Biotechnology and Biological Sciences Research Council
BRIC PhD, BBSRC Reference BB/K02034X/1
United Kingdom
Citation
Journal: Vaccine / Year: 2020 Title: An assessment of the use of Hepatitis B Virus core protein virus-like particles to display heterologous antigens from Neisseria meningitidis. Authors: Sebastian Aston-Deaville / Emil Carlsson / Muhammad Saleem / Angela Thistlethwaite / Hannah Chan / Sunil Maharjan / Alessandra Facchetti / Ian M Feavers / C Alistair Siebert / Richard F ...Authors: Sebastian Aston-Deaville / Emil Carlsson / Muhammad Saleem / Angela Thistlethwaite / Hannah Chan / Sunil Maharjan / Alessandra Facchetti / Ian M Feavers / C Alistair Siebert / Richard F Collins / Alan Roseman / Jeremy P Derrick / Abstract: Neisseria meningitidis is the causative agent of meningococcal meningitis and sepsis and remains a significant public health problem in many countries. Efforts to develop a comprehensive vaccine ...Neisseria meningitidis is the causative agent of meningococcal meningitis and sepsis and remains a significant public health problem in many countries. Efforts to develop a comprehensive vaccine against serogroup B meningococci have focused on the use of surface-exposed outer membrane proteins. Here we report the use of virus-like particles derived from the core protein of Hepatitis B Virus, HBc, to incorporate antigen domains derived from Factor H binding protein (FHbp) and the adhesin NadA. The extracellular domain of NadA was inserted into the major immunodominant region of HBc, and the C-terminal domain of FHbp at the C-terminus (CFHbp), creating a single polypeptide chain 3.7-fold larger than native HBc. Remarkably, cryoelectron microscopy revealed that the construct formed assemblies that were able to incorporate both antigens with minimal structural changes to native HBc. Electron density was weak for NadA and absent for CFHbp, partly attributable to domain flexibility. Following immunization of mice, three HBc fusions (CFHbp or NadA alone, NadA + CFHbp) were able to induce production of IgG1, IgG2a and IgG2b antibodies reactive against their respective antigens at dilutions in excess of 1:18,000. However, only HBc fusions containing NadA elicited the production of antibodies with serum bactericidal activity. It is hypothesized that this improved immune response is attributable to the adoption of a more native-like folding of crucial conformational epitopes of NadA within the chimeric VLP. This work demonstrates that HBc can incorporate insertions of large antigen domains but that maintenance of their three-dimensional structure is likely to be critical in obtaining a protective response.
History
Deposition
Sep 18, 2019
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Header (metadata) release
Jul 29, 2020
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Map release
Jul 29, 2020
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Update
Nov 25, 2020
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Current status
Nov 25, 2020
Processing site: PDBe / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Supramolecule #1: VLP fusion sequence expressed in E.coli.
Supramolecule
Name: VLP fusion sequence expressed in E.coli. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: HBcS-NadA-CFHbp Fusion protein with linker regions. HBc - Ordered core of VLP: from HBV core protein (NCBI taxonomy ID 10407). NadA - Partially ordered surface displayed domain: ...Details: HBcS-NadA-CFHbp Fusion protein with linker regions. HBc - Ordered core of VLP: from HBV core protein (NCBI taxonomy ID 10407). NadA - Partially ordered surface displayed domain: extracellular domain of NadA from Neisseria meningitidis (NCBI taxonomy ID 487). CFHbp - Unresolved/disordered component : C-terminal domain of factor H binding protein from Neisseria meningitidis (NCBI taxonomy ID 487).
Cryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV Details: 3 ul applied for 30s at room temperature, then 4-5 s blot..
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Electron microscopy
Microscope
FEI TITAN KRIOS
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Sampling interval: 14.0 µm / Digitization - Frames/image: 3-36 / Number grids imaged: 1 / Number real images: 2367 / Average exposure time: 4.0 sec. / Average electron dose: 79.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Particle selection
Number selected: 10119 Details: Initially ~44,000 particles were initially picked and extracted in EMAN. Approx 12,000 were shell fragments or incomplete. Others were intact but lower resolution, so damaged or affected by ...Details: Initially ~44,000 particles were initially picked and extracted in EMAN. Approx 12,000 were shell fragments or incomplete. Others were intact but lower resolution, so damaged or affected by charging, or other issue. After filtering/cleaning by EMAN2 2D classification 10119 particles were taken forwards for initial 3D reconstruction in EMAN2. This set of 10119 was then 2D cleaned by CryoSPARC to give 9145 particles.
CTF correction
Software - Name: EMAN2 (ver. 2.01) / Software - details: then CTF applied by CryoSPARC
Initial angle assignment
Type: OTHER / Software - Name: cryoSPARC (ver. v0.65)
Final 3D classification
Number classes: 3 / Software - Name: cryoSPARC (ver. v0.65) / Details: model #0 94.2 % mode l#1 4.6 % model #2 1.2%
Final angle assignment
Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v0.65)
Final reconstruction
Applied symmetry - Point group: I (icosahedral) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v0.65) / Number images used: 8598
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