title: Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422.
abstract: The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape.
  
  
  OBJECTIVE
  Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic variant of the epitope bind to soluble E2 with a lower affinity than other blocking antibodies and do not neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together with new structural data showing the linear peptide bound to neutralizing MAbs in extended conformations, suggests that the epitope displays a conformational flexibility that contributes to neutralization escape. Such features can be of major importance for the design of epitope-based anti-HCV vaccines.
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy.
authors.forename: Annamaria
authors.initials: A
authors.lastname: Sandomenico
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Leonardi
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Berisio
authors.affiliation: Department of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Naples, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Sanguigno
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy Department of Pharmacy, University of Naples Federico II, Naples, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: Focà
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy Department of Pharmacy, University of Naples Federico II, Naples, Italy.
authors.forename: Annalia
authors.initials: A
authors.lastname: Focà
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy.
authors.forename: Alessia
authors.initials: A
authors.lastname: Ruggiero
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy.
authors.forename: Nunzianna
authors.initials: N
authors.lastname: Doti
authors.affiliation: Kedrion SpA, Lucca, Italy.
authors.forename: Livio
authors.initials: L
authors.lastname: Muscariello
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy Second University of Naples, Naples, Italy.
authors.forename: Daniela
authors.initials: D
authors.lastname: Barone
authors.affiliation: Kedrion SpA, Lucca, Italy.
authors.forename: Claudio
authors.initials: C
authors.lastname: Farina
authors.affiliation: Medical Research Council-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom.
authors.forename: Ania
authors.initials: A
authors.lastname: Owsianka
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy.
authors.forename: Luigi
authors.initials: L
authors.lastname: Vitagliano
authors.affiliation: Medical Research Council-University of Glasgow Centre for Virus Research, Glasgow, United Kingdom arvind.patel@glasgow.ac.uk menotti.ruvo@unina.it.
authors.forename: Arvind H
authors.initials: AH
authors.lastname: Patel
authors.affiliation: Institute of Biostructures and Bioimaging, CNR and CIRPeB, University of Naples Federico II, Naples, Italy arvind.patel@glasgow.ac.uk menotti.ruvo@unina.it.
authors.forename: Menotti
authors.initials: M
authors.lastname: Ruvo
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D018985:Epitopes, B-Lymphocyte / Q000276:immunology*; D016174:Hepacivirus / Q000276:immunology*; D018937:Hepatitis C Antibodies / Q000737:chemistry / Q000276:immunology* / Q000302:isolation & purification*; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D009500:Neutralization Tests; D011485:Protein Binding; D011487:Protein Conformation; D014759:Viral Envelope Proteins / Q000737:chemistry / Q000276:immunology*
chemical_list: D000911:Antibodies, Monoclonal; D018985:Epitopes, B-Lymphocyte; D018937:Hepatitis C Antibodies; D014759:Viral Envelope Proteins; C091015:glycoprotein E2, Hepatitis C virus
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