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Structural and functional approaches to the study of protein antigenicity
ImmunologyToday,Vol. 10, No. 8, 1989
-brier 6 Kimura, H. and Wilson, D. (1984) Nature 308, 463-464 7 Kappler, J., Roehm, N. and Marrack, P. (1987) Ce1149, 273-280
B Kisielow, P., Bl(~thmann, H., Staerz, U.D. etal. (1988) Nature
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333, 742-746 9 Qin, S., Cobbold, S., Benjamin, R, and Waldmann, H. (1989) J. Exp. Meal. 169, 779-794 10 Lo, L)., 8urkly. L.C.,Widera, G. etal.(1988) Cell 53, 159-168 11 Miller, J.F.A.P.,Morahan, G., Allison, J. etal. (1989) Irnmunol. Rev. 107, 109-123 12 Miller, J.F.A.P.,Morahan, G. and Allison, J. (1989) ImmunoL Today ~O, 53-57 13 Sakaguchi,S., Takahashi,T. and Nishizuka,Y. (1982) J. Exp. Med. 156, 1577-1586 14 Taguchi. O., Takahashi,T., Seto, M, etal.(1986)J. Exp. Meal. 164, 60-71 15 Taguchi, O. and Nishizuka,Y. (1987)]. Exp. Meal. 165,
Repressionof academicsin China Sir,
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The repression by communist party hard-liners of the pro-democracy movement initiated by students in Beijing has led to grave concern for
the safety and intellectual freedom of academics in China. As scientists who are committed to freedom of thought and expression, you must be concerned about the imminent loss of these freedoms and the personal safety of our counterparts in China. We beg you to express, in
Strudural
the strongest possible terms, your abhorrence of any physically or intellectually repressive action that might be taken against academics in China.
Staffof the ImmunologySection,Department of Pathology,Universityof HongKong. (Received5 June1989)
andfundionalapproachesto the studyof proteinantigenid
Structural and functional approaches to the study of protein antigeniC1 lead to two different perceptions of the nature of protein epitopes. The structural approach concentrates on the pure~/ spatial arrangements of atoms found in the antigenantibody complex and shows that at least 15 amino acid residues may be implicated in each epitope. The fun~'onal approach, which introduces the additional dimension of time, takes the form of cross-reactive binding measurements and leads to the view that a smaller number of residues are implicated in each epitope. Furthermore, as functional binding assaysare irretrievably operational in character, different types of epitopes are identified by the use of different probes. Three current controversies i~~ the field of protein antigenicity are discussed here: the mechanism of antigen-antibody interaction, the suitability of peptides as synthetic vaccines and the valuP of prediction methods for locating epitopes in proteins. "Sdence is not just the making of observations: it is the making of inferences on the basis of observations within the framework of a theory "1. This remark by the philosopher of science, D.L Hull is particularly apt for the science of immunology which is replete with inductive
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t6 Bandeira,A., Coutinho, A., Carnaud, C., Jacquemart, F. and Fomi, L. (1989) Proc NatlAcad. Sci. USA 86, 272-276 17 Roser,B. (1989) ImmunoL Rev. 107, 179-202 18 Pereira, P., Bandeira, A., Coutinho, A. etal. (1989) Annu. Rev. Immunol. 7, 209-249 19 Lundkvist, I., Coutinho, A., Varela, F. and Holmberg, D. Proc Natl Acad. Sci. USA (in press) 20 8andeira, A., Larsson,E-L.,Forni, L. eta/. (1987) Eur. ]. ImrnunoL 17, 901-908 21 Pereira,P., Larsson,E-L.,Forni, L. etaL (1985) Proc. Nat/ Acad. 5eL USA 82, 7691-7695 22 Coutinho, A. Immuno/. Rev.(in press) 23 Holmberg, D., Forsgren,S., Ivars,F. and Coutinho, A. (1984) Eur. J. ImmunoL 14, 435-441 24 Forni, L., Heusser,C. and Coutinho, A. (1988)Ann. Inst. PasteurImrnunol. 139, 245-256
Laboratoired'lmrnunochirnie,Institutde BiologieMoldc.ulaireet Cellulaire, 15 ~JeDesca,'tes,67084Strasbourg,France.
MarcB.V.Vm R c mrtd inferences that are far from deductively certain. In the field of protein antigen!city there are currently three major controversies: first, the mechanism of antigen-antibody interaction; second, the suitability of synthetic peptides for mimicking the antigenic sites of proteins and acting as potential vaccines, and third, the ability of various algorithms to predict the location of antigenic sites in proteins solely on the basis of amino acid sequence data. To resolve these cont;oversies, it is important to differentiate clearly between observation and inference. This in turn requires that the operational constraints of immunochernical observations are recognized and that the concepts and theoretical assumptions underlying the inferences are clearly defined. In this review, Marc Van Regenmortel analyses the observations and inferences underlying these three controversies. It will become clear that a major problem lies in the conceptual jump needed to 9o frum str~cture to function and from function to structure. Translating observations of structures into biological processes and functions turns out to be as difficult in immunology as in the rest of biology. 1989, ElsevierScience Publishers Ltd. UK. 0167-4919189/$02.00
Immunology Today, Vol. 10, No. 8, 1989
The mechanismof antigeHntibody interaction There are two opposing views of the mechanism of antigenic recognition. According to the lock-and-key model, the binding sites of antibody molecules simply recognize patches at the surface of antigens that are complementary to them in shape and chemical properties. This model stresses the fact that antibodies must have access to an antigenic site in order to bind and it has led to the view that antibody accessibility is the primary intrinsic determinant of antigenicity2,3. Proponents of this model hold that the binding process does not lead to any significant conformational changes in the antigen 4. In contrast the induced fit model proposes that the mechanism of binding occurs through a process of induced complementarity involving significant side-chain movements of the antigen and possibly minor movements of the backbone conformation S. This second model stresse~ the importance of the observed correlation between antigenicity and local mobility of the peptide main chain 6-8 and may be described by the analogy of the adaptation between hand and glove. In order to distinguish iF, each of the two models what is exper~,~nental observation from what is interpretation, it is helpful to recall how immunologists define antigenic sites and localize them in proteins.
o
Table 1. Methodsusedto localizeepitopesin proteins Method
Typeof epitope recognized
Criterionfor Average allocatingresiduesto nu~berof epitope residues iden~ecl in epitope
1 X-ray Discontinuous VanderWaal's crystallographyof epitopereactingwith conta~in epitopeantigen-Fab homologousantib0dy paratopeinterface complexes
15
2 Studyof crossreactivebinding of peptide fragmentswith anti-protein antibodies
Continuousepitope Residualbindingof cross-reactingwith linearfragmentabove heterologous thresholdof assay antibody
3-8
3 Studyofcrossrea~ivebinding of proteinwith anti-peptide antibodies
Continuousepitope Inductionof crosscross-reactingwith reactiveantibodies heterologous antibody
3-8
Determinationof Continuous,cross- Abrogationof crossMethods usedto localizeepitopesin proteins criticalresiduesin rea~ngepitope reactivityby The antigenic sites or epitopes of a protein correspond peptideby containing critical substitutionof critical to those parts of the molecule that are specificafly systematic residuesinterspersed andfunctionally recognized by the binding sites or paratopes of antibody replacementwith withirrelevant relevantresidues molecules. Epitope:. Jre relational entities since they can otheraminoacids residues be recognized only in an operational sense by the binding of complcmentary paratopes. Observation of Studyof cross- Discontinuous Abrogation of crossspace-filling models of globular proteins shows that very reactivity epitope reactivityby few linear stretches of residues are present at the surface between substitutiono~jitica~ of a protein. If it is assumed that all epitope-paratope homologous residue interfaces in proteins involve a surface of about 700 ~2 proteinsor point (as shown in the case of lysozyme9,1°), it follows from mutants the known folding pattern of many globular proteins that no surface epitope region is likely to contain only atoms from a continuous stretch of residues ~1. Antibody molecules are thus likely to recognize a set of residues in short peptides. As a result, our knowledge of protein the antigen that are not contiguous in the sequence, but epitopes concerns mainly incomplete epitopes that have are brought together by the folding of the p~-ptide chain retained only some of the original structural features or by the juxtaposition of two separate chains. Such an present before fragmentation of the ~rotein 12. This epitope is usually referred to as a 'discontinuous' or knowledge can be obtained because cT the polyspeci'assembled epitope', as opposed to the term 'con- ficity of antibodies, that is their ability to cross-react with tinuous epitope', which describes the situation where a a variety of more or less closely related epitopes. short linear stretch of residues is recognized by the However, it should be appreciated that, by their very antibody. It is customary to give the label 'continuous nature, these studies can identify only those structures epitope' to any linear peptide fragment of a protein that that cross-react (in most cases rather poorly) and that is found to react with antibodies raised against the intact they cannot specify, all the features responsible for the molecule. This practice can lead to confusion since what interaction between the antigen and its homologous is called a continuous (or sequential or linear) epitope of antibody. The different approaches 1~-1s that have been used for the protein, in fact, usually represents only a portion of a larger discontinuous epitope. In generat, antibodies in- mapping protein epitopes are summ3rized in Table 1. It duced by a native protein and directed to a complex should be noted that only method 1 (Table 1) is based on discontinuous epitope cross-react only weakly with an a structural analysis, since it is the only method that gives epitope fragment constituted by a linear peptide; an selective attention to the visual appearance of the spatial additional reason for this weak cross-reaction is that the arrangements found in the antigen-antibody complex at peptide usually has a conformation different from the a specific time. in contrast, all other methods correscorresponding region in the protein Most studies aimed pond to a functional analysis taking the form of binding at unravelling the antigenic structure of proteins have measurements. Since the criteria used for allocating focused not on antigenicity per se but on the phenom- residues to an epitope by method 1 are purely structural enon of cross-reactive antigenicity betvveen prc:eins and and spatial, this method cannot provide direct evidence
3-S
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Immunology Today, Vol. 10, No. 8, 1989
B
~A~-.....-----~A~ 1
2
rig. 1 . . ~ a ~ c representa~bnof S/TUctural(AJ and funct/0nal(8) ana~/s of antJ'genan~x~/interaclfon. In A, ~.a spa~alarrangementof the ep/tope-paratopeinterfaceis mualized and a total of 12 hydrogen bonds (--) are identified as in the complex betweer antibody and lysozymc~. This number of hydrogen bonds corresponds to more than 20 kcal moll of binding energy, equivalentto an affinity constant of morn than 10is. Since the affin;Cyob.~,n,adby Amit et a~9 was only about 10s, the difference is dueto thebreaking of hydrogen bonds ~at existed between the residues and solvent molecules prior to the assoda~on of epitope and paratope. This dynamic aspect cannot be represented in a static structural picture. In B, the arrow indicates the additional dimension of time which is an e~enEal ingredient in func~'onalana,~. Sincethis fourth dimensioncannot be easilyvisualiz~ in a spatial representa~on,one usual~refers to states I and 2 of the sy~em.
