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Our immune system counters the invasion by viruses, bacteria, parasites and
other pathogens, with specialized molecules and cells that eliminate or neutralize
those invaders or the toxins that they produce. An important molecular arm
of the immune system is composed of the antibodies which bind tightly and
specifically to foreign substances (antigens). The specificity of an antibody
molecule is so exquisite that it can distinguish between, for example, two
protein antigens that differ by only one amino acid residue.
Very importantly, our immune system has memory and we are able to mount an
immediate response to infection by a pathogen that we had encountered before.
Indeed, we are able to prevent many diseases by vaccination, i.e., by exposing
our immune system to non-infective versions of the pathogen, or to its antigen(s).
However, many pathogens, like the flu virus, the cold virus, and the virus
that causes AIDS, are able to evade the immune response by constantly mutating.
Sometimes, evasion is accomplished by a single nucleotide change in an antigen
that is coded by more than a thousand bases.
Our immune system is capable of reacting to essentially all parts of an antigen,
although some parts, the so-called immunodominant epitopes, elicit most of
the response. The clever pathogens, of course, localize their mutations in
those immunodominant epitopes. A strategy for thwarting those pathogens then
is to use as vaccines versions of their antigens, in which the antigenicity
(the ability to attract an antibody response) of their immunodominant epitopes
had been reduced so that our immune system would react vigorously to the other
(less reactive) parts of the antigens also. Implementation of the strategy
is done in three steps: first, the immunodominant epitopes are located; second,
the residues which are responsible for the high antigenicity of those epitopes
are identified; and third, those residues are replaced with amino acids that
are expected to contribute less to the antigenicity, while preserving structure.
The strategy is illustrated here using the design of a possible vaccine against
an H3N2 flu virus as an example.
The
antigen
The antigen is the hemagglutinin of the influenza A virus, (A/New York/55/2004(H3)),
(entry ABO37541 in the NCBI database (http://www.ncbi.nlm.nih.gov)).
A three-dimensional model for this molecule was generated using the protein
modeling software, SWISS-MODEL (Schwede et al. 2003) (http://swissmodel.expasy.org//SWISS-MODEL.html),
and using the Protein Data Bank entry 1HA0 (Chen et al. 2002) as template.
Location
of the immunodominant epitopes
The strength of the binding of antibodies to antigens is determined by the
reactivities of both the paratope (the antigen-binding site) of the antibody
and of the epitope (the site to which the antibody binds) of the antigen.
In the case of protein antigens, the antigenicity of an epitope is ultimately
determined by the physicochemical properties of the amino acids which constitute
the epitope. Several measures of the physicochemical properties of the various
naturally-occurring amino acids are available (for example, Grantham 1974,
Sandberg et al. 1998) and such measures can be used to estimate the antigenicity
of protein antigens (see, for example, Padlan 1985).
Here, an epitope is defined as the cluster of amino acids within a certain
distance from a chosen point in the three-dimensional structure of an antigen
(for example, the alpha-carbon position nearest the geometric center of the
cluster) and the antigenicity of the epitope is defined as the sum of the
contributions of the constituent amino acids. The contribution of each amino
acid residue is the chosen physicochemical measure weighted by the solvent
exposure of the residue.
Figure 1.
Plot of antigenicity vs. position
for the hemagglutinin of (A/New York/55/2004(H3)) virus before (top), after
one cycle (middle), and after two cycles (bottom), of de-antigenization using
Sandberg et al. (1998) values. Antigenicities are in arbitrary units.
The antigenicity values of the epitopes of (A/New York/55/2004(H3)), calculated
using parameters proposed by Sandberg et al. (1998), are plotted against residue
position in Figure 1 (top plot). Several peaks are observed to be significantly
above the average; they are taken to represent the "immunodominant epitopes".
Amino
acid replacements and recalculations
The residues, which contribute significantly to the "immunodominant epitopes",
were replaced by amino acids that are expected to reduce antigenicity. A model
of the molecule, with the suggested sequence changes incorporated, was built
using SWISS-MODEL and the antigenicities recalculated. After two cycles of
amino-acid replacements, remodeling, and recalculation of antigenicities,
no additional replacements were suggested. The antigenicity plots resulting
from these two cycles are included in Figure 1 (middle and bottom plots).
It is observed that after one cycle (middle plot), many of the prominent peaks
in the original plot (top) are greatly reduced in size; the "immunodominant"
peaks are essentially all gone after the second cycle (bottom plot)
Possible vaccines against this particular influenza A virus
Either of the sequences (not shown), which produced the middle and bottom
plots in Figure 1, could represent a possible vaccine against influenza A
virus, (A/New York/55/2004(H3)).
Further
applications of the strategy
The de-antigenization of immunodominant epitopes can also be used in designing
hypoallergenic molecules useful in allergy desensitization (allergy shots).
Allergy is caused by the binding of allergens to a type of antibody, IgE,
that is bound to mast cells in connective tissue and basophils in the blood.
Allergen-IgE binding triggers the cells to release histamines and other compounds
responsible for allergy symptoms including anaphylaxis. Allergens also have
immunodominant epitopes, called dominant IgE epitopes, to which specific IgE
binds. Allergy desensitization is done by introducing increasing amounts of
allergen into the patient to elicit an IgG rather than an IgE response. De-antigenization
of the dominant IgE epitopes will render the existing specific IgE incapable
of binding to the modified allergen, so that larger doses of the modified
allergen could be administered with lessened chance of anaphylaxis.
Figure 2.
Plot of antigenicity vs. position for the Der p 1 antigen from European house
dust mites before (top), after one cycle (middle), and after two cycles (bottom),
of de-antigenization using Sandberg et al. (1998) values. Antigenicities are
in arbitrary units.
Applying the strategy of de-antigenization to Der p 1 (NCBI entry P08176 (Chua
et al. 1988)), the major allergen of the European house dust mite, D.
pteronyssinus, results in the antigenicity plots shown in Figure 2. Either
of the sequences which generated the middle and bottom plots in Figure 2 and
those which would result from the application of the strategy to the other
allergens of D. pteronyssinus represent molecules that should be
useful in the desensitization against house dust mite allergy.
Patent applications for the use of the strategy in designing vaccines against
constantly mutating pathogens and in designing molecules useful in allergy
desensitization have been submitted (US11/645,448 and US11/823,330, respectively).
