Stabilization of H5 highly pathogenic avian influenza hemagglutinin improves vaccine-elicited neutralizing antibody responses

Abstract

Transmission of highly pathogenic avian influenza from H5 clade 2.3.4.4b has expanded in recent years to infect large populations of birds and mammals, heightening the risk of a human pandemic. Influenza viruses adapted to transmission in birds and some other animals tend to have a less stable hemagglutinin (HA) than seasonal influenza viruses, enabling membrane fusion at comparatively high pH levels. Here, we combine five mutations within H5 HA that dramatically increase its melting temperature and promote stable closure of the HA trimer. Structural analysis by cryo-electron microscopy revealed that the stabilizing mutations create several new hydrophobic interactions, while maintaining local HA structure. We found that vaccinating mice with stabilized H5 HA immunogens resulted in higher hemagglutination inhibition and neutralization titers than non-stabilized comparators. Epitope mapping of vaccine-elicited polyclonal antibody responses using negative stain electron microscopy and deep mutational scanning showed that site E on the side of the HA receptor binding domain was immunodominant across all groups; however, the stabilized immunogens shifted responses toward the receptor binding site (RBS), eliciting a higher proportion of neutralizing antibodies. These findings highlight that H5 HA-stabilizing mutations enhance the quality of antibody responses across different vaccine formats, underscoring their potential to improve pandemic preparedness vaccines targeting viruses from this widely circulating clade.

Fig. 1. Stabilizing mutations within H5 increase thermostability and reduce binding of internally-directed mAb.

(A) Stabilizing mutations H355F/K380M/R397M/L418I/E432L (green) highlighted within the A/Texas/37/2024 crystal structure (PDB ID: 9DIQ). Key interacting side chains are shown in gray. (B) Surface models of HA foldon trimer and HA RC_I_1 one-component nanoparticle. (C) Melting temperatures of H5 HA constructs at pH 8.0 and pH 5.5 as measured by nanoDSF. (D) SEC chromatograms of H5 HA constructs. Same legend as panel (C). (E) BLI of H5 HA constructs against anti-stem mAb MEDI8852 and anti-trimer interface mAb FluA-20. Same legend as panel (C). (F) Flow cytometry plots of CR9114 and FluA-20 binding to membrane-anchored H5 HA constructs expressed on the surface of HEK293F cells.

Fig. 2. Cryo-EM of H5-FMLMI-foldon.

(A) Cryo-EM reconstruction of H5-FMLMI-foldon. (B) Superimposition of the cryo-EM model of H5-FMLMI-foldon and the A/Texas/37/2024 crystal structure (PDB ID: 9DWE) for the H5 HA ectodomain (left), the central stem epitope (center), and the RBS epitope (right). (C) Close-ups of density for the FMLMI mutations in the H5-FMLMI-foldon reconstruction with the built model. (D) Close-ups of the FMLMI mutations and interacting residues in the cryo-EM model of H5-FMLMI-foldon (orange) superimposed with the A/Texas/37/2024 crystal structure (PDB ID: 9DWE; gray).

Fig. 3. Vaccine-elicited Antibody Responses in Mice Immunized with H5 protein- and mRNA-LNP-delivered constructs

(A) H5 mouse immunization schedule and groups. Molecular models of immunogens are not to scale. (B-C) ELISA binding titers against vaccine-matched TX24 and vaccine-mismatched IN05 in immune sera at B. week 2 and C. week 6. (D) HAI titers in immune sera at week 6. (E-F) Microneutralization titers in immune sera at E. week 2 and F. week 6. Each symbol represents an individual animal, and the geometric mean of each group is indicated by the bar (N = 9-10 mice/group). Statistical significance was determined using one-way ANOVA with Tukey’s multiple comparisons test; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.

Fig. 4. ns-EMPEM of Vaccine-elicited Antibody Responses

(A) SEC chromatograms of TX24 HA complexed with polyclonal Fabs from H5 mouse study at week 6. (B) Locations of conventional H3 HA antigenic sites (37). (C)Representative 2D class averages of purified immune complexes in (A). H5-RC_I_1 and the H5-membrane-anchored 10 mL fraction contain a cartoon schematic of a likely 3D model, while all other groups are composite 3D models from ns-EMPEM analysis. Scale bars = 15 nm.

Fig. 5. DMS Epitope Mapping of Vaccine-elicited Neutralizing Antibody Responses

(A) Escape from serum antibody neutralization for all tolerated amino-acid mutations at each site in HA. Average escape for all animals in each group is shown with the number of sera from each group of animals analyzed indicated in brackets. See https://dms-vep.org/Flu_H5_American-Wigeon_South-Carolina_2021-H5N1_DMS/htmls/Stabilized_HA_sera_escape_faceted.html for individual animal escape measurements and interactive plots. (B) Average serum escape per position for animals from the H5-RC_I_1 and H5-FMLMI-RC_I_1 groups overlaid on HA surface representation (PBD ID: 4KWM). Numbering based on mature H3 HA.