that every residue contributes to the binding reaction. On the other hand, the criteria used for identifying antigenic residues by methods 2-5 (Table 1) are functional and these methods which are based on binding measurements introduce the additional dimension of time and therefore analyse function and not structure (Fig. 1). As pointed ou~ by Lambert and Hughes is, structuralism and functionalism are two "different ways of seeing" and these two approaches represent one of the major dlchotomies in biology. As shown in Table 1, the structuralist perspective inherent in method 1 leads to the view that an epitope con~sts of anproximate!y 15 residues that are in contact with the paratope9,1o,~7. As pointed out by Getzoff et aL s, various crystallographic groups use different definitions for residues involved in the interface; whatever the defin!tion of interface (for example, residues within van der Waals contact distance, residues buried to a certain radius probe sphere, residues with side chains interacting directly), it is clear that the visual observation of a contact between epitope and paratope still necessitates a decision as to what constitutes a significant contact needed for the binding interaction to occur. Residues in the interface that a:ually contribute to the binding energy are identifiable only in binding measurements. As indicated in Table 1, methods 2-5 which operate within a functionalist perspective actually analyze cross-reactivity instead of antigenic reactivity; these methods lead to the conclusion that a much smaller number of residues (between 1 and 5) are critical to antibody binding and thus that the epitope defined in a functional sense involves fewer residues than the epitope defined in structural terms. AS discussed elsewhere ~8-2o, binding measurements are necessarily of an operational nature: depending on the type of probe used for assessing immunochemical binding (free peptide versus conjugated peptide, sh3rt peptide versus a more constrained long peptide, liquid versus solid-phase immunoassay, monoclonal versus polyclonal antibody reagent), it is unavoidable that the same residues of the antigen wili not always be scored as
participating in different types of binding assays. If epitopes are defined in a binding assay, it is to be expected that the experimental conditions of the assay could, for instance, influence the conformation of the peptide; assay conditions will in fact determine whether binding is observed or not, and thus whether certain residues are scored as belonging to the functional epitope. Although some authors s maintain that this definition of epitope is inconsistent, it simply reflects the operational nature of the concept of functional epitopes. An operational detmition gives meaning to a term, because it enables us to decide when the term applies by observing a response under specified test conditions21. The relevance of operationalism in science is linked to its insistence that many scientific objects of study can be properly defined only through the description of the experimental conditions that allow these objects to be observed. Attempts to define the inherent epitopes of a protein on a purely structural basis, for instance by saying that they are synonymous with surface exposure or with accessibility to a spherical probe 2, go counter to the immunological and functional nature of the epitope concept. Such an approach disregards the fact that a structural analysis is purely spatial, while a functional analysis is spatio-temporal. Recently, Getzoff et al. s suggested that the practice of defining epitopes operationally results in confusion because of the difficulty of integrating results from different experimental techniques. These a!,thors suggested that by making a distinction between critical residues (those identified in binding experiments) and contact residues (those defined crystallographically) it would be pcssible to remove the inconsistency arising from the use of different methods and assays. Unfortunately, structural and functional approaches to the study of protein antigenicity unavoidably lead to results that are incommensurate with each other (with respect to the dimension of time) and, in addition, functional binding assays are irretrievably operational in character. From binding measurementsto inferred mecharJsm "When it comes to understanding the mechanism of antigen-antibody interaction, one needs to extrapolate from a static structural picture to a spatio-temporal and dynamic process that cannot be frozen in time (Fig. 1). For instance, an X-ray crystallographic study of a peptide-antibody complex could demonstrate that a certain peptide had adopted a native-liKe conformation when bound to an anti-protein antibody; however, as pointed out by Crumnton 22, this structural information could not establish if the observed complex resulted from the antibody having selected one conform~;tion from a mixture of d;fferent conformations, or if the recognition process involved an induced complementarity subsequent to *~',einitial peptide--antibody interaction. Argume ,t:~ in favour of a contribution of induced complementarity to antigen-7,,: recognition have been developed an the basis of results obtained with the protein myc.hemerythrin. Using a method in which all possible overlapping he;'~-peptides were synthesized on a linear polymer of polyacrylic acid and tested for antigenic activity while still bound to the solid phase, seven continuous epitopes were identified in myohemerythrin 23. In order to assesswhich amino acids
Immunology'Today,Vol. 10, No. 8, 1989
within each synthesized peptide are essential for antibody binding, each residue was replaced, in turn, by the other 19 amino acids. Certain residues could be replaced by all common amino acids without affecting binding by antibodies; however, a total of 35 residues were found to be critical for binding and could not be replaced at all without impairing the antigenic reactivity5. When the position of these 35 critical residues in the native myohemerythrin structure was examined, each antigenic site was found to contain one or more buried critical side chains that were inaccessible to the solvent. The authors, therefore, proposed a reaction mechanism in which antibody binding to solvent-exposed amino acids causes a rearrangement (breaking of salt bridges or hydrogen bonds) followed by side-chain rotations which uncover previously buried atoms of the antigen. The examples of myohemerythrin epitopes discdssed by Getzoff et al. s are very suggestive and make the proposed mechanism of interaction by induced complementarity highly plausible. However, it should be noted that the contribution of critical residues was assessed from the measurement of cross-reactions between short peptides and anti-protein antibodies and that no direct evidence was presented that these residues are also critical for antibody binding to the native protein antigen. If the binding constant of the antibody for the peptide is much lower than that for the protein, it is conceivable that certain substitutions in the peptide could simply reduce binding below the measurable threshold while this would not be the case with the native protein. Experiments with myohemerythrin analogues obtained by site-directed mutagenesis will clarify this point. A possible role for induced fit in the reaction mechanism has also been inferred from the observed correlation between regions of local mobility in the protein and the position of short peptide fragments that cross-rea~ with the protein s-7,23. The cross-reactivity between intact protein and short peptides has been investigated both with anti-protein and with anti-peptide antibodies and both types ef study indicated that there was a frequent bias for continuous epitopes being located in regions of local mobility. However, since it is unlikely that the short peptide probes used in these studies can mimic constrained elements of secondary structure, this type of analysis will not necessarily reveal the presence of antibodies that do recognize secondary structure. It would indeed be unwarranted to conclude that the immune system is biased against the recognition of ordered structures on the basis of measurements that exclude long peptides that are likely to be more structured. Local mobility in the protein could help to make the corresponding region more like a short peptide and thus enhances cross-reactive antigenicity; at the same time, a longer peptide might resemble more closely a constrained feature of the secondary structure present in the protein and show good cross-reactivity for that reason. Either type of study is biased operationally and should not be used to make generalizations about which intrinsic structural feature determines all possible forms of protein antigenicity including cross-reactive antigenicityl s.19. Some authors reiec~. ~he functional significance of movements of a few angstroms and do not concede that this might facilitate the mutual adjustment of epitopes and paratopes4. They point out that no major confor-
mational changes have been observed in the lysozyme molecule after binding to antibody, and suggest that the location of epitopes simply correlates with the most exposed regions of the protein. ~urface. The search for a primary correlate with antigenLcLty, such as accessibility, appears to proceed from a type of causal thinking that should be abohshed from biology. It is now generally recognized that the systems under investigation in the biological sciences exhibit such a high degree of complexity that traditional notions of linear causality do not apply z4. A more appropriate analytical framework is 'network causality' in which a large number of factors or variables are seen to influence a particular feature of the system instead of causing it. No single factor can be equated with the cause of antigenicity since a great many factors may determine it.
