*4.6. Virus Neutralization*

Virus neutralization with antibodies was carried out in accordance with the generally accepted method, as described [25]. The titer of the infectious virus was expressed in TCPD50/mL (tissue cytopathic dose of the virus causing a 50% cytopathic effect on the infected cells). It was found by titrating the viral preparation on the Vero cells monolayer (African green monkey kidney cell culture) grown in 96-well culture plates (Corning, Glendale, AZ, USA). Before use, blood serum was inactivated by heating at 56 ◦C for 30 min to inactivate the antiviral effect of complement proteins. Before applying it to a monolayer of cells, blood serum ranging from 1 to 20 with two dilutions was preincubated with the infectious SARS-CoV-2/human/RUS/Nsk-FRCFTM-1/2020 strain in a titer of 103 TCPD50/mL (50% tissue cytopathic doses in mL) for 1 h at 37 ◦C in a nutrient medium containing a 2% blood serum (heated at 56 ◦C for 30 min) of cattle. Then, it was applied to a monolayer of cell culture in three replicates. After the incubation of the mixture of antibodies with the virus for 1 h at 37 ◦C, the monolayer of cells was washed and left in a nutrient medium containing 2% cattle blood sera until a cytopathogenic effect was observed in control wells containing infected cells. To observe the infected and control cells in dynamics, an inverted microscope Mikromed I (Mikromed, Sankt Petersburg, Russia) was used at 10× magnification. The cells were fixed for 30 minutes with a formaldehyde solution and a 0.05% crystalline violet solution with 20% alcohol, as described [26]. Then, the liquid from the wells was removed and washed with water. The results were recorded visually. The neutralizing antibodies titer was considered the final dilution of blood serum at which cells were protected from the cytopathogenic effect in 50% of the wells. The neutralizing antibodies titer, which was detected in three repetitions in two independent experiments, was calculated using the median results.

**Supplementary Materials:** The following are available online at https://www.mdpi.com/article/ 10.3390/pathogens10111421/s1, Table S1: Specific interaction of inactivated viruses and recombinant proteins in IgG ELISA of convalescents and patients died from COVID-19; Table S2: The neutralizing activity of the blood serum of convalescents and patients with COVID-19; Table S3. The neutralizing activity of the blood serum of patients who subsequently died in hospital from COVID-19.

**Author Contributions:** Conceptualization, A.S.; methodology, E.K., D.S. (Dmitry Shcherbakov) and A.C.; validation, A.C., A.S. and E.K.; formal analysis, O.K. and D.S. (Daniil Shanshin); investigation, A.C., T.S., Y.K. and V.R.; resources, M.V.; data curation, E.K.; writing—original draft preparation, E.K.; writing—review and editing, M.G. and E.K.; supervision, M.G. and M.V.; project administration, A.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** The reported study was funded by RFBR according to the research project No 20-54-80012.

**Institutional Review Board Statement:** The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Ethics Committee of the Federal Research Center for Fundamental and Translational Medicine (Protocol No. 17 as of 17 June 2020) and from the Infectious Disease Hospital #1 (Protocol No. S/025 as of 12 May 2020).

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Data Availability Statement:** The data presented in this study are available in the main text, figures, tables and supplementary material.

**Acknowledgments:** We gratefully acknowledge FBHF City Infectious Diseases Clinical Hospital No. 1 (Novosibirsk) and its staff for providing sera from patients used in experimental analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.
