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Stabilization of the trimeric pre-fusion structures of #influenza #H1 and #H9 #hemagglutinins by mutations in the stem helices

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By G.M.

 


Abstract

Stabilizing the pre-fusion structures of antigenic proteins can enhance the effectiveness of antiviral vaccines. The pre-fusion form of hemagglutinin (HA) from the influenza virus typically adopts a stable trimeric structure. However, the recombinant ectodomain of HA from the A/California/04/2009 (H1N1) influenza virus formed a monomer in solution rather than the expected trimer. To promote trimer formation in the pre-fusion conformation, we redesigned five amino acid residues in the stem region of HA that are involved in trimerization. The engineered HA protein formed a stable trimer at both pH 8.0 and pH 5.5. Additionally, the thermal stability of the modified protein improved, as indicated by an approximately ten-degree increase in its denaturation temperature. Cryo-EM analysis at 2.2 angstrom resolution confirmed that the mutant HA protein adopted the pre-fusion structure. Furthermore, the stabilized mutant exhibited enhanced immunogenicity in mice. We applied the same optimization strategy to the HA proteins from A/Malaysia/1706215/2007 (H1N1) and A/swine/Hong Kong/2106/98 (H9N2). These engineered proteins demonstrated increased thermal stability and retained a trimeric pre-fusion structure, as confirmed by cryo-EM analysis. Extending this optimization strategy to the equivalent five residues in hemagglutinins from six additional group 1 influenza viruses successfully stabilized their trimeric structures.


Competing Interest Statement

The authors have declared no competing interest.


Funder Information Declared

National Research Foundation of Korea, https://ror.org/013aysd81, RS-2024-00344154

National Research Facilities & Equipment Center, RS-2024-00436298

Technology Innovation Program, 20019707

Source: BioRxIV, https://www.biorxiv.org/content/10.1101/2025.08.27.672506v1

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