Universal stabilization of the influenza hemagglutinin by structure-based redesign of the pH switch regions

Abstract

For an efficacious vaccine immunogen, influenza hemagglutinin (HA) needs to maintain a stable quaternary structure, which is contrary to the inherently dynamic and metastable nature of class I fusion proteins. In this study, we stabilized HA with three substitutions within its pH-sensitive regions where the refolding starts. An X-ray structure reveals how these substitutions stabilize the intersubunit β-sheet in the base and form an interprotomeric aliphatic layer across the stem while the native prefusion HA fold is retained. The identification of the stabilizing substitutions increases our understanding of how the pH sensitivity is structurally accomplished in HA and possibly other pH-sensitive class I fusion proteins. Our stabilization approach in combination with the occasional back mutation of rare amino acids to consensus results in well-expressing stable trimeric HAs. This repair and stabilization approach, which proves broadly applicable to all tested influenza A HAs of group 1 and 2, will improve the developability of influenza vaccines based on different types of platforms and formats and can potentially improve efficacy.

Keywords: fusion; influenza; protein design; protein stability; vaccine.

Fig. 1.

Influenza HA protein structure. (A) Schematic representation of HA with indicated fusion peptide (FP), refolding regions 1 and 2 (RR1 and RR2), heptad repeat (HR) motif, central helix (CH), transmembrane (TM) domain, and cytoplasmic domain (CD). (B) Structure of prefusion trimeric WT H3-HK68 [PDB identifier 4FNK (22)] in surface representation (gray). The internal protein structure is outlined in the cartoon with one monomer colored according to A. (C) Bend of the helical structure between helices D and E. (D) Conformation of the postfusion monomer [PDB identifier 1QU1 (50)]. (E) Location of histidine switches 1 and 2 (pHS1 and pHS2). The histidines and charged residues forming the switches are shown in space, filling representation in orange for pH switch 1 (pHS1) and yellow for pH switch 2 (pHS2). Fusion inhibitor Arbidol is plotted in blue based on PDB identifier 5T6N (18). pHS1, pHS2, and Arbidol binding to the other protomers were plotted as outline. (F) Details of the structure of the histidine switches pHS1 and pHS2 in the WT HA. H106-K51-E103 linked by hydrogen bonds in pHS2 (Top) and H26-R153-E150 triad in pHS1 (Bottom). Residues belonging to pHS1 and pHS2 are outlined in the same color as in E.

Fig. 2.

Characterization of HA-containing stabilizing mutations. (A) Analytical SEC profiles of cell culture supernatant of Expi293F cells expressing H1-CA09 HA with combinations of stabilizing mutations; WT, H26W substitution, K51I + E103I substitution, and fully stabilized H26W, K51I + E103I. Data are representing mean of three independent transfections in one experiment. Peaks representing monomeric and trimeric species are indicated by an “M” and “T,” respectively. (B) Analytical SEC culture supernatant profiles of Expi293F cells expressing WT HA including foldon trimerization domain and fully stabilized HA (H26W, K15I, and E103I) for H1-MI15, H3-HK68, and H3-IN11. (C) Temperature stability of purified HA determined by DSF. Shown are Tm₅₀ values for WT H1-CA09 and stabilized H1-CA09 HA and for WT foldon-fused and stabilized HA of H1-MI15, H3-HK68, and H3-IN11. (D) Two-week pH stability at 4 °C of purified HA analyzed by analytical SEC. Shown are profiles of WT and stabilized HA of H1-MI15 (pH 5.5 and 7.4) and H3-HK68 (pH 4.7, 5.5, and 7.4). (E) Negative-stained EM of WT H1-CA09 (Left) and stabilized HA (Right) with representative 2D-averaged classes. (F) Negative-stained EM of WT H3-HK68 HA foldon-fused (Left) and stabilized HA (Right) with representative 2D-averaged classes (each box is 27.4 nm) and 3D density map (white mesh) superimposed on the X-ray structure of H3-HK68 HA trimer (PDB identifier 4FNK) represented in blue spheres with one monomer plotted in dark blue.

Fig. 3.

Crystal structure and suggested mechanism of action of the HA-stabilizing mutations. (A) Changes in the sidechain orientation induced by the H26W mutation in pHS1 (Left). The structure of the WT pHS1 is plotted in orange. The structure of the mutant is colored according to the color scheme introduced in Fig. 1A. The mutated pHS1 is also plotted in space-filling representation (Right). W26 sidechain tightly packs between the β-sheet (blue) and R153. (B) Changes in the sidechain orientation induced by the K51I mutation in pHS2 (Left). The structure of the WT pHS2 is plotted in yellow. The structure of the mutant is colored according to the color scheme introduced in Fig. 1A. pHS2 together with two neighboring residues I103 and I29 is also shown in a top cross-section view (Right). All four aliphatic residues form a tight hydrophobic cluster with H106 turning away. (C) The hydrophobic cluster from B is shown for the whole trimer (black outline), with the other two protomers colored in gray. The cluster encompasses the three H106 residues facing each other (magenta outline) and stabilized by negatively charged D109 residues (cyan outline). The histidines form most tight interactions with I29 (Right), shown in cross-section perpendicular to the threefold axis from the top.

Fig. 4.

Stabilizing mutations applied to group 1 and 2 representative HAs. (A) Phylogeny of influenza A and B adapted from ref. ; maximum likelihood tree representing amino acid sequences of HA. Analytical SEC profiles of culture supernatant of Expi293F cells expressing selected group 1 and 2 WT and fully stabilized HA (H26W, K15I, and E103I) subtype HAs. Peaks representing monomeric and trimeric species are indicated by an “M” and “T,” respectively. Due to differences in experimental setup, retention times are different from B, C, and D. (B) Analytical SEC profiles of culture supernatant of Expi293F cells expressing WT, stabilized and repaired of selected H3 HAs; H3N2 A/Netherlands/179/1993 (H3-NE93), H3N2 A/Singapore/INFIMH/16/0019/2016 (H3-SI16), H3N2 A/Wisconsin/67/2005 (H3-WI05), and H3N2 A/Brisbane/10/2007 (H3-BR07). (C) Analytical SEC profiles of Expi293F cell culture supernatants of H1N1 A/South Carolina/1/1918 I53_dn5B (H1-SC18), H1-CA09-I53_dn5B, H3-HK68-I53_dn5B, and H3-WI05-I53_dn5B. For the H1 HAs, the molecular weight determined by MALS is shown. (D) Analytical SEC profiles of purified WT and stabilized H1-CA09 HA-I53_dn5B kept at pH 5.5 and 7.4 for 2 wk (Top Left). Temperature stability as determined by DSF for WT and stabilized (Top Right). Negative-stained EM micrograph of WT and stabilized (Bottom) HA-I53_dn5B fusions. Representative 2D-averaged classes (box is 35 nm) could be generated for the stabilized variant.

Fig. 5.

Stabilization of pH switch regions prevents low-pH–triggered cell–cell fusion. HA fusogenicity as measured in a cell–cell fusion assay in HEK293 cells by cotransfection of plasmids encoding WT or stabilized HA (H1-SC18, H1-MI05, H3-WI05, and H3-HK68), human TMPRSS2, and GFP. Overlays of GFP and brightfield channels 24 h after transfection are shown before (pH 7.4) and 1 h after (pH 5.0) exposure to low-pH medium to trigger fusion.