Epitopemapping Angela C. Horsfall, Frank C. Hay, Andy J. Soltys and Meinir G. Jones
The correct identification of antigenic epitopes will greatly aid the diagnosis and prognosis of disease and the identification of targets for immunization/vaccination and immunosuppression. Historically, empirical epitope mapping of protein antigens relied upon either enzymic digestion or cyanogen bromide cleavage into successively smaller fragments retaining epitopic specificity. Later, advances in peptide synthesis technology1,2 and the advent of molecular biology techniques brought epitope mapping more sharply into focus (see Fig. 1). The successful cloning and sequencing of many protein antigens paved the way for the generation of small fragments overlapping in sequence, and the construction of synthetic peptides corresponding to different areas of the protein, which may be probed for reactivity with T or B cells. More recently, a novel development of peptide synthesis on polyethylene pins has enabled use of an enzyme-linked immunosorbant assay (ELISA) technique to screen antibody binding to the covalently linked peptides 3 on the pins. One initial attraction of this technique was the regeneration of the pins by removal of bound antibody and the repeated use of each peptide. In his introduction to the workshop, M. Geysen (Victoria, Australia) presented results on the new synthetic technique that allows pin peptides to be cleaved into physiological solutions for T-cell epitope mapping4 and high-affinity soluble phase interactions with antibodies (see Fig. 1).
The immune response to extrinsic and intrinsic antigens involves specificantigen receptors on T and B cells. The precise antigenic determinants, or epitopes, recognized by these receptors are discrete sequences within the native antigen. The ability to identify and manufacture key epitopes in the immune response has important implications for disease diagnosis and immunointervention. Consequently, increasingly sophisticated technologies are being applied to epitope mapping. This report from a recent workshop* gives a balanced view of progress to date and the challenges ahead.
T-cell epitopes
tors (TCRs) when presented in the context of the appropriate major histocompatibility complex (MHC) molecule. However, several contributors commented on the importance of amino acids that flank the epitope; they may influence processing, MHC binding and presentation. Critical residues can be identified by replacement synthesis. Algorithms exist predicting that T-cell epitopes either bear motifs of polar and hydrophobic residues s or are regions of amphipathic helices6. However, the limitations of epitope predictions were emphasized by the failure of either algorithm to predict a major T-cell epitope on the 19 kDa protein of M. tuberculosis (D. Harris, London). What are the mechanics of identifying T-cell epitopes? Ideally, a simple and low cost approach to T-cell epitope scanning is used to localize epitopes to a particular region of the molecule. Sequences of interest may then be expanded with overlapping synthetic peptides, for which the more expensive pin technology is appropriate. However, it should be borne in mind that post-
Recognition of antigen by T cells requires processing and presentation of short linear peptides. The consensus of opinion is that peptides of less than 12 (probably about 8-9) amino acids can bind to T-cell recep-
':The workshop on Epitope Mapping, organized by A. Horsfall, was held in London on Friday 12 April under the auspices of the British Society for Immunology.
transcriptional modification of proteins may significantly affect antigenicity: for example, some T-cell clones from patients with myasthenia gravis proliferate in response to native acetylcholine receptor (AChR) antigen but not to recombinant fragments or synthetic peptides (G. Harcourt, Oxford). Additionally, T cells may be stimulated by structures that are more complex than simple peptides. Mouse T-cell hybridomas that recognize iodinated thyroglobulin were shown to be stimulated by a nine amino acid peptide containing thyroxine (B. Champion, London). Replacement of the iodinated residue with any other naturally-occurring amino acid abolished stimulation, emphasizing a critical role for iodination of the peptide. As iodine molecules are extremely large and represent almost 50% of the molecular mass of the 9-mer sequence, future studies will aim to explain how this epitope interacts with the MHC and the TCR. Proliferation of CD4 ÷ T cells from healthy individuals to some 15-mer sequences from the human immunodeficiency virus 1 (HIV-1) gag p24 sequence has been observed. Compared with those directed against recall antigens such as tetanus toxoid these responses were low, and low frequencies of responder T cells were identified in the 'memory cell' population. Such results may reflect exposure to crossreactive sequences and indicate the need for caution in assigning 'pathogenic' epitopes (A. Vyakarnam and C. Michie, London). It is notable that MHC class-IIrestricted T cells exhibit diversity of epitope recognition - examples of virus, parasite and AChR antigens were given, whereas class-lrestricted T cells generally obey the 'one haplotype, one epitope' rule. Responses to streptococcal cell wall
© 1991, Elsevier Science Publishers Ltd, UK. 0167 -4919/91/$02.00
lmmunology Today
211
Vol. 12 No. 71991
ill !'iiii
!ii!!iii!
