Identifying B‐cell Epitopes On a Protein - pediagenosis
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Monday, July 6, 2020

Identifying B‐cell Epitopes On a Protein

Identifying B‐cell Epitopes On a Protein
How many epitopes are there on a single protein? This depends upon how one defines an epitope. For the small protein lysozyme (molecular weight 14 300 daltons), the structures of three non-competing monoclonal antibodies in complex with the protein antigen have been determined. They have minimally overlapping footprints that cover just under half of the surface of the protein (Figure 5.7). One could extrapolate that a small protein such as this could have of the order of between three and six non‐overlapping epitopes recognized by noncompeting antibodies.

Figure 5.7 Three epitopes on the small protein lysozyme. The crystal structures of lysozyme bound to three antibodies (HyHEL‐5, HyHEL‐10, and D1.3) have been determined. In the figure, the Fv fragment of each antibody is shown separated from lysozyme to reveal the footprint of interaction in each case. The three epitopes are nearly non‐overlapping with only a small overlap between HyHEL‐10 and D1.3.

The specificity of a given antibody could then be defined by its ability to compete with the three to six “prototype” antibodies. In practice, this is often done; an antibody is said to be directed against a given epitope if it competes with a prototype antibody of known specificity. This is, of course, a rather simplistic view as many antibodies will compete with more than one prototype antibody allowing a more sophisticated B‐cell epitope map to be constructed. An even more sophisticated map can be constructed by scanning mutagenesis of the antigen. In the latter case, single positions in the antigen can be substituted by differing amino acids (usually alanine – hence the term “alanine scanning mutagenesis”) and the effects on antibody binding measured (see Figure 5.10). At this greater level of precision, it is likely that no two antibodies will give exactly the same footprint, and therefore no two antibodies recognize exactly the same epitope.

Figure 5.10 Energetic map of an antibody–antigen interface. The antibody D1.3 (single chain Fv (sFv) shown here) binds with high affinity to hen egg‐white lysozyme (HEL) and the crystal structure of the complex has been solved (see Figure 5.7). The energetic contribution of contact residues for both antibody and antigen can be estimated by substituting the residue with the relatively “neutral” residue alanine. The effect can be expressed in terms of the loss of free energy of binding for the interaction on alanine substitution (ΔΔG). A large positive value for ΔΔG shows that the alanine substitution has had a strong detrimental effect on binding and implies that the residue substituted forms a crucial contact in the interface between antibody and antigen. Clearly, most contact residues, particularly on the antibody, contribute little to the overall binding energy. There are clear “hotspots” on both antibody and antigen and the hotspot residues on the antibody side of the interaction correspond to those on the antigen side.

What determines the strength of the antibody response to a given epitope on a protein? There appear to be a number of factors involved. Perhaps the most important is the accessibility of the epitope on the protein surface. Loops that protrude from the surface of the folded protein tend to elicit particularly good antibody responses. The surface of influenza virus is decorated by the hemagglutinin protein (HA) (Figure 5.8a). On infection with the virus or vaccination with materials containing HA, antibodies are elicited, particularly to the “top” of the structure that neutralize the virus and protect against re‐infection or even infection itself in the case of a vaccine. However, mutations in the targeted regions allow the virus to “escape” from neutralizing antibodies and infect human hosts who were protected against the original form of the virus. Influenza epidemics thus directly reflect antibody targeting to certain preferred epitopes. Furthermore, vaccination tends to afford protection only against some strains of influenza virus and is typically administered on an annual basis. However, recently monoclonal antibodies have been described that neutralize many different strains of influenza virus (Figure 5.8a), so‐called broadly neutralizing antibodies, and the epitopes recognized by theses anti-bodies might be targeted by a suitable designed “universal flu vaccine.”

Figure 5.8 Antibodies bound to the surface glycoproteins of influenza virus and HIV. (a) A model of broadly neutralizing antibodies targeting relatively conserved epitopes on influenza virus hemagglutinin (HA). Natural infection and vaccination typically result in antibodies directed to highly variable epitopes on the top of the structure. However some antibodies (green) are able to recognize conserved elements associated with the sialic acid‐binding site in this region. Other antibodies (pink) recognize conserved epitopes in the stem of HA. The antibodies shown are Fab fragments. N‐linked glycans in blue. (b) A model of broadly neutralizing antibodies targeting conserved epitopes on the HIV envelope spike. Again natural infection typically elicits antibodies directed to highly variable epitopes toward the top of the structure, leading to strain‐specific antibodies. The spike is very densely coated with sugars that hinder antibody recognition. Nevertheless, some antibodies do bind to conserved epitopes as shown. N‐linked glycans in blue.

HIV is another virus that exploits the tendency of the anti-body system to respond to highly exposed variable regions on the viral surface protein to evade immune control. Following primary infection, it takes some time (weeks) for neutralizing antibodies to reach a level where they begin to inhibit virus replication. These antibodies are typically elicited to exposed regions on the virus. While these antibodies are being elicited, the virus has diversified (i.e., it has become a swarm of related viruses) through the errors associated with RNA to DNA transcription of this retrovirus. Among this swarm is a virus that has sequence changes in the epitopes targeted by the neutralizing antibody response that allow it to escape from the response. This new virus becomes predominant. Eventually a response is mounted to this virus and a second new virus emerges and so on. The antibody response chases the virus over many years but never appears to gain control. Nevertheless, again broadly neutralizing antibodies to HIV have been identified and are being intensely investigated for clues as to how to design an HIV vaccine, since such antibodies are precisely those that should offer protection against global circulating strains of HIV (Figure 5.8b). One point worthy of note is that accessible loops on protein structures tend to be flexible. Therefore epitope dominance has also been associated with flexible regions of a protein antigen.

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