Epitope mapping: the first step in developing epitope-based vaccines

Abstract

Antibodies are an effective line of defense in preventing infectious diseases. Highly potent neutralizing antibodies can intercept a virus before it attaches to its target cell and, thus, inactivate it. This ability is based on the antibodies' specific recognition of epitopes, the sites of the antigen to which antibodies bind. Thus, understanding the antibody/epitope interaction provides a basis for the rational design of preventive vaccines. It is assumed that immunization with the precise epitope, corresponding to an effective neutralizing antibody, would elicit the generation of similarly potent antibodies in the vaccinee. Such a vaccine would be a 'B-cell epitope-based vaccine', the implementation of which requires the ability to backtrack from a desired antibody to its corresponding epitope. In this article we discuss a range of methods that enable epitope discovery based on a specific antibody. Such a reversed immunological approach is the first step in the rational design of an epitope-based vaccine. Undoubtedly, the gold standard for epitope definition is x-ray analyses of crystals of antigen:antibody complexes. This method provides atomic resolution of the epitope; however, it is not readily applicable to many antigens and antibodies, and requires a very high degree of sophistication and expertise. Most other methods rely on the ability to monitor the binding of the antibody to antigen fragments or mutated variations. In mutagenesis of the antigen, loss of binding due to point modification of an amino acid residue is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping are also useful. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For epitope mapping, computational algorithms have been developed, such as Mapitope, which has recently been found to be effective in mapping conformational discontinuous epitopes. The pros and cons of various approaches towards epitope mapping are also discussed.

Fig. 1

Mutagenic comparison of serine-containing statistically significant pairs (SSPs) versus amino acid pairs. (a) Amino acid sequences of b12 monoclonal antibody (mAb) affinity purified phages: C10 (wild-type phage), S4 and S8 in which Ser-4 and Ser-8 are converted to alanine. The LWSDL segment of phage C10, determined to contain SSPs which are important for b12 binding, is underlined. (b) Two-fold dilutions of equal amounts of phages were dot-blotted onto a nitrocellulose membrane filter and reacted with the b12 mAb. The fth-1 phage, containing no insert, was used as a negative control, and signals were produced using enhanced chemiluminescence (from Bublil et al.,[87] with permission).

Fig. 2

Space-filling representation of Mapitope predictions of (a) the monoclonal antibody 13b5 epitope on the surface of HIV-1 p24 antigen and (b) the trastuzumab epitope on the surface of the HER-2/neu receptor.[78] The number of amino acids comprising each antigen’s surface, genuine and predicted epitopes are given. Also given is the number of correctly predicted residues (indicated in green), over-predicted residues (indicated in red) and residues that were missed (indicated in blue). The p-values express a hyper-geometric distribution (the probability of randomly predicting the epitope) and were calculated as described by Mayrose et al.[85]