ИСТИНА

Creation of epitope vaccines - vaccines, the active components of which are small protein epitopes of an infectious agent - is one of the most promising modern approaches in immunoprophylaxis. These vaccines do not have the disadvantages typical of live vaccines (reversal of pathogenic properties, residual virulence, incomplete inactivation, etc.). They are distinguished by a high degree of standardization, have a weak reactogenicity, with their help it is possible to avoid both the development of autoimmune processes during immunization and the formation of non-protective antibodies and antibodies that contribute to the development of antibody-dependent intensification of infection. Six epitopes (144-153, 337-346, 414-425, 452-494, 470-491, 496-507) were selected from the sequence of the Spike protein of the SARS-CoV-2, which implement protein-protein interactions in complexes with neutralizing antibodies and ACE2 (angiotensin converting enzyme 2). All epitopes, except for one with the alpha-helical conformation (337-346), have a loop-like conformation with close N- and C-terminus. To fix the conformation of the selected epitopes, an approach using protein (epitope) scaffolds was used. As an epitope scaffold, a homologue of the Rop protein form Escherichia coli was used, the structure of which contains a “helix – turn – helix” motif. Loop-shaped epitopes were inserted directly into the turn, and the alpha-helical epitope was inserted using flexible glycine-containing spacers. In the case of two epitopes, 452-494 and 470-491, the conformation was additionally fixed by a disulfide bond formed between the cysteine residues present in the epitopes. The presence of a disulfide bond was proved by mass spectrometry. For the purpose of multimerization, either an aldolase from Thermotoga maritima, which forms a trimer in solution, or an α-helical trimerizer of the SARS-CoV-2 Spike protein was attached to the epitopes inserted into the Rop-like protein. All obtained proteins (10 variants) showed a high level of immunogenicity after three parenteral administrations to mice with an interval of 2 weeks. Sera of mice immunized with fusion proteins containing epitopes with disulfide bonds (452-494, 470-491) interacted in a high titer with both the inactivated SARS-CoV-2 virus and the RBD (Receptor Binding Domain) domain of the Spike protein. Also, the interaction of sera with RBD was demonstrated in the case of the protein with the epitope 414-425. A study was carried out on laboratory animals (mice) of activation of cellular immunity, detected by the level of cytokine synthesis by splenocytes of immunized mice. The most pronounced increase of cytokines’ level was observed in the case of a response to proteins, including epitopes with disulfide bonds (452-494, 470-491). The presented approach can be used in the future to create new epitope vaccines for the prevention of viral infections.