Synthetic peptide vaccines The possibility of using synthetic peptides as vaccines against viral, bacterial and parasitic diseases has evoked considerable interestzs,26 and remains a major driving force that stimulates much current research in the field of synthetic polypeptides27. Although it is frequently stated that the potential usefulness of synthetic peptides as vaccines was discovered around 1980, it is the German virologist F.A. Anderer who should be credited with this discoverj which was made about 15 years earlier. Anderer and his colleagues, working with tobacco mosaic virus (TMV), showed in 1963-1965 (for a review see Ref. 15) that short synthetic peptides corresponding to the carboxyterminal region of the viral protain were able to induce antibodies that neutralized viral infectivity2~. Some later workers failed to appreciate that the neutralization of this plant virus by antibodies followed most of the characteristic features of animal virus neutralization 29. Because of the host-based compartmentalization of virologists, it took 15 years for the approach shown by Anderer to be successful with a plant viru~ to be applied to animal viruses. Within a few years, it was then shown that vaccination of animals with synthetic paptides could lead to protective immunity against foot-and-mouth disease, influenza, hepatitis B, diphtheria and choler.~zs,z6.3°. In order to be effective, a synthetic peptide vaccine must possess a high level of immunogenicity and induce antibodies that cross-react extensively with the pathogen. The immunogenic capacity of a peptide refers to its ability to induce an immune response and depends on extrinsic host factors such as the immunoglobulin gene repertoire, self-tolerance and various cellular regulatory mechanisms 31.3z. These factors, as well as the role of helper T cells33, are beyond the scope of this review. The claim that immunization with peptides leads to a very high frequency of induction of antibodies that can recognize the native protein 34-36 gave rise to the view that it is very easy to obtain anti-peptide antibodies of so-called predetermined specificity that cross-react with a related epitope of the cognate protein 36. Recently, this claim has been challenged on the basis of findings showing that intact cytochrome c activates only a small fraction of peptide-primed B lymphocytes37. The contention that antibodies against a hlghiy disordered state (the peptide) recognize the highly ordered state (the folded protein) has been called the order-disorder paradox 38.
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Immunology Today, Vol. 10, No. 8, 1989
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rig, 2. Predictedsegmentaltr~bility profile of rnyoglobin calculated~th the scale of Karplus and ~huId z according to the smoothing procedure deso~bedin Ref. 49. The solid squares conesx~d to amino adds that are part of known continuousepitopesof myoglob~n.Theopen squaresrepresentthe averagevalueof the mobility parameterover the entire sequenceand the two hoEzontalbroken linesrepresent+_0.7 so from the mean. Suchan interval correspondsto 50% of the population. Whenthe cut-off level for correctprediction wasset at the mean +0.7 so, the meff~d iden~ed 45% of the known antigenic residues (from Van Regenrnortelet
al.29.
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As discussed elsewhere 27, to show that anti-peptide antibodies react with a truly native protein, the protein antigen should preferably not be adsorbed to plastic in a solid-phase immunoassay since this is known to at !ea~ partly denature proteins39-41. However, if a solid-phase assay is used, it is then necessary to include contro!s In which the liquid-phase protein is allowed to compete with plate-bound antigen and inhibition to a level approaching 100% occurs 42. Furthermore, the antigen should preferably not be labelled since this treatment could also induce some denaturation. In the experiment~ that the ,~bove-mentioned views were based on, these conditions were not met. For instance, in experiments carried out with 20 synthetic peptides representing over 75% of the influenza haemagglutinin sequence, it was observed that 18 of the 20 peptides elicited antibodies that reacted with purified haemagglutinin or intact virus, but that none of the peptides reacted with anti-haemagglutinin antibodies present in influenza virus antisera 34. It was also shown that the cross-reacting antipeptide antibodies were not directed againsta rare native-like conformation of the peptide 3s. Since the correct peptide conformation was not a rare occurrence, there was no explanation for the observation that none of the 20 peptides reacted with anti-haemagglutinin antibodies. Subsequently it has been suggested that this type of result is obtained because peptides show a marked conformational preference in solution, and that such a preferred conformation may be stabilized on the surface of the carrier protein or when the peptide binds to the B-cell receptor during
immune stimulation 38, Since no evidence was presented that the anti-peptide antibodies actually recognized the native haemagglutinin, it seems more plausible to infer that the haemagglutinin had been denatured by adsorption to the plastic and by the methanol incubation step that was used in the ~ssay. As far as the extremely low level of cross-reactivity observad with influenza virus is concerned 34, this is probably due to the presence of a small number of denatured haemagglutinin molecules in the preparation 43. In conclusion, these observations do not support the notion that when synthetic peptides are used as immunogens, antibodies cross-reactive with the native protein are easily raised against almost any part of the molecule. Nor do the reported observations justify the view that the correct tertiary conformation of an epitope need not be reproduced in a synthetic peptide in orde: to have an effective synthetic vaccine. Although it seems probable that neutralization epitopes ~n a pathogen (that is those inducing neutralizing antibodies) are preserved only in antigens with an intact tertiary or quaternary structure, it remains unclear at present to what extent it will be necessary to mimic the native conformation of neutralizing epitopes in order to achieve protective immunity by means of synthetic peptides, in view of the intense research activity in this area26,44, the answer to this question should be available soon. Developing peptides suitable for vaccination is a more difficult task than selecting peptides able to induce antibodies that simply cross-react with the cognate protein. Although anti-peptide antibodies are extremely useful for isolatiJ,=l and characterizing gene products 36.43, the high success rate in this case is due in part to the fact that the cross-reactive antibodies need not necessarily be specific for the native conformation of the protein to detect the antigen in many of the currently used imrnunoassays. !:h.ep._red_i~ionof epi~-,~s in proteins In an attempt to understand the structural basis of protein antigenicity many investigators analyze model proteins of known three-dimensional structure and look for a correlation between certain structural parameters and the location of epitopes6.7. In most cases, the tertiary structure information is reduced to a linear scale of amino acid sequence, and an attempt is made to correlate Lhe location of continuous epitopes with structural properties such as accessibility, protrusion, mobility and hydrophilicity2.3.7.a,4s.Although it is generally recognized that most protein epitopes are discontinuous, the emphasis on continuous epitopes in such studies is due to the potential usefulness of predicting which linear peptide fragments are likely to possess cross-reactive antigenicity with the intact protein. Since for most proteins the only available structural information today concerns their amino acid sequence, it is clearly the prediction of cross-reactive antigenicity of short linear peptides which is of practical interest to most investigators. Few studies have addressed the issue of mimicking discontinuous epitopes by synthesis 14,~. Relative scales describing the structural propensity of each of the 20 common amino acids have been derived and these are commonly used for constructing prediction profiles and for locating the position of cross-raactive, continuous epitopes46-48. In a recent study, the predictive value of algorithms based on eight propensity scales
ImmunologyToday, Vol. 10, No. 8, 1989
of the amino acids has been compared, using as a criterion of effectiveness the number of residues correctly predicted to be antigenic in four well-studied proteins a9. None of the methods achieved a high level of correct precliction, although two algorithms based on a segmenta! mobility scale47 and a hydrophilicity scale48 gave slightly better results than the others. Figure 2 illustrates the predicted mobility plot for myoglobin based on the scale of Karplus and Schulz47; when the cut-off level for correct prediction of antigenic residues was set at the mean value of mobility + 0.7 SD, the method identified only 45% of the antigenic residues49. In another recent study of three proteins s, the success of three predictive algorithms was assessed at two peptide lengths (7 and 15 residues respectively) and the proportion of rabbit antisera responding to each peptide was used to analyze the validity of these predictions by statistical Monte Carlo trials s. This type of simulation determines the probability of finding an epitope in a randomly chosen peptide of a given length. It was found that none of the three algorithms predicted better than random at a peptide length of seven residues, but that the predictions were somewhat better for 15-residue peptides. Since the three studied proteins (myoglobin, myohemerythrin and TMV protein) are relatively small and the entire surf3ce of the molecules is known to harbour residues that are part of continuous epitopes 13.1s, it is expected that 15 residue peptides will have a reasonably good probability of inducing crossreactive antibodies s,27. When the analysis is restricted to seven residue peptides (the usual length of continu-us epitopes), the low success rate of prediction methods no doubt reflects the inadequacy of a unidimensional analysis for describing the three-dimensional reality of discontinuous epitopes. Furthermore, the usual inclusion of non-critical replaceable residues within the boundaries of continuous epitopes tends to falsify the calculated propensity scales of amino acids used for the construction of prediction profiles 5o. In conclusion, our limited understanding of the structural basis of cross-reactive antigenicity is responsible for the limited success in predicting the location of seven residue continuous epitopes. However, since most workers use longer peptides for evaluating functional cross-reactivity, the impression is that prediction methods are fairly efficient. A final example will illustrate the vagaries of extrapolating from immunochemical observation to inference 1. In a recent study of the frequency with which the different amino acids are found in known epitopes, it was observed that one of the few clear-cut trends was that arginine was greatly under-represented as an antigenic residue s°. This led to the suggestion that the low propensity factor for arginine to occur as a critical residue partially accounts for the poor immunogenicity of histones - prr reins rich in this amino acid s. This inference, however, .s probably invalidated by the presence of sample bias in the original data base, since extensive studies of the antigenic structure of histones have shown the presence in these molecules of four continuous epitopes containing no less than eight arginine residuess~. In summary, this analysis of three current controversies in the field of protein antigenicity illustrates the conten-
