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- EMDB-42048: H3N2 A/Victoria/3/1975 complexed with mouse polyclonal Fab - wk 7... -

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Basic information

Entry
Database: EMDB / ID: EMD-42048
TitleH3N2 A/Victoria/3/1975 complexed with mouse polyclonal Fab - wk 7, control immunogen
Map data
Sample
  • Complex: H3N2 A/Victoria/3/1975 immune complex with mouse polyclonal Fab - week 7, control immunogen group
    • Complex: H3N2 A/Victoria/3/1975
    • Complex: Polyclonal Fab
    • Protein or peptide: H3N2 A/Victoria/3/1975
KeywordsComplex / Fab / Hemagglutinin / polyclonal / IMMUNE SYSTEM
Biological speciesInfluenza A virus (A/Victoria/3/1975(H3N2)) / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 17.6 Å
AuthorsCarter JJ / Barnes CO
Funding support United States, 1 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)GT15335 United States
CitationJournal: Nat Chem Biol / Year: 2024
Title: Vaccine design via antigen reorientation.
Authors: Duo Xu / Joshua J Carter / Chunfeng Li / Ashley Utz / Payton A B Weidenbacher / Shaogeng Tang / Mrinmoy Sanyal / Bali Pulendran / Christopher O Barnes / Peter S Kim /
Abstract: A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily ...A major challenge in creating universal influenza vaccines is to focus immune responses away from the immunodominant, variable head region of hemagglutinin (HA-head) and toward the evolutionarily conserved stem region (HA-stem). Here we introduce an approach to control antigen orientation via site-specific insertion of aspartate residues that facilitates antigen binding to alum. We demonstrate the generalizability of this approach with antigens from Ebola, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and influenza viruses and observe enhanced neutralizing antibody responses in all cases. We then reorient an H2 HA in an 'upside-down' configuration to increase the exposure and immunogenicity of HA-stem. The reoriented H2 HA (reoH2HA) on alum induced stem-directed antibodies that cross-react with both group 1 and group 2 influenza A subtypes. Electron microscopy polyclonal epitope mapping (EMPEM) revealed that reoH2HA (group 1) elicits cross-reactive antibodies targeting group 2 HA-stems. Our results highlight antigen reorientation as a generalizable approach for designing epitope-focused vaccines.
History
DepositionSep 20, 2023-
Header (metadata) releaseJan 24, 2024-
Map releaseJan 24, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42048.png

Downloads & links

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Map

FileDownload / File: emd_42048.map.gz / Format: CCP4 / Size: 844.7 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
7.05 Å/pix.
x 60 pix.
= 423. Å		7.05 Å/pix.
x 60 pix.
= 423. Å		7.05 Å/pix.
x 60 pix.
= 423. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 7.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.714
Minimum - Maximum-1.2504086 - 4.1241817
Average (Standard dev.)0.00029388897 (±0.10916694)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 423.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_42048_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

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Histogram (log scale)

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Half map: #2

Fileemd_42048_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : H3N2 A/Victoria/3/1975 immune complex with mouse polyclonal Fab -...

EntireName: H3N2 A/Victoria/3/1975 immune complex with mouse polyclonal Fab - week 7, control immunogen group
Components
  • Complex: H3N2 A/Victoria/3/1975 immune complex with mouse polyclonal Fab - week 7, control immunogen group
    • Complex: H3N2 A/Victoria/3/1975
    • Complex: Polyclonal Fab
    • Protein or peptide: H3N2 A/Victoria/3/1975

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Supramolecule #1: H3N2 A/Victoria/3/1975 immune complex with mouse polyclonal Fab -...

SupramoleculeName: H3N2 A/Victoria/3/1975 immune complex with mouse polyclonal Fab - week 7, control immunogen group
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Recombinantly expressed H3HA with polyclonal Fabs generated from pooled H2HA-immunized mouse antisera

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Supramolecule #2: H3N2 A/Victoria/3/1975

SupramoleculeName: H3N2 A/Victoria/3/1975 / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Influenza A virus (A/Victoria/3/1975(H3N2))

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Supramolecule #3: Polyclonal Fab

SupramoleculeName: Polyclonal Fab / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: H3N2 A/Victoria/3/1975

MacromoleculeName: H3N2 A/Victoria/3/1975 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Influenza A virus (A/Victoria/3/1975(H3N2))
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKTIIALSYI FCLVFAQDLP GNDNNSTATL CLGHHAVPNG TLVKTITNDQ IEVTNATELV QSSSTGKICN NPHRILDGIN CTLIDALLGD PHCDGFQNEK WDLFVERSKA FSNCFPYDVP DYASLRSLVA SSGTLEFINE GFNWTGVTQN GGSSACKRGP DSGFFSRLNW ...String:
MKTIIALSYI FCLVFAQDLP GNDNNSTATL CLGHHAVPNG TLVKTITNDQ IEVTNATELV QSSSTGKICN NPHRILDGIN CTLIDALLGD PHCDGFQNEK WDLFVERSKA FSNCFPYDVP DYASLRSLVA SSGTLEFINE GFNWTGVTQN GGSSACKRGP DSGFFSRLNW LYKSGSTYPV QNVTMPNNDN SDKLYIWGVH HPSTDKEQTN LYVQASGKVT VSTKRSQQTI IPNVGSRPWV RGLSSRISIY WTIVKPGDIL VINSNGNLIA PRGYFKMRTG KSSIMRSDAP IGTCSSECIT PNGSIPNDKP FQNVNKITYG ACPKYVKQNT LKLATGMRNV PEKQTRGIFG AIAGFIENGW EGMIDGWYGF RHQNSEGTGQ AADLKSTQAA IDQINGKLNR VIEKTNEKFH QIEKEFSEVE GRIQDLEKYV EDTKIDLWSY NAELLVALEN QHTIDLTDSE MNKLFEKTRR QLRENAEDMG NGCFKIYHKC DNACIGSIRN GTYDHDVYRD EALNNRFQIK GSMKQIEDKI EEILSKIYHI ENEIARIKKL IGEVASSSGL NDIFEAQKIE WHEAHHHHHH G

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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
StainingType: NEGATIVE / Material: Uranyl Formate

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 15.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: Stochastic gradient descent ab initio in cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 17.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 211909
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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