Citation
- Authors: Troyer Z. et al.
- Year: 2021
- Journal: J Extracell Vesicles 10
- Applications: in vitro / DNA / jetOPTIMUS
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Method
Production of spike(+) EVs and spike(-) EVs:
HEK293T cells were plated in 60.8 cm2 tissue culture dishes.
The following day, each dish was transfected with 6 μg of pCAGGS SARS-CoV-2 spike plasmid (or mock transfected) using JetOptimus. The media on the cells was replaced with fresh EV-depleted DMEM 4 h post-transfection.
Three days post-transfection, conditioned media from 12 dishes was filtered and extracellular vesicles were purified using tangential flow filtration (TFF).
Production of spike-pseudotype lentivirus (S-LVs) for CCF2-AM entry assay:
HEK293T cells were plated in 60.8 cm2 tissue culture dishes.
The following day, each dish was transfected with 5 μg of pNL4-3ΔEnvΔVpr-EGFP HIV core plasmid, 3 μg of β-lactamase-Vpr plasmid, and 3 μg of pCAGGS SARS-CoV-2 D614G spike plasmid using JetOptimus. The media on the cells was replaced with fresh EV-depleted DMEM 4 h post-transfection.
Three days post-transfection, conditioned media from 6 dishes was filtered, and LVs were concentrated and purified using TFF.
ACE2 transient transfection:
HEK293Ts were plated in 60.8 cm2 tissue culture dishes.
The following day, each dish was transfected with 8 μg of hACE2-KanR plasmid using JetOptimus. The media on the cells was replaced with fresh media 4 h post-transfection.
The cells were detached using trypsin, counted, and re-plated for use in the LV entry assay 48 h post-transfection.
Abstract
In late 2019, a novel coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan, China. SARS-CoV-2 and the disease it causes, coronavirus disease 2019 (COVID-19), spread rapidly and became a global pandemic in early 2020. SARS-CoV-2 spike protein is responsible for viral entry and binds to angiotensin converting enzyme 2 (ACE2) on host cells, making it a major target of the immune system - particularly neutralizing antibodies (nAbs) that are induced by infection or vaccines. Extracellular vesicles (EVs) are small membraned particles constitutively released by cells, including virally-infected cells. EVs and viruses enclosed within lipid membranes share some characteristics: they are small, sub-micron particles and they overlap in cellular biogenesis and egress routes. Given their shared characteristics, we hypothesized that EVs released from spike-expressing cells could carry spike and serve as decoys for anti-spike nAbs, promoting viral infection. Here, using mass spectrometry and nanoscale flow cytometry (NFC) approaches, we demonstrate that SARS-CoV-2 spike protein can be incorporated into EVs. Furthermore, we show that spike-carrying EVs act as decoy targets for convalescent patient serum-derived nAbs, reducing their effectiveness in blocking viral entry. These findings have important implications for the pathogenesis of SARS-CoV-2 infection in vivo and highlight the complex interplay between viruses, extracellular vesicles, and the immune system that occurs during viral infections.