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- EMDB-72062: Polyclonal immune complex of Fab from mice sera binding the head ... -

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Basic information

Entry
Database: EMDB / ID: EMD-72062
TitlePolyclonal immune complex of Fab from mice sera binding the head of H5 HA after immunization with inactivated split A/bald eagle/FL/W22-134-OP/2022 Influenza virus vaccine adjuvanted with CpG
Map dataNegative stain map of polyclonal Fab from mice sera binding the head of H5 HA after immunization with inactivated split A/bald eagle/FL/W22-134-OP/2022 Influenza virus vaccine adjuvanted with CpG
Sample
  • Complex: Polyclonal immune complex of Fab from mice sera binding H5 HA after immunization with inactivated split A/bald eagle/FL/W22-134-OP/2022 Influenza virus vaccine adjuvanted with CpG
Keywordsinfluenza / hemagglutinin / Flu A / polyclonal / complex / fab complex / VIRAL PROTEIN
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsAndrade TG / Rodriguez AJ / Han J / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)75N39019C00051 United States
CitationJournal: bioRxiv / Year: 2025
Title: A clade 2.3.4.4b H5N1 virus vaccine that elicits cross-protective antibodies against conserved domains of H5 and N1 glycoproteins.
Authors: Eduard Puente-Massaguer / Thales Galdino Andrade / Michael J Scherm / Kirill Vasilev / Hassanein Abozeid / Alesandra J Rodriguez / Josh Yueh / Disha Bhavsar / John D Campbell / Dong Yu / ...Authors: Eduard Puente-Massaguer / Thales Galdino Andrade / Michael J Scherm / Kirill Vasilev / Hassanein Abozeid / Alesandra J Rodriguez / Josh Yueh / Disha Bhavsar / John D Campbell / Dong Yu / Richard J Webby / Yoshihiro Kawaoka / Gabriele Neumann / Julianna Han / Andrew B Ward / Florian Krammer /
Abstract: The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness. ...The continuous evolution and widespread dissemination of highly pathogenic avian influenza (HPAI) H5N1 viruses, particularly clade 2.3.4.4b, pose critical challenges to global pandemic preparedness. In this study, we assessed a low-dose inactivated split virus vaccine derived from clade 2.3.4.4b H5N1, formulated with an Alum/CpG adjuvant, using a preclinical mouse model. This vaccine induced potent humoral and cellular immune responses, generating high titers of cross-reactive antibodies targeting both hemagglutinin (HA) and neuraminidase (NA) glycoproteins across homologous and heterologous H5 clades. The Alum/CpG adjuvant enabled significant antigen dose-sparing while promoting a balanced Th1/Th2 immune profile. Functional analyses demonstrated strong virus neutralization, neuraminidase inhibition, and potent antibody-dependent cellular cytotoxicity activity. Additionally, the vaccine elicited robust antigen-specific CD4 and CD8 T cell responses and effectively controlled viral replication in the lungs, accompanied by reduced lung inflammation. Importantly, vaccinated mice were fully protected against lethal challenges with both the homologous clade 2.3.4.4b and heterologous clade 1 H5N1 viruses, despite low hemagglutination inhibition titers. Electron microscopy polyclonal epitope mapping revealed serum antibodies targeting multiple epitopes on homologous HA and NA, with some cross-reacting to conserved epitopes on heterologous proteins, underscoring broad immune recognition. Collectively, these results highlight the potential of this vaccine candidate to provide broad, multifunctional, and durable immunity against both current and emerging H5N1 threats, supporting its further development for pandemic preparedness.
History
DepositionAug 9, 2025-
Header (metadata) releaseDec 10, 2025-
Map releaseDec 10, 2025-
UpdateDec 17, 2025-
Current statusDec 17, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_72062.png

Downloads & links

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Map

FileDownload / File: emd_72062.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain map of polyclonal Fab from mice sera binding the head of H5 HA after immunization with inactivated split A/bald eagle/FL/W22-134-OP/2022 Influenza virus vaccine adjuvanted with CpG
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 208 pix.
= 416. Å		2 Å/pix.
x 208 pix.
= 416. Å		2 Å/pix.
x 208 pix.
= 416. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0263
Minimum - Maximum-0.03430944 - 0.116731204
Average (Standard dev.)0.000077326564 (±0.003596677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 416.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Negative stain map from 3D classification

Fileemd_72062_additional_1.map
AnnotationNegative stain map from 3D classification
Projections & Slices
AxesZYX

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Half map: Negative stain half map A

Fileemd_72062_half_map_1.map
AnnotationNegative stain half map A
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Negative stain half map B

Fileemd_72062_half_map_2.map
AnnotationNegative stain half map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Polyclonal immune complex of Fab from mice sera binding H5 HA aft...

EntireName: Polyclonal immune complex of Fab from mice sera binding H5 HA after immunization with inactivated split A/bald eagle/FL/W22-134-OP/2022 Influenza virus vaccine adjuvanted with CpG
Components
  • Complex: Polyclonal immune complex of Fab from mice sera binding H5 HA after immunization with inactivated split A/bald eagle/FL/W22-134-OP/2022 Influenza virus vaccine adjuvanted with CpG

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Supramolecule #1: Polyclonal immune complex of Fab from mice sera binding H5 HA aft...

SupramoleculeName: Polyclonal immune complex of Fab from mice sera binding H5 HA after immunization with inactivated split A/bald eagle/FL/W22-134-OP/2022 Influenza virus vaccine adjuvanted with CpG
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 280 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.015 mg/mL
BufferpH: 7.4
StainingType: NEGATIVE / Material: 2% w/v uranyl formate
GridModel: Homemade / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeTFS TALOS F200C
Image recordingFilm or detector model: FEI CETA (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionType: NONE
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6cfg

Final reconstructionResolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 4.0) / Number images used: 3039
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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