Pseudotyped HIV virus incorporating the SARS-CoV-2 spike protein [Wuhan-1 and delta (B.1.617.2)] were produced as previously described (45 (link), 46 (link)). Serial dilutions of heat-inactivated plasma were prepared with DMEM (10%FBS and 1%Pen/Strep) and incubated with pseudotype virus for 1hr at 37°C in 96-well plates. Next, HeLa cells stably expressing the ACE2 receptor (provided by Dr James Voss, The Scripps Research Institute) were added (10,000 cells/25µL per well) and the plates were left for 72hr. Infection level was assessed in lysed cells with the Bright-Glo luciferase kit (Promega), using a Victor™ X3 multilabel reader (Perkin Elmer). Measurements were performed in duplicates used to calculate the ID50.
Rakshit S., Adiga V., Ahmed A., Parthiban C., Chetan Kumar N., Dwarkanath P., Shivalingaiah S., Rao S., D’Souza G., Dias M., Maguire T.J., Doores K.J., Zoodsma M., Geckin B., Dasgupta P., Babji S., van Meijgaarden K.E., Joosten S.A., Ottenhoff T.H., Li Y., Netea M.G., Stuart K.D., De Rosa S.C., McElrath M.J, & Vyakarnam A. (2022). Evidence for the heterologous benefits of prior BCG vaccination on COVISHIELD™ vaccine-induced immune responses in SARS-CoV-2 seronegative young Indian adults. Frontiers in Immunology, 13, 985938.
Corresponding Organization : King's College London
Other organizations :
PES University, St. John's National Academy of Health Sciences, St.John's Medical College Hospital, Center for Experimental and Clinical Infection Research, Helmholtz Centre for Infection Research, Medizinische Hochschule Hannover, Centre for Individualised Infection Medicine, Radboud University Medical Center, Radboud University Nijmegen, Christian Medical College & Hospital, Leiden University Medical Center, Infectious Disease Research Institute, Fred Hutch Cancer Center, University of Washington
Pseudotyped HIV virus incorporating the SARS-CoV-2 spike protein [Wuhan-1 and delta (B.1.617.2)]
Serial dilutions of heat-inactivated plasma
dependent variables
Infection level assessed in lysed cells with the Bright-Glo luciferase kit (Promega), using a Victor™ X3 multilabel reader (Perkin Elmer)
control variables
DMEM (10%FBS and 1%Pen/Strep)
HeLa cells stably expressing the ACE2 receptor (provided by Dr James Voss, The Scripps Research Institute)
controls
Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.
Annotations
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