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TitleStabilization of the H5 clade 2.3.4.4b hemagglutinin improves vaccine-elicited neutralizing antibody responses in mice.
Journal, issue, pagesSci Transl Med, Vol. 18, Issue 839, Page eaea8770, Year 2026
Publish dateMar 4, 2026
AuthorsAnnie Dosey / Bernadeta Dadonaite / Rebecca A Gillespie / Elizabeth M Leaf / Matthew J Vukovich / Jackson McGowan / Emily Grey / Hiromi Muramatsu / Rachel H J Jun / Norbert Pardi / Masaru Kanekiyo / Jesse D Bloom / Neil P King /
PubMed AbstractTransmission 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. ...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 that are adapted to transmission in birds and a variety of mammals tend to have a less stable hemagglutinin (HA) than seasonal influenza viruses, enabling membrane fusion at comparatively higher pH levels. Here, we combined five mutations in the H5 HA that increased its melting temperature and promoted stable closure of the HA trimer. Structural analysis by cryo-electron microscopy revealed that the stabilizing mutations create several new hydrophobic interactions while maintaining the local HA structure. We found that vaccinating mice with stabilized H5 HA immunogens resulted in higher hemagglutination inhibition and neutralization titers than nonstabilized 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, which elicited a higher proportion of neutralizing antibodies. Consistent with these findings, stabilized H5 HA immunogens delivered as messenger RNA-lipid nanoparticle (mRNA-LNP) vaccines protected mice against H5N1 challenge. 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.
External linksSci Transl Med / PubMed:41779867
MethodsEM (single particle)
Resolution15.16 - 19.97 Å
Structure data

EMDB-71459: Negative stain EM map 3 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-RC_I_1 in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 18.99 Å

EMDB-71460: Negative stain EM map 2 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-RC_I_1 in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 17.83 Å

EMDB-71461: Negative stain EM map 1 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-RC_I_1 in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 15.16 Å

EMDB-71462: Negative stain EM map of polyclonal serum from mouse immunized with H5 TX24-foldon in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 16.43 Å

EMDB-71463: Negative stain EM map 1 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-foldon in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 19.41 Å

EMDB-71464: Negative stain EM map 2 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-foldon in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 18.44 Å

EMDB-71465: Negative stain EM map 3 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-foldon in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 18.96 Å

EMDB-71466: Negative stain EM map 1 of polyclonal serum from mouse immunized with H5 TX24-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 17.1 Å

EMDB-71467: Negative stain EM map 2 of polyclonal serum from mouse immunized with H5 TX24-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 19.1 Å

EMDB-71468: Negative stain EM map 3 of polyclonal serum from mouse immunized with H5 TX24-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 18.59 Å

EMDB-71469: Negative stain EM map 4 of polyclonal serum from mouse immunized with H5 TX24-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 19.97 Å

EMDB-71470: Negative stain EM map 1 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 16.16 Å

EMDB-71471: Negative stain EM map 2 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 17.08 Å

EMDB-71472: Negative stain EM map 3 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 15.82 Å

EMDB-71473: Negative stain EM map 4 of polyclonal serum from mouse immunized with H5 TX24-FMLMI-membrane anchored in complex with TX24-I53_dn5B.
Method: EM (single particle) / Resolution: 16.71 Å

Source
  • Mus musculus (house mouse)
  • Influenza A virus

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