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tion that it is easier for scientists to agree on the contents of an observation than on its meaning and relevance for a particular problem. It reminds us that the conceptual component in scientific research is paramount and that our explanatory models are in constant need of revision and further refinement. Refelences
1 Hull, D.L. (1965) in The British Journal for the PMosophy of Science 16, 1-18 2 Novotny, J. and Haber. E. (1986)Biochem.:stry25, 6748-6754 3 Novotny,J., Bruccoleri, R.E.,Carlson, W.D.o Handschumacher, M. and Hal0er,E. (1987) Science 238, 1584-1586 4 Mariuzza, R.A., Phillips, S.E.V.and Poljack, RJ. (1987)Annu. Rev. Biophys. Biophys. Chem. 16, 139-159 5 Getzoff, E.D.,Tainer,J.A., Lerner, R.A. and Geysen,H.M. (1988)Adv. Immunol. 43, 1-98 6 Westhof, E., Altschuh, D., Moras, D. eta/. (1984) Nature311, 123-125 7 Tainer, J.A., Getzoff, E.D., Paterson,Y., Olson, A.J. and Lerner, R.A. (1985)Annu. Rev.Immuno/. 3, 501-535 8 Geysen,H.M., Rodda, SJ., Mason, T.J.eta/. (1987)Science 238, 1584-1586 9 Amit, A.G., Mariuzza, R.A., Phillips, S.E.V.and Poljak, RJ. (1986) Science 233, 747-753 10 Sheriff, S., Silverton, E.W., Padlan, E.A. etaL(1987)Proc. Natl Acad. Sci. USA 84, 8075-8079 11 Barlow, D.J., Edwards, M.S. and Thornton, J.M. (1986) Nature 322, 747-748 12 Van Regenmortel, M.H.V. (1987) TrendsBiocher.~ Sci. 12, 237-240 13 Benjamin, D.C., Berzo~,ky,JA., East,I.J. eta/. (1984)Annu. Rev./mmunol. 2, 67-101 14 Atassi, M.Z. (1984) Eur. J. Biochem. 145, 1-20 15 Van Regenmortel, M.H.V. (1989)Philos. Trans. R. Sac. London, Ser. B 320, 451-466 !6 L a m ~ , D.M. and Hughes,A.J. (1988)J. Theor. BioL 133, 133-145
11 Colman, P.M. (1988)Adv. Immuno/. 43, 99-132 18 AI Moudallal, Z., Briand, J.P.and Van Regenmortel, M.H.V. (1985) EMBOJ. 4, 1231-1235 19 Van Regenmortel, M.H.V. (1986) TrendsBiochem. Sci 11, 36-39 20 Van Regenmortel, M.H.V., Muller, S., Quesniaux,V.F., Altschuh, D. and Briand, I.P. (1988) in Vaccines: New Concepts and Developments (Ko,~Jer,H. and LoVerde, P.T.,eds), pp. 113-122, Longman 21 Hull, D.L. (1968) Syst. Zoo/. 17, 438-457 22 Crumpton, M.J. (1986) in Synthetic Peptides asAntigens (Symposium 119) pp. 93-106, Ciba Foundation 23 Geysen,H.M., Tainer,J.A., Rodda, S.J.et al. (1987)Science 235, 1184-1190 24 Sat-tier,R. (1986) Bid-philosophy. Analytic and Holistic Perspectives, Springer-Verlag 25 Arnon, R. (1987)Synthetic Vaccines(Vollandll), CRCPress Inc. 26 Brown, F., Chanock, R.M. and Lerner, R.A. (1986) Vaccines 1986. New Approaches to Immunization, Cold Spring Harbor Laboratory 27 Van Regenmortel, M.H.V., Briand, J.P., Muller, S. and Plaue, S. (1988) Laboratory Techniques in Biochemistry and Molecular BiologyVol. 19 (Burdon, R.H.and Van Knippenberg, P.H.,eds), Elsevier 28 Anderer, F.A. and Schlumberger, H.D. (1965) Biochim. Biophys. Acta 97, 503-509 29 Rappaport, I. (1965)Adv. Virus Res. 11,223-275 30 Steward, M.W. and Howard, C.R. (1987)lmmunol. Today8, 51-58
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31 Berzc.~sky,J.A. (1985) Science229, 932-940
Sercarz, E.E.and Berzofsky,J.A. (1987) Immunogenicityof Protein Antigens: Repertoireand Regulation (Vol I a~d II), CRC PressInc. 33 Berzofsky,J.A., Cease, K.B., Cornette, J.L. etaL (1987) ImmunoL Rev.98, 9-52 ]4 Green, N., Alexander, H., nlson, A. etal. (1982) Ce1128, 477-487 35 Niman, H.L., Houghten, R.A., Walker, L.A. etal. (1983)Proc Natl Acad. Sci. USA 80, 4949-4953 36 Lerner,R.A. (1984)Adv. Immunol. 36, 1-44 37 Jemmerson,R. and Blankenfeld, R. (1989)MoL ImmunoL 26, 301-307 38 Dyson, H.J., Lerner, R.A. and Wright, P.E.(1988)Annu. Rev. Biophys. Biophys. Chem. 17, 305-324 39 Soderquist, M.E. and Wa Iton, A.G. (1980) J. Colloid Interface Sci. 75, 386-397 40 Friguet, B., Djavadi-Ohaniance,L. and Goldberg, M.E. (1984) MoL ImmunoL 21,673-677 41 Darst, S.A., Robertson,C.R. and Berzofsky,J.A. (1988)
Biophys. J. 53, 533-539 42 Jemmersen, R. (1987) Proc. NatIAcad. 5ci. USA 84, 9180-9184 43 Walter, G. (1986)J. Immunol. Methods 88, 149-161 44 Mutter, M., Altmann, K.H., Muller, K., Vuilleumier, S. and Votherr, T. (1986) Helv. Chim.Acta. 69, 985-995 45 Sasaki,A., Mikawa, Y., Sakamoto, Y. etal. (1988) MoL Immunol. 25, 157-163 46 Hopp, T.P. (1986) J. Immunol. Methods 88, 1-18 47 Karplus, P.A. and Schulz,G.E. (1985)NatutwissensoSaften 72,212-213 48 Parker,J.M.R., Guo, D. and Hodges, R.S.(1986) Biochemistry 25, 5425-5432 46 Van Regenmortel, M.H.V. and Daneyde Marcillac, G. (1988) ImmunoL Lett. 17, 95-108 50 Geysen,H.M., Mason, TJ. and Rodda, S.J.(1988) J. Mol. Recognition 1, 32-41 51 Van Regenmortel, M.H.V., Muller, S. and Briand, J.P.(1985) in Protidesof the BiologicalFluids(VoL 33) (Peeters,H., ed.), pp. 265-268, Pergamon Press
HIV-1, ilTLV-I and normal T-cell growth: transcriptional strategies and In this review, Warner Greene and colleagues discuss recent studies that have revealed an intriguing mo!ecular interplay between two pathogenic human retroviruses, HIV-1 and HTLV1, and certain cellular genes that normally control T-cell growth. Activation of T cells during an immune responseresults in the induction of select transcription factors that bind specifical~, to KB enhancer elements present in both the IL-2R~ and IL-2 genes. Normal T-ceil growth is in part regulated by the transient expression of these genes. The Taxprotein of HTLV-1 induces these same ~B-specific proteins, but in contrast to immune ~imulation, HTLV-I infection of T ceils leads to constitutive IL-2R, gene expression and immortalization. A second human retrovirus, HIV-1, can subvert the normal action of the ~-binding factors induced by these immune stimuli. Rather t,Sanpromoting T-cellgrowth, these factors may augment viral replication and promote T-celldeath. Antigen-induced qrowth of T lympiiocytes is normally controlled by the transient activation of a select set of cellular genes including those encoding the growthpromoting lymphokine, interleukin 2 (IL-2), and its specific membrane receptor (IL-2R)1-3. The interaction of IL-2 with the functional high affinity form of IL-2R triggers intracellular events that culminate in T-cell proliferation. The high affinity IL-2R corresponds to a membrane complex comprising at least two distinct IL-2 binding subunits including IL-2Re (Tac, p55)4,s and IL-2RI3 (p70/75) 6-~°. As some, and perhaps all, resting T cells constitutively express IL-2R~ (Refs 10-12), high affinity IL-2R display and T-cell proliferation are significantly
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surprises
WarnerC. Greene,ErnstBiihnidnand Dean W. Ballard regulated by the de-novo induction of the IL-2Ro~gene. Cell surface iL-2R~ expression is transiently induced by a variety of immune stimuli including antigen, certain cytokines like IL-1 (Ref. 13) and tumor necrosis factor-~ (TNF-oL)14.1s, and by various nonspecific mitogens such as phytohemagglutinin (PHA)16 and the tumor promoter, phorbol 12-myristate 13-acetate (PMA) 16. The normally transient nature of IL-2 and IL-2Re gene expression appears to contribute noz only to the development of the T-cell immune respon3e, but also to its physiological termination fn contra.~t to these T-cell stimuli, infection of human CD4 ~- T cells with the type 1 human T-cell le;ikemia virus (HTLV° 1) is associated with a persistent state of high level cell surface IL-2R expression17-2o. This pathogenic retrovirus, which has been implicated as the cause of the aggressive and often fatal adult T-cell leukemia (ATL), encodes a 40 kD~ tr~,",~ acting ~agu!aton/protein termed Tax21. In addition to activating the HTLV-1 long terminal repeat (LTR), and consequently the expression of viral genes under its control, ]ax shares with T-cell mitogens the ability to induce the transcription of various cellular genes including IL-2Rcx and IL-2 (Refs 22-28). These effects of Tax, which bypass the nermal intracellular controls that ensure transient IL-2Rcx gene expression, provide one explanation for the sustained expression of IL-2Ra observed in HTLV-1 infected T-cell lines. In addition, the effects of Tax protein on the IL-2 gene may promote autocrine proliferation of infected cells during © 1989, ElsevierScienceP[ ,~lishorsLtd, UK. 0167-4919189/$02.00
-brier 6 Kimura, H. and Wilson, D. (1984) Nature 308, 463-464 7 Kappler, J., Roehm, N. and Marrack, P. (1987) Ce1149, 273-280
B Kisielow, P., Bl(~thmann, H., Staerz, U.D. etal. (1988) Nature
_ _
333, 742-746 9 Qin, S., Cobbold, S., Benjamin, R, and Waldmann, H. (1989) J. Exp. Meal. 169, 779-794 10 Lo, L)., 8urkly. L.C.,Widera, G. etal.(1988) Cell 53, 159-168 11 Miller, J.F.A.P.,Morahan, G., Allison, J. etal. (1989) Irnmunol. Rev. 107, 109-123 12 Miller, J.F.A.P.,Morahan, G. and Allison, J. (1989) ImmunoL Today ~O, 53-57 13 Sakaguchi,S., Takahashi,T. and Nishizuka,Y. (1982) J. Exp. Med. 156, 1577-1586 14 Taguchi. O., Takahashi,T., Seto, M, etal.(1986)J. Exp. Meal. 164, 60-71 15 Taguchi, O. and Nishizuka,Y. (1987)]. Exp. Meal. 165,
Repressionof academicsin China Sir,
_ _
The repression by communist party hard-liners of the pro-democracy movement initiated by students in Beijing has led to grave concern for
the safety and intellectual freedom of academics in China. As scientists who are committed to freedom of thought and expression, you must be concerned about the imminent loss of these freedoms and the personal safety of our counterparts in China. We beg you to express, in
Strudural
the strongest possible terms, your abhorrence of any physically or intellectually repressive action that might be taken against academics in China.
