|Entry||Database: EMDB / ID: EMD-21181|
|Title||EM-Based Polyclonal Epitope Mapping. ConM-SOSIP Complexed with Purified Polyclonal Fab (Grp 2, ID r2383 Wk22)|
|Sample||ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immunized rabbit (Grp 2, ID r2383, Wk22):|
|Biological species||synthetic construct (others)|
|Method||single particle reconstruction / negative staining / Resolution: 17.11 Å|
|Authors||Ward AB / Antanasijevic A|
|Funding support|| United States, 1 items |
|Citation||Journal: PLoS Pathog / Year: 2020|
Title: Structural and functional evaluation of de novo-designed, two-component nanoparticle carriers for HIV Env trimer immunogens.
Authors: Aleksandar Antanasijevic / George Ueda / Philip J M Brouwer / Jeffrey Copps / Deli Huang / Joel D Allen / Christopher A Cottrell / Anila Yasmeen / Leigh M Sewall / Ilja Bontjer / Thomas J ...Authors: Aleksandar Antanasijevic / George Ueda / Philip J M Brouwer / Jeffrey Copps / Deli Huang / Joel D Allen / Christopher A Cottrell / Anila Yasmeen / Leigh M Sewall / Ilja Bontjer / Thomas J Ketas / Hannah L Turner / Zachary T Berndsen / David C Montefiori / Per Johan Klasse / Max Crispin / David Nemazee / John P Moore / Rogier W Sanders / Neil P King / David Baker / Andrew B Ward /
Abstract: Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and ...Two-component, self-assembling nanoparticles represent a versatile platform for multivalent presentation of viral antigens. Computational design of protein nanoparticles with differing sizes and geometries enables combination with antigens of choice to test novel multimerization concepts in immunization strategies where the goal is to improve the induction and maturation of neutralizing antibody lineages. Here, we describe detailed antigenic, structural, and functional characterization of computationally designed tetrahedral, octahedral, and icosahedral nanoparticle immunogens displaying trimeric HIV envelope glycoprotein (Env) ectodomains. Env trimers, based on subtype A (BG505) or consensus group M (ConM) sequences and engineered with SOSIP stabilizing mutations, were fused to an underlying trimeric building block of each nanoparticle. Initial screening yielded one icosahedral and two tetrahedral nanoparticle candidates, capable of presenting twenty or four copies of the Env trimer. A number of analyses, including detailed structural characterization by cryo-EM, demonstrated that the nanoparticle immunogens possessed the intended structural and antigenic properties. When the immunogenicity of ConM-SOSIP trimers presented on a two-component tetrahedral nanoparticle or as soluble proteins were compared in rabbits, the two immunogens elicited similar serum antibody binding titers against the trimer component. Neutralizing antibody titers were slightly elevated in the animals given the nanoparticle immunogen and were initially more focused to the trimer apex. Altogether, our findings indicate that tetrahedral nanoparticles can be successfully applied for presentation of HIV Env trimer immunogens; however, the optimal implementation to different immunization strategies remains to be determined.
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_21181.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.05 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immu...
|Entire||Name: ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immunized rabbit (Grp 2, ID r2383, Wk22)|
Number of components: 2
-Component #1: protein, ConM-SOSIP.v9 complexed with purified polyclonal Fab fro...
|Protein||Name: ConM-SOSIP.v9 complexed with purified polyclonal Fab from an immunized rabbit (Grp 2, ID r2383, Wk22)|
Recombinant expression: No
|Source||Species: synthetic construct (others)|
|Source (engineered)||Expression System: Homo sapiens (human) / Vector: pPPI4 / Strain: HEK293F|
-Component #2: protein, ConM-SOSIP.v9
|Protein||Name: ConM-SOSIP.v9 / Recombinant expression: No|
|Source||Species: synthetic construct (others)|
|Source (engineered)||Expression System: Homo sapiens (human)|
|Specimen||Specimen state: Particle / Method: negative staining|
|Sample solution||Specimen conc.: 0.05 mg/mL / Buffer solution: TBS buffer, pH 7.4 / pH: 7.4|
|Staining||Sample diluted to 0.05 mg/mL. 3 uL was applied onto the grid, blotted off, and then stained with 2% uranyl formate for 60 seconds.|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
Model: Tecnai Spirit / Image courtesy: FEI Company
|Imaging||Microscope: FEI TECNAI SPIRIT|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 nm|
|Specimen Holder||Model: OTHER|
|Camera||Detector: TVIPS TEMCAM-F416 (4k x 4k)|
|Processing||Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 61933|
|3D reconstruction||Algorithm: BACK PROJECTION / Software: RELION / Resolution: 17.11 Å / Resolution method: FSC 0.143 CUT-OFF|
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