Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2025 Feb 18;122(7):e2416628122.
doi: 10.1073/pnas.2416628122. Epub 2025 Feb 12.

Structural characterization of influenza group 1 chimeric hemagglutinins as broad vaccine immunogens

Yen Thi Kim Nguyen #  1 , Xueyong Zhu #  1 , Julianna Han #  1 , Alesandra J Rodriguez  1 , Weina Sun  2 , Wenli Yu  1 , Peter Palese  2   3 , Florian Krammer  2   4   5   6 , Andrew B Ward  1 , Ian A Wilson  1   7
Affiliations

Structural characterization of influenza group 1 chimeric hemagglutinins as broad vaccine immunogens

Yen Thi Kim Nguyen et al. Proc Natl Acad Sci U S A. .

Abstract

Chimeric hemagglutinins (cHA) appear to be promising for the design and development of universal influenza vaccines. Influenza A group 1 cHAs, cH5/1, cH8/1, and cH11/1, comprising an H1 stem attached to either an H5, H8, or H11 globular head, have been used sequentially as vaccine immunogens in human clinical trials and induced high levels of broadly protective antibodies. Using X-ray crystallography and negative-stain electron microscopy, we determined structures of cH5/1, cH8/1, and cH11/1 HAs in their apo (unliganded) and antibody Fab-bound states. Stem-reactive antibodies 3E1 and 31.b.09 recognize their cognate epitopes in cH5/1, cH8/1, and cH11/1 HAs. However, with cH5/1, the head domains are rotated by 35 to 45° around the threefold axis of the HA trimer compared to native HA with a more splayed-open conformation at the stem base. cH11/1 with 3E1 is structurally more native-like but resembles cH5/1 with 31.b.09, whereas cH8/1 with 31.b.09 exhibited a range of closed-to-open stem configurations with some separation of head and stem domains. Furthermore, all of these group 1 cHAs effectively bound a broad head trimer interface antibody and other broad stem antibodies. Thus, the cHAs exhibit structural plasticity without compromising the stem and head trimer interface epitopes for elicitation of influenza A group 1 cross-reactive antibodies.

Keywords: HA trimer interface and stem; X-ray crystallography; chimeric group 1 influenza hemagglutinin; negative-stain electron microscopy; universal vaccine design.

PubMed Disclaimer

Conflict of interest statement

Competing interests statement:W.S. and F.K. are co-founders and scientific advisory board members of CastleVax. F.K. has consulted for Merck, Curevac, Seqirus, GlaxoSmithKline (GSK) and Pfizer and is currently consulting for 3rd Rock Ventures, Gritstone and Avimex. The Krammer laboratory is collaborating with Dynavax on influenza vaccine development. Mount Sinai has spun out a company, Kantaro, to market serological tests for SARS-CoV-2 and another company, CastleVax, to develop SARS-CoV-2 vaccines. The Icahn School of Medicine at Mount Sinai has filed patent applications relating to influenza virus vaccines, which list P.P. and F.K. as co-inventors. The Icahn School of Medicine at Mount Sinai has also filed patent applications relating to SARS-CoV-2 serological assays, NDV-based SARS-CoV-2 vaccines, and influenza virus therapeutics which list F.K. as co-inventor. W.S. is listed as co-inventor of NDV-based SARS-CoV-2 vaccines.

