Abstract

Hybrid Proteins with Short Conformational Epitopes of the Receptor-Binding Domain of SARS-CoV-2 Spike Protein Promote Production of Virus-Neutralizing Antibodies When Used for Immunization

Anna S. Karyagina^1,2,3,a, Alexander V. Gromov¹, Tatyana M. Grunina^1,2, Alexander M. Lyaschuk¹, Maria S. Poponova¹, Denis A. Kleymenov¹, Natalia V. Strukova¹, Maria S. Generalova¹, Anna V. Ryazanova¹, Zoya M. Galushkina¹, Olga Yu. Dobrynina¹, Tatyana N. Bolshakova¹, Maria V. Sergeeva⁴, Ekaterina A. Romanovskaya-Romanko⁴, Igor V. Krasilnikov⁵, Marina E. Subbotina^1,2,b, and Vladimir G. Lunin^1,2

¹Gamaleya National Research Center of Epidemiology and Microbiology, Ministry of Health of the Russian Federation, 123098 Moscow, Russia

²All-Russia Research Institute of Agricultural Biotechnology, 127550 Moscow, Russia

³Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

⁴Institute of Influenza, Ministry of Health of the Russian Federation, 197376 St. Petersburg, Russia

⁵Saint Petersburg Institute of Vaccines and Sera, FMBA, 198320 St. Petersburg, Russia

^* To whom correspondence should be addressed.

Received January 31, 2022; Revised February 16, 2022; Accepted February 16, 2022
Based on the previously developed approach, hybrid recombinant proteins containing short conformational epitopes (a.a. 144-153, 337-346, 414-425, 496-507) of the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein (S protein) were synthesized in Escherichia coli cells as potential components of epitope vaccines. Selected epitopes are involved in protein–protein interactions in the S protein complexes with neutralizing antibodies and ACE2 (angiotensin-converting enzyme 2). The recombinant proteins were used for immunization of mice (three doses with 2-week intervals), and the immunogenicity of protein antigens and ability of the resulting sera to interact with inactivated SARS-CoV-2 and RBD produced in eukaryotic cells were examined. All recombinant proteins showed high immunogenicity; the highest titer in the RBD binding assay was demonstrated by the serum obtained after immunization with the protein containing epitope 414-425. At the same time, the titers of sera obtained against other proteins in the RBD and inactivated virus binding assays were significantly lower than the titers of sera obtained with the previously produced four proteins containing the loop-like epitopes 452-494 and 470-491, the conformation of which was fixed with a disulfide bond. We also studied activation of cell-mediated immunity by the recombinant proteins that was monitored as changes in the levels of cytokines 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), 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 obtained can be used to develop epitope vaccines for prevention of COVID-19 and other viral infections.
KEY WORDS: SARS-CoV-2, S protein, RBD, RBM, epitope vaccine, epitope, aldolase
DOI: 10.1134/S0006297922040022