immunogenicity and antigenic specificity in molecular and structural terms. Even with our present knowledge, which is still largely empirical, we have shown that the activity of the peptide can be increased by several orders of magnitude by simple manipulations. REFERENCES 1 2 3
4
5 6
7
8
9
10 11
12
Jenner, E. An Enquiry into the Causes and Effects of the Variolae Vaccinae. Sampson Low, London, 1798 Pasteur, L. Methode pour prevenir la rage apres morsure. C. R. Acad. Sci. 1885, 101,765-772 Semple, D. The preparation of a safe and efficient antirabic vaccine. Scientific Memorandum Officers' Medical and Sanitary Depts, No. 44, Govt of India, Calcutta, 1911 Frenkel, H.S. La culture du virus de la fievre aphteuse sur I'epithelium de la langue des bovides. Bull. Off. Int. Epiz. 1947, 28, 155 162 Salk, J.E. A concept of the mechanism of immunity for preventing paralysis in poliomyelitis. Ann. NY Acad. Sci. 1955, 61, 1023-1036 Beck, E. and Strohmaier. K. Subtyping of European foot-and-mouth disease virus strains by nucleotide sequence determination. J. Virol. 1987, 61. 1621 1629 Brown, F. and Crick. J. Application of agar gel diffusion analysis to a study of the antigenic structure of inactivated vaccines prepared from the virus of foot-and-mouth disease. J. Immun. 1958. 82, 444 447 Bahnemann, H.G. Inactivation of virus antigens for vaccine preparation with particular reference to the application of binary ethyleneimine. Vaccine 1990, 8, 75 88 Rowlands, D.J., Sangar, D.V. and Brown, F. A comparative chemical and serological study of the full and empty particles of foot-and-mouth disease virus. J. Gen. Virol. 1975, 26, 227 238 Wild. T.F. and Brown, F. Nature of the inactivating action of trypsin on foot-and-mouth disease virus. J. Gen. Virol. 1967, 1,247 250 Laporte, J., Grosclaude, J., Wantyghem, J., Bernard, S. and Rouze, P. Neutralisation en culture cellulaire du pouvoir infectieux du virus de la fievre aphteuse par des s~rums provenant de porcs immunises & I'aide d'une proteine virale purifiee. CR. Acad. Sci. Paris 1973, 276, 3399 3401 Kleid, D.G_ Yansura, D., Small, B., Dowbenko. D., Moore, D.M.. Grubman, M.J. et al. Cloned viral protein vaccine for foot-and-mouth disease; responses in cattle and swine. Science 1981, 21& 1125 1129
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17
18
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22
23
24
25
26
27
28
29
30
Rweyemamu, M.M.. Terry. G. and Pay, T.W.F. Stability and immunogenicity of empty particles of foot-and-mouth disease vrrus Arch. Virol. 1979, 59. 69 79 Lewis. S.A, Morgan, D. and Grubman, M.J. Expression, processing and assembly of foot-and-mouth disease virus capsid structures in heterologous systems: Induction of a neutralizing antibody response in guinea pigs. J. Virol. 1991, 65, 6572 6580 Strohmaier. K., Franze, R. and Adam, K.-H. Localization and characterisation of the antigenic portion of the foot-and-mouth disease virus protein. J. Gen. Virol. 