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the mice subcutaneously or intradermally with 100 micrograms of peptides in adjuvant
The animals are sacrificed at 42 days, and the spleno-
three times, at 2-week intervals.
cytes are obtained for use in T-cell (ELISpot) assays.
One can expect to find that these assays confirm that the original epitopes (wild
type) are more immunogenic than the modified epitopes. Measurable T-cell re-
sponses seen in mice given the wild-type epitopes, and not seen in modified peptide
or placebo-vaccinated mice, validate the effect of the modifications.
6.4.6 Evaluating the Effect on Protein Structure and Function
Some amino acid substitutions may have a profound effect on the functionality of the
protein. Several methods of assessing the effect of deimmunizing changes on the
function target protein exist. A comparison of the sequence containing the proposed
changes with the sequences of other similar proteins can indicate areas of variability
that may be tolerant of change. Molecular modeling can also help elucidate the im-
pact of the proposed changes on the structure of the protein. These two approaches
are explained in more detail in the next paragraphs.
6.4.6.1 Evaluate by Comparison with Other Similar Proteins
Within a particular protein, amino acid sequences that are conserved in the course
of evolution can be presumed to be required for the proper function of the protein.
For example, an analysis of 92 variants of cytochrome c derived from eukaryotic
species shows that 21 of 104 residues are exactly conserved and another 14 are
mainly restricted to amino acids that share common properties such as I, L, V or F,
and Y. Thus, modifications of the cytochrome c protein aiming to de-immunize the
protein should not be performed at any of these 35 residues, as they could eliminate
monomers that are critical to the function and/or structure of the protein. This ap-
proach was recently described in a paper by Kersh, Miley, Nelson, Grakoui,
Horvath, Donermeyer, Kappler, Allen, and Fremont (2001).
6.4.6.2 Evaluate the Effect on Structure Using Modeling
Structural analyses comparing wild-type and modified protein sequences can also be
performed. In general these methods start by modeling the observed structure of the
wild-type protein. Interactions between the individual atoms that make up the struc-
ture are measured and an overall energy value is calculated. Modified proteins are
then overlaid on top of the wild-type structure and reiterative modeling of the protein
is performed until a minimum energy configuration is achieved. The difference in
overall energy between the wild-type protein and its homologue, as measured by
calculating the root mean square (RMS) deviation of the positions of the alpha-
carbons in the protein chains, can be used as a predictor of the structural deviation
between the two proteins.
Several
in silico
methods are available for evaluating the effect of amino acid
substitution on protein structure. A number of computational chemistry software
programs such as InsightII (Accelrys, Inc.), Cerius2 (Accelrys, Inc.), Sybyl (Tripos, Inc.),