Staffof the ImmunologySection,Department of Pathology,Universityof HongKong. (Received5 June1989)
andfundionalapproachesto the studyof proteinantigenid
Structural and functional approaches to the study of protein antigeniC1 lead to two different perceptions of the nature of protein epitopes. The structural approach concentrates on the pure~/ spatial arrangements of atoms found in the antigenantibody complex and shows that at least 15 amino acid residues may be implicated in each epitope. The fun~'onal approach, which introduces the additional dimension of time, takes the form of cross-reactive binding measurements and leads to the view that a smaller number of residues are implicated in each epitope. Furthermore, as functional binding assaysare irretrievably operational in character, different types of epitopes are identified by the use of different probes. Three current controversies i~~ the field of protein antigenicity are discussed here: the mechanism of antigen-antibody interaction, the suitability of peptides as synthetic vaccines and the valuP of prediction methods for locating epitopes in proteins. "Sdence is not just the making of observations: it is the making of inferences on the basis of observations within the framework of a theory "1. This remark by the philosopher of science, D.L Hull is particularly apt for the science of immunology which is replete with inductive
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t6 Bandeira,A., Coutinho, A., Carnaud, C., Jacquemart, F. and Fomi, L. (1989) Proc NatlAcad. Sci. USA 86, 272-276 17 Roser,B. (1989) ImmunoL Rev. 107, 179-202 18 Pereira, P., Bandeira, A., Coutinho, A. etal. (1989) Annu. Rev. Immunol. 7, 209-249 19 Lundkvist, I., Coutinho, A., Varela, F. and Holmberg, D. Proc Natl Acad. Sci. USA (in press) 20 8andeira, A., Larsson,E-L.,Forni, L. eta/. (1987) Eur. ]. ImrnunoL 17, 901-908 21 Pereira,P., Larsson,E-L.,Forni, L. etaL (1985) Proc. Nat/ Acad. 5eL USA 82, 7691-7695 22 Coutinho, A. Immuno/. Rev.(in press) 23 Holmberg, D., Forsgren,S., Ivars,F. and Coutinho, A. (1984) Eur. J. ImmunoL 14, 435-441 24 Forni, L., Heusser,C. and Coutinho, A. (1988)Ann. Inst. PasteurImrnunol. 139, 245-256
Laboratoired'lmrnunochirnie,Institutde BiologieMoldc.ulaireet Cellulaire, 15 ~JeDesca,'tes,67084Strasbourg,France.
MarcB.V.Vm R c mrtd inferences that are far from deductively certain. In the field of protein antigen!city there are currently three major controversies: first, the mechanism of antigen-antibody interaction; second, the suitability of synthetic peptides for mimicking the antigenic sites of proteins and acting as potential vaccines, and third, the ability of various algorithms to predict the location of antigenic sites in proteins solely on the basis of amino acid sequence data. To resolve these cont;oversies, it is important to differentiate clearly between observation and inference. This in turn requires that the operational constraints of immunochernical observations are recognized and that the concepts and theoretical assumptions underlying the inferences are clearly defined. In this review, Marc Van Regenmortel analyses the observations and inferences underlying these three controversies. It will become clear that a major problem lies in the conceptual jump needed to 9o frum str~cture to function and from function to structure. Translating observations of structures into biological processes and functions turns out to be as difficult in immunology as in the rest of biology. 1989, ElsevierScience Publishers Ltd. UK. 0167-4919189/$02.00
Immunology Today, Vol. 10, No. 8, 1989
The mechanismof antigeHntibody interaction There are two opposing views of the mechanism of antigenic recognition. According to the lock-and-key model, the binding sites of antibody molecules simply recognize patches at the surface of antigens that are complementary to them in shape and chemical properties. This model stresses the fact that antibodies must have access to an antigenic site in order to bind and it has led to the view that antibody accessibility is the primary intrinsic determinant of antigenicity2,3. Proponents of this model hold that the binding process does not lead to any significant conformational changes in the antigen 4. In contrast the induced fit model proposes that the mechanism of binding occurs through a process of induced complementarity involving significant side-chain movements of the antigen and possibly minor movements of the backbone conformation S. This second model stresse~ the importance of the observed correlation between antigenicity and local mobility of the peptide main chain 6-8 and may be described by the analogy of the adaptation between hand and glove. In order to distinguish iF, each of the two models what is exper~,~nental observation from what is interpretation, it is helpful to recall how immunologists define antigenic sites and localize them in proteins.
o
Table 1. Methodsusedto localizeepitopesin proteins Method
Typeof epitope recognized
Criterionfor Average allocatingresiduesto nu~berof epitope residues iden~ecl in epitope
1 X-ray Discontinuous VanderWaal's crystallographyof epitopereactingwith conta~in epitopeantigen-Fab homologousantib0dy paratopeinterface complexes
15
2 Studyof crossreactivebinding of peptide fragmentswith anti-protein antibodies
Continuousepitope Residualbindingof cross-reactingwith linearfragmentabove heterologous thresholdof assay antibody
3-8
3 Studyofcrossrea~ivebinding of proteinwith anti-peptide antibodies
Continuousepitope Inductionof crosscross-reactingwith reactiveantibodies heterologous antibody
3-8
Determinationof Continuous,cross- Abrogationof crossMethods usedto localizeepitopesin proteins criticalresiduesin rea~ngepitope reactivityby The antigenic sites or epitopes of a protein correspond peptideby containing critical substitutionof critical to those parts of the molecule that are specificafly systematic residuesinterspersed andfunctionally recognized by the binding sites or paratopes of antibody replacementwith withirrelevant relevantresidues molecules. Epitope:. Jre relational entities since they can otheraminoacids residues be recognized only in an operational sense by the binding of complcmentary paratopes. Observation of Studyof cross- Discontinuous Abrogation of crossspace-filling models of globular proteins shows that very reactivity epitope reactivityby few linear stretches of residues are present at the surface between substitutiono~jitica~ of a protein. If it is assumed that all epitope-paratope homologous residue interfaces in proteins involve a surface of about 700 ~2 proteinsor point (as shown in the case of lysozyme9,1°), it follows from mutants the known folding pattern of many globular proteins that no surface epitope region is likely to contain only atoms from a continuous stretch of residues ~1. Antibody molecules are thus likely to recognize a set of residues in short peptides. As a result, our knowledge of protein the antigen that are not contiguous in the sequence, but epitopes concerns mainly incomplete epitopes that have are brought together by the folding of the p~-ptide chain retained only some of the original structural features or by the juxtaposition of two separate chains. Such an present before fragmentation of the ~rotein 12. This epitope is usually referred to as a 'discontinuous' or knowledge can be obtained because cT the polyspeci'assembled epitope', as opposed to the term 'con- ficity of antibodies, that is their ability to cross-react with tinuous epitope', which describes the situation where a a variety of more or less closely related epitopes. short linear stretch of residues is recognized by the However, it should be appreciated that, by their very antibody. It is customary to give the label 'continuous nature, these studies can identify only those structures epitope' to any linear peptide fragment of a protein that that cross-react (in most cases rather poorly) and that is found to react with antibodies raised against the intact they cannot specify, all the features responsible for the molecule. This practice can lead to confusion since what interaction between the antigen and its homologous is called a continuous (or sequential or linear) epitope of antibody. The different approaches 1~-1s that have been used for the protein, in fact, usually represents only a portion of a larger discontinuous epitope. In generat, antibodies in- mapping protein epitopes are summ3rized in Table 1. It duced by a native protein and directed to a complex should be noted that only method 1 (Table 1) is based on discontinuous epitope cross-react only weakly with an a structural analysis, since it is the only method that gives epitope fragment constituted by a linear peptide; an selective attention to the visual appearance of the spatial additional reason for this weak cross-reaction is that the arrangements found in the antigen-antibody complex at peptide usually has a conformation different from the a specific time. in contrast, all other methods correscorresponding region in the protein Most studies aimed pond to a functional analysis taking the form of binding at unravelling the antigenic structure of proteins have measurements. Since the criteria used for allocating focused not on antigenicity per se but on the phenom- residues to an epitope by method 1 are purely structural enon of cross-reactive antigenicity betvveen prc:eins and and spatial, this method cannot provide direct evidence
3-S
1-3
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B
~A~-.....-----~A~ 1
2
rig. 1 . . ~ a ~ c representa~bnof S/TUctural(AJ and funct/0nal(8) ana~/s of antJ'genan~x~/interaclfon. In A, ~.a spa~alarrangementof the ep/tope-paratopeinterfaceis mualized and a total of 12 hydrogen bonds (--) are identified as in the complex betweer antibody and lysozymc~. This number of hydrogen bonds corresponds to more than 20 kcal moll of binding energy, equivalentto an affinity constant of morn than 10is. Since the affin;Cyob.~,n,adby Amit et a~9 was only about 10s, the difference is dueto thebreaking of hydrogen bonds ~at existed between the residues and solvent molecules prior to the assoda~on of epitope and paratope. This dynamic aspect cannot be represented in a static structural picture. In B, the arrow indicates the additional dimension of time which is an e~enEal ingredient in func~'onalana,~. Sincethis fourth dimensioncannot be easilyvisualiz~ in a spatial representa~on,one usual~refers to states I and 2 of the sy~em.