Conventional synthesis
Pepscan
Phage libraries
Peptides used for T end B ceils
Many peptides per synthesis
Many peptides per library Cheap once phage library is established
Cleavable peptides allow T-cell work Peptides can be reused
One peplide per synthes~s Costly for large numbers Peptides cam~t be reused
Can utilize unnatural amino acids Costly for initial screening of large proteins
I
I
Soluble peptides for recognition by antibodies or T cells
Cannot use unnatural amino acids Requires molecular techniques
B-cell peptides often give high noise levels Costly for initial screening of large proteins
lTl'r Rxed ~ d e s
~y
~r
reco~tion
Oeavab~ pep~des for
T-!~ r ~ t i e n
~Antibody recognition
./1
Purification and amplification of phage with relevant Peptide
Relevant peptide
) Replacement analysis
Window analysis Further studies
QRP I~TI YGK
ep leeRTIVGre P IRRRTIVGKnR
JRRTYJ6KRRR
Optimum peptide le~agth Optimum peptide composition Optimum peptide
)
Fig. 1. Epitope mapping using peptides. proteins also showed that T- and B-cell epitopes can overlap (J. Robinson, Newcastle-upon-Tyne).
B-cell epitopes Information on the nature of the protein epitopes recognized by B cells is more complex. Based upon molecular modelling of antibodies reacting with antigens 7, over 90% of B-cell epitopes are thought to be conformational. This may vary depending upon the nature of the antigen: some cell surface proteins, for example bacterial cell wall or viral capsid antigens, may have accessible linear sequences whereas epitopes on intracellular and extracellular proteins are likely to be formed by discontinuous surface sequences. Attempts to map B-cell epitopes have met with mixed success, depending largely on the system under investigation. For example, immunized animals have generally given
clear-cut results irrespective of whether polyclonal or monoclonal antibodies were used. By contrast, the analysis of human sera raised several problems: using the pin technique, the noise:signal ratio is high in human polyclonal sera (normally screened at a dilution of 1:100). Increasing this dilution may improve results as may the use of affinitypurified antibodies. Noise:signal ratios may also be reduced by examining reactivity with pins before and after incubation of sera with native antigen. Serum antibodies that react with native antigen but not with denatured antigen may recognize only conformational epitopes. The problems encountered using human antibodies were shown by a study of both pre- and postimmunization sera from healthy volunteers orally immunized with cholera toxin and analysed using the pin technique. The 'epitopes' identified
ImmunologyToday
212
were identical before and after immunization (D. Lewis, London). Similarly, sera from patients with autoantibodies to an intracellular ribonucleoprotein (La/SS-B) showed peaks of binding to overlapping octapeptides on pins throughout the entire sequence but these areas were mirrored by healthy sera, albeit at lower levels of binding. Pooled healthy IgG (from more than 1000 donors) showed a similar profile and binding was increased or decreased in proportion to the concentration of IgG added to the pins (A. Horsfall, London). As autoimmune sera are frequently hypergammaglobulinaemic, many arbitrarily assigned epitopes may be nonspecific. Affinity-purified antibodies may prove to be the only reliable source of antibodies for this kind of analysis. Further complexities were introduced by data that showed that epitopes recognized by antibodies to
vol. 12 No. 7 1991
synthetic peptides may be masked by glycosylation (D. Davis, Cambridge).
