SARS-CoV-2 Pseudovirus Production

The B.1.1.7, B.1.351, and P.1 pseudovirus constructs contained the same mutations as the S constructs. They were ordered as gBlock gene fragments (Integrated DNA Technologies) and cloned Sac I/Apa I in the pCR3 SARS-CoV-2–S_Δ19 expression plasmid (GenBank: MT449663.1) (44 (link)) using Gibson Assembly (Thermo Fisher Scientific). The D614G pseudovirus construct was made using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies). All constructs were verified by Sanger sequencing. Pseudovirus was produced by cotransfecting the pCR3 SARS-CoV-2–S_Δ19 expression plasmid with the pHIV-1_NL43 ΔEnv-NanoLuc reporter virus plasmid in HEK293T cells (American Type Culture Collection, CRL-11268) (44 (link), 45 (link)). Cell supernatant containing the pseudovirus was harvested 48 hours after transfection and stored at −80°C until further use.

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Caniels T.G., Bontjer I., van der Straten K., Poniman M., Burger J.A., Appelman B., Lavell H.A., Oomen M., Godeke G.J., Valle C., Mögling R., van Willigen H.D., Wynberg E., Schinkel M., van Vught L.A., Guerra D., Snitselaar J.L., Chaturbhuj D.N., Cuella Martin I., Moore J.P., de Jong M.D., Reusken C., Sikkens J.J., Bomers M.K., de Bree G.J., van Gils M.J., Eggink D, & Sanders R.W. (2021). Emerging SARS-CoV-2 variants of concern evade humoral immune responses from infection and vaccination. Science Advances, 7(36), eabj5365.

Publication 2021

gBlock gene fragments Gibson Assembly QuikChange Site-Directed Mutagenesis Kit HEK293T cells

B 1 1 7 B 1 351 Cell Mutations Nanoluc Plasmid Sars cov 2 Site directed mutagenesis Transfection Virus

Corresponding Organization : Cornell University

Other organizations : Vrije Universiteit Amsterdam, Public Health Service of Amsterdam

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Variable analysis

independent variables

SARS-CoV-2 spike protein constructs: B.1.1.7, B.1.351, P.1, and D614G

dependent variables

Characteristics of the SARS-CoV-2 pseudovirus constructs

control variables

Sanger sequencing to verify all pseudovirus constructs
Cotransfection of SARS-CoV-2 spike protein expression plasmid and pHIV-1NL43 ΔEnv-NanoLuc reporter virus plasmid in HEK293T cells to produce pseudovirus

controls

Positive control: Wild-type SARS-CoV-2 spike protein construct
Negative control: Not explicitly mentioned

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