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

Daniela Pagnozzi, Francesca Tamarozzi, Anna Maria Roggio, Vittorio Tedde, Maria Filippa Addis, Salvatore Pisanu, Gabriella Masu, Cinzia Santucciu, Ambra Vola, Adriano Casulli, Giovanna Masala, Enrico Brunetti, Sergio Uzzau

Research output: Contribution to journalArticle

4 Citations (Scopus)

Abstract

Background: 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. 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 enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: 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. 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.

Original languageEnglish
Pages (from-to)1342-1351
Number of pages10
JournalClinical Infectious Diseases
Volume66
Issue number9
DOIs
Publication statusPublished - May 1 2018

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Echinococcus granulosus
Synthetic Vaccines
Cysts
Antigens
Enzyme-Linked Immunosorbent Assay
Albendazole
Echinococcosis
Serologic Tests
Serum
Western Blotting

Keywords

  • AgB
  • Cyst viability
  • Cystic echinococcosis
  • Immunological characterization
  • Serodiagnosis
  • Structural

ASJC Scopus subject areas

  • Microbiology (medical)
  • Infectious Diseases

Cite this

Structural and immunodiagnostic characterization of synthetic antigen B subunits from echinococcus granulosus and their evaluation as target antigens for cyst viability assessment. / 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.

In: Clinical Infectious Diseases, Vol. 66, No. 9, 01.05.2018, p. 1342-1351.

Research output: Contribution to journalArticle

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. / Structural and immunodiagnostic characterization of synthetic antigen B subunits from echinococcus granulosus and their evaluation as target antigens for cyst viability assessment. In: Clinical Infectious Diseases. 2018 ; Vol. 66, No. 9. pp. 1342-1351.
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abstract = "Background: 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. 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 enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: 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. 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.",
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AU - Pagnozzi, Daniela

AU - Tamarozzi, Francesca

AU - Roggio, Anna Maria

AU - Tedde, Vittorio

AU - Addis, Maria Filippa

AU - Pisanu, Salvatore

AU - Masu, Gabriella

AU - Santucciu, Cinzia

AU - Vola, Ambra

AU - Casulli, Adriano

AU - Masala, Giovanna

AU - Brunetti, Enrico

AU - Uzzau, Sergio

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N2 - Background: 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. 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 enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: 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. 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.

AB - Background: 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. 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 enzyme-linked immunosorbent assay (ELISA), AgB1 and AgB2 were further tested in two ELISA setups and extensively validated on 422 human serum samples. Results: 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. 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.

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KW - Immunological characterization

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