Volume 26, Issue 2, Pages 438-446.e5 (January 2019) Double Lock of a Human Neutralizing and Protective Monoclonal Antibody Targeting the Yellow Fever Virus Envelope  Xishan Lu, Haixia Xiao, Shihua Li, Xuefei Pang, Jian Song, Sheng Liu, Huijun Cheng, Yan Li, Xiangxi Wang, Chaobin Huang, Tianling Guo, Jan ter Meulen, Stephane Daffis, Jinghua Yan, Lianpan Dai, Zihe Rao, Hans-Dieter Klenk, Jianxun Qi, Yi Shi, George F. Gao  Cell Reports  Volume 26, Issue 2, Pages 438-446.e5 (January 2019) DOI: 10.1016/j.celrep.2018.12.065 Copyright © 2018 The Authors Terms and Conditions

Cell Reports 2019 26, 438-446.e5DOI: (10.1016/j.celrep.2018.12.065) Copyright © 2018 The Authors Terms and Conditions

Figure 1 Binding, Neutralization, and Protection of mAb 5A against YFV (A) BIAcore diagram of 5A IgG bound to YFV-17D sE protein. 5A IgG binds to the YFV-17D sE protein with high affinity and slow kinetics. The KD value was calculated using BIAcore 3000 analysis software (BIAevaluation Version 4.1). (B) BIAcore diagram of 5A IgG bound to YFV-China sE protein. (C) YFV-17D amplified in Vero cells was mixed with threefold serial dilutions of the indicated mAbs, and neutralization activity was then evaluated by a FACS-based neutralization assay using Vero cells. Data are presented as mean ± SEM and representative of two independent experiments. (D) Neutralization activity of the indicated mAbs against YFV-China. Data are presented as mean ± SEM and representative of two independent experiments. (E) Therapeutic effect of mAb 5A against YFV-17 lethal challenge. BALB/c mice were randomly assigned into groups of five animals. For treatment, mice were challenged with YFV-17D 24 hr before the administration of the same volume of mAb 5A or PBS as a control or 2G4 as an antibody isotype control. Mouse survival was recorded daily. (F) Body weight was recorded daily. Survival and body-weight-loss curves are plotted using GraphPad Prism 5. See also Figure S4. Cell Reports 2019 26, 438-446.e5DOI: (10.1016/j.celrep.2018.12.065) Copyright © 2018 The Authors Terms and Conditions

Figure 2 Structures of YFV-17D sE in Both Pre- and Post-fusion States (A) Schematic representation of YFV E protein. Domain I (DI) is in red, DII is in yellow, and DIII is in blue. A 52-residue “stem” region (light blue) is sandwiched between the DIII and the C-terminal transmembrane anchor (dark blue). (B) Ribbon diagram of the sE dimer. This is the conformation of YFV sE in its pre-fusion state in solution above the fusion pH. See also Figures S1 and S2. (C) Conformational transition of sE from its pre-fusion state to post-fusion state. DIII and DII rotate 62.7 and 23.5 degrees toward DI, respectively, which brings DIII closer to the fusion loop (FL). (D) Ribbon diagram of the sE trimer. The hydrophobic FLs are exposed on the tip of the trimer, and key residue W101 (dark yellow) protrudes ahead. See also Figures S1–S3. Cell Reports 2019 26, 438-446.e5DOI: (10.1016/j.celrep.2018.12.065) Copyright © 2018 The Authors Terms and Conditions

