SARS-CoV-2 and Epstein–Barr Virus-like Particles Associate and Fuse with Extracellular Vesicles in Virus Neutralization Tests
Abstract
:1. Introduction
2. Material and Methods
2.1. Cell Lines and Cell Culture
2.2. Generation and Purification of SARS-CoV-2- and EBV-like Particles and Engineered EVs
2.3. Western Blot
2.4. ELISA
2.5. NTA Analysis
2.6. Nano Flow Analysis of VLPs and EVs
2.7. VLP Neutralization Test (VLPNT) for SARS-CoV-2
2.8. Cell-Free VLPNT (cfVLPNT) for SARS-CoV-2
2.9. SARS-CoV-2 Neutralization Test (VNT100)
2.10. EBV Neutralization Test
2.11. Cell-Free EBV Neutralization Test
2.12. Statistical Analysis
2.13. Patients and Serum Specimens
3. Results
3.1. Manufacture of SARS-CoV-2 VLPs and Recipient EVs
3.2. Association of SARS-CoV-2 VLPs and ACE2 Vesicles
3.3. Fusion of SARS-CoV-2 VLPs and ACE2 Vesicles
3.4. Cell-Free SARS-CoV-2 Neutralization Test
3.5. Comparison of Virus Neutralization Tests
3.6. Adaptation to SARS-CoV-2 Variants
3.7. Adaptation to EBV
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
% | Percent |
°C | Degree Celsius |
×g | Gravitational force equivalent |
ACE2 | Angiotensin-converting enzyme 2 |
BSL | Biosafety level |
c | Concentration |
cfVLPNT | Cell-free VLP neutralization test |
CN | Concordant negative |
conc. | Concentrated |
COVID-19 | Coronavirus disease 2019 |
CP | Concordant positive |
CPE | Cytopathic effect |
CTSL | Cathepsin L |
CTV | CellTraceViolet (Thermo Fisher Scientific) |
CTY | CellTraceYellow (Thermo Fisher Scientific) |
CV | Coefficient of variation |
cVNT | Conventional virus neutralization test |
D | Discrepant |
Da | Dalton |
dd | Double distilled |
DMEM | Dulbecco’s Modified Eagle’s Medium |
DMSO | Dimethyl sulfoxide |
DNA | Deoxyribonucleic acid |
E | Envelope protein |
e.g. | exempli gratia |
EB-VLP | Epstein-Barr virus-like particle |
EBV | Epstein-Barr virus |
ELISA | Enzyme-linked immunosorbent assay |
EV | Extracellular vesicle |
FBS | Fetal bovine serum |
FL | Full length |
FN | False negative |
FP | False positive |
FSC | Forward scatter |
g | Gram |
GMO | Genetically modified organism |
GMT | Geometric mean titer |
h | Human |
HIV | Human immunodeficiency viruses |
HRP | Horseradish peroxidase |
IC50 | Half maximal inhibitory concentration |
IgG | Immunoglobulin G |
IU | International units |
k | kilo |
kb | kilo base pair |
L | Liter |
LOD | Limit of detection |
log10 | Decadic logarithm |
Luc | Luciferase |
M | Membrane protein |
m | Meter |
m | milli |
M | mol L−1 |
mAb | Monoclonal antibody |
MERS-CoV | Middle East respiratory syndrome Coronavirus |
min | Minutes |
MLV | Murine leukemia virus |
MMP | Matrix metalloproteinase |
MNA | Microneutralization assay |
mol | Mole |
mRNA | Messenger RNA |
N | Nucleoprotein |
n | nano |
n | Sample number |
n.a. | Not applicable |
na | Not available |
NAb | Neutralizing antibody |
NIBSC | UK National Institute for Biological Standards and Control |
nLuc | nano-Luciferase |
NM | N-myristoly |
NmAb | Neutralizing monoclonal antibody |
non-red. | Non-reducing |
NPV | Negative predictive value |
ns | Not significant |
NTA | Nanoparticle tracking analysis |
p | p-value |
PAGE | Polyacrylamide gel electrophoresis |
PBS | Phosphate buffered saline |
PCR | Polymerase chain reaction |
Pen | Penicillin |
PFU | Plaque-forming units |
PPV | Positive predictive value |
PRNT | Plaque reduction neutralization test |
PRNT50 | 50% plaque reduction neutralization titer |
pVNT | Pseudotyped virus neutralization test |
r | Pearson correlation coefficient |
RBD | Receptor binding domain |
RBM | Receptor binding motif |
red. | Reducing |
RLU | Relative light units |
RNA | Ribonucleic acid |
RT | Room temperature |
RT-qPCR | Reverse transcription quantitative real-time PCR |
S | Spike protein |
SARS-CoV | Severe acute respiratory syndrome Coronavirus |
SARS-CoV-2 | Severe acute respiratory syndrome Coronavirus 2 |
SD | Standard deviation |
SDS | Sodium dodecyl sulfate |
SpO2 | Peripheral oxygen saturation |
SSC-H | Side scatter height |
Strep | Streptomycin |
sVNT | Surrogate virus neutralization tests |
TBS | Tris-buffered saline |
TBST | Tris-buffered saline with 0.1% Tween-20 |
TMPRSS2 | Transmembrane protease serine 2 |
TN | True negative |
TP | True positive |
Tris | Tris(hydroxymethyl)aminomethane |
V | Volt |
VLP | Virus-like-particle |
VLPN50 | 50% VLP neutralization titer |
VLPNT | VLP neutralization test |
VNT | Virus neutralization tests |
VNT100 | 100% virus neutralization titer |
VNT50 | 50% virus neutralization titer |
VOC | Variant of concern |
VSV | Vesicular stomatitis virus |
w/v | Weight per volume |
WB | Western-blot |
WHO | World Health Organization |
µ | micro |
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Roessler, J.; Pich, D.; Krähling, V.; Becker, S.; Keppler, O.T.; Zeidler, R.; Hammerschmidt, W. SARS-CoV-2 and Epstein–Barr Virus-like Particles Associate and Fuse with Extracellular Vesicles in Virus Neutralization Tests. Biomedicines 2023, 11, 2892. https://0-doi-org.brum.beds.ac.uk/10.3390/biomedicines11112892
Roessler J, Pich D, Krähling V, Becker S, Keppler OT, Zeidler R, Hammerschmidt W. SARS-CoV-2 and Epstein–Barr Virus-like Particles Associate and Fuse with Extracellular Vesicles in Virus Neutralization Tests. Biomedicines. 2023; 11(11):2892. https://0-doi-org.brum.beds.ac.uk/10.3390/biomedicines11112892
Chicago/Turabian StyleRoessler, Johannes, Dagmar Pich, Verena Krähling, Stephan Becker, Oliver T. Keppler, Reinhard Zeidler, and Wolfgang Hammerschmidt. 2023. "SARS-CoV-2 and Epstein–Barr Virus-like Particles Associate and Fuse with Extracellular Vesicles in Virus Neutralization Tests" Biomedicines 11, no. 11: 2892. https://0-doi-org.brum.beds.ac.uk/10.3390/biomedicines11112892