Figures

Fig. 1.
Fig. 1.
Crystal and nsEM structures of cH5/1 HA with 3E1 Fab. (A) Overall structure of cH5/1 HA with 3E1 Fab. One HA–Fab protomer is colored green for cH5/1 head domain, pink for stem domain, light yellow for 3E1 Fab LC, and light cyan for 3E1 Fab HC. The other cH5/1 HA and Fab protomers are in lavender. (B and C) Surface representation of cH5/1 HA from its complex with 3E1 Fab, H1 from CA04 HA structure (PDB ID: 4M4Y), and H5 from Viet04 HA structure (PDB ID: 3GBM), after alignment of the corresponding stem domains in standard side (B) and top (C) views. The stem domains are colored pink, and the three head domains of each HA trimer are in green, blue, and magenta. The more open stem is highlighted in a dashed red double arrow. (D) Representative 3D reconstructions of apo cH5/1 HA in one single sample using nsEM. The X-ray structure of cH5/1 HA (gray) was fitted into each nsEM map. The Upper panel depicts an averaged reconstruction of all cH5/1 HA particles in state 1: a more closed but rotated head as well as a splayed open stem. The Lower panel depicts a cH5/1 HA reconstruction that shows state 2: more open and rotated heads as well as a splayed open stem. The nsEM maps of cH5/1 are shown in light green (state 1) and light cyan (state 2) represent 13% (4,398 particles) and 6% (1,819 particles) of the total cH5/1 particles (32,600), respectively. As many particles are transitioning from trimer to dimer, fewer particles were similar enough to converge in 3D reconstructions.
Fig. 2.
Fig. 2.
Crystal structure of cH5/1 HA with 31.b.09 Fab. (A) Overall structure of cH5/1 HA with 31.b.09 Fab. One HA–Fab protomer is colored green for H5 HA head, pink for H1 HA stem, yellow for 31.b.09 Fab LC, and cyan for 31.b.09 Fab HC. The other HA and Fab protomers are in lavender. (B and C) Surface representation of cH5/1 HA from its complex with 31.b.09 Fab, H1 from CA04 HA structure (PDB ID: 4M4Y), and H5 from Viet04 HA structure (PDB ID: 3GBM), after alignment of the corresponding stem domains in standard side (B) and top (C) views. The stem domains are colored pink, and the three head domains of all HA are in green, blue, and magenta. The more open stem domain is highlighted in a dashed red double arrow. (D) Comparison of cH5/1 in the two complex structures with 31.b.09 and 3E1. The complex structures are represented by ribbons. cH5/1 HAs are colored in blue and gray with 31.b.09 and 3E1, respectively, while 31.b.09 and 3E1 are in pink and teal, respectively. (E) Alignment of cH5/1 HA with 31.b.09 to cH5/1 HA with 3E1 suggests a steric clash between Ser291 and Ser72 from cH5/1 HA and LC of 31.b.09, respectively (highlighted in a light orange ellipse). Ser291 residues in cH5/1 HA with 31.b.09 and cH5/1 HA with 3E1 are shown in blue and gray sticks, respectively. The clash would appear to cause a shift of the loop containing Ser291 that is marked in a red curved arrow connector.
Fig. 3.
Fig. 3.
Crystal and nsEM structures of cH11/1 HA and complex with 3E1 Fab. (A) Overall structure of cH11/1 HA with 3E1 Fab. One HA-Fab protomer is colored green for cH11/1 HA head, pink for cH11/1 HA stem, light yellow for LC of 3E1 Fab, and light cyan for HC of 3E1 Fab. The other HA and Fab protomers are in lavender. (B and C) Surface representation of cH11/1 HA from its complex with 3E1 Fab, H1 from CA04 HA structure (PDB ID: 4M4Y), and cH5/1 HA from its complex with 3E1, after alignment of the corresponding stem domains in standard side (B) and top (C) views. The stem domains are colored pink, and the three head domains of each HA trimer are in green, blue, and magenta. The more open stem is highlighted in a dashed red double arrow. (D) Representative 3D reconstructions of apo cH11/1 HA by nsEM. The X-ray structure of cH11/1 HA (gray) was fitted into each nsEM map. The particle reconstructions show that cH11/1 HA is state 1, representing a more closed conformation. The nsEM map of cH11/1 HA shown in a wheat color represents 45% of the trimeric HA particles that were selected for the 2D class averages.
