Structural and Immunodiagnostic Characterization of Synthetic Antigen B Subunits From Echinococcus granulosus and Their Evaluation as Target Antigens for Cyst Viability Assessment

• Published in: Clinical infectious diseases 2018-05, Vol.66 (9), p.1342-1351
• Main Authors: Pagnozzi, Daniela, Tamarozzi, Francesca, Roggio, Anna Maria, Tedde, Vittorio, Addis, Maria Filippa, Pisanu, Salvatore, Masu, Gabriella, Santucciu, Cinzia, Vola, Ambra, Casulli, Adriano, Masala, Giovanna, Brunetti, Enrico, Uzzau, Sergio
• Format: Article
• Language: English
• Subjects: Albendazole; and Commentaries; Antigens; Conformation; Cysts; Diagnostic systems; Echinococcosis; Enzyme-linked immunosorbent assay; Evaluation; Immunoblotting; Immunoreactivity; Interfaces; Oligomerization; Oligomers; Patients; Sensitivity; Structural analysis; Viability
• ISSN: 1058-4838; 1537-6591
• DOI: 10.1093/cid/cix1006

Several tools have been proposed for serodiagnosis of cystic echinococcosis (CE), but none seems promising for cyst viability assessment. Antigens with stage-specific diagnostic value have been described, but few studies with well-characterized antigens and human serum samples have been performed. Antigen B (AgB) proteoforms hold promise as markers of viability, due to their differential stage-related expression and immunoreactivity. Four AgB subunits (AgB1, AgB2, AgB3, AgB4) were synthesized and structurally characterized. Based on the preliminary evaluation of the subunits by western immunoblotting and enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. All subunits showed a high degree of spontaneous oligomerization. Interacting residues within oligomers were identified, showing that both the 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 sensitivities 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 postsurgical patients (44%). Interestingly, 19 of 20 patients with spontaneously inactive cysts and 6 of 9 treated with albendazole >5 years earlier were negative on the AgB1 assay. 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.