Optimized pseudotyping conditions for the SARS COV-2 Spike glycoprotein.

The SARS COV-2 Spike glycoprotein is solely responsible for binding to the host cell receptor and facilitating fusion between the viral and host membranes. The ability to generate viral particles pseudotyped with SARS COV-2 Spike is useful for many types of studies, such as characterization of neutralizing antibodies or development of fusion-inhibiting small molecules. Here we characterized the use of a codon-optimized SARS COV-2 Spike glycoprotein for the generation of pseudotyped HIV-1, MLV, and VSV particles. The full-length Spike protein functioned inefficiently with all three systems but was enhanced over 10-fold by deleting the last 19 amino acids of the cytoplasmic tail of Spike. Infection of 293FT target cells was only possible if the cells were engineered to stably express the human ACE-2 receptor, but stably introducing an additional copy of this receptor did not further enhance susceptibility. Stable introduction of the Spike activating protease TMPRSS2 further enhanced susceptibility to infection by 5-10 fold. Substitution of the signal peptide of the Spike protein with an optimal signal peptide did not enhance or reduce infectious particle production. However, modifications D614G and R682Q further enhanced infectious particle production. With all enhancing elements combined, the titer of pseudotyped HIV-1 particles reached almost 106 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS COV-2 Spike was successfully used to detect neutralizing antibodies in plasma from COVID-19 patients, but not plasma from uninfected individuals. IMPORTANCE When working with pathogenic viruses, it is useful to have rapid quantitative tests for viral infectivity that can be performed without strict biocontainment restrictions. A common way of accomplishing this is to generate viral pseudoparticles that contain the surface glycoprotein from the pathogenic virus incorporated into a replication-defective viral particle that contains a sensitive reporter system. These pseudoparticles enter cells using the glycoprotein from the pathogenic virus leading to a readout for infection. Conditions that block entry of the pathogenic virus, such as neutralizing antibodies, will also block entry of the viral pseudoparticles. However, viral glycoproteins often are not readily suited for generating pseudoparticles. Here we describe a series of modifications that result in the production of relatively high titer SARS COV-2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.