Conformational epitopes Techniques for the mapping of conformational epitopes are currently being developed. Where threedimensional structure is available, predicted discontinuous surface determinants in close contact can be synthesized as two linear sequences that are, subsequently, covalently linked. An alternative is to use the 'mimotope' strategy. This requires affinity-purified antibody and involves the sequential synthesis of peptides starting from a dipeptide and increasing the length with different variations of amino acid sequences until maximal binding to antibody is achieved. This procedure could well be beyond the financial budget of many laboratories and, although success has been reported for monoclonal antibodies to two viral proteins3,8, no data are available for polyclonal responses. An alternative approach to solid phase peptide synthesis has recently been described 9-11. By incorporating mixtures of bases at each stage of synthesis, random oligonucleotides encoding all possible peptides can easily be produced. Use of the appropriate molecular techniques enables insertion of oligonucleotides into phages and the resultant peptide is expressed within the filament protein on the phage surface. As each phage takes up a single oligonucleotide, the expressed peptide will be unique to that phage, enabling screening with the antibody of interest. DNA from the selected phage can then be amplified by polymerase chain reaction and the peptide sequence deduced. So far 6-mer to 15-mer peptides have been produced. A library of peptides expressed in phages would be unlimited in supply and available for all antibody systems (see Fig. 1). By this method, the elucidation of both linear epitopes and conformational mimotopes are theoretically possible, although in its present form this technique is not directly applicable to T-cell epitopes. I. Roitt (London) summarized the salient points to emerge from the workshop. The relevance of using stimulation indices as a measure of T-cell recognition was questioned:
analysis of precursor frequency would be more useful. There was no doubt that pin synthesis of cleavable peptides would be useful for T-cell epitope mapping but the environment surrounding peptides on pins was thought to be unsatisfactory for scanning B-cell epitopes. The newer technology of making biotinlabelled peptides that could be cleaved and immobilized on streptavidin coated microtiter plates may be a more promising alternative (M. Geysen) and data on the phage libraries are eagerly awaited.
Angela Horsfall is at the Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW, UK; Frank Hay, Andy Soltys and Meinir Jones are in the Division of Immunology, Dept of Cellular and Molecular Sciences, St George's Hospital Medical School, London SW17 ORE, UK.
Immunology Today
References 1 Merrifield,R.B. (1963) J. Am. Chem. Soc. 85, 2149-2154 2 Houghten, R.A. (1985) Proc. Natl Acad. Sci. USA 82, 5131-5135 3 Geysen,H.M., Rodda, S.J., Mason, T.M., Tribbick, G. and Schoofs,P.G. (1987) J. Immunol. Methods 102, 259-274 4 Maeji, N.J., Bray,A.M. and Geysen, H.M. (1990) J. Immunol. Methods 134, 23-33 5 Rothbard, J.B. and Taylor, W.R. (1988) EMBO J. 7, 93-100 6 Margalit, H., Spouge,J.L., Cornette, J.L. et al. (1987)J. Immunol. 138, 2213-2229 7 Blundell,T.L, Sibanda, B.L., Sternberg, M.J.E. and Thornton, J.M. (1987) Nature 326, 347-352 8 Geysen,H.M., Rodda, S.J. and Mason, T.J. (1986) Mol. Immunol. 23, 709-715 9 Scott, J.K. and Smith, G.P. (1990) Science 249, 386-390 10 Cwirla, S.E., Peters, E.A., Barrett, R.W. and Dower, W.J. (1990) Proc. Natl Acad. Sci. USA 87, 6378-6382 11 Devlin, J.J., Panganibar, L.C. and Devlin, P.E. (1990) Science 249, 404-406
* I m m u n e parameters in HIV infection - a practical
213
rot 12 No. 7 1991
The correct identification of antigenic epitopes will greatly aid the diagnosis and prognosis of disease and the identification of targets for immunization/vaccination and immunosuppression. Historically, empirical epitope mapping of protein antigens relied upon either enzymic digestion or cyanogen bromide cleavage into successively smaller fragments retaining epitopic specificity. Later, advances in peptide synthesis technology1,2 and the advent of molecular biology techniques brought epitope mapping more sharply into focus (see Fig. 1). The successful cloning and sequencing of many protein antigens paved the way for the generation of small fragments overlapping in sequence, and the construction of synthetic peptides corresponding to different areas of the protein, which may