Volume 20, Issue 12, Pages 2935-2943 (September 2017) Antibody 27F3 Broadly Targets Influenza A Group 1 and 2 Hemagglutinins through a Further Variation in VH1-69 Antibody Orientation on the HA Stem  Shanshan Lang, Jia Xie, Xueyong Zhu, Nicholas C. Wu, Richard A. Lerner, Ian A. Wilson  Cell Reports  Volume 20, Issue 12, Pages 2935-2943 (September 2017) DOI: 10.1016/j.celrep.2017.08.084 Copyright © 2017 The Author(s) Terms and Conditions

Cell Reports 2017 20, 2935-2943DOI: (10.1016/j.celrep.2017.08.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 1 Antibody 27F3 Neutralizes Heterotypic Influenza Viruses and Recognizes the Conserved HA Stem (A) Neutralization of 27F3 antibodies against representative strains of influenza viruses (with half maximal effective concentration [EC50] values). (B) The H1 HA (A/California/04/2009) trimer is shown as solid gray surface with one HA protomer colored with its HA1 in blue and HA2 in green. The 27F3 heavy chain is in pink, and light chain in light gray. Only two Fabs can be seen in this view as the third Fab is hidden behind the trimer at the back. Glycans are not included in the model due to lack of electron density. (C) The 27F3 epitope on H1 HA. The 27F3 heavy chain dominates contacts with the HA stem. The heavy-chain CDR loops that contact the HA stem are shown in pink as a backbone trace with side chains of contact residues as sticks. The epitope residues on the HA surface that interact with 27F3 are colored in blue (HA1) and green (HA2) and labeled. Cell Reports 2017 20, 2935-2943DOI: (10.1016/j.celrep.2017.08.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 2 Interaction of the HA Stem of A/California/04/2009 H1N1 by 27F3 Heavy-Chain CDRs and FR3 (A) CDR H1 contacts HA2 mainly though hydrogen bonds. (B) Interactions between CDR H2 and HA are mediated by hydrophobic contacts (side chain) and hydrogen bonding (main chain) of VH1-69 germline (GL) conserved residues Pro52a, Ile53, and Phe54. (C) The somatically mutated Ile73 contacts the hydrophobic HA surface, creating a buried surface area of ∼80 Å2. Additionally, the GL conserved Asp72 hydrogen bonds to HA1 Ser291. (D) Tyr100a dominates the CDR H3 contacts with the HA stem via aromatic stacking and hydrogen bonding. Hydrogen bonds are in dotted lines. Cell Reports 2017 20, 2935-2943DOI: (10.1016/j.celrep.2017.08.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 3 Comparison of VH1-69-Derived, Anti-HA Stem Antibodies to the HA Stem (A) Amino acid sequence comparison of the 27F3 VH domain with equivalent CR6261, CR9114, and F10 and VH1-69 germline sequences, with residues buried in the antibody-antigen interface marked in red. The heavy chain CDRs and FR3 of 27F3, CR9114, CR6261, and F10 that interact with the HA stem (HA1 blue, HA2 green) are shown in a backbone trace with side chains in sticks. To calculate the approach angles of different antibodies compared to 27F3, the HAs in the complexes were aligned and the rotation required to superimpose the VH domain of individual antibodies with 27F3 VH were calculated in Coot. A dotted line is drawn from FR3 residue 73 and the structurally conserved Tyr on CDR H3 to more readily visualize the different relative antibody rotations. (B–E) The VH domains of CR9114, CR6261, and F10 are rotated by 13°, 29°, and 52° away from the HA2 A-helix compared to the 27F3 VH domain (B). The CR6261, CR9114, and F10 complex coordinates used here are from PDB: 3GBN, PDB: 4FQI, and PDB: 3FKU, respectively. Cell Reports 2017 20, 2935-2943DOI: (10.1016/j.celrep.2017.08.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 4 Different Targeting Mechanisms of CDR H2 to the HA Stem (A) In complex with 27F3, His38 (HA1) shifts from the downward apo-conformation (gray) to the upward conformation (cyan) to form a hydrogen bond with the CDR H2 P52a main-chain carbonyl oxygen. Phe54 mainly interacts with His18 (HA1) and Trp21 (HA2). (B) The CR9114-bound HA shows two alternative conformations for His38 (PDB: 4FQI). Hydrogen bonds are shown as dotted lines. (C and D) CDR H2 of group 1 specific bnAbs CR6261 and F10 target His38 (HA1) and Trp21 (HA2), and His38 points downward to contact Phe54 of CDR H2 (PDB: 3GBN, PDB: 3FKU). Cell Reports 2017 20, 2935-2943DOI: (10.1016/j.celrep.2017.08.084) Copyright © 2017 The Author(s) Terms and Conditions

Figure 5 Different Conformations of CDR H2 in VH1-69 Anti-HA Stem Antibodies Pro52a, Ile53, and Phe54 of CDR H2 in various VH1-69 antibodies to the HA stem are shown in sticks with main-chain carbonyl oxygens labeled. (A) The group 1 and 2 binding antibodies 27F3 and CR9114 share similar main-chain conformations that enable CDR H2 carbonyl oxygens to H-bond with the HA stem. In addition, Phe54 stacks on Trp21 in a relatively planar orientation. (B) The group 1 antibodies CR6261 and F10 have their CDR H2 main chains oriented differently, making it less favorable to form H-bonds with the HA stem. Cell Reports 2017 20, 2935-2943DOI: (10.1016/j.celrep.2017.08.084) Copyright © 2017 The Author(s) Terms and Conditions