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

Fin J Milder, Mandy Jongeneelen, Tina Ritschel, Pascale Bouchier, Ilona J M Bisschop, Martijn de Man, Daniel Veldman, Lam Le, Baerbel Kaufmann, Mark J G Bakkers, Jarek Juraszek, Boerries Brandenburg, Johannes P M Langedijk

Research output: Contribution to journalArticleAcademicpeer-review


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.

Original languageEnglish
Number of pages8
JournalProceedings of the National Academy of Sciences of the United States of America
Issue number6
Publication statusPublished - 7 Feb 2022
Externally publishedYes


  • amino acids/genetics
  • cell line
  • hemagglutinin glycoproteins, influenza virus/genetics
  • hemagglutinins/genetics
  • humans
  • hydrogen-ion concentration
  • influenza vaccines/genetics
  • influenza, human/virology
  • mutation/genetics
  • protein conformation, beta-Strand/genetics


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