be probed for reactivity with T or B cells. More recently, a novel development of peptide synthesis on polyethylene pins has enabled use of an enzyme-linked immunosorbant assay (ELISA) technique to screen antibody binding to the covalently linked peptides 3 on the pins. One initial attraction of this technique was the regeneration of the pins by removal of bound antibody and the repeated use of each peptide. In his introduction to the workshop, M. Geysen (Victoria, Australia) presented results on the new synthetic technique that allows pin peptides to be cleaved into physiological solutions for T-cell epitope mapping4 and high-affinity soluble phase interactions with antibodies (see Fig. 1).
The immune response to extrinsic and intrinsic antigens involves specificantigen receptors on T and B cells. The precise antigenic determinants, or epitopes, recognized by these receptors are discrete sequences within the native antigen. The ability to identify and manufacture key epitopes in the immune response has important implications for disease diagnosis and immunointervention. Consequently, increasingly sophisticated technologies are being applied to epitope mapping. This report from a recent workshop* gives a balanced view of progress to date and the challenges ahead.
T-cell epitopes
tors (TCRs) when presented in the context of the appropriate major histocompatibility complex (MHC) molecule. However, several contributors commented on the importance of amino acids that flank the epitope; they may influence processing, MHC binding and presentation. Critical residues can be identified by replacement synthesis. Algorithms exist predicting that T-cell epitopes either bear motifs of polar and hydrophobic residues s or are regions of amphipathic helices6. However, the limitations of epitope predictions were emphasized by the failure of either algorithm to predict a major T-cell epitope on the 19 kDa protein of M. tuberculosis (D. Harris, London). What are the mechanics of identifying T-cell epitopes? Ideally, a simple and low cost approach to T-cell epitope scanning is used to localize epitopes to a particular region of the molecule. Sequences of interest may then be expanded with overlapping synthetic peptides, for which the more expensive pin technology is appropriate. However, it should be borne in mind that post-
Recognition of antigen by T cells requires processing and presentation of short linear peptides. The consensus of opinion is that peptides of less than 12 (probably about 8-9) amino acids can bind to T-cell recep-
':The workshop on Epitope Mapping, organized by A. Horsfall, was held in London on Friday 12 April under the auspices of the British Society for Immunology.
transcriptional modification of proteins may significantly affect antigenicity: for example, some T-cell clones from patients with myasthenia gravis proliferate in response to native acetylcholine receptor (AChR) antigen but not to recombinant fragments or synthetic peptides (G. Harcourt, Oxford). Additionally, T cells may be stimulated by structures that are more complex than simple peptides. Mouse T-cell hybridomas that recognize iodinated thyroglobulin were shown to be stimulated by a nine amino acid peptide containing thyroxine (B. Champion, London). Replacement of the iodinated residue with any other naturally-occurring amino acid abolished stimulation, emphasizing a critical role for iodination of the peptide. As iodine molecules are extremely large and represent almost 50% of the molecular mass of the 9-mer sequence, future studies will aim to explain how this epitope interacts with the MHC and the TCR. Proliferation of CD4 ÷ T cells from healthy individuals to some 15-mer sequences from the human immunodeficiency virus 1 (HIV-1) gag p24 sequence has been observed. Compared with those directed against recall antigens such as tetanus toxoid these responses were low, and low frequencies of responder T cells were identified in the 'memory cell' population. Such results may reflect exposure to crossreactive sequences and indicate the need for caution in assigning 'pathogenic' epitopes (A. Vyakarnam and C. Michie, London). It is notable that MHC class-IIrestricted T cells exhibit diversity of epitope recognition - examples of virus, parasite and AChR antigens were given, whereas class-lrestricted T cells generally obey the 'one haplotype, one epitope' rule. Responses to streptococcal cell wall
© 1991, Elsevier Science Publishers Ltd, UK. 0167 -4919/91/$02.00
lmmunology Today
211
Vol. 12 No. 71991
ill !'iiii
!ii!!iii!