1982, 59, 295 306 Bittle, J.L., Houghten, R.A., Alexander, H., Shinnick, T.M., Sutcliffe, J.C., Lerner, R.A. et ~,. Protection against foot-and-mouth disease by immunization with a chemically synthesised peptide predicted from the viral nucleotide sequence. Nature (London) 1982, 298, 30 33 Pfaff, E., Mussgay, M., Bohm, H.O., Schulz, E.G. and Schaller. H. Antibodies against a preselected peptide recognize and neutralize foot-and-mouth disease virus. EMBO J. 1982, 1,669 674 Anderer, F . A . Versuche zur Bestimmung der serologisch Terminanten Gruppen des Tobakmosaikvirus. Z. Naturforsch. B 1963, 188, 1010 1014 Langebeheim, H., Arnon, R. and Sela, M. Antiviral effect on MS2 coliphage obtained with a synthetic antigen. Proc. Natl. Acad. Sci. USA 1976, 73, 3636--3640 Acharya, R., Fry, E., Stuart, D., Fox, G., Rowlands, D. and Brown, F. The three-dimensional structure of foot-and-mouth disease virus at 2.9A resolution. Nature (London) 1989, 337, 709 711 Geysen, H.M., Meloen, R.H. and Barteling, S.J. Use of peptide synthesis to probe viral antigens for epitopes to a resolution of a single amino acid. Proc. Natl. Acad. Sci. USA 1984, 81, 3998-4002 Parry, N.R., Ouldridge, EJ., Barnett, P.V., Rowlands, D.J, Brown, F., Bittle, J.L. et al. Identification of neutralizing epitopes of foot-and-mouth disease virus. In: Vaccines 85. Molecular and Chemical Basis of Resistance to Parasitic, Bacterial and Viral Diseases. (Eds Lerner, R.A., Chanock, R.M. and Brown, F.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1985, pp 211 216 DiMarchi, R., Brooke, G., Gale, C., Cracknell, V., Doel, T. and Mowat. N. Protection of cattle against foot-and-mouth disease by a synthetic peptide. Science 1986, 232, 639 641 Tam, J.P. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Natl. Acad. Sci. USA 1988, 65, 5409 5413 Francis, M.J., Hastings, G.Z., Brown, F., McDermed, J., Lu, Y.-A. and Tam, J. Immunological evaluation of the multiple antigen peptide (MAP) system using the major immunogenic site of foot-and-mouth virus. Immunology 1991, 73, 249 254 Clarke, B.E.. Newton, S.E., Carroll, A.R., Francis, M.J., Appleyard. G.. Syred, A.D. et al. Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein. Nature 1987, 330, 381 384 Francis, M.J., Hastings, GZ., Syred, A.D., McGinn. B., Brown, F. and Rowlands, DJ. Non-responsiveness to a foot-and-mouth disease virus peptide overcome by addition of foreign helper T-cell determinants. Nature 1987. 330, 168 170 Collen, T., DiMarchi. R. and Doel, T.R. A T-cell epitope in VP1 of foot-and-mouth disease virus is immunodominant for vaccinated cattle. J. Immunol. 1991, 146, 749 755 Siligardi, G., Drake, A.F., Mascagni, P., Rowlands, D.J., Brown, F. and Gibbons, W.A. A CD strategy for the study of polypeptide folding/unfolding. Int. J. and Pept. Protein Res. 1991, 38, 519 527 Siligardi, G., Drake, A.F., Mascagni, P., Rowlands, D.. Brown, F. and Gibbons, W.A. Correlations between the conformations elucidated by DC spectroscopy and the antigenic properties of four peptides of the foot-and-mouth disease virus. Eur. J. Biochem. 1991, 199, 445 451
The economically important foot-and-mouth disease has been successfully controlled in Western Europe by comprehensive immunization using killed vaccine. The author discusses the wisdom of abandoning this policy, and outlines research into alternative vaccines using recombinant DNA technology, in particular using synthetic peptides.