Figure 3 Complexes and Footprints of mAb 5A scFv on YFV-17D sE in Both Pre- and Post-fusion States (A) Overall view of the 5A scFv complexed with YFV sE in its pre-fusion dimer. The sE moiety is colored by Ds (DI is in red, DII is in yellow, and DIII is in blue); the scFv molecules are colored in dark green and light gray for heavy and light chains, respectively. (B) Zoom of the 5A-YFV sE (pre-fusion) interactions to show the recognition sites. Hydrogen bonds are shown as dotted lines, and the residues involved are labeled. (C) Overall view of 5A scFv complexed with YFV sE in its post-fusion trimer. (D) Overall view of 5A scFv bound to YFV sE in its post-fusion monomer. (E) Zoom of the 5A-YFV sE (post-fusion) interactions to show the recognition sites. Hydrogen bonds are shown as dotted lines, and the residues involved are labeled. See also Figure S4. (F and G) Footprints of 5A on a surface representation of YFV sE in pre-fusion (F) and post-fusion (G). The two subunits of sE in the dimer are in light and dark gray. Relevant antigenic sE regions are labeled (FLs are in red, b strands are in yellow, ij loops are in pink, bc loops are in green, the 150 loop is in orange, and kl loops are in blue). (H and I) Footprints of dengue virus (DENV) and/or ZIKV EDE antibodies C8 (H) and A11 (I) on a surface representation of DENV-2 sE in the pre-fusion state. Relevant antigenic sE regions are labeled (sites in DIII are in cyan; color coding in other regions is as in F and G). See also Figures S3 and S4 and Tables S1 and S2. Cell Reports 2019 26, 438-446.e5DOI: (10.1016/j.celrep.2018.12.065) Copyright © 2018 The Authors Terms and Conditions

Figure 4 Docking IgG onto the Complex of 5A scFv-YFV sE Trimer IgG molecules (PDB: 1HZH) are docked to the 5A scFv-YFV sE trimer (shown as a ribbon). Color coding in YFV sE is the same as in Figure 3. Three IgG molecules are colored in green. See also Figure S5. Cell Reports 2019 26, 438-446.e5DOI: (10.1016/j.celrep.2018.12.065) Copyright © 2018 The Authors Terms and Conditions

Figure 5 5A Neutralize YFV by Inhibiting Both Attachment and Fusion of YFV (A) The amount of virus attached on the cell surface was detected by RT-PCR. Viruses were exposed to the indicated concentrations of 5A before incubation with BHK-21 cells. Ebola-specific mAb 13C6 (2.5 μg/mL) was used as an unrelated antibody for control. Values are means with SD of four samples per group. p values were analyzed by Student’s t test (n.s., p > 0.05; ∗p < 0.05; ∗∗∗∗p < 0.001). Data are representative of two independent experiments. (B) Neutralization test of YFV-China when 5A is added pre- or post-attachment of virus to cells that were incubated at 4°C. Results show no obvious difference in the neutralization profile. Data are presented as mean ± SD and representative of two independent experiments. (C) 5A partially blocks low-pH-induced fusion of YFV-17D with liposomes. Liposomes were loaded with self-quenching concentrations of the fluorescent dye calcein. (Perturbation of the bilayer causes the release of calcein, resulting in dilution and a consequent increase in its fluorescence.) Fusion of YFV with liposomes occurred at pH 4.9. Fusion was measured in real time as described in the STAR Methods. Representative viral fusion curves are from two independent experiments. Cell Reports 2019 26, 438-446.e5DOI: (10.1016/j.celrep.2018.12.065) Copyright © 2018 The Authors Terms and Conditions

Figure 6 Proposed Neutralization Mechanism of 5A Antibody against YFV mAb 5A is proposed to neutralize YFV by interfering 3 stages during virus entry and double locking the E protein in both pre- and post-fusion states. (1) By binding to the pre-fusion E protein in YFV, 5A alters the spatial distance between the virus surface and the host cell membrane, thereby hindering virus attachment. (2) Virus-bound or unbound 5A antibodies can be internalized into the early endosome (pH > 5.5) by endocytosis and then inhibit the dimer-to-timer transition of E protein in the late endosome (pH < 5.5). (3) By binding to the E trimers in their fusion intermediate state, 5A causes steric hindrance for insertion of the FL into the endosomal membrane and thereby blocks virus fusion and the release of genetic material. Cell Reports 2019 26, 438-446.e5DOI: (10.1016/j.celrep.2018.12.065) Copyright © 2018 The Authors Terms and Conditions