Isolation of antibodies specific to a single conformation-dependant antigenic determinant on the EG95 hydatid vaccine

Abstract

EG95 is a recombinant vaccine protein that elicits protection against hydatid disease in sheep. Previous studies have shown that the host-protective epitopes on EG95 depend on correct conformation and cannot be represented by simple "linear" peptides. By screening random peptide phage display libraries with polyclonal antibodies directed against conformation-dependant epitopes of EG95, we have selected a number of peptides that mimic these epitopes. The selected peptides did not show sequence homology to EG95. Antigen binding assays involving these peptides have provided evidence of at least four conformationally-dependant epitope regions on EG95. One of the selected peptides, E100, has been used to purify antibodies from anti-sera raised in sheep vaccinated with EG95. This yielded monospecific antibodies capable of recognizing recombinant EG95 in ELISA and native EG95 in Western blot assays. This antibody was demonstrated to be effective in antibody-dependant complement-mediated in vitro killing of Echinococcus granulosus oncospheres. Peptide E100 may represent the basis for a quality control assay for EG95 production, and has the potential to become a component of a synthetic peptide-based vaccine against E. granulosus.

Fig. 1

Indirect ELISA showing the enrichment of specific phage during the affinity selection process. Phages were affinity selected on anti-cEG95 using Library 1 (A) and Library 2 (B). Pooled phages were amplified and 10¹⁰ TU/ml were added to microtitre wells preadsorbed with anti-cEG95, anti-GST or skim milk powder. Phages were detected with anti-M13 HRP monoclonal antibody. R1 phage is a clone selected randomly from the library without any selection pressure.

Fig. 2

Binding of phage clones to anti-cEG95 (●), anti-GST (▾) and no antibody (○). The binding was quantified by the Phage Capture ELISA. Abscissa values are the common log of the phage concentration.

Fig. 3

Competitive phage indirect ELISA. Anti-cEG95 antibodies were adsorbed onto microtitre plates and a mixture of phage and protein competitor (EG95-MBP or GST) at a final concentration of 250 μg/ml in PBS with 5% skim milk powder. Phage binding was measured. Displayed values are the mean ± SEM of duplicate wells.

Fig. 4

Effect of synthetic peptides on the binding of phage clones to anti-cEG95 antibody. Individual panels each represent a single phage clone. The abscissas correspond to thirteen synthetic peptides. The ordinates are the percentage of inhibition that individual peptides cause relative to the addition of no inhibitor. Putative Epitopes A–F represent phages displaying similar patterns of inhibition by synthetic free peptides.

Fig. 5

Antibody capture ELISA results of affinity purification fractions. Affinity purified antibody fractions were examined for ability to bind EG95-MBP (●) or GST (○). The antibodies affinity purified to E100 peptide are represented in panel A. The antibodies affinity purified to G1 peptide are represented in panel B.

Fig. 6

Dot blot assay. Recombinant proteins EG95-GST, GST and EG95-MBP were blotted on to nitrocellulose membrane. The proteins were probed with pooled anti-sera from sheep immunised with EG95-GST (column A), naïve pooled sera (column B), antibodies affinity purified on E100-NHS-Sepharose (column C) and antibodies affinity purified on G1-NHS-Sepharose (column D).

Fig. 7

Western blots of E. granulosus oncosphere antigens reacted with sheep anti-sera to EG95-GST (lane A), naïve sera (lane B), antibodies affinity purified on E100-NHS-Sepharose (lane C) and antibodies affinity purified on G1-NHS-Sepharose (lane D). Lane E is a Coomassie stained SDS-PAGE of the oncosphere antigens.

Fig. 8

Survival of E. granulosus oncospheres in vitro in the presence of pooled anti-EG95-GST anti-sera (hatched bars), pre-immune sera (white bars), antibodies affinity purified on peptide E100 (black bars) and peptide G1 (grey bars). Sera in the test wells were diluted from 1:2.5 through to 1:20. Affinity purified antibodies were diluted from 6.8 μg/ml to 0.85 μg/ml. Assays for each dilution were performed in duplicate. Each individual bar represents the number of oncospheres (mean ± SEM) surviving after 10 days of culture.