Keywords:Foot-and-mouth disease : immunization policy; recombinant DNA technology; peptide HISTORICAL
INTRODUCTION
Vaccination against infectious diseases has been one of the more successful stories in the history of human and veterinary medicine. Not only has it contributed in large measure to the eradication of the devastating disease of smallpox but it has also been instrumental in the control of other human diseases such as diphtheria, tetanus, measles, poliomyelitis, rubella and yellow fever. Equally important has been the development in veterinary medicine of vaccines to control leptospirosis, rinderpest and foot-and-mouth disease in cattle, clostridial diseases in sheep, cattle and pigs, diarrhoea caused by Escherichia coil in piglets, and Newcastle and M a r e k ' s disease in poultry. Nevertheless, the empiricism underlying these vaccines leaves much to be desired. Although variolation was practised in many Far Eastern countries as long ago as the tenth century, the introduction by Jenner ~ of cowpox virus as a vaccine for the prevention of smallpox is generally regarded as the start of vaccination. Almost a century later, Pasteur and his colleagues developed live vaccines for chicken cholera, anthrax and rabies in rapid succession (see, for example, Pasteur-'). All these vaccines consisted either of a naturally occurring variant or relative {in the case of smallpox t of the virulent organism or one which had been 'weakened" by serendipitous laboratory manipulations. The products, which infected the host for the most part without causing clinical disease, elicited protective immune responses. It was established shortly afterwards, however, that United States Department of Agriculture, PO Box 848, Greenport, New York, 11944-0848, USA 0264410)(,.'92/1410224:)5 ~3 1992 Butterworth-HeinemannLtd 1022 Vaccine, Vol. 10, Issue 14, 1992
multiplication of the organism was not a necessary requirement for vaccination because secreted proteins, appropriately treated (toxoided) would afford protection against diphtheria and tetanus. Moreover, the demonstration by Scruple 3 that protection 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 + and poliomyelitis s respectively. The application of comprehensive vaccination policies against foot-and-mouth disease ( F M D ) in Western Europe, starting with Holland in 1952. has been so successful in controlling the disease, with tlo outbreaks since 1989, that the European Community countries, perhaps misguidedly, have now ceased to wtccinate against the disease. The information in Figures I and 2 shows how devastating the disease can be and emphasizes the impact of vaccination in three of the countries in Western Europe. Since the whole purpose of vaccination is to prevent the occurrence of a diseasc, rather than merely to control it, the effect of abandoning vaccination in Western Europe will be observed keenly throughout the veterinary and farming communities. The reasons for the abandonment of the vaccination programmes against foot-and-mouth disease appear to have been based on first, cost and second, the indisputable fact that most of the outbreaks in recent years have been caused either by escape of virus front vaccine production units or the use of improperly inactivated vaccines ~'. With modern methods of containment and the proven efl'ectiveness of imines for inactivating the virus ++s, this failure to ensure safety is indefensible. It ix regrettable, therefore, that alternative vaccines emerging from the new recombinant D N A technology are not yet available, first, to obviate the need to produce the quantities of virus that are needed for the production of conventional inactivated vaccines, and second, to eliminate entirely the need to use virus in the vaccines. Nevertheless, research on alternative vaccines continues becausc it is perceived that such products may be required in the less developed countries. In considering such products, it is worthwhile at the outset to list the commercial criteria to be met by any new wtccine. The new product must be innocuous and it must also be as
Vaccination against foot-and-mouth disease: F. Brown 100000
80000
L 60 000
0 40000
.Q E Z
20 000 I
0 1880
1890
1900
a
1910
1920
1930
1910
1920
1930
Year 80000
60 000 L X~
o
40000
..Q
E Z
20000
0 1880
b
,
i 1890
1900
Year
Figure 1 Farms affected by foot-and-mouth disease in (a) Holland and (b) Germany between 1880 and 1930
a
good or better than the current vaccines. If it is also cheaper, then the only remaining concern would be to make it attractive to the market. Experience in Western Europe has demonstrated that the current inactivated F M D vaccines are very efficient when properly administered. Moreover, a single inoculation-will elicit a protective immune response against as severe a challenge as 10 000 IDso of virus inoculated directly into the tongue, or close contact with an infected animal. This is an important feature of the current vaccines, which is a credit to the manufacturers who produce them; this feature is often overlooked by those who make inactivated vaccines against other diseases where multiple inoculations are used to elicit the level of response which will protect. Consequently, the scientific and technical challenges are great. However, the scientific rewards, as well as the commercial rewards, are also great. Our approaches to vaccination have lacked adventure for far too long and the new technology affords many opportunities to remedy that situation. The emergence of recombinant DNA technology and techniques for nucleic acid sequencing, monoclonal antibody production and the more rapid solving of protein structures by X-ray crystallography, allied to the major advances which are being made in our understanding of the immune response at the molecular level, have provided those involved in vaccination with the opportunity to design and develop entirely new products for foot-and-mouth disease bearing in mind that protection is directly related to the level of neutralizing antibody in the serum. Several avenues are being explored which are based on this new technology. The basic information on the virus on which these approaches are being made is next described. VIRUS AND VIRUS-SPECIFIED ANTIGENS
Io2F
/
~
.~ lO5 _/~b 1o3 -
Four virus-specified antigens are found in harvests of virus grown in tissue culture cells: (a) the infectious virus particle; (b) the so-called 'empty' particle; (c) the 12S protein sub-unit, and (d) the virus infection-associated (VIA) antigen. All four particles have been purified and analysed. Their properties are given in Table 1. The four antigens can readily be separated and these fractionation studies have shown that most of the neutralizing activity is elicited by the intact virus particles. However, the empty particles also elicit neutralizing antibody, provided they are stabilized by a cross-linking agent ~. In contrast, the 12S protein sub-unit elicits only very low levels of neutralizing antibody and the VIA antigen is devoid of this activity.
