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.
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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.
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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.”
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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.
