2.6. Immunogenicity Assessment
RBD
VIRUS, SEPARATION, CLOT
For serum separation, blood was collected into Vacuette tubes with clot activator and serum separator gel. Serum was separated via centrifugation, aliquoted into 1.7 mL tubes and stored frozen for further analysis for anti-SARS-CoV-2 antibodies.Humoral immunity against SARS-CoV-2 was assessed in a virus neutralization test (NT). NT was performed using SARS-CoV-2 strain PIK35 in Vero cells as described previously [Serum samples from randomly picked participants were additionally tested in ELISA for total antibodies against RBD with CoronaPass Total (Genetico, Russia) and for total antibodies to S protein trimer (Vector-Best, Russia).Interferon-gamma (IFN-γ) production in response to stimulation with SARS-CoV-2 S-protein peptides was assessed using QuantiFERON SARS-CoV-2 Starter Set (Qiagen, Germany), using two SARS-CoV-2 S protein peptide pools (Ag1 and Ag2) designed to stimulate the production of IFN-γ by S-protein-specific CD4 (Ag1) and CD4/CD8 (Ag2) T cells, which was detected by ELISA according to the manufacturer’s protocol. A Mitogen tube served as a positive control. The baseline IFN-γ level in the Nil tube (unstimulated lymphocytes) was subtracted from the IFN-γ level in the antigen tubes and the Mitogen tube to correct for background or non-specific IFN-γ signal. The results were reported in international units of IFN-γ per milliliter of whole blood (IU/mL) using a dilution curve for the standard supplied with the kit. A calibration curve was generated at each assay run. Samples were considered reactive for an IFN-γ response if the IFN-γ levels obtained from the tubes with stimulus were 0.01 IU/mL above baseline levels obtained from the unstimulated control.