A Prevalent Focused Human Antibody Response to the Influenza Virus Hemagglutinin Head Interface

Abstract

Novel animal influenza viruses emerge, initiate pandemics, and become endemic seasonal variants that have evolved to escape from prevalent herd immunity. These processes often outpace vaccine-elicited protection. Focusing immune responses on conserved epitopes may impart durable immunity. We describe a focused, protective antibody response, abundant in memory and serum repertoires, to a conserved region at the influenza virus hemagglutinin (HA) head interface. Structures of 11 examples, 8 reported here, from seven human donors demonstrate the convergence of responses on a single epitope. The 11 are genetically diverse, with one class having a common, IGκV1-39, light chain. All of the antibodies bind HAs from multiple serotypes. The lack of apparent genetic restriction and potential for elicitation by more than one serotype may explain their abundance. We define the head interface as a major target of broadly protective antibodies with the potential to influence the outcomes of influenza virus infection. IMPORTANCE The rapid appearance of mutations in circulating human influenza viruses and selection for escape from herd immunity require prediction of likely variants for an annual updating of influenza vaccines. The identification of human antibodies that recognize conserved surfaces on the influenza virus hemagglutinin (HA) has prompted efforts to design immunogens that might selectively elicit such antibodies. The recent discovery of a widely prevalent antibody response to the conserved interface between two HA "heads" (the globular, receptor-binding domains at the apex of the spike-like trimer) has added a new target for these efforts. We report structures of eight such antibodies, bound with HA heads, and compare them with each other and with three others previously described. Although genetically diverse, they all converge on a common binding site. The analysis here can guide immunogen design for preclinical trials.

Keywords: X-ray crystallography; conserved epitope; human antibody repertoire; influenza vaccines.

FIG 1

Structures of head interface antibodies bound to HA head domains. (A) HA trimer from A/Bangkok/01/1979(H3N2), shown with a yellow surface. The HA head domain of a single protomer is shown in a cartoon representation in gray. The H3 and H1 head constructs used for crystallization experiments are shown to the right. Fabs are colored according to the V_H (darker color) and V_L (lighter color) gene usage. HA heads are all shown in gray. H2214, S5V2-29, and FluA-20 have been previously reported, and their PDB accession numbers are indicated (9, 10). (B) The six antibodies (two columns of three) that do not use the IGκV1-39 gene. (C) One column showing the five antibodies that use IGκV1-39 (cyan). (D, left) Contact heat map on the HA head surface. The numbers of Fabs (from a total of 11) contacting each residue were tallied and are colored according to the key. Contacts for IGκV1-39 Fabs and non-IGκV1-39 Fabs and their relationship with conserved residues are shown in Fig. S1C in the supplemental material. (Right) Two key points of contact, the 220 loop (purple) and residues 99 to 105 (dark gray), are shown on the HA surface.

FIG 2

Stereotyped HA engagement by IGκV1-39 antibodies. (A) Superposition on the HA head domain of the five IGκV1-39 antibodies. The HA heads are in gray, IGκV1-39 is in cyan, and heavy chains are colored according to V_H gene usage and are the same as those in Fig. 1. (B) Zoomed-in view of the IGκV1-39 interaction of D2 H1-1/H3-1 with HA from the bottom view of panel A. Key residues are shown in sticks. Light chain residues that contact HA are in yellow, and those that buttress/permit a curled HCDR3 conformation are in orange. A common acidic residue in HCDR3 is highlighted in green. (C) Partial sequence alignment of the germ line IGκV1-39 sequence and the five antibodies. Shading is colored as described above for panel B. (D) Sequence alignment of the antibody HCDR3s. The acidic residue at the sixth position of HCDR3 is shaded in green.

FIG 3

Diversity of non-IGκV1-39 head interface antibodies. (A) All six structures are shown with a common orientation for the HA head domain. Fabs are colored according to V_H (darker color) and V_L (lighter color) gene usage; HA heads are in gray. Some V-gene usages are shared among these antibodies, but they do not dictate how those Fabs contact HA. (B) Top views of the structures in panel A. Panels C to H are details of this view. Fab fragments are faded except for selected complementarity determining regions (CDR) or framework regions (FR) to highlight the diversity of Fab-HA contacts. (C) S1V2-51. HA residue Trp222 is coordinated in a hydrophobic vault by HCDR2 and -3 and LCDR3 that is similar to mouse antibody 8H10. (D) 8H10 (8) (PDB accession number 6N5B) and its interaction with Trp222. (E) S8V2-47. HCDR2 and -3 and LCDR1 and -3 all contribute to the interaction with HA. (F) S8V2-37. HCDR3 occupies the position of LCDR2 in the IGVκ1-39 antibody complexes and creates an intertwined antigen-combining site with HCDR3, LCDR1 and -2, and LFR3. (G) S8V2-18. Among the interface antibodies, HCDR2 and -3 contact the most recessed surfaces of the HA head. (H) S1V2-83. Most of the interaction with HA is through HCDR3.

FIG 4

Breadth of binding by head interface antibodies (Abs). (A) Dissociation constants from ELISA measurements. The pan-influenza A virus-binding stem-directed antibody FI6v3 (12) was used as a positive control, and an influenza B virus-specific RBS-directed antibody, CR8033 (31), was used as a negative control for influenza A virus isolates. Boxes are colored according to the key at the bottom. HAs used are as follows: H1 A/California/04/2009(H1N1), H2 A/Japan/305/1957(H2N2), H3 A/Hong Kong/JY2/1968(H3N2), H4 A/American black duck/New Brunswick/00464/2010(H4N6), H5 A/Viet Nam/1203/2004(H5N1), H6 A/Taiwan/02/2013(H6N1), H7 A/Taiwan/01/2017(H7N9), H8 A/northern shoveler/California/HKWF1204/2007(H8N4), H9 A/Beijing/1/2017(H9N2), H10 A/Jiangxi/IPB13/2013(H10N8), H13 A/gull/Maryland/704/1977(H13N6), A/mallard/Wisconsin/10OS3941/2010(H14N6), B-Phuket B/Phuket/3073/2013, H3-BK-79 A/Bangkok/01/1979(H3N2), and gHA^shield (8). (B) Phylogram of 16 HA serotypes. The division of HA groups 1 and 2 is shown by a dotted line. (C) Structure of the 220 loop of A/Bangkok/01/1979(H3N2). The HA head is faded except for the 220 loop to emphasize this feature. Residues Arg220, Pro221, and Gly229 are shown in sticks. (D) Comparison with A/Aichi/02/1968(H3N2) (PDB accession number 2VIU) (gray), which has an Arg at position 229.