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- EMDB-45328: Polyclonal immune complex of Fab binding the H1 HA from serum of ... -

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Entry
Database: EMDB / ID: EMD-45328
TitlePolyclonal immune complex of Fab binding the H1 HA from serum of donor 2 at day 14
Map dataNegative stain map of global refinement of particle stack used to sort polyclonal responses to H1 HA from human donor 2 at day 14
Sample
  • Complex: Polyclonal immune complex of Fab binding the H1 HA from serum of donor 2 at day 14
Keywordsinfluenza / hemagglutinin / H1 / polyclonal complex / Fab complex / viral fusion protein / VIRAL PROTEIN / VIRAL PROTEIN-IMMUNE SYSTEM complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / negative staining / Resolution: 25.0 Å
AuthorsLeon AN / Rodriguez AJ / Richey ST / Torrents de la Pena A / Jackson AM / Han J / Ward AB
Funding support United States, 1 items
OrganizationGrant numberCountry
Other privateHuman Vaccines Project, Inc. United States
CitationJournal: mBio / Year: 2025
Title: Structural mapping of polyclonal IgG responses to HA after influenza virus vaccination or infection.
Authors: André Nicolás León / Alesandra J Rodriguez / Sara T Richey / Alba Torrents de la Pena / Rachael M Wolters / Abigail M Jackson / Katherine Webb / C Buddy Creech / Sandra Yoder / Philip A ...Authors: André Nicolás León / Alesandra J Rodriguez / Sara T Richey / Alba Torrents de la Pena / Rachael M Wolters / Abigail M Jackson / Katherine Webb / C Buddy Creech / Sandra Yoder / Philip A Mudd / James E Crowe / Julianna Han / Andrew B Ward /
Abstract: Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. ...Cellular and molecular characterization of immune responses elicited by influenza virus infection and seasonal vaccination have informed efforts to improve vaccine efficacy, breadth, and longevity. Here, we use negative stain electron microscopy polyclonal epitope mapping (nsEMPEM) to structurally characterize the humoral IgG antibody responses to hemagglutinin (HA) from human patients vaccinated with a seasonal quadrivalent flu vaccine or infected with influenza A viruses. Our data show that both vaccinated and infected patients had humoral IgGs targeting highly conserved regions on both H1 and H3 subtype HAs, including the stem and anchor, which are targets for universal influenza vaccine design. Responses against H1 predominantly targeted the central stem epitope in infected patients and vaccinated donors, whereas head epitopes were more prominently targeted on H3. Responses against H3 were less abundant, but a greater diversity of H3 epitopes were targeted relative to H1. While our analysis is limited by sample size, on average, vaccinated donors responded to a greater diversity of epitopes on both H1 and H3 than infected patients. These data establish a baseline for assessing polyclonal antibody responses in vaccination and infection, providing a context for future vaccine trials and emphasizing the need for further characterization of protective responses toward conserved epitopes. (201 words)IMPORTANCESeasonal influenza viruses cause hundreds of thousands of deaths each year and up to a billion infections; under the proper circumstances, influenza A viruses with pandemic potential could threaten the lives of millions more. The variable efficacies of traditional influenza virus vaccines and the desire to prevent pandemic influenzas have motivated work toward finding a universal flu vaccine. Many promising universal flu vaccine candidates currently focus on guiding immune responses to highly conserved epitopes on the central stem of the influenza hemagglutinin viral fusion protein. To support the further development of these stem-targeting vaccine candidates, in this study, we use negative stain electron microscopy to assess the prevalence of central stem-targeting antibodies in individuals who were exposed to influenza antigens through traditional vaccination and/or natural infection during the 2018-2019 flu season.
History
DepositionJun 12, 2024-
Header (metadata) releaseFeb 5, 2025-
Map releaseFeb 5, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45328.png

Downloads & links

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Map

FileDownload / File: emd_45328.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain map of global refinement of particle stack used to sort polyclonal responses to H1 HA from human donor 2 at day 14
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.06 Å/pix.
x 160 pix.
= 329.6 Å		2.06 Å/pix.
x 160 pix.
= 329.6 Å		2.06 Å/pix.
x 160 pix.
= 329.6 Å

Surface

Projections

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Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.06 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0174
Minimum - Maximum-0.034084976 - 0.09470577
Average (Standard dev.)-0.00020506636 (±0.004845564)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 329.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Negative stain map of polyclonal Fab binding the...

Fileemd_45328_additional_1.map
AnnotationNegative stain map of polyclonal Fab binding the H1 HA tophead and central stem epitopes from human donor 2 at day 14
Projections & Slices
AxesZYX

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Half map: Negative stain map of half map B for...

Fileemd_45328_half_map_1.map
AnnotationNegative stain map of half map B for global refinement of particle stack used to sort polyclonal responses to H1 HA from human donor 2 at day 14
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Negative stain map of half map A for...

Fileemd_45328_half_map_2.map
AnnotationNegative stain map of half map A for global refinement of particle stack used to sort polyclonal responses to H1 HA from human donor 2 at day 14
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 binding the H1 HA from serum of ...

EntireName: Polyclonal immune complex of Fab binding the H1 HA from serum of donor 2 at day 14
Components
  • Complex: Polyclonal immune complex of Fab binding the H1 HA from serum of donor 2 at day 14

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Supramolecule #1: Polyclonal immune complex of Fab binding the H1 HA from serum of ...

SupramoleculeName: Polyclonal immune complex of Fab binding the H1 HA from serum of donor 2 at day 14
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
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 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Image recordingFilm or detector model: FEI EAGLE (4k x 4k) / Average electron dose: 25.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD / Nominal defocus max: -2.0 µm / Nominal defocus min: -2.0 µm
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4m4y


Details: generated 40A map in chimera
Final reconstructionResolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 22800
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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