Comprehensive and systemic optimization for improving the yield of SARS-CoV-2 spike pseudotyped virus

Molecular Therapy: Methods & Clinical Development

Xinping Fu¹  Lihua Tao¹  Xiaoliu Zhang¹ 

[1] Department of Biology and Biochemistry and Center for Nuclear Receptor and Cell Signaling, University of Houston, Houston, TX 77204, USA;

关键词: SARS-CoV-2;    COVID-19;    pseudovirus;    lentiviral vector;    neutralization assay;   

DOI  :  

来源: DOAJ

【 摘 要 】

Virus neutralization assay is principally conducted by measuring the ability of the antibodies in patient sera to prevent the infection of susceptible cells by the virus. As SARS-CoV-2 is classified as a risk group 3 pathogen, neutralization assay using a live virus needs to be handled in a biosafety level 3 laboratory. To overcome this limitation, pseudotyped viruses have been developed as an alternative for the live SARS-CoV-2. However, one of the issues that we and others have encountered during the production of pseudotyped virus with SARS-CoV-2 spike protein was the low virus yield. In our own experience, we were only able initially to produce a stock with a virus titer that is more than two orders of magnitude lower than what we usually get with a vesicular stomatitis virus glycoprotein (VSV-G) pseudotyped lentiviral vector. We have conducted a series of improvements, including using a C-terminal truncated form of spike protein and a D614G mutated spike. Together, these have led to a significant improvement in the yield of the pseudotyped virus. Finally, our data show that using a high-affinity ACE2-expressing cell line resulted in a reduction in detection sensitivity of the neutralization assay.

【 授权许可】

Unknown