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Comprehensive and systemic optimization for improving the yield of SARS-CoV-2 spike pseudotyped virus
Xinping Fu1, Lihua Tao1, Xiaoliu Zhang1
1Department of Biology and Biochemistry and Center for Nuclear Receptor and Cell Signaling, University of Houston, Houston, TX 77204, USA.
Molecular Therapy. Methods & Clinical Development
|February 1, 2021
Summary
Improving SARS-CoV-2 neutralization assays involves enhancing pseudotyped virus production. Modifications to the spike protein and viral vector significantly increased virus yield, optimizing this critical diagnostic tool.
Area of Science:
- Virology
- Immunology
- Biotechnology
Background:
- Virus neutralization assays detect antibodies by measuring their ability to block viral infection.
- Live SARS-CoV-2 neutralization assays require BSL-3 containment, posing logistical challenges.
- Pseudotyped viruses offer a safer alternative but often suffer from low production yields.
Purpose of the Study:
- To improve the production yield of SARS-CoV-2 pseudotyped viruses.
- To optimize SARS-CoV-2 pseudotyped virus for use in neutralization assays.
- To investigate the impact of cell line choice on assay sensitivity.
Main Methods:
- Production of SARS-CoV-2 pseudotyped lentiviral vectors.
- Optimization of spike protein constructs, including C-terminal truncation and D614G mutation.
- Comparison of virus yields using different spike protein variants and production methods.
- Evaluation of neutralization assay sensitivity using varying ACE2-expressing cell lines.
Main Results:
- Significant improvement in pseudotyped virus yield achieved through spike protein engineering.
- C-terminal truncated and D614G mutated spike proteins enhanced virus production.
- High-affinity ACE2-expressing cell lines reduced the sensitivity of neutralization assays.
Conclusions:
- Optimized pseudotyped virus production protocols enhance yield for SARS-CoV-2 neutralization assays.
- Spike protein modifications are crucial for efficient pseudotyped virus generation.
- Careful selection of cell lines is necessary to maintain assay sensitivity and accuracy.