Conventional synthesis
Pepscan
Phage libraries
Peptides used for T end B ceils
Many peptides per synthesis
Many peptides per library Cheap once phage library is established
Cleavable peptides allow T-cell work Peptides can be reused
One peplide per synthes~s Costly for large numbers Peptides cam~t be reused
Can utilize unnatural amino acids Costly for initial screening of large proteins
I
I
Soluble peptides for recognition by antibodies or T cells
Cannot use unnatural amino acids Requires molecular techniques
B-cell peptides often give high noise levels Costly for initial screening of large proteins
lTl'r Rxed ~ d e s
~y
~r
reco~tion
Oeavab~ pep~des for
T-!~ r ~ t i e n
~Antibody recognition
./1
Purification and amplification of phage with relevant Peptide
Relevant peptide
) Replacement analysis
Window analysis Further studies
QRP I~TI YGK
ep leeRTIVGre P IRRRTIVGKnR
JRRTYJ6KRRR
Optimum peptide le~agth Optimum peptide composition Optimum peptide
)
Fig. 1. Epitope mapping using peptides. proteins also showed that T- and B-cell epitopes can overlap (J. Robinson, Newcastle-upon-Tyne).
B-cell epitopes Information on the nature of the protein epitopes recognized by B cells is more complex. Based upon molecular modelling of antibodies reacting with antigens 7, over 90% of B-cell epitopes are thought to be conformational. This may vary depending upon the nature of the antigen: some cell surface proteins, for example bacterial cell wall or viral capsid antigens, may have accessible linear sequences whereas epitopes on intracellular and extracellular proteins are likely to be formed by discontinuous surface sequences. Attempts to map B-cell epitopes have met with mixed success, depending largely on the system under investigation. For example, immunized animals have generally given
clear-cut results irrespective of whether polyclonal or monoclonal antibodies were used. By contrast, the analysis of human sera raised several problems: using the pin technique, the noise:signal ratio is high in human polyclonal sera (normally screened at a dilution of 1:100). Increasing this dilution may improve results as may the use of affinitypurified antibodies. Noise:signal ratios may also be reduced by examining reactivity with pins before and after incubation of sera with native antigen. Serum antibodies that react with native antigen but not with denatured antigen may recognize only conformational epitopes. The problems encountered using human antibodies were shown by a study of both pre- and postimmunization sera from healthy volunteers orally immunized with cholera toxin and analysed using the pin technique. The 'epitopes' identified
ImmunologyToday
212
were identical before and after immunization (D. Lewis, London). Similarly, sera from patients with autoantibodies to an intracellular ribonucleoprotein (La/SS-B) showed peaks of binding to overlapping octapeptides on pins throughout the entire sequence but these areas were mirrored by healthy sera, albeit at lower levels of binding. Pooled healthy IgG (from more than 1000 donors) showed a similar profile and binding was increased or decreased in proportion to the concentration of IgG added to the pins (A. Horsfall, London). As autoimmune sera are frequently hypergammaglobulinaemic, many arbitrarily assigned epitopes may be nonspecific. Affinity-purified antibodies may prove to be the only reliable source of antibodies for this kind of analysis. Further complexities were introduced by data that showed that epitopes recognized by antibodies to
vol. 12 No. 7 1991
synthetic peptides may be masked by glycosylation (D. Davis, Cambridge).
