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Development of a Platform for Producing Recombinant Protein Components of Epitope Vaccines for the Prevention of COVID-19

Anna S. Karyagina1,2,3,a*, Alexander V. Gromov1, Tatyana M. Grunina1,2, Alexander M. Lyaschuk1, Alexander V. Grishin1, Natalia V. Strukova1, Maria S. Generalova1, Zoya M. Galushkina1, Lyubov’ A. Soboleva1, Olga Yu. Dobrinina1, Tatyana N. Bolshakova1, Marina E. Subbotina1,2,b*, Ekaterina A. Romanovskaya-Romanko4, Igor V. Krasilnikov5, Nikita B. Polyakov1,6, Andrey I. Solovyev1, Daniil A. Grumov1, Vladimir G. Zhukhovitsky1, Ekaterina I. Ryabova1, Vladimir V. Prokofiev1, and Vladimir G. Lunin1,2

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

6Vernadsky Institute of Geochemistry and Analytical Chemistry, Russian Academy of Sciences, 119991 Moscow, Russia

* To whom correspondence should be addressed.

Received June 8, 2021; Revised July 11, 2021; Accepted July 28, 2021
A new platform for creating anti-coronavirus epitope vaccines has been developed. Two loop-like epitopes with lengths of 22 and 42 amino acid residues were selected from the receptor-binding motif of the Spike protein from the SARS-CoV-2 virus that participate in a large number of protein-protein interactions in the complexes with ACE2 and neutralizing antibodies. Two types of hybrid proteins, including one of the two selected epitopes, were constructed. To fix conformation of the selected epitopes, an approach using protein scaffolds was used. The homologue of Rop protein from the Escherichia coli ColE1 plasmid containing helix-turn-helix motif was used as an epitope scaffold for the convergence of C- and N-termini of the loop-like epitopes. Loop epitopes were inserted into the turn region. The conformation was additionally fixed by a disulfide bond formed between the cysteine residues present within the epitopes. For the purpose of multimerization, either aldolase from Thermotoga maritima, which forms a trimer in solution, or alpha-helical trimerizer of the Spike protein from SARS-CoV-2, was attached to the epitopes incorporated into the Rop-like protein. To enable purification on the heparin-containing sorbents, a short fragment from the heparin-binding hemagglutinin of Mycobacterium tuberculosis was inserted at the C-terminus of the hybrid proteins. All the obtained proteins demonstrated high level of immunogenicity after triplicate parenteral administration to mice. Sera from the mice immunized with both aldolase-based hybrid proteins and the Spike protein SARS-CoV-2 trimerizer-based protein with a longer epitope interacted with both the inactivated SARS-CoV-2 virus and the Spike protein receptor-binding domain at high titers.
KEY WORDS: SARS-CoV-2, Spike protein, RBD, RBM, epitope vaccine, epitope, epitope scaffold, aldolase, hybrid protein

DOI: 10.1134/S0006297921100096