Protective human antibodies against a conserved epitope in pre- and postfusion influenza hemagglutinin

Abstract

Phylogenetically and antigenically distinct influenza A and B viruses (IAV and IBV) circulate in human populations, causing widespread morbidity. Antibodies (Abs) that bind epitopes conserved in both IAV and IBV hemagglutinins (HAs) could protect against disease by diverse virus subtypes. Only one reported HA Ab, isolated from a combinatorial display library, protects against both IAV and IBV. Thus, there has been so far no information on the likelihood of finding naturally occurring human Abs that bind HAs of diverse IAV subtypes and IBV lineages. We have now recovered from several unrelated human donors five clonal Abs that bind a conserved epitope preferentially exposed in the postfusion conformation of IAV and IVB HA2. These Abs lack neutralizing activity in vitro but in mice provide strong, IgG subtype-dependent protection against lethal IAV and IBV infections. Strategies to elicit similar Abs routinely might contribute to more effective influenza vaccines.

Keywords: ADCC; influenza vaccines; memory B cell.

Fig. 1.

S1V2-72 and K06.18 are broadly binding HA stem mAbs whose epitope is distinct from those of other standard stem mAbs. (A) Results of a Luminex multiplex binding assay conducted with culture supernatant IgG from human Bmem clones S1V2-72 and K06.18. Binding mean fluorescence intensities (MFIs) are color-coded by magnitude: yellow (≥1 × 10³), orange (≥1 × 10⁴), red (≥1 × 10⁵), black: not done. LOD: below limit of detection. (B) Serially diluted recombinant IgG forms of S1V2-72, K06.18, their UAs, CR9114, and an irrelevant mAb were screened by Luminex assay for binding to full-length, trimeric, soluble prefusion rHA ectodomains (FLsE). (C) Flow cytometry histograms depicting recombinant IgG binding to K530 cell lines expressing recombinant, native HA on the cell surface. (D–F) Ab epitope overlap determined by competitive inhibition of binding in a Luminex assay. Each curve depicts the binding MFI of K06.18 (D) or S1V2-72 (E and F) to HA-conjugated microbeads in the presence of serially diluted competitor mAbs. HAs used: H1/X-181 (D), H1/CA09 (E; for CR9114, 222-1C06, and S1V2-72 filled diamonds) or H3/X-31 (E; for CR8020 and S1V2-72 empty diamonds), or H5/VN04 (F). (G) ELISA measurement of recombinant IgG binding to a truncated peptide encompassing the LAH of prefusion HA2. LAH5 and LAH31 are mAbs reported (18, 21) to bind the LAH.

Fig. 2.

S1V2-72 binds a conserved β-hairpin exposed in EHA2. (A) 5Å-resolution cryo-EM map for S1V2-72 Fab bound to B/MY04 EHA2. (B) Docked model showing the AF2-predicted structures (ribbon models) of two S1V2-72 Fabs and trimeric B/MY04 EHA2 docked into the cryo-EM map shown in (A). (C) Left: ribbon model showing S1V2-72 Fab (heavy chain in orange; light chain in yellow) complexed with B/MY04 EHA2 monomer (cyan). Right: Rotated, close-up view of the S1V2-72:EHA2 interface. Also shown is the position of the HA1 strand (dashed green line) that would be linked by disulfide bond (black line) to HA2 in the postfusion structure of authentic HA1+HA2. (D) A simplified close-up view of the S1V2-72:EHA2 interface, showing only EHA2, the HCDRs, and LCDR3. (E) Alignment of the amino acid sequences encoding the β-hairpin in different HA subtypes. The boxed residues form the loop of the hairpin. Dashed positions indicate identity with the consensus residue. CON: consensus sequence. B-V: Victoria lineage of IBV. B-Y: Yamagata lineage of IBV.

Fig. 3.