Conformational epitopes Techniques for the mapping of conformational epitopes are currently being developed. Where threedimensional structure is available, predicted discontinuous surface determinants in close contact can be synthesized as two linear sequences that are, subsequently, covalently linked. An alternative is to use the 'mimotope' strategy. This requires affinity-purified antibody and involves the sequential synthesis of peptides starting from a dipeptide and increasing the length with different variations of amino acid sequences until maximal binding to antibody is achieved. This procedure could well be beyond the financial budget of many laboratories and, although success has been reported for monoclonal antibodies to two viral proteins3,8, no data are available for polyclonal responses. An alternative approach to solid phase peptide synthesis has recently been described 9-11. By incorporating mixtures of bases at each stage of synthesis, random oligonucleotides encoding all possible peptides can easily be produced. Use of the appropriate molecular techniques enables insertion of oligonucleotides into phages and the resultant peptide is expressed within the filament protein on the phage surface. As each phage takes up a single oligonucleotide, the expressed peptide will be unique to that phage, enabling screening with the antibody of interest. DNA from the selected phage can then be amplified by polymerase chain reaction and the peptide sequence deduced. So far 6-mer to 15-mer peptides have been produced. A library of peptides expressed in phages would be unlimited in supply and available for all antibody systems (see Fig. 1). By this method, the elucidation of both linear epitopes and conformational mimotopes are theoretically possible, although in its present form this technique is not directly applicable to T-cell epitopes. I. Roitt (London) summarized the salient points to emerge from the workshop. The relevance of using stimulation indices as a measure of T-cell recognition was questioned:
analysis of precursor frequency would be more useful. There was no doubt that pin synthesis of cleavable peptides would be useful for T-cell epitope mapping but the environment surrounding peptides on pins was thought to be unsatisfactory for scanning B-cell epitopes. The newer technology of making biotinlabelled peptides that could be cleaved and immobilized on streptavidin coated microtiter plates may be a more promising alternative (M. Geysen) and data on the phage libraries are eagerly awaited.
Angela Horsfall is at the Kennedy Institute of Rheumatology, Bute Gardens, London W6 7DW, UK; Frank Hay, Andy Soltys and Meinir Jones are in the Division of Immunology, Dept of Cellular and Molecular Sciences, St George's Hospital Medical School, London SW17 ORE, UK.
Immunology Today
References 1 Merrifield,R.B. (1963) J. Am. Chem. Soc. 85, 2149-2154 2 Houghten, R.A. (1985) Proc. Natl Acad. Sci. USA 82, 5131-5135 3 Geysen,H.M., Rodda, S.J., Mason, T.M., Tribbick, G. and Schoofs,P.G. (1987) J. Immunol. Methods 102, 259-274 4 Maeji, N.J., Bray,A.M. and Geysen, H.M. (1990) J. Immunol. Methods 134, 23-33 5 Rothbard, J.B. and Taylor, W.R. (1988) EMBO J. 7, 93-100 6 Margalit, H., Spouge,J.L., Cornette, J.L. et al. (1987)J. Immunol. 138, 2213-2229 7 Blundell,T.L, Sibanda, B.L., Sternberg, M.J.E. and Thornton, J.M. (1987) Nature 326, 347-352 8 Geysen,H.M., Rodda, S.J. and Mason, T.J. (1986) Mol. Immunol. 23, 709-715 9 Scott, J.K. and Smith, G.P. (1990) Science 249, 386-390 10 Cwirla, S.E., Peters, E.A., Barrett, R.W. and Dower, W.J. (1990) Proc. Natl Acad. Sci. USA 87, 6378-6382 11 Devlin, J.J., Panganibar, L.C. and Devlin, P.E. (1990) Science 249, 404-406
* I m m u n e parameters in HIV infection - a practical
213
rot 12 No. 7 1991
