PEPOP: computational design of immunogenic peptides

Abstract

Background: Most methods available to predict protein epitopes are sequence based. There is a need for methods using 3D information for prediction of discontinuous epitopes and derived immunogenic peptides.

Results: PEPOP uses the 3D coordinates of a protein both to predict clusters of surface accessible segments that might correspond to epitopes and to design peptides to be used to raise antibodies that target the cognate antigen at specific sites. To verify the ability of PEPOP to identify epitopes, 13 crystallographically defined epitopes were compared with PEPOP clusters: specificity ranged from 0.75 to 1.00, sensitivity from 0.33 to 1.00, and the positive predictive value from 0.19 to 0.89. Comparison of these results with those obtained with two other prediction algorithms showed comparable specificity and slightly better sensitivity and PPV. To prove the capacity of PEPOP to predict immunogenic peptides that induce protein cross-reactive antibodies, several peptides were designed from the 3D structure of model antigens (IA-2, TPO, and IL8) and chemically synthesized. The reactivity of the resulting anti-peptides antibodies with the cognate antigens was measured. In 80% of the cases (four out of five peptides), the flanking protein sequence process (sequence-based) of PEPOP successfully proposed peptides that elicited antibodies cross-reacting with the parent proteins. Polyclonal antibodies raised against peptides designed from amino acids which are spatially close in the protein, but separated in the sequence, could also be obtained, although they were much less reactive. The capacity of PEPOP to design immunogenic peptides that induce antibodies suitable for a sandwich capture assay was also demonstrated.

Conclusion: PEPOP has the potential to guide experimentalists that want to localize an epitope or design immunogenic peptides for raising antibodies which target proteins at specific sites. More successful predictions of immunogenic peptides were obtained when a peptide was continuous as compared with peptides corresponding to discontinuous epitopes. PEPOP is available for use at http://diagtools.sysdiag.cnrs.fr/PEPOP/.

Figure 1

PEPOP. (A) Description of PEPOP. Step 1: The solvent accessible surface area is calculated from the 3D structure of a protein. Step 2: Segments of accessible and contiguous amino acids are listed, and the shortest Euclidian distance matrix between segments is calculated. Step 3: The segments are clustered according to the matrix (clusters can be displayed on the 3D structure of the Ag). Step 4: Peptides are designed according to the clustering analyses. (B) The PEPOP interface. Segments and clusters can interactively be displayed on a 3D view of the Ag.

Figure 2

Comparison of known epitopes with the predicted major cluster. Amino acids in the epitope and in the major cluster are in green; those outside of the major cluster are in red; those non-accessible are in yellow; amino acids in the major cluster but not in the epitope are in blue. (A) Epitope on HEL recognized by mAb D44.1 (B) Epitope on HPr recognized by mAb Jel42.

Figure 3

Example of peptides designed from the 3D structure of IA-2. The two peptides, #5 (A) and #6 (B) of Table 3, were constructed from the longest segment (segment 1: reference segment) to which were added the nearest neighbor segments (segment 2 to respectively segment 4 and segment 5), according to two different methods (respectively method NN and NNd). Segments indicated in the IA-2 sequence are in the same color as in the 3D structure.

Figure 4

Sandwich immunoassay design for IA-2. (A) 3D view. Peptide #1 (in red) and peptide #4 (in blue) are represented on the 3D structure of IA-2. (B) Graphic representation. The graph indicates the reactivity of the polyclonal (Abs) with IA-2, in a sandwich-type ELISA format. The Ag, captured by the immobilized Ab, is detected by a biotinylated Ab, followed by addition of streptavidin-peroxidase. Reactivity of×, anti-peptide #4 with IA-2, detected by anti-peptide #1; ▲, anti-peptide #4 with TPO, detected by anti-peptide #1; ■, anti-peptide #15 with IA-2, detected by anti-peptide #1; ◆, anti-peptide #15 with TPO, detected by anti-peptide #1.