Exploring peptide mimics for the production of antibodies against discontinuous protein epitopes

Abstract

Peptide "mimics" (mimotopes) of linear protein epitopes and carbohydrate epitopes have been successfully used as immunogens to elicit cross-reactive antibodies against their cognate epitopes; however, immunogenic mimicry has been difficult to achieve for discontinuous protein epitopes. To explore this, we developed from phage-displayed peptide libraries optimized peptide mimics for three well-characterized discontinuous epitopes on hen egg lysozyme and horse cytochrome c. The peptides competed with their cognate antigens for antibody binding, displayed affinities in the nM range, and shared critical binding residues with their native epitopes. Yet, while immunogenic, none of the peptides elicited antibodies that cross-reacted with their cognate antigens. We analyzed the 3-D structure of the site within each discontinuous epitope that shared critical binding residues with its peptide mimic, and observed that in each case it formed a ridge-like patch on the epitope; in no case did it cover most or all of the epitope. Thus, the peptides' lack of immunogenic mimicry could be attributed to their inability to recapitulate the topological features of their cognate epitopes. Our results suggest that direct peptide immunizations are not a practical strategy for generating targeted antibody responses against discontinuous epitopes.

Figure 1

Overlapping 10-mer peptides covering the HEL amino acid sequence probed with anti-HEL sera reveals common patterns of reactivity. Shown are images from x-ray film exposed to membrane incubated with two different anti-HEL serum samples (A & B). The top and bottom images are duplicates of each set of HEL overlapping peptides. For corresponding peptide sequences see Supplemental Table 3.

Figure 2

Average half-maximum Ab titers after four immunizations against phage, synthetic peptide, and cognate antigen produced by mice immunized with phage clones D1.3p-E, D1.3p-C, HyHEL-5p and E8p. nd: Not done, this synthetic peptide could not be made.

Figure 3

Defining CBRs in peptides D1.3p-E and E8p. (A) Binding of D1.3 Fab and IgG with phage clones bearing Ala replacements in the D1.3-E phage peptide. (B) Competition for E8 Fab binding by E8 phage, CytC and synthetic E8 peptides bearing Ala or Ser replacements at each amino acid position versus immobilized E8 phage and CytC. A lack of competition by a peptides (i.e., high ELISA signal) shows that its replaced residue is critical to E8 Fab binding. ELISA readings were taken at 30 minutes, and HRP-conjugated-goat-(anti-mouse-IgG-H+L) Ab was used as the secondary reagent for both experiments.

Figure 3

Defining CBRs in peptides D1.3p-E and E8p. (A) Binding of D1.3 Fab and IgG with phage clones bearing Ala replacements in the D1.3-E phage peptide. (B) Competition for E8 Fab binding by E8 phage, CytC and synthetic E8 peptides bearing Ala or Ser replacements at each amino acid position versus immobilized E8 phage and CytC. A lack of competition by a peptides (i.e., high ELISA signal) shows that its replaced residue is critical to E8 Fab binding. ELISA readings were taken at 30 minutes, and HRP-conjugated-goat-(anti-mouse-IgG-H+L) Ab was used as the secondary reagent for both experiments.

Figure 4

Surface topology of the D1.3 (A, B), HyHEL-5 (C, D), and E8 (E, F) epitopes and paratopes. Surface of the Ag epitopes (A, C and E) with residues shared between the epitope and the selected peptides shown in green, red and blue, with the remainder of the epitope shown in orange. Oxygen and nitrogen atoms that make direct contacts with Ab are shown in red and blue, respectively. Surface representation of the Ab paratopes (B, D and F) in complex with Ag (ribbon and epitopes in α-carbon backbone representation). Residues in the paratope that contact residues shared between the epitope and the peptide are shown in green with oxygen and nitrogen contacts in red and blue; the remainder of the paratope is orange. Shared residues in the epitopes are labeled and shown as ball-and-sticks. The paratopes by themselves are shown in Supplemental Figure 5. Images were generated using Protein Data Bank file 1FDL for the HEL:D1.3 complex, 1YQV for the HEL:HyHEL complex and 1WEJ for the CytC:E8 complex.