Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C 1 Virus E2 Epitope 412-422. 2

234 words 24 Abstract (max 250 words, currently 234) 27 Hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved 28 region of E2 encompassing amino acids 412-423 (epitope I) and containing Trp420, a residue critical for 29 virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I 30 sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) 31 AP33 and HCV-1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide 32 containing E2 residues 412-422 (C-Epitope I) but not to the linear counterpart. These MAbs bound to 33 purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural 34 analysis of the complex between C-Epitope I and one of our MAbs (C2) show that the Trp420 side chain 35 is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and 36 solvent-exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic 37 peptide in the antibody combining site is rotated by 180° compared to other complexes. All these 38 structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to 39 the high selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the 40 epitope in more flexible and extended conformations, which recent data suggest play a role in the 41 mechanisms of neutralization escape. 42 43 Importance (max 150 words, currently 149) 44 Hepatitis C virus (HCV) remains a major health care burden affecting almost 3% of the global population. 45 The conserved epitope I comprising residues 412-423 of the viral E2 glycoprotein, is a valid vaccine 46 candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope 47 adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic 48 peptides mimicking this structure to elicit anti-HCV antibodies. MAbs that specifically recognize a cyclic 49 variant of the epitope bind to soluble E2 with lower affinity than other blocking antibodies and don't 50 3 neutralize virus. The structure of the complex between one such MAb and the cyclic epitope, together 51 with new structural data showing the linear peptide bound to neutralizing MAbs in extended 52 conformations, suggests that …