Work

The project is a systematic and integrated study aimed at elucidating the role of vitamin D-auto/paracrine system in the mechanisms of genomic and/or non-genomic regulation of cellular effects of SARS-CoV-2 S protein. The project is divided into 2 stages: stage 1 will be performed in vitro on epithelial cell lines with ACE2 overexpression (MA-104 and/or ST-cells); stage 2 will be realized in vivo – on a rat model of alimentary vitamin D deficiency. At the initial stage we plan to develop a construct to obtain recombinant SARS-CoV-2 S protein using pET24a or pET28a vectors, to transform the E. coli BL21(DE3) by the plasmid and purify it by affinity chromatography. By immunocytochemical methods and flow cytometry we will investigate the binding of the SARS-CoV-2 S protein, conjugated with a fluorescent label, to the membrane of epithelial cell lines with ACE2 overexpression under the action of different doses of hormonally active form of vitamin D3 (1,25(OH)2D3). Next, using immunocytochemical methods, confocal microscopy, western blot analysis and RT-PCR, the efficiency of nuclear translocation of S protein under the action of 1,25(OH)2D3 will be determined. We also will study the indirect mechanisms of vitamin D3 protective action with the involvement of NF-κB-associated signaling pathways on the lines of epithelial cells with ACE2 overexpression. At the second stage, the rats will be immunized with SARS-CoV-2 S protein. We will evaluate the dose-dependent effect of vitamin D3 on the synthesis of key components of vitamin D-auto/paracrine system (RT-PCR and western blot analysis) after immunization and determine antibody titers to S protein (ELISA) depending on the D-status of rats. The new data will be obtained related to the link between the intensity of the immune response after immunization with the level of vitamin D3 (serum 25OHD) and the state of D-auto/paracrine system in target organs (content of vitamin D receptor – VDR, enzymes CYP27B1 and CYP24A1).