Structural and immunodiagnostic characterization of synthetic Antigen B subunits from Echinococcus granulosus and their evaluation as target antigens for cyst viability assessment

BACKGROUND: Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none appears promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human sera have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. METHODS: Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and ELISA, AgB1 and AgB2 were further tested in two ELISA setups, and extensively validated on 422 human sera. RESULTS: All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both N-Terminal and C-Terminal of each subunit are involved in homo-oligomer contact interfaces. No hetero-oligomer was identified. AgB1 and AgB2 ELISAs revealed different sensitivity relative to cyst stage. Of note, besides high specificity (97.2%), AgB1 revealed a higher sensitivity for active-transitional cysts (100% for CE1, 77.8% for CE2, 81.5% for CE3a, and 86.3% for CE3b) than for inactive cysts (41.7% for CE4 and 11.1% for CE5) and post-surgery patients (44%). Interestingly, 19/20 patients with spontaneously inactive cysts and 6/9 treated with albendazole over 5 years earlier were negative on the AgB1 assay. CONCLUSIONS: The structural characterization of subunits provides insights into the synthetic antigen conformation. The stage-related sensitivity of synthetic AgB1 holds promise as part of a multiantigen setting and deserves further longitudinal evaluation as marker of cyst viability.