View All Abstracts for Session 40
227b Hen Eggwhite Lysozyme as a Model Antigen for Producing Epitope-Targeted Vaccines
M.B. Irving*, A. Menendez and J.K. Scott
Simon Fraser University, Burnaby, BC, Canada
Background: Hen eggwhite lysozyme (HEL), a protein whose structure and antigenicity have been well characterized, is used in a vaccine model system designed to elicit antibodies (Abs) that are targeted against a specific epitope.
Methods and Results: Using D1.3 Fab, a murine monoclonal (M)Ab fragment that binds a discontinuous epitope on HEL, a panel of phage-displayed random peptide libraries was screened, and 2 distinct consensus sequences emerged. Two sublibraries were built from those sequences to optimize each consensus sequence. From the sublibraries, several phage clones were selected that bound D1.3 Fab and could compete with HEL for binding to D1.3. Further analysis showed that each of the selected peptides bore 2 cysteine residues and that intact intramolecular disulfide bridges are required for binding by D1.3. Synthetic peptide was prepared for 1 sequence from each of the sublibraries. Affinity analyses revealed that the peptides bind D1.3 with micromolar affinities. The 2 synthetic peptides in this study have 2 and 5 residues in common with the high-energy contacts that D1.3 makes with HEL. We suspect that these residues may account for a significant portion of the peptides’ binding energy, and that they form the basis of the peptides’ structural mimicry with the D1.3 epitope on HEL. Our goal is to develop a peptide-conjugate or recombinant phage vaccine that induces the production of high-titer “D1.3-like” Abs in BALB/c mice and to compare the structures and affinity of those Abs with those of the “native” D1.3 MAb. We tested several immunization strategies, including direct immunizations with the peptide and a prime-boost approach. In the latter, HEL was used in priming immunizations to provide multiple antigenic sites, including the “native” D1.3 epitope, whereas boosts were carried out with peptide conjugated to carrier protein; this was meant to amplify the “D1.3-like” Abs that had been produced by HEL in the priming. Mice immunized with phage displaying recombinant peptide (with and without HEL in the prime) produced a stronger antipeptide response than those immunized with peptide conjugated to carrier.
Conclusion: ELISA analysis suggests that Abs that cross-react with HEL and the peptide mimics are present in some mouse sera. Splenic B-cells from those mice will be isolated and their Ab genes characterized to determine if “D1.3-like” Abs have indeed been produced and amplified by the prime-boost immunizations.