\
[
~
\
lO 2
,o°
,
,
-;-
C 105
Capsid protein VPI
104~
103 102 10 0 1950
I 1960
1970
_ 1980
Year Figure 2 Effect of mass vaccination on the number of outbreaks of foot-and-mouth disease in (a) Holland, (b) France and (c) the former Federal Republic of Germany. Arrows indicate the start of mass vaccination
With some strains of the virus the immunogenic activity is greatly reduced by treatment with trypsin 1°. Since only VP1 is affected by this treatment the immunodominant region has been assumed to be located on this protein. Moreover, VPI isolated from virus particles possesses low but significant immunogenic activity whereas the other proteins are devoid of activity1 Although these results do not provide decisive evidence that there are no immunogenic sites on VP2, VP3 or VP4, the marked loss of activity which occurs when virus
Vaccine, Vol. 10, Issue 14, 1992
1023
Vaccination against foot-and-mouth disease. F. Brown Table 1
Antigenic components of foot-and-mouth disease virus
Components
Sedimentation constant (S)
Virus particle
146
Empty particle
75
Protein subunit
12
Virus infection-associated antigen
3.8
Composition
Immunogenicity
1 molecule ssRNA ( M 2.6 x 106). 60 copies each of VPI-3 (M 24 x 103) and VP4 (M,: 8 x 103)
High
60 copies each of VPO", VP1 and VP3
High
Pentamer ofVP1 3
Low
RNA polymerase
Nil
( M r : - 5 6 x 103)
aVPO comprises VP2 and VP4 covalently linked
of serotype O is treated with trypsin, when these proteins are apparently unaffected, provides good evidence that the major activity is associated with VPI. These observations led to considerable effort by Biogen and Genentech about a decade ago to express this protein in Escherichia coli cells with the objective of producing a sub-unit vaccine 12. However, the low immunogenic activity of the expressed protein, compared with that of the virus particle, led to its abandonment as a commercial proposition.
Empty particles Although early experiments had indicated that the empty particles had very low immunogenicity compared with the intact virus particles, it was found that 'empties' were as efficient as the full particles in absorbing neutralizing antibody from immune serum 9. The problem was overcome by stabilizing the empties with formaldehyde 9. Subsequent experiments showed that with some strains of the virus the untreated empty particles were as immunogenic as the full particles ~3. Recent experiments by Grubman and his colleagues 14 have shown that empty particles produced by expression of the DNA corresponding to that part of the viral RNA coding for the capsid protein region will elicit neutralizing antibody in guinea-pigs and in pigs. Although the levels of neutralizing activity are low, it is anticipated that stabilization of the product will lead to enhanced activity.
