Design of an HA2-based Escherichia coli expressed influenza immunogen that protects mice from pathogenic challenge

Abstract

Influenza HA is the primary target of neutralizing antibodies during infection, and its sequence undergoes genetic drift and shift in response to immune pressure. The receptor binding HA1 subunit of HA shows much higher sequence variability relative to the metastable, fusion-active HA2 subunit, presumably because neutralizing antibodies are primarily targeted against the former in natural infection. We have designed an HA2-based immunogen using a protein minimization approach that incorporates designed mutations to destabilize the low pH conformation of HA2. The resulting construct (HA6) was expressed in Escherichia coli and refolded from inclusion bodies. Biophysical studies and mutational analysis of the protein indicate that it is folded into the desired neutral pH conformation competent to bind the broadly neutralizing HA2 directed monoclonal 12D1, not the low pH conformation observed in previous studies. HA6 was highly immunogenic in mice and the mice were protected against lethal challenge by the homologous A/HK/68 mouse-adapted virus. An HA6-like construct from another H3 strain (A/Phil/2/82) also protected mice against A/HK/68 challenge. Regions included in HA6 are highly conserved within a subtype and are fairly well conserved within a clade. Targeting the highly conserved HA2 subunit with a bacterially produced immunogen is a vaccine strategy that may aid in pandemic preparedness.

Fig. 1.

Structures of the HA ectodomain. (A) Regions that are included in HA6 are shown in either maroon (HA1) or yellow (HA2). The rest of the molecule is shown in cyan. This figure was derived from the X-ray structure of HA from the (H3N2) isolate A/HK/68 (PDB ID code 1HGD) (7) and was drawn using the program Rasmol (41). Conformation of residues (45–110) HA2 in the neutral pH (B) and low pH (C) structures of HA. The stretch 57–98 is shown in pink and the HA2 residues 63F and 73V, which are buried in the low pH form but are exposed in the neutral pH form, are shown in green and blue, respectively. Only a portion of the HA2 trimer (residues 45–110) is shown here for clarity.

Fig. 2.

CD studies on the WT and mutant (57–98) HA2 peptides. (A) Far UV CD spectra of WT (57–98) HA2 peptide at pH 7.0 (•) and 4.5 (○), 25 °C, depicting the random coil to helix transition upon acidification. (B) pH titration of WT and mutant (57–98) HA2 peptides. The θ₂₂₂ of the WT (•), 63D (○), 73 D (▾), and 63D, 73D (▵) mutant peptides are plotted as a function of pH. 73D mutant and 63D, 73D double mutant peptides are highly destabilized and do not form a coiled coil at any pH. Lines through the points are only for visual clarity.

Fig. 3.

Spectroscopic analysis of HA6. (A) Far UV CD spectrum of 5 μM HA6 in PBS, pH 7.4 at 25 °C. (B) Mean residue ellipticity of HA6 as a function of pH in 5 mM citrate glycine HEPES buffer, 25 °C. The molecule loses its helicity as the pH is lowered, confirming that HA6 does not form the extended coiled coil structure observed at the low pH for WT HA2 (6). (C) Isothermal Chemical Denaturation curve at pH 8.0, 25 °C for 2 μM HA6. The denaturation of the protein was monitored by the fluorescence intensity at 338 nm as a function of denaturant concentration.

Fig. 4.

Immunogenicity and protection against lethal challenge (A/HK/68) by HA6 in mice. Mice of 10 per group were immunized with indicated vaccines either twice (× 2) or once (× 1) and challenged intranasally with an LD90 of mouse adapted A/HK/68. Weight changes (A) and mortality (B) were monitored for 20 days post challenge. Convalescent mice from intranasal A/HK/68 inoculation were included as a positive control, and mice receiving CpG7909 alone as adjuvant control. (C) Binding of HA6 antiserum to viral infected cells. HA6 antiserum (red), sera from CpG7909 adjuvant control group (green), and convalescent sera from A/HK/68 infected mice (blue) were tested by FACS for binding to mock infected (i), A/HK/68 infected (ii), or A/PR/8/34 infected (iii) MDCK cells.