A protective and broadly binding antibody class engages the influenza virus hemagglutinin head at its stem interface

Abstract

Influenza infection and vaccination impart strain-specific immunity that protects against neither seasonal antigenic variants nor the next pandemic. However, antibodies directed to conserved sites can confer broad protection. Here we identify and characterize a class of human antibodies that engage a previously undescribed, conserved epitope on the influenza hemagglutinin (HA) protein. Prototype antibody S8V1-157 binds at the normally occluded interface between the HA head and stem. Antibodies to this HA head-stem interface epitope are non-neutralizing in vitro but protect against lethal influenza infection in mice. Antibody isotypes that direct clearance of infected cells enhance this protection. Head-stem interface antibodies bind to most influenza A serotypes and seasonal human variants, and are present at low frequencies in the memory B cell populations of multiple human donors. Vaccines designed to elicit these antibodies might contribute to "universal" influenza immunity.

Figure 1:. Identification of a novel, broadly binding, HA head-directed antibody class.

A. Luminex screening of Bmem-cell Nojima culture supernatants identified four antibodies that broadly react with influenza A HA FLsEs and heads. The mean fluorescence intensity (MFI) values are colored according to the key. B. In a Luminex competitive binding assay, the four antibodies from (A) that share a pattern of reactivity did not compete with antibodies that engage known HA epitopes, but compete with each other for HA binding. Structures of Fab-HA complexes were aligned on an HA trimer from A/American black duck/New Brunswick/00464/2010(H4N6) (PDB: 5XL2). Fab structures include HC19 (PDB 2VIR), S5V2–29 (PDB 6E4X), HC45 (PDB 1QFU), CR9114 (PDB 4FQY), CR8020 (PDB 3SDY) and FI6v3 (PDB 3ZTJ). SARS-CoV antibody CR3022 was used as an HA non-binding control. C. The cross-competing HA antibodies share genetic signatures.

Figure 2:. Human antibodies engage a recessed surface at the head-stem interface of the influenza HA molecule.

A. Structure of antibody S8V1–157 complexed with the HA head of A/American black duck/New Brunswick/00464/2010(H4N6) colored in gray. The heavy chain is colored darker blue and the light chain is lighter blue. Engagement of this site is incompatible with the defined prefusion H4 HA trimer, colored in white (PDB: 5XL2) or with individual HA monomers. B. A surface projection showing the degree of amino acid conservation among HAs engaged by this antibody class (see Figure 3). The head-stem epitope is circumscribed in blue in the rightmost panel. Conservation scores were produced using ConSurf ^,. C. Key S8V1–157 contacts. The orientation relative to panel A is indicated.

Figure 3:. Breadth of HA binding by HA head-stem epitope antibodies.

A. Equilibrium dissociation constants (K_d), determined by ELISA. Broadly binding influenza A HA antibody FI6v3 and influenza B HA antibody CR8071 served as binding controls. B. Phylogenetic relationships of HAs used in our panel. HAs bound by HA head-stem epitope antibodies are indicated. Binding data from Figure S4 are incorporated into panel B.

Figure 4:. HA head-stem epitope antibodies bind cell surface-anchored HA.

Flow cytometry histograms depict the fluorescence intensities of recombinant IgG binding to K530 cell lines expressing recombinant, native HA on the cell surface. K530 cells were labeled with 400 ng/ml of the four head-stem epitope antibodies or control antibodies targeting the HA receptor binding site (HC19, K03.12 and H5.3), the head interface (S5V2–29), a lateral head epitope (HC45), stem (FI6v3), or SARS-CoV spike protein (CR3022).

Figure 5:. HA head-stem epitope antibodies protect against lethal influenza virus infection and severe disease.

C57BL/6 mice (n = 7 per group) were intraperitoneally injected with 150 μg of recombinant antibody via intraperitoneal injection three hours prior to intranasal challenge with 5xLD50 of A/Aichi/02/1968(H3N2)(X31). Mice were weighed daily and euthanized at a humane endpoint of 25% loss of body weight. Antibodies passively transferred included musinized IgG1 and IgG2c versions of HA head-stem epitope antibodies S8V1–157 and S1V2–65, neutralizing antibody HC19, head interface antibody S5V2–29 and SARS-CoV antibody CR3022. Mice injected with PBS were included as an additional control. A. Post-infection survival rate. B-D. Body weight curves for infected mice administered S8V1–157 (B), S1V2–65 (C), or S5V2–29 (D) antibodies, compared with controls. *p < 0.05 and ***p < 0.001 compared with isotype control CR3022. Not significant (n.s.), p 0.05; † p < 0.05, †† p < 0.01, and †††† p < 0.0001 IgG2c compared with IgG1.

Figure 6:. The HA head-stem epitope epitope is immunogenic in humans.

A. An additional 528 Nojima culture supernatants from donors K01, K03, S1, S5, S8, S9 and S12 were screened for competition with a recombinant musinized S8V1–157 IgG1 for HA binding. Culture supernatants that inhibited S8V1–157 binding by >90% are colored and specified. B. HA reactivity of S8V1–157-competing Nojima culture supernatants, as determined by multiplex Luminex assay. N.D.: not determined.