Peptides A third approach has been provided by the observation that a portion of VPI will elicit neutralizing antibodylS 17. About 30 years ago Andercr 18 showed that fragments of the coat protein of tobacco mosaic virus would elicit the production of antibodies which neutralized virus infectivity. Synthesis of the fragment in an active form using peptide chemistry led to the expectation that totally synthetic vaccines could be produced and the work of Sela and his colleagues with the bacteriophage MS2 a9 and a variety of microorganisms reinforced this view. Not only would such an approach be aesthetically pleasing but it would also have considerable practical advantages (Table 2), not least of which would be the stability of a peptide, so that it could be stored indefinitely at ambient temperatures. Moreover, provided adequate delayed release mechanisms can be designed, booster doses of the peptide could be delivered from implants. This approach depends on being able to identify the active fragment or fragments on the virus
1024
Vaccine, Vol. 10, Issue 14, 1992
Table 2 1 2 3 4 5 6 7
Advantages of a peptide vaccine
Product chemically defined Stable indefinitely No infectious agent present No large-scale production plant required No downstream processing required Can be designed to stimulate appropriate immune responses Provides opportunity to use delayed release mechanisms
particle. Four methods havc bcen used to identify putative immunogenic sequences on the capsid protein VP1 of FMDV. Strohmaier and his colleagues ~5 used the classical method of hydrolysing the protein with cyanogen bromide or various proteolytic enzymes and testing the fragments for tlteir ability to clicit neutralizing antibody. This approach allowed the identification of two regions 146 154 and 200 213 which they predicted would elicit such a response. In an indirect approach which took advantage of the occurrence of the virus as a multiplicity of antigenic variants, Bittle et al. ~ reasoned that such variation would be reflected in sequence differences in the immunogenic protein. Comparison of the sequences of VP1 molecules from viruses belonging to three different serotypes showed that, while much of the sequence is conserved, there is considerable variability at residues 42-61, 131 160 and 193 204. Antibody against the 42 61 region did not react with intact virus particles suggesting that this region was not on the surface. In contrast the 131 160 and 193 204 regions elicited neutralizing antibody although the 131 160 region was considerably more active than the 193 204 sequence. Subsequent studies by X-ray crytallography 2° have shown that both these regions are on the surface of the virus particle whereas 42 61 lies underneath the promincnt GH surface loop. The "Pepscan" method is a more direct serological approach, in which ovcrlapping peptides corresponding to the entire sequence of VP1 were synthesized on polyethylene pins, which were then allowed to react with neutralizing antibody 2~. Those peptides which reacted with the antibody were then detected by screening with anti-species antibody against the virus immune serum. This approach also pinpointed a region within the 141-160 sequence of VPI. The fourth approach ~v was based on the reasoning that a good candidate would possess a strong helical structure with hydrophobic and hydrophilic zones on
Vaccination against foot-and-mouth disease: F. Brown
opposite sides of the helix. Such a structure was found at residues 144-159. The fact that all four approaches highlighted the importance of the 141-160 region resulted in a concentration of effort on this sequence. The recently solved structure of the virus 2° has revealed that the dominant immunogenic site is located within a prominent disordered loop, which is in accord with the contention that immunogenicity is greater in those segments of protein molecules which are flexible. Additionally, the attachment site of the virus is also located within the same loop region so that antibody directed against this sequence will prevent virus attachment to susceptible cells. Moreover, the C-terminus of VP1 is located close to the loop region, providing structural evidence for the enhanced immunogenicity which has been reported for the hybrid peptide comprising residues 141-160 and 200-213 referred to above z2'23. P R E S E N T A T I O N O F T I l E 141-160 P E P T I D E TO T H E H O S T Because of the cost of experiments in the natural hosts, most of the work with experimental vaccines is done in mice and guinea-pigs. These experiments have provided interesting and significant insights into the response of the host animal to the peptide antigen. For example, it is frequently stated that peptides alone are not immunogenic. In our first experiments with the 141 - 160 peptide we found that if it was linked to keyhole limpet haemocyanin, a single inoculation of ~100/~g was sufficient to protect guinea-pigs against a severe challenge infection 16. However, the peptide was also immunogenic in the absence of a carrier protein if it was polymerized either with glutaraldehyde or air-oxidized after adding a cysteine residue at each terminus 17. Moreover, subsequent work has shown that the unconjugated peptide is immunogenic when delivered in liposomes or when allowed to form disulphide dimers via an added non-natural cysteine at the C-terminus. This carrierindependent activity is due to the presence of sites in the 20 amino acid sequence which not only elicited neutralizing antibody (B-cell epitopes) but also sites which provided T-cell help for antibody