that every residue contributes to the binding reaction. On the other hand, the criteria used for identifying antigenic residues by methods 2-5 (Table 1) are functional and these methods which are based on binding measurements introduce the additional dimension of time and therefore analyse function and not structure (Fig. 1). As pointed ou~ by Lambert and Hughes is, structuralism and functionalism are two "different ways of seeing" and these two approaches represent one of the major dlchotomies in biology. As shown in Table 1, the structuralist perspective inherent in method 1 leads to the view that an epitope con~sts of anproximate!y 15 residues that are in contact with the paratope9,1o,~7. As pointed out by Getzoff et aL s, various crystallographic groups use different definitions for residues involved in the interface; whatever the defin!tion of interface (for example, residues within van der Waals contact distance, residues buried to a certain radius probe sphere, residues with side chains interacting directly), it is clear that the visual observation of a contact between epitope and paratope still necessitates a decision as to what constitutes a significant contact needed for the binding interaction to occur. Residues in the interface that a:ually contribute to the binding energy are identifiable only in binding measurements. As indicated in Table 1, methods 2-5 which operate within a functionalist perspective actually analyze cross-reactivity instead of antigenic reactivity; these methods lead to the conclusion that a much smaller number of residues (between 1 and 5) are critical to antibody binding and thus that the epitope defined in a functional sense involves fewer residues than the epitope defined in structural terms. AS discussed elsewhere ~8-2o, binding measurements are necessarily of an operational nature: depending on the type of probe used for assessing immunochemical binding (free peptide versus conjugated peptide, sh3rt peptide versus a more constrained long peptide, liquid versus solid-phase immunoassay, monoclonal versus polyclonal antibody reagent), it is unavoidable that the same residues of the antigen wili not always be scored as
participating in different types of binding assays. If epitopes are defined in a binding assay, it is to be expected that the experimental conditions of the assay could, for instance, influence the conformation of the peptide; assay conditions will in fact determine whether binding is observed or not, and thus whether certain residues are scored as belonging to the functional epitope. Although some authors s maintain that this definition of epitope is inconsistent, it simply reflects the operational nature of the concept of functional epitopes. An operational detmition gives meaning to a term, because it enables us to decide when the term applies by observing a response under specified test conditions21. The relevance of operationalism in science is linked to its insistence that many scientific objects of study can be properly defined only through the description of the experimental conditions that allow these objects to be observed. Attempts to define the inherent epitopes of a protein on a purely structural basis, for instance by saying that they are synonymous with surface exposure or with accessibility to a spherical probe 2, go counter to the immunological and functional nature of the epitope concept. Such an approach disregards the fact that a structural analysis is purely spatial, while a functional analysis is spatio-temporal. Recently, Getzoff et al. s suggested that the practice of defining epitopes operationally results in confusion because of the difficulty of integrating results from different experimental techniques. These a!,thors suggested that by making a distinction between critical residues (those identified in binding experiments) and contact residues (those defined crystallographically) it would be pcssible to remove the inconsistency arising from the use of different methods and assays. Unfortunately, structural and functional approaches to the study of protein antigenicity unavoidably lead to results that are incommensurate with each other (with respect to the dimension of time) and, in addition, functional binding assays are irretrievably operational in character. From binding measurementsto inferred mecharJsm "When it comes to understanding the mechanism of antigen-antibody interaction, one needs to extrapolate from a static structural picture to a spatio-temporal and dynamic process that cannot be frozen in time (Fig. 1). For instance, an X-ray crystallographic study of a peptide-antibody complex could demonstrate that a certain peptide had adopted a native-liKe conformation when bound to an anti-protein antibody; however, as pointed out by Crumnton 22, this structural information could not establish if the observed complex resulted from the antibody having selected one conform~;tion from a mixture of d;fferent conformations, or if the recognition process involved an induced complementarity subsequent to *~',einitial peptide--antibody interaction. Argume ,t:~ in favour of a contribution of induced complementarity to antigen-7,,: recognition have been developed an the basis of results obtained with the protein myc.hemerythrin. Using a method in which all possible overlapping he;'~-peptides were synthesized on a linear polymer of polyacrylic acid and tested for antigenic activity while still bound to the solid phase, seven continuous epitopes were identified in myohemerythrin 23. In order to assesswhich amino acids
Immunology'Today,Vol. 10, No. 8, 1989
within each synthesized peptide are essential for antibody binding, each residue was replaced, in turn, by the other 19 amino acids. Certain residues could be replaced by all common amino acids without affecting binding by antibodies; however, a total of 35 residues were found to be critical for binding and could not be replaced at all without impairing the antigenic reactivity5. When the position of these 35 critical residues in the native myohemerythrin structure was examined, each antigenic site was found to contain one or more buried critical side chains that were inaccessible to the solvent. The authors, therefore, proposed a reaction mechanism in which antibody binding to solvent-exposed amino acids causes a rearrangement (breaking of salt bridges or hydrogen bonds) followed by side-chain rotations which uncover previously buried atoms of the antigen. The examples of myohemerythrin epitopes discdssed by Getzoff et al. s are very suggestive and make the proposed mechanism of interaction by induced complementarity highly plausible. However, it should be noted that the contribution of critical residues was assessed from the measurement of cross-reactions between short peptides and anti-protein antibodies and that no direct evidence was presented that these residues are also critical for antibody binding to the native protein antigen. If the binding constant of the antibody for the peptide is much lower than that for the protein, it is conceivable that certain substitutions in the peptide could simply reduce binding below the measurable threshold while this would not be the case with the native protein. Experiments with myohemerythrin analogues obtained by site-directed mutagenesis will clarify this point. A possible role for induced fit in the reaction mechanism has also been inferred from the observed correlation between regions of local mobility in the protein and the position of short peptide fragments that cross-rea~ with the protein s-7,23. The cross-reactivity between intact protein and short peptides has been investigated both with anti-protein and with anti-peptide antibodies and both types ef study indicated that there was a frequent bias for continuous epitopes being located in regions of local mobility. However, since it is unlikely that the short peptide probes used in these studies can mimic constrained elements of secondary structure, this type of analysis will not necessarily reveal the presence of antibodies that do recognize secondary structure. It would indeed be unwarranted to conclude that the immune system is biased against the recognition of ordered structures on the basis of measurements that exclude long peptides that are likely to be more structured. Local mobility in the protein could help to make the corresponding region more like a short peptide and thus enhances cross-reactive antigenicity; at the same time, a longer peptide might resemble more closely a constrained feature of the secondary structure present in the protein and show good cross-reactivity for that reason. Either type of study is biased operationally and should not be used to make generalizations about which intrinsic structural feature determines all possible forms of protein antigenicity including cross-reactive antigenicityl s.19. Some authors reiec~. ~he functional significance of movements of a few angstroms and do not concede that this might facilitate the mutual adjustment of epitopes and paratopes4. They point out that no major confor-
mational changes have been observed in the lysozyme molecule after binding to antibody, and suggest that the location of epitopes simply correlates with the most exposed regions of the protein. ~urface. The search for a primary correlate with antigenLcLty, such as accessibility, appears to proceed from a type of causal thinking that should be abohshed from biology. It is now generally recognized that the systems under investigation in the biological sciences exhibit such a high degree of complexity that traditional notions of linear causality do not apply z4. A more appropriate analytical framework is 'network causality' in which a large number of factors or variables are seen to influence a particular feature of the system instead of causing it. No single factor can be equated with the cause of antigenicity since a great many factors may determine it.