Fig. 4.
Fig. 4.
Crystal structure of cH11/1 HA with 31.b.09 Fab. (A) Overall structure of cH11/1 HA with 31.b.09 Fab. One HA–Fab protomer is colored green for the cH11/1 HA head, pink for the cH11/1 HA stem, yellow for 31.b.09 Fab LC, and cyan for 31.b.09 Fab HC. The other HA and Fab protomers are in lavender. (B and C) Surface representation of cH11/1 HA from its complex with 31.b.09 Fab, H1 from CA04 HA structure (PDB ID: 4M4Y), and cH5/1 HA from its complex with 31.b.09, after alignment of the corresponding stem domains in standard side (B) and top (C) views. The stem domains are colored pink, and the three head domains of each HA trimer are in green, blue, and magenta. The more opened stem is highlighted by dashed red double arrows. (D) Comparison of cH11/1 HA in the two complex structures with 3E1 and 31.b.09 as represented by ribbons. cH11/1 HAs are colored in green and gray with 31.b.09 and 3E1, respectively. 31.b.09 and 3E1 are colored in pink and teal, respectively. (E) Alignment of cH11/1 HA with 31.b.09 to cH11/1 with 3E1 suggests a steric clash between Ser291 and Ser72 from cH11/1 HA and LC of 31.b.09, respectively. The Ser291 residues in cH11/1 HA with 31.b.09 and cH11/1 HA with 3E1 are shown in green and gray sticks, respectively. The clash region is highlighted in a light orange ellipse. The clash would appear to cause a shift of the loops containing Ser291 and marked in a red curved arrow connector.
Fig. 5.
Fig. 5.
3D reconstructions and docking of cHAs into representative nsEM maps of apo cH8/1 from a single sample. Models of cH8/1 monomers were generated by grafting the cH5/1 stem from the crystal structure of cH5/1 with antibody 31.b.09 onto the H8 HA head from IAV A/blue-winged teal/Guatemala/CIP049-14/2010 (H8N4) (PDB ID: 6ZRK) and fitted using ChimeraX into each protomer density of the 3D reconstructions. The cH8/1 trimers range from wild-type like trimers (state 1) to intermediate-open trimers (states 2 to 3) to more open trimers (state 4), which are shown in three different side and one top view.
Fig. 6.
Fig. 6.
The stem region of cH5/1 HA with 31.b.09 resembles the headless mini-HA #4900 immunogen (28) rather than native H1 HA. (A) Superimposition of the cH5/1 HA stem with CA04 H1 HA stem (PDB 4M4Y). (B) Superimposition of the cH5/1 stem with mini-HA #4900 (PDB ID: 5CJQ). The cH5/1 stem splayed-open conformation is similar to the mini-HA #4900 headless immunogen. cH5/1, CA04 HA, and mini-HA #4900 HA proteins are colored in blue, lavender, and wheat, respectively. The splayed-open stem domain is highlighted by a dashed red double arrow.
See this image and copyright information in PMC

References

    1. Wiggins K. B., Smith M. A., Schultz-Cherry S., The nature of immune responses to influenza vaccination in high-risk populations. Viruses 13, 1109 (2021). - PMC - PubMed
    1. Grohskopf B. L., et al. , Prevention and control of seasonal influenza with vaccines: Recommendations of the advisory committee on immunization practices–United States, 2023–24 Influenza season. MMWR. Recomm. Rep. 72, 1–25 (2023). - PMC - PubMed
    1. Agor J. K., Özaltın O. Y., Models for predicting the evolution of influenza to inform vaccine strain selection. Hum. Vaccin. Immunother. 14, 678–683 (2018). - PMC - PubMed
    1. Krammer F., García-Sastre A., Palese P., Is it possible to develop a “universal” influenza virus vaccine? Potential target antigens and critical aspects for a universal influenza vaccine. Cold Spring Harb. Perspect. Biol. 10, a028845 (2018). - PMC - PubMed
    1. Wu N. C., et al. , Preventing an antigenically disruptive mutation in egg-based H3N2 seasonal influenza vaccines by mutational incompatibility. Cell Host Microbe 25, 836–844.e5 (2019). - PMC - PubMed

MeSH terms

LinkOut - more resources