production (Th-cell epitopes). The marked immunogenicity of the peptide without the need for carrier protein and the observation that its administration as a polymer enhances its activity made it an ideal candidate for evaluating Tam's multiple antigenic peptide (MAP) system z4. This system, which provides for a direct solid-phase synthesis of a peptide antigen on a lysine backbone, was first used as an octamer. |n an experiment with the 141 160 peptide to compare the responses to monomer, dimer, tetramer and octamer constructs built on the same principle, we have found that neutralizing antibody responses known to protect guinea-pigs against challenge infection were obtained with a single inoculation of 0.8 4.0/~g of peptide, presented as a tetramer or octamer, whereas 20/~g of dimer were required to elicit a similar level of antibody 25. The important message to emerge from the work, therefore, was that presentation of the peptide as multiple copies gives a greatly enhanced response. This conclusion amply confirms the demonstration that presentation of the peptide fused to the N-terminus of hepatitis B core antigen, which spontaneously
assembles into 27 nm particles with approximately 100 copies of the peptide repeated over the surface, is also highly immunogenic, with as little as 0.2 #g of peptide eliciting protective levels of neutralizing antibody 26. THE NEED FOR T-CELL EPITOPES Although the neutralizing antibody response to the uncoupled peptide in guinea-pigs approached that obtained with the virus particle, cattle and pigs gave a very much poorer response. This non-responsiveness has been studied in a genetically defined population of inbred mice 2v. Congenic B10 mice, genetically identical at all loci except those of the H-2 complex, were compared for their neutralizing antibody response to the peptide. Of the strains studied only B10.BR and B10.RIII(7INS) responded, with the first of these strains producing a higher response than that ofoutbred Charles River mice. Among the non-responding congenic mice the B10.D2 strain (which is H-2 d haplotype) was chosen because several H-2d-restricted T-cell epitopes had been characterized previously. It was found that sequences from ovalbumin and sperm whale myoglobin, synthesized as hybrid peptides at the C-terminus of the FMDV peptide, overcame the non-responsiveness of the B10.D2 mice. More recently, synthetic peptides corresponding to regions of VP1 have been identified which stimulate the proliferation of peripheral blood lymphocytes of cattle immunized with an inactivated FMDV vaccine. A peptide corresponding to amino acids 21-40 induced a population of T cells which recognized the native protein and provided help when linked to the 141 160 sequence28. THE PROBLEM OF ANTIGENIC VARIATION The occurrence of FMDV as seven distinct serotypes poses considerable problems in the control of the disease because animals recovered from infection with a virus from one serotype, or vaccinated with a monvalent vaccine, are still susceptible to infection with viruses from the remaining six serotypes. Moreover, antigenic variation within a serotype is so great that a vaccine prepared from one isolate may not afford protection against infection with other isolates from the s a m e serotype. Sequencing the gene coding for the capsid protein region of the virus has shown that the greatest variability occurs within the sequence coding for amino acids 141-160 of VP1. One example will serve to illustrate the importance of this sequence in determining specificity. Four viruses, isolated from a single sample of bovine tongue epithelium from an animal infected with a virus of serotype A, were found to have the same amino acid sequence across the entire capsid protein region except for substitutions at positions 148 and 153 of VP1. These viruses can be distinguished by cross-neutralization and radioimmunoprecipitation tests using antibody against the virus particles and the corresponding peptides. Since antigenic variation is determined by the configuration of the epitope, this sequence variation has provided the opportunity to study this problem with a small molecule possessing immunological activity. Circular dichroism and nuclear magnetic resonance studies have shown that configurational differences in the peptides can be detected 29'3°. This correlation of configuration with
Vaccine, Vol. 10, Issue 14, 1992 1025
V a c c i n a t i o n a g a i n s t f o o t - a n d - m o u t h d i s e a s e . F. B r o w n
serological properties means that this type of structural analysis will be valuable for studying the folding of the peptides and may eventually lead to the design of molecules with defined antigenic properties. CONCLUSIONS There would be clear advantages if it were possible to construct a peptide, with the appropriate T-cell epitope, that not only provided a wide spectrum of protection against the antigenic variants that occur in the field but also presented none of the dangers, perceived or otherwise, associated with the current vaccines. Moreover, such a molecule, which would be free from the variability found with biological products, would offer the opportunity to investigate the basis of immunogenicity and antigenic specificity in molecular and structural terms. Even with our present knowledge, which is still largely empirical, we have shown that the activity of the peptide can be increased by several orders of magnitude by simple manipulations. REFERENCES 1 2 3
4
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7
8
9
10 11
12
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