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- EMDB-6077: Characterization of a broadly neutralizing monoclonal antibody th... -

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Entry
Database: EMDB / ID: EMD-6077
TitleCharacterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin
Map dataNegative stain reconstruction of Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1/1968 (H3N2), initiated with common lines, refined with projection matching against single particles, under C3 symmetry
Sample
  • Sample: Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1/1968 (H3N2)
  • Protein or peptide: hemagglutinin
  • Protein or peptide: Fab of monoclonal antibody 9H10
Keywordsgroup 2 influenza A virus / broadly neutralizing antibody
Biological speciesInfluenza A virus / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 24.6 Å
AuthorsTan GS / Lee PS / Hoffman RMB / Mazel-Sanchez B / Krammer F / Leon PE / Ward AB / Wilson IA / Palese P
CitationJournal: J Virol / Year: 2014
Title: Characterization of a broadly neutralizing monoclonal antibody that targets the fusion domain of group 2 influenza A virus hemagglutinin.
Authors: Gene S Tan / Peter S Lee / Ryan M B Hoffman / Beryl Mazel-Sanchez / Florian Krammer / Paul E Leon / Andrew B Ward / Ian A Wilson / Peter Palese /
Abstract: Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can ...Due to continuous changes to its antigenic regions, influenza viruses can evade immune detection and cause a significant amount of morbidity and mortality around the world. Influenza vaccinations can protect against disease but must be annually reformulated to match the current circulating strains. In the development of a broad-spectrum influenza vaccine, the elucidation of conserved epitopes is paramount. To this end, we designed an immunization strategy in mice to boost the humoral response against conserved regions of the hemagglutinin (HA) glycoprotein. Of note, generation and identification of broadly neutralizing antibodies that target group 2 HAs are rare and thus far have yielded only a few monoclonal antibodies (MAbs). Here, we demonstrate that mouse MAb 9H10 has broad and potent in vitro neutralizing activity against H3 and H10 group 2 influenza A subtypes. In the mouse model, MAb 9H10 protects mice against two divergent mouse-adapted H3N2 strains, in both pre- and postexposure administration regimens. In vitro and cell-free assays suggest that MAb 9H10 inhibits viral replication by blocking HA-dependent fusion of the viral and endosomal membranes early in the replication cycle and by disrupting viral particle egress in the late stage of infection. Interestingly, electron microscopy reconstructions of MAb 9H10 bound to the HA reveal that it binds a similar binding footprint to MAbs CR8020 and CR8043.
IMPORTANCE: The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, ...IMPORTANCE: The influenza hemagglutinin is the major antigenic target of the humoral immune response. However, due to continuous antigenic changes that occur on the surface of this glycoprotein, influenza viruses can escape the immune system and cause significant disease to the host. Toward the development of broad-spectrum therapeutics and vaccines against influenza virus, elucidation of conserved regions of influenza viruses is crucial. Thus, defining these types of epitopes through the generation and characterization of broadly neutralizing monoclonal antibodies (MAbs) can greatly assist others in highlighting conserved regions of hemagglutinin. Here, we demonstrate that MAb 9H10 that targets the hemagglutinin stalk has broadly neutralizing activity against group 2 influenza A viruses in vitro and in vivo.
History
DepositionAug 28, 2014-
Header (metadata) releaseSep 24, 2014-
Map releaseSep 24, 2014-
UpdateNov 12, 2014-
Current statusNov 12, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 24
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 24
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6077.png

Downloads & links

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Map

FileDownload / File: emd_6077.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNegative stain reconstruction of Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1/1968 (H3N2), initiated with common lines, refined with projection matching against single particles, under C3 symmetry
Voxel sizeX=Y=Z: 6.15 Å
Density
Contour LevelBy AUTHOR: 24.0 / Movie #1: 24
Minimum - Maximum-23.517362590000001 - 134.20054626000001
Average (Standard dev.)0.16498315 (±6.23728752)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-32-32-32
Dimensions646464
Spacing646464
CellA=B=C: 393.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.156.156.15
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z393.600393.600393.600
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-32-32-32
NC/NR/NS646464
D min/max/mean-23.517134.2010.165

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Supplemental data

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Sample components

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Entire : Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1...

EntireName: Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1/1968 (H3N2)
Components
  • Sample: Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1/1968 (H3N2)
  • Protein or peptide: hemagglutinin
  • Protein or peptide: Fab of monoclonal antibody 9H10

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Supramolecule #1000: Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1...

SupramoleculeName: Fab 9H10 bound to hemagglutinin (H3) from Influenza A/Hong Kong/1/1968 (H3N2)
type: sample / ID: 1000
Oligomeric state: one hemagglutinin homotrimer binds to three 9H10 Fabs
Number unique components: 2
Molecular weightTheoretical: 300 KDa

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Macromolecule #1: hemagglutinin

MacromoleculeName: hemagglutinin / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Oligomeric state: trimer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Influenza A virus / Strain: A/Hong Kong/1/1968 (H3N2) / synonym: flu virus
Molecular weightTheoretical: 150 KDa

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Macromolecule #2: Fab of monoclonal antibody 9H10

MacromoleculeName: Fab of monoclonal antibody 9H10 / type: protein_or_peptide / ID: 2 / Name.synonym: antigen-binding fragment / Number of copies: 3 / Oligomeric state: monomer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Mus musculus (house mouse) / Strain: BALB/c / synonym: house mouse
Molecular weightTheoretical: 50 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

Concentration0.01 mg/mL
BufferpH: 7.4 / Details: 150 mM NaCl, 20 mM Tris
StainingType: NEGATIVE
Details: Protein adsorbed for 20 seconds, blotted, stained with 2% uranyl formate for 20 seconds, and blotted again.
GridDetails: 400 mesh copper grid coated with nitrocellulose and a thin layer of carbon
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI SPIRIT
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus min: 1.0 µm / Nominal magnification: 52000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Tilt angle min: -55
TemperatureAverage: 298 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism monitored/corrected using continuously acquired FFTs at 52000X.
DateFeb 1, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 283
Tilt angle max0
Experimental equipment
Model: Tecnai Spirit / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 24.6 Å / Resolution method: OTHER / Software - Name: EMAN2, Sparx / Number images used: 4791
DetailsInitial model determined by common lines from reference-free class averages. Final model refined against single particles using projection matching.

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