Structure-based design of hepatitis C virus E2 glycoprotein improves serum binding and cross-neutralization

Brian G. Pierce; Zhen-Yong Keck; Ruixue Wang; Patrick Lau; Kyle Garagusi; Khadija Elkholy; Eric A. Toth; Richard A. Urbanowicz; Johnathan D. Guest; Pragati Agnihotri; Melissa C. Kerzic; Alexander Marin; Alexander K. Andrianov; Jonathan K. Ball; Roy A. Mariuzza; Thomas R. Fuerst; Steven K.H. Foung

The copyright holder for this preprint (which was not peer-reviewed) is the . https://doi.org/10.1101/2020.04.15.044073 doi: bioRxiv preprint 4D, HC33.1, AR3A, HC33.1), and X-ray structural characterization studies (AR3A, HEPC74, HC84.1, HC33.1, HCV1) (32, 36-39). The HC84.26.WH.5DL is an affinity matured clone of the parental HC84.26 antibody with improved affinity and neutralization breadth over the parental antibody (31). The binding site of CD81 has been mapped to E2 residues in antigenic domains B, D, and E (35), thus CD81 binding provides additional assessment of antigenicity of that E2 supersite (6) . Binding experiments with this panel showed nanomolar binding affinities to wildtype sE2, which were largely maintained for sE2 designs. A 10-fold increase in binding affinity of sE2 design H445P for domain D HMAb HC84.26.WH.5DL was observed, showing that this design, located within antigenic domain D, not only maintained affinity, but improved engagement in that case; a steady-state binding fit for that interaction is shown in Figure 3A . However, this effect was not observed for combinations of designs including H445P, suggesting possible interplay between designed sites. As expected, domain A hyperglycosylation designs Y632NS, ΔHVR1-Y632NS, and Triple (ΔHVR1-H445P-Y632NS) showed loss of binding (>5-fold for each) to antigenic domain A HMAb CBH-4G (Y632NS-CBH-4G binding measurement is shown in Figure 3B ), though we did not observe disruption of binding to CBH-4D. Additionally, design ΔHVR1-Y632NS showed moderate (6-fold) loss of CD81 binding, which was not the case for other designs. As domain A HMAbs have distinct, albeit similar, binding determinants on E2 (28), differential effects on domain A antibody binding by Y632NS variants reflect likely differences in HMAb docking footprints on E2. Measurements of glycan occupancy at residue 632 using mass spectroscopy showed partial levels of glycosylation at that site for Y632NS and combinations (Table 4) , which may be responsible for incomplete binding ablation to the tested antigenic domain A HMAbs. As alanine substitution at Y632 was previously found to disrupt binding of domain A antibodies (28), it is possible that the Y632N amino acid substitution in the Y632NS author/funder. All rights reserved. No reuse allowed without permission.