Proteins utilized for immunization, formaldehyde-inactivated SARS-CoV-2, and RBD were adsorbed within the microplates and used while antigens in ELISA. recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was shown from the serum acquired after immunization with the protein comprising epitope 414-425. At the same time, the titers of sera acquired against other proteins in the RBD and inactivated computer virus binding assays were significantly lower than the titers of sera acquired with the previously produced four proteins comprising the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed having a disulfide relationship. We also analyzed activation of cell-mediated immunity from the recombinant proteins that was monitored as changes in the levels of cytokines in the splenocytes of immunized mice. Probably the most pronounced increase in the cytokine synthesis was observed in response to the proteins comprising epitopes with disulfide bonds (452-494, 470-491), as well as epitopes 414-425 and 496-507. For some recombinant proteins with short conformational epitopes, adjuvant optimization allowed to acquired mouse sera showing virus-neutralizing activity in the GNE-900 microneutralization assay with live SARS-CoV-2 (hCoV-19/Russia/StPetersburg-3524/2020 EPI_ISL_415710 GISAID). The results acquired can be used to develop epitope vaccines for prevention of COVID-19 and additional viral infections. Rop-like protein in order to Rabbit Polyclonal to MRGX3 bring together the N- and C-termini and to preserve the loop-like conformation of the epitopes (i.e., the Rop-like protein served mainly because an epitope scaffold). The synthesis of these proteins in cells and the following purification resulted in the disulfide relationship formation in the protein structure . The cross proteins also contained the heparin-binding website (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. GNE-900 Large immunogenicity and ability of antibodies created in response to immunization with these proteins to interact with the S protein receptor-binding website (RBD) and inactivated SARS-CoV-2 were demonstrated for all four hybrid proteins. The objectives of this study were production of other cross proteins transporting conformational epitopes of the SARS-CoV-2 S protein according to the previously developed protocol, investigation of immunogenicity of the produced proteins and connection of induced antibodies with the RBD and inactivated SARS-CoV-2, characterization of cytokine response in splenocytes after immunization with the synthesized proteins, investigation of computer virus neutralization from the sera acquired with most encouraging cross proteins in the microneutralization assay with the medical isolate of SARS-CoV-2. MATERIALS AND METHODS Strain and vectors. BL21 (DE3) (B FC (DE3) cells (Agilent GNE-900 Systems, USA), altered plasmid vector pQE6 (Qiagen, USA) comprising 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 . These proteins contained partial D2 and D3 sequences (a.a. 470-490 and 453-494, respectively) of the SARS-CoV-2 S protein (UniProtKB: locus SPIKE_SARS2, accession “type”:”entrez-protein”,”attrs”:”text”:”P0DTC2″,”term_id”:”1835922048″,”term_text”:”P0DTC2″P0DTC2) put between two -helices of the Rop-like protein from (1st Rop helix: a.a. 2-34; second Rop helix: a.a. 34-65; PDB: 2JS5_A, GNE-900 RefSeq “type”:”entrez-protein”,”attrs”:”text”:”WP_010959602″,”term_id”:”499262062″,”term_text”:”WP_010959602″WP_010959602). Next, sequences coding for either trimerization-mediating -helix of S protein (Tri) (a.a. 958-991, UniProtKB: locus SPIKE_SARS2, accession “type”:”entrez-protein”,”attrs”:”text”:”P0DTC2″,”term_id”:”1835922048″,”term_text”:”P0DTC2″P0DTC2) or ALD (a.a. 2-201, PDB: 1WA3) were launched. All recombinant proteins contained the HBD (a.a. 160-174, UniProtKB/Swiss-Prot: “type”:”entrez-protein”,”attrs”:”text”:”A1KFU9″,”term_id”:”146286201″,”term_text”:”A1KFU9″A1KFU9.1) of the heparin-binding hemagglutinin (HBHA) from in the C-terminus. 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 explained by Karyagina et al. . The sequences for the S protein fragments D4 (a.a. 496-507), D6 (a.a. 144-153), D8 (a.a. 337-346), D13 (a.a. 414-425), and RBM (a.a. 433-511) were synthesized after codon optimization with the JCat system (http://www.jcat.de/) and correction with the purpose of improving the secondary structure of the transcribed RNAs for further facilitation of their manifestation in cells (DINAMelt web server; http://www.unafold.org/Dinamelt/applications/two-state-melting-folding.php). All sequences were synthesized by Evrogen (Russia) and cloned into the GNE-900 pL1003 plasmid encoding the Rop-D2-Rop-Tri-HBD protein and pL989 plasmid encoding the Rop-D2-Rop-ALD-HBD protein  by substituting the D2 fragment. Cultivation of maker strains and disruption of O55:B5 (Sigma, USA),.