Synthetic peptide vaccines The possibility of using synthetic peptides as vaccines against viral, bacterial and parasitic diseases has evoked considerable interestzs,26 and remains a major driving force that stimulates much current research in the field of synthetic polypeptides27. Although it is frequently stated that the potential usefulness of synthetic peptides as vaccines was discovered around 1980, it is the German virologist F.A. Anderer who should be credited with this discoverj which was made about 15 years earlier. Anderer and his colleagues, working with tobacco mosaic virus (TMV), showed in 1963-1965 (for a review see Ref. 15) that short synthetic peptides corresponding to the carboxyterminal region of the viral protain were able to induce antibodies that neutralized viral infectivity2~. Some later workers failed to appreciate that the neutralization of this plant virus by antibodies followed most of the characteristic features of animal virus neutralization 29. Because of the host-based compartmentalization of virologists, it took 15 years for the approach shown by Anderer to be successful with a plant viru~ to be applied to animal viruses. Within a few years, it was then shown that vaccination of animals with synthetic paptides could lead to protective immunity against foot-and-mouth disease, influenza, hepatitis B, diphtheria and choler.~zs,z6.3°. In order to be effective, a synthetic peptide vaccine must possess a high level of immunogenicity and induce antibodies that cross-react extensively with the pathogen. The immunogenic capacity of a peptide refers to its ability to induce an immune response and depends on extrinsic host factors such as the immunoglobulin gene repertoire, self-tolerance and various cellular regulatory mechanisms 31.3z. These factors, as well as the role of helper T cells33, are beyond the scope of this review. The claim that immunization with peptides leads to a very high frequency of induction of antibodies that can recognize the native protein 34-36 gave rise to the view that it is very easy to obtain anti-peptide antibodies of so-called predetermined specificity that cross-react with a related epitope of the cognate protein 36. Recently, this claim has been challenged on the basis of findings showing that intact cytochrome c activates only a small fraction of peptide-primed B lymphocytes37. The contention that antibodies against a hlghiy disordered state (the peptide) recognize the highly ordered state (the folded protein) has been called the order-disorder paradox 38.
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rig, 2. Predictedsegmentaltr~bility profile of rnyoglobin calculated~th the scale of Karplus and ~huId z according to the smoothing procedure deso~bedin Ref. 49. The solid squares conesx~d to amino adds that are part of known continuousepitopesof myoglob~n.Theopen squaresrepresentthe averagevalueof the mobility parameterover the entire sequenceand the two hoEzontalbroken linesrepresent+_0.7 so from the mean. Suchan interval correspondsto 50% of the population. Whenthe cut-off level for correctprediction wasset at the mean +0.7 so, the meff~d iden~ed 45% of the known antigenic residues (from Van Regenrnortelet
al.29.
270
As discussed elsewhere 27, to show that anti-peptide antibodies react with a truly native protein, the protein antigen should preferably not be adsorbed to plastic in a solid-phase immunoassay since this is known to at !ea~ partly denature proteins39-41. However, if a solid-phase assay is used, it is then necessary to include contro!s In which the liquid-phase protein is allowed to compete with plate-bound antigen and inhibition to a level approaching 100% occurs 42. Furthermore, the antigen should preferably not be labelled since this treatment could also induce some denaturation. In the experiment~ that the ,~bove-mentioned views were based on, these conditions were not met. For instance, in experiments carried out with 20 synthetic peptides representing over 75% of the influenza haemagglutinin sequence, it was observed that 18 of the 20 peptides elicited antibodies that reacted with purified haemagglutinin or intact virus, but that none of the peptides reacted with anti-haemagglutinin antibodies present in influenza virus antisera 34. It was also shown that the cross-reacting antipeptide antibodies were not directed againsta rare native-like conformation of the peptide 3s. Since the correct peptide conformation was not a rare occurrence, there was no explanation for the observation that none of the 20 peptides reacted with anti-haemagglutinin antibodies. Subsequently it has been suggested that this type of result is obtained because peptides show a marked conformational preference in solution, and that such a preferred conformation may be stabilized on the surface of the carrier protein or when the peptide binds to the B-cell receptor during
immune stimulation 38, Since no evidence was presented that the anti-peptide antibodies actually recognized the native haemagglutinin, it seems more plausible to infer that the haemagglutinin had been denatured by adsorption to the plastic and by the methanol incubation step that was used in the ~ssay. As far as the extremely low level of cross-reactivity observad with influenza virus is concerned 34, this is probably due to the presence of a small number of denatured haemagglutinin molecules in the preparation 43. In conclusion, these observations do not support the notion that when synthetic peptides are used as immunogens, antibodies cross-reactive with the native protein are easily raised against almost any part of the molecule. Nor do the reported observations justify the view that the correct tertiary conformation of an epitope need not be reproduced in a synthetic peptide in orde: to have an effective synthetic vaccine. Although it seems probable that neutralization epitopes ~n a pathogen (that is those inducing neutralizing antibodies) are preserved only in antigens with an intact tertiary or quaternary structure, it remains unclear at present to what extent it will be necessary to mimic the native conformation of neutralizing epitopes in order to achieve protective immunity by means of synthetic peptides, in view of the intense research activity in this area26,44, the answer to this question should be available soon. Developing peptides suitable for vaccination is a more difficult task than selecting peptides able to induce antibodies that simply cross-react with the cognate protein. Although anti-peptide antibodies are extremely useful for isolatiJ,=l and characterizing gene products 36.43, the high success rate in this case is due in part to the fact that the cross-reactive antibodies need not necessarily be specific for the native conformation of the protein to detect the antigen in many of the currently used imrnunoassays. !:h.ep._red_i~ionof epi~-,~s in proteins In an attempt to understand the structural basis of protein antigenicity many investigators analyze model proteins of known three-dimensional structure and look for a correlation between certain structural parameters and the location of epitopes6.7. In most cases, the tertiary structure information is reduced to a linear scale of amino acid sequence, and an attempt is made to correlate Lhe location of continuous epitopes with structural properties such as accessibility, protrusion, mobility and hydrophilicity2.3.7.a,4s.Although it is generally recognized that most protein epitopes are discontinuous, the emphasis on continuous epitopes in such studies is due to the potential usefulness of predicting which linear peptide fragments are likely to possess cross-reactive antigenicity with the intact protein. Since for most proteins the only available structural information today concerns their amino acid sequence, it is clearly the prediction of cross-reactive antigenicity of short linear peptides which is of practical interest to most investigators. Few studies have addressed the issue of mimicking discontinuous epitopes by synthesis 14,~. Relative scales describing the structural propensity of each of the 20 common amino acids have been derived and these are commonly used for constructing prediction profiles and for locating the position of cross-raactive, continuous epitopes46-48. In a recent study, the predictive value of algorithms based on eight propensity scales
ImmunologyToday, Vol. 10, No. 8, 1989
