Immunoinformatics may lead to a reappraisal of the nature of B cell epitopes and of the feasibility of synthetic peptide vaccines

The terms we use to describe the experimental objects we study have an uncanny way of shaping the way we think about them. The B cell epitopes of proteins, which correspond to the regions recognized by the binding sites (or paratopes) of soluble and membrane-bound antibody molecules, have been described as either continuous or discontinuous depending on whether the amino acids included in the epitope are contiguous in the polypeptide chain or not. This terminology leads one to accept that the units of recognition operative in epitope-paratope interactions are individual amino-acids whereas it is, of course, at the level of individual atoms that the so-called ‘recognition’ takes place. The distinction between continuous and discontinuous epitopes is actually fuzzy since discontinuous epitopes often contain continuous stretches of a few contiguous residues (that may bind to certain antibodies). Furthermore, continuous epitopes usually contain a number of indifferent residues that are not implicated in the binding interaction and can be replaced by any other amino acid without impairing antigenic activity. Such continuous epitopes may thus be considered as discontinuous in the functional sense. Some authors have challenged the view that native proteins possess continuous epitopes recognized by antiprotein antibodies and suggested that so-called continuous epitopes are actually ‘unfoldons’, i.e., unfolded regions of the protein antigen that cross-react with antibodies specific for the denatured protein. It is argued that such antibodies are present in antiprotein sera because some of the molecules used for immunization have become denatured before or after being injected in the animal. Such arguments are difficult to refute since it is impossible to verify the exact conformation of the immunogen when it is interacting with a B cell receptor in the immunized host (the so-called black box conundrum). In the reciprocal situation where antibodies raised to peptides are allowed to react with the cognate protein, it also cannot be discounted that the antibodies recognize the protein only because the antigen preparation used in the assay contained some denatured molecules. It is now recognized that earlier erroneous claims that immunization with peptides always elicits high levels of antibodies that cross-react with the native, cognate protein arose because it was not realized that the protein used in solidphase immunoassays had become denatured by adsorption to plastic. Whereas the reactivity of a discontinuous epitope is clearly dependent on the native conformation of the molecule, it is less obvious that a continuous epitope is also conformation-dependent. This is shown, however, by the fact that an antibody will not recognize in the same manner, a peptide when it is free in solution, conjugated to a carrier or adsorbed to a solid-phase. It must be stressed that an epitope is not an intrinsic feature of a protein molecule but is a relational entity that can be defined only by its ability to react with the paratope of an antibody molecule. This means that epitopes, in addition to being regions of an antigen defined in structural terms, are also entities endowed with a specific recognition activity that can be assessed only in a functional binding assay. Binding assays, therefore, lead to a functional description of epitopes, usually in terms of which alterations induced by chemical manipulation or site-directed mutagenesis, are able to affect the binding reactivity. This functional description, which involves the four dimensions of space-time is complementary to the information derived from the static structure of the epitope revealed by X-ray crystallography of antigen-antibody complexes. It is generally assumed that crystallographic analysis of immune complexes gives the most reliable information on the structure of epitopes although this is available for only a limited number of antigens. A further important limitation is that the structure seen in the complex is usually different from the structure of the respective binding sites in the free antigen and antibody molecules, i.e., before they have been altered by the induced fit and mutual adaptation of the two partners that occur during the interaction. The conformation of the epitope before complexation is probably the one that is relevant for understanding which structure is recognized by the immune system. Most of our knowledge of protein epitopes has been obtained by studying the antigenic cross-reactivity between the intact protein and peptide fragments. Such studies lead to the identification of linear peptides, that only partly mimic the configuration of the atoms in the immunogen which initially interacted with the membrane-bound antibodies of B-cells. These cross-reactive peptides are detected because the relationship between an epitope and a paratope is never of an exclusive nature. As a result the exact boundaries and minimum size of the continuous epitopes delineated with cross-reactive peptides remain generally illdefined. It must be emphasized that studies of protein antigenicity using peptide fragments only gives information on the phenomenon of cross-reactivity between what are actually dissimilar epitopes in the peptide and protein. It is well-established that the structure of a peptide bound to antibody is usually different from the structure of the corresponding region in the intact, cognate protein.