Optimized pseudotyping conditions for the SARS-COV2 Spike glycoprotein
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Abstract
The SARS-COV2 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-COV2 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-COV2 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, modification of a single amino acid in the furin cleavage site of Spike (R682Q) enhanced infectious particle production another 10-fold. With all enhancing elements combined, the titer of pseudotyped particles reached almost 10 6 infectious particles/ml. Finally, HIV-1 particles pseudotyped with SARS-COV2 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-COV2 pseudoparticles that are suitable for detection of neutralizing antibodies from COVID-19 patients.
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SciScore for 10.1101/2020.05.28.122671: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells were rinsed with PBS one hour after infection and replace with complete media supplemented with 2 microliters of mouse hybridoma supernatant containing anti-VSV-G antibody I1 (Kerafast) to neutralize input virus. anti-VSV-Gsuggested: NoneExperimental Models: Cell Lines Sentences Resources The ACE2 cell lines were generated by transfecting 293FT cells with 500 ng MLV GagPol expression vector, 400 ng of retroviral transfer vector pQCXIP-ACE2 or … SciScore for 10.1101/2020.05.28.122671: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Cells were rinsed with PBS one hour after infection and replace with complete media supplemented with 2 microliters of mouse hybridoma supernatant containing anti-VSV-G antibody I1 (Kerafast) to neutralize input virus. anti-VSV-Gsuggested: NoneExperimental Models: Cell Lines Sentences Resources The ACE2 cell lines were generated by transfecting 293FT cells with 500 ng MLV GagPol expression vector, 400 ng of retroviral transfer vector pQCXIP-ACE2 or pQCXIH-ACE2, and 100 ng of VSV-G expression vector. 293FTsuggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)The TMPRSS2 cell line was generated by transfecting 293FT cells with 500 ng MLV GagPol expression vector, 400 ng of retroviral transfer vector pQCXIH-TMPRSS2, and 100 ng of VSV-G expression vector. TMPRSS2suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)Viral media was used to transduce 293FT, 293FT/ACE2, or 293/Cre-sensor/ACE2 cells were selected with hygromycin (200 micrograms/ml) beginning 2 days post-transduction and maintained until control treated cells were all eliminated. 293/Cre-sensor/ACE2suggested: NoneResults from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- No funding statement was detected.
- No protocol registration statement was detected.
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