Sequences coding for linkers and restriction nuclease sites were added to the constructs. Rop-D4-Rop-Tri-HBD, Rop-D4-Rop-ALD-HBD, Rop-D6-Rop-Tri-HBD, Rop-D6-Rop-ALD-HBD, Rop-D8-Rop-Tri-HBD, Rop-D8-Rop-ALD-HBD, Rop-D13-Rop-Tri-HBD, Rop-D13-Rop-ALD-HBD, and Rop-RBM-Rop-Tri-HBD proteins were produced as described by Karyagina et al. in the splenocytes of immunized mice. The most pronounced increase in the cytokine synthesis was observed in response to the proteins containing epitopes with disulfide bonds (452-494, 470-491), CUDC-907 (Fimepinostat) as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to obtained mouse sera displaying virus-neutralizing activity in the microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results CUDC-907 (Fimepinostat) obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections. Keywords: SARS-CoV-2, S protein, RBD, RBM, epitope vaccine, epitope, aldolase INTRODUCTION Development of new vaccines for prevention of COronaVIrus Disease 2019 (COVID-19) and other viral infections is a very important task. Whole-virus vaccines, protein subunit and full-size protein vaccines, vector-based vaccines, and RNA vaccines (which ensure the synthesis of viral proteins in an organism) induce generation of a wide array of antibodies, although not all of these antibodies are protective [1]. In some cases, such antibodies can promote antibody-dependent enhancement of infection when the disease is contracted after vaccination [2-4]. Vaccines containing small epitopes of infectious agent proteins can avoid the abovementioned problems. They are also easily standardized and exhibit low reactogenicity. The drawbacks of epitope vaccines are low immunogenicity and inability to induce formation of neutralizing antibodies, as well as possible capacity of new virus variants to avoid immune response developed to these vaccines. Some of the approaches used to increase vaccine immunogenicity and to facilitate the synthesis of virus-neutralizing antibodies involve epitope multimerization [5] and adjuvant optimization [6]. Another promising approach is selection of continuous conformational epitopes (elements of protein surface capable of effective induction of antibody synthesis) instead of linear epitopes, since the structure of the former is more similar to the structure of viral protein. Based on the contacts identified in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2), we selected two determinants in the S protein receptor-binding motif (RBM) (a.a. 452-494 and 470-491) that participated in a large number of contacts between the S protein and neutralizing antibodies or ACE2 [7]. Both determinants have a loop-like conformation and include two cysteine residues that form a disulfide bond in the S protein. For immunization, we produced two hybrid proteins, in CUDC-907 (Fimepinostat) which the selected antigenic determinants were inserted into the turn of the helix-turn-helix motif of Rop-like protein in order to bring together the N- and C-termini and to preserve the loop-like conformation of the epitopes (i.e., the Rop-like protein served as an epitope scaffold). The synthesis of these proteins in cells and the following purification resulted in the disulfide bond formation in the protein structure [7]. The hybrid proteins also contained the heparin-binding domain (HBD) to facilitate protein purification and either aldolase (ALD) or -helical fragment (a.a. 958-991) of the SARS-CoV-2 S protein to ensure protein trimerization. High immunogenicity and ability of antibodies formed in response to immunization with these proteins to interact with the S protein receptor-binding domain (RBD) and inactivated SARS-CoV-2 were demonstrated for all four hybrid proteins. The objectives of this study were production of other hybrid proteins carrying conformational epitopes of the SARS-CoV-2 S protein according to the previously developed protocol, investigation of immunogenicity of the produced proteins and interaction of induced antibodies with the RBD GRIA3 and inactivated SARS-CoV-2, characterization of cytokine response in splenocytes after immunization with the synthesized proteins, investigation of virus neutralization by the sera obtained with most promising hybrid proteins in the microneutralization assay with the clinical isolate of SARS-CoV-2. MATERIALS AND METHODS Strain and vectors. BL21 (DE3) (B FC (DE3) cells (Agilent Technologies, CUDC-907 (Fimepinostat) USA), modified plasmid vector pQE6 (Qiagen, USA) containing T7 promoter instead of T5 promoter, and pRep4 plasmid from M15 [pRep4] (Qiagen) were used in the study. Design of constructs encoding Rop-D2-Rop-Tri-HBD, Rop-D3-Rop-Tri-HBD, Rop-D2-Rop-ALD-HBD, and Rop-D3-Rop-ALD-HBD proteins was described in detail in.