of the amino acids has been compared, using as a criterion of effectiveness the number of residues correctly predicted to be antigenic in four well-studied proteins a9. None of the methods achieved a high level of correct precliction, although two algorithms based on a segmenta! mobility scale47 and a hydrophilicity scale48 gave slightly better results than the others. Figure 2 illustrates the predicted mobility plot for myoglobin based on the scale of Karplus and Schulz47; when the cut-off level for correct prediction of antigenic residues was set at the mean value of mobility + 0.7 SD, the method identified only 45% of the antigenic residues49. In another recent study of three proteins s, the success of three predictive algorithms was assessed at two peptide lengths (7 and 15 residues respectively) and the proportion of rabbit antisera responding to each peptide was used to analyze the validity of these predictions by statistical Monte Carlo trials s. This type of simulation determines the probability of finding an epitope in a randomly chosen peptide of a given length. It was found that none of the three algorithms predicted better than random at a peptide length of seven residues, but that the predictions were somewhat better for 15-residue peptides. Since the three studied proteins (myoglobin, myohemerythrin and TMV protein) are relatively small and the entire surf3ce of the molecules is known to harbour residues that are part of continuous epitopes 13.1s, it is expected that 15 residue peptides will have a reasonably good probability of inducing crossreactive antibodies s,27. When the analysis is restricted to seven residue peptides (the usual length of continu-us epitopes), the low success rate of prediction methods no doubt reflects the inadequacy of a unidimensional analysis for describing the three-dimensional reality of discontinuous epitopes. Furthermore, the usual inclusion of non-critical replaceable residues within the boundaries of continuous epitopes tends to falsify the calculated propensity scales of amino acids used for the construction of prediction profiles 5o. In conclusion, our limited understanding of the structural basis of cross-reactive antigenicity is responsible for the limited success in predicting the location of seven residue continuous epitopes. However, since most workers use longer peptides for evaluating functional cross-reactivity, the impression is that prediction methods are fairly efficient. A final example will illustrate the vagaries of extrapolating from immunochemical observation to inference 1. In a recent study of the frequency with which the different amino acids are found in known epitopes, it was observed that one of the few clear-cut trends was that arginine was greatly under-represented as an antigenic residue s°. This led to the suggestion that the low propensity factor for arginine to occur as a critical residue partially accounts for the poor immunogenicity of histones - prr reins rich in this amino acid s. This inference, however, .s probably invalidated by the presence of sample bias in the original data base, since extensive studies of the antigenic structure of histones have shown the presence in these molecules of four continuous epitopes containing no less than eight arginine residuess~. In summary, this analysis of three current controversies in the field of protein antigenicity illustrates the conten-
O
tion that it is easier for scientists to agree on the contents of an observation than on its meaning and relevance for a particular problem. It reminds us that the conceptual component in scientific research is paramount and that our explanatory models are in constant need of revision and further refinement. Refelences
1 Hull, D.L. (1965) in The British Journal for the PMosophy of Science 16, 1-18 2 Novotny, J. and Haber. E. (1986)Biochem.:stry25, 6748-6754 3 Novotny,J., Bruccoleri, R.E.,Carlson, W.D.o Handschumacher, M. and Hal0er,E. (1987) Science 238, 1584-1586 4 Mariuzza, R.A., Phillips, S.E.V.and Poljack, RJ. (1987)Annu. Rev. Biophys. Biophys. Chem. 16, 139-159 5 Getzoff, E.D.,Tainer,J.A., Lerner, R.A. and Geysen,H.M. (1988)Adv. Immunol. 43, 1-98 6 Westhof, E., Altschuh, D., Moras, D. eta/. (1984) Nature311, 123-125 7 Tainer, J.A., Getzoff, E.D., Paterson,Y., Olson, A.J. and Lerner, R.A. (1985)Annu. Rev.Immuno/. 3, 501-535 8 Geysen,H.M., Rodda, SJ., Mason, T.J.eta/. (1987)Science 238, 1584-1586 9 Amit, A.G., Mariuzza, R.A., Phillips, S.E.V.and Poljak, RJ. (1986) Science 233, 747-753 10 Sheriff, S., Silverton, E.W., Padlan, E.A. etaL(1987)Proc. Natl Acad. Sci. USA 84, 8075-8079 11 Barlow, D.J., Edwards, M.S. and Thornton, J.M. (1986) Nature 322, 747-748 12 Van Regenmortel, M.H.V. (1987) TrendsBiocher.~ Sci. 12, 237-240 13 Benjamin, D.C., Berzo~,ky,JA., East,I.J. eta/. (1984)Annu. Rev./mmunol. 2, 67-101 14 Atassi, M.Z. (1984) Eur. J. Biochem. 145, 1-20 15 Van Regenmortel, M.H.V. (1989)Philos. Trans. R. Sac. London, Ser. B 320, 451-466 !6 L a m ~ , D.M. and Hughes,A.J. (1988)J. Theor. BioL 133, 133-145
11 Colman, P.M. (1988)Adv. Immuno/. 43, 99-132 18 AI Moudallal, Z., Briand, J.P.and Van Regenmortel, M.H.V. (1985) EMBOJ. 4, 1231-1235 19 Van Regenmortel, M.H.V. (1986) TrendsBiochem. Sci 11, 36-39 20 Van Regenmortel, M.H.V., Muller, S., Quesniaux,V.F., Altschuh, D. and Briand, I.P. (1988) in Vaccines: New Concepts and Developments (Ko,~Jer,H. and LoVerde, P.T.,eds), pp. 113-122, Longman 21 Hull, D.L. (1968) Syst. Zoo/. 17, 438-457 22 Crumpton, M.J. (1986) in Synthetic Peptides asAntigens (Symposium 119) pp. 93-106, Ciba Foundation 23 Geysen,H.M., Tainer,J.A., Rodda, S.J.et al. (1987)Science 235, 1184-1190 24 Sat-tier,R. (1986) Bid-philosophy. Analytic and Holistic Perspectives, Springer-Verlag 25 Arnon, R. (1987)Synthetic Vaccines(Vollandll), CRCPress Inc. 26 Brown, F., Chanock, R.M. and Lerner, R.A. (1986) Vaccines 1986. New Approaches to Immunization, Cold Spring Harbor Laboratory 27 Van Regenmortel, M.H.V., Briand, J.P., Muller, S. and Plaue, S. (1988) Laboratory Techniques in Biochemistry and Molecular BiologyVol. 19 (Burdon, R.H.and Van Knippenberg, P.H.,eds), Elsevier 28 Anderer, F.A. and Schlumberger, H.D. (1965) Biochim. Biophys. Acta 97, 503-509 29 Rappaport, I. (1965)Adv. Virus Res. 11,223-275 30 Steward, M.W. and Howard, C.R. (1987)lmmunol. Today8, 51-58
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Sercarz, E.E.and Berzofsky,J.A. (1987) Immunogenicityof Protein Antigens: Repertoireand Regulation (Vol I a~d II), CRC PressInc. 33 Berzofsky,J.A., Cease, K.B., Cornette, J.L. etaL (1987) ImmunoL Rev.98, 9-52 ]4 Green, N., Alexander, H., nlson, A. etal. (1982) Ce1128, 477-487 35 Niman, H.L., Houghten, R.A., Walker, L.A. etal. (1983)Proc Natl Acad. Sci. USA 80, 4949-4953 36 Lerner,R.A. (1984)Adv. Immunol. 36, 1-44 37 Jemmerson,R. and Blankenfeld, R. (1989)MoL ImmunoL 26, 301-307 38 Dyson, H.J., Lerner, R.A. and Wright, P.E.(1988)Annu. Rev. Biophys. Biophys. Chem. 17, 305-324 39 Soderquist, M.E. and Wa Iton, A.G. (1980) J. Colloid Interface Sci. 75, 386-397 40 Friguet, B., Djavadi-Ohaniance,L. and Goldberg, M.E. (1984) MoL ImmunoL 21,673-677 41 Darst, S.A., Robertson,C.R. and Berzofsky,J.A. (1988)
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HIV-1, ilTLV-I and normal T-cell growth: transcriptional strategies and In this review, Warner Greene and colleagues discuss recent studies that have revealed an intriguing mo!ecular interplay between two pathogenic human retroviruses, HIV-1 and HTLV1, and certain cellular genes that normally control T-cell growth. Activation of T cells during an immune responseresults in the induction of select transcription factors that bind specifical~, to KB enhancer elements present in both the IL-2R~ and IL-2 genes. Normal T-ceil growth is in part regulated by the transient expression of these genes. The Taxprotein of HTLV-1 induces these same ~B-specific proteins, but in contrast to immune ~imulation, HTLV-I infection of T ceils leads to constitutive IL-2R, gene expression and immortalization. A second human retrovirus, HIV-1, can subvert the normal action of the ~-binding factors induced by these immune stimuli. Rather t,Sanpromoting T-cellgrowth, these factors may augment viral replication and promote T-celldeath. Antigen-induced qrowth of T lympiiocytes is normally controlled by the transient activation of a select set of cellular genes including those encoding the growthpromoting lymphokine, interleukin 2 (IL-2), and its specific membrane receptor (IL-2R)1-3. The interaction of IL-2 with the functional high affinity form of IL-2R triggers intracellular events that culminate in T-cell proliferation. The high affinity IL-2R corresponds to a membrane complex comprising at least two distinct IL-2 binding subunits including IL-2Re (Tac, p55)4,s and IL-2RI3 (p70/75) 6-~°. As some, and perhaps all, resting T cells constitutively express IL-2R~ (Refs 10-12), high affinity IL-2R display and T-cell proliferation are significantly
272
Howard HughesMedical Institute, Duke UniversityMedical Center, Durham,NC27710, USA.
surprises
WarnerC. Greene,ErnstBiihnidnand Dean W. Ballard regulated by the de-novo induction of the IL-2Ro~gene. Cell surface iL-2R~ expression is transiently induced by a variety of immune stimuli including antigen, certain cytokines like IL-1 (Ref. 13) and tumor necrosis factor-~ (TNF-oL)14.1s, and by various nonspecific mitogens such as phytohemagglutinin (PHA)16 and the tumor promoter, phorbol 12-myristate 13-acetate (PMA) 16. The normally transient nature of IL-2 and IL-2Re gene expression appears to contribute noz only to the development of the T-cell immune respon3e, but also to its physiological termination fn contra.~t to these T-cell stimuli, infection of human CD4 ~- T cells with the type 1 human T-cell le;ikemia virus (HTLV° 1) is associated with a persistent state of high level cell surface IL-2R expression17-2o. This pathogenic retrovirus, which has been implicated as the cause of the aggressive and often fatal adult T-cell leukemia (ATL), encodes a 40 kD~ tr~,",~ acting ~agu!aton/protein termed Tax21. In addition to activating the HTLV-1 long terminal repeat (LTR), and consequently the expression of viral genes under its control, ]ax shares with T-cell mitogens the ability to induce the transcription of various cellular genes including IL-2Rcx and IL-2 (Refs 22-28). These effects of Tax, which bypass the nermal intracellular controls that ensure transient IL-2Rcx gene expression, provide one explanation for the sustained expression of IL-2Ra observed in HTLV-1 infected T-cell lines. In addition, the effects of Tax protein on the IL-2 gene may promote autocrine proliferation of infected cells during © 1989, ElsevierScienceP[ ,~lishorsLtd, UK. 0167-4919189/$02.00