S1V2-72 protects against lethal IAV or IAB challenge by IgG subtype–dependent mechanisms. (A and B) Body weight and survival of mAb-infused mice after infection with H3N2 (A) or HBNB (B) influenza virus. Asterisks denote significant differences (P < 0.05) in survival at day 13 compared to 222-1C06-treated controls. (C) In vitro microneutralization IC₅₀ values. (D) Results of an in vitro ADCC proxy assay. Mouse FcγRIV-expressing effector cells and HA-expressing target cells were cocultured in the presence of serially diluted IgG2c mAbs. FcγRIV activation was measured as luminescence output, as described in Materials and Methods.

Fig. 4.

Characterization of mAbs identified by their S1V2-72-like genetic signature. (A and B) ELISA results showing rIgG binding to EHA2s (A) or HA0s (B). (C) Flow cytometry histograms depicting rIgG binding to K530 cell lines expressing recombinant, native HA on the cell surface. (D) Ab epitope overlap determined by competitive inhibition of binding in an ELISA. Each curve depicts the binding of S1V2-72 to B/MY04 EHA2-coated microplates in the presence of serially diluted competitor mAbs.

Fig. 5.

B/MY04 EHA2 vaccination elicits potent GC and serum Ab responses against the S1V2-72 epitope. (A) Schematic depicting the schedule for vaccinating mice and analyzing their immune response. (B) Representative flow cytometry dot plots depicting the frequencies of GC B cells and PCs in draining popliteal LNs from naive, primed (18 d postprime), primed and boosted (8 d postboost), or primed but unboosted (same timepoint as boosted) mice. (C and D) Aggregate frequencies and absolute numbers of GC B cells (C) and PCs (D) from mice treated as described in (A) and (B). Data were pooled from three independent experiments with two to three animals per group per experiment. (E) Luminex MFI values for culture supernatant IgG binding. Each symbol represents clonal IgG from a single GC B cell isolated 18 d postprime with B/MY04 EHA2. Data are from one experiment with three mice, representative of two independent experiments comprising five mice total. Horizontal red lines denote the binding threshold (mean plus 6 SD of signal from control wells containing no B cells). (F) Competitive inhibition by S1V2-72. S1V2-72 human IgG or irrelevant human IgG was preincubated with B/MY04 EHA2 Luminex beads, followed by addition of Nojima culture supernatant (mouse) IgGs. Competitive inhibition was calculated as mouse IgG binding in the presence of S1V2-72 as a percentage of mouse IgG binding in the presence of irrelevant human IgG. Values below the dotted horizontal line are >90% inhibited. We tested only clonal supernatant IgGs whose (uninhibited) binding MFI for B/MY04 EHA2 was ≥10³ (E). (G) Luminex MFI values for B/MY04 EHA2 binding by serially diluted serum IgG from mice treated as in (A). Each curve shows the geometric mean ± SEM. Binding by recombinant S1V2-72 mouse IgG1 standard (2 μg/mL initial, then serially threefold diluted) is shown for comparison. Data are from one experiment, representative of three independent experiments with similar results. (H) Concentrations of B/MY04 EHA2-reactive serum IgG, normalized to S1V2-72 mouse IgG1 standard Ab [as shown in (G)]. Data were pooled from three independent experiments with two to three mice per group per experiment. (I) Competitive inhibition by immune serum. Serially diluted serum was preincubated with B/MY04 EHA2 or H3/X31 EHA2 Luminex beads; then, S1V2-72 human IgG was added. Inhibition was calculated as the percentage of human IgG binding signal relative to control samples lacking mouse Ig. Each curve depicts the geometric mean ± SEM of the groups shown in (G). Values below the dotted horizontal line are >90% inhibited. Inhibition by recombinant S1V2-72 mouse IgG1 standard (2 μg/mL initial, then serially threefold diluted) is shown for comparison. (J) Total S1V2-72-competing B/MY04 EHA2-reactive serum Ig, normalized to S1V2-72 mouse IgG1 standard Ab [as shown in (I)]. Each symbol represents a single animal (C, D, F, H, and J). Solid black horizontal lines denote geometric means (C, D, H, and J). Asterisks (C, D, H, and J) denote statistically significant differences at P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***). See SI Appendix, Materials and Methods for details of statistical analyses.