The receptor-binding domain (RBD) of the protein S SARS-CoV-2 is considered to be one of the appealing targets for developing a vaccine against COVID-19. The choice of an expression system is essential when developing subunit vaccines, as it ensures the effective synthesis of the correctly folded target protein, and maintains its antigenic and immunogenic properties. Here, we describe the production of a recombinant RBD protein using prokaryotic (pRBD) and mammalian (mRBD) expression systems, and compare the immunogenicity of prokaryotic and mammalian-expressed RBD using a BALB/c mice model. An analysis of the sera from mice immunized with both variants of the protein revealed that the mRBD expressed in CHO cells provides a significantly stronger humoral immune response compared with the RBD expressed in E.coli cells. A specific antibody titer of sera from mice immunized with mRBD was ten-fold higher than the sera from the mice that received pRBD in ELISA, and about 100-fold higher in a neutralization test. The data obtained suggests that mRBD is capable of inducing neutralizing antibodies against SARS-CoV-2.

Currently, SARS-CoV-2 spike receptor-binding-domain (RBD)-based vaccines are considered one of the most effective weapons against COVID-19. During the first step of assessing vaccine immunogenicity, a mouse model is often used. In this paper, we tested the use of five experimental animals (mice, hamsters, rabbits, ferrets, and chickens) for RBD immunogenicity assessments. The humoral immune response was evaluated by ELISA and virus-neutralization assays. The data obtained show hamsters to be the least suitable candidates for RBD immunogenicity testing and, hence, assessing the protective efficacy of RBD-based vaccines.

For the first time, the chymosin gene (CYM) of a maral was characterized. Its exon/intron organization was established using comparative analysis of the nucleotide sequence. The CYM mRNA sequence encoding a maral preprochymosin was reconstructed. Nucleotide sequence of the CYM maral mRNA allowed developing an expression vector to ensure production of a recombinant enzyme. Recombinant maral prochymosin was obtained in the expression system of Escherichiacoli [strain BL21 (DE3)]. Total milk-coagulation activity (MCA) of the recombinant maral chymosin was 2330 AU/ml. The recombinant maral prochymosin relative activity was 52955 AU/mg. The recombinant maral chymosin showed 100-81% MCA in the temperature range 30-50°C, thermal stability (TS) threshold was 50°C, and the enzyme was completely inactivated at 70°C. Preparations of the recombinant chymosin of a single-humped camel and recombinant bovine chymosin were used as reference samples. Michaelis–Menten constant (K_m), turnover number (k_cat), and catalytic efficiency (k_cat/K_m) of the recombinant maral chymosin, were 1.18 ± 0.1 µM, 2.68 ± 0.08 s⁻¹ and 2.27± 0.10 µm M⁻¹·s⁻¹, respectively.

Moose (Alces alces) recombinant chymosin with a milk-clotting activity of 86 AU/mL was synthesized in the Kluyveromyces lactis expression system. After precipitation with ammonium sulfate and chromatographic purification, a sample of genetically engineered moose chymosin with a specific milk-clotting activity of 15,768 AU/mg was obtained, which was used for extensive biochemical characterization of the enzyme. The threshold of the thermal stability of moose chymosin was 55 °C; its complete inactivation occurred after heating at 60 °C. The total proteolytic activity of moose chymosin was 0.332 A₂₈₀ units. The ratio of milk-clotting and total proteolytic activities of the enzyme was 0.8. The K_m, k_cat and k_cat/K_m values of moose chymosin were 4.7 μM, 98.7 s⁻¹, and 21.1 μM⁻¹ s⁻¹, respectively. The pattern of change in the coagulation activity as a function of pH and Ca²⁺ concentration was consistent with the requirements for milk coagulants for cheese making. The optimum temperature of the enzyme was 50–55 °C. The introduction of Mg²⁺, Zn²⁺, Co²⁺, Ba²⁺, Fe²⁺, Mn²⁺, Ca²⁺, and Cu²⁺ into milk activated the coagulation ability of moose chymosin, while Ni ions on the contrary inhibited its activity. Using previously published data, we compared the biochemical properties of recombinant moose chymosin produced in bacterial (Escherichia coli) and yeast (K. lactis) producers.

Although the incidence and mortality of SARS-CoV-2 infection has been declining during the pandemic, the problem related to designing novel antiviral drugs that could effectively resist viruses in the future remains relevant. As part of our continued search for chemical compounds that are capable of exerting an antiviral effect against the SARS-CoV-2 virus, we studied the ability of triterpenic acid amides to inhibit the SARS-CoV-2 main protease. Molecular modeling suggested that the compounds are able to bind to the active site of the main protease via non-covalent interactions. The FRET-based enzyme assay was used to reveal that compounds 1e and 1b can inhibit the SARS-CoV-2 main protease at micromolar concentrations.

Although the COVID-19 pandemic caused by SARS-CoV-2 viruses is officially over, the search for new effective agents with activity against a wide range of coronaviruses is still an important task for medical chemists and virologists. We synthesized a series of thiazolo-thiophenes based on (+)- and (−)-usnic acid and studied their ability to inhibit the main protease of SARS-CoV-2. Substances containing unsubstituted thiophene groups or methyl- or bromo-substituted thiophene moieties showed moderate activity. Derivatives containing nitro substituents in the thiophene heterocycle—just as pure (+)- and (−)-usnic acids—showed no anti-3CL^pro activity. Kinetic parameters of the most active compound, (+)-3e, were investigated, and molecular modeling of the possible interaction of the new thiazolo-thiophenes with the active site of the main protease was carried out. We evaluated the binding energies of the ligand and protein in a ligand–protein complex. Active compound (+)-3e was found to bind with minimum free energy; the binding of inactive compound (+)-3g is characterized by higher values of minimum free energy; the positioning of pure (+)-usnic acid proved to be unstable and is accompanied by the formation of intermolecular contacts with many amino acids of the catalytic binding site. Thus, the molecular dynamics results were consistent with the experimental data. In an in vitro antiviral assay against six strains (Wuhan, Delta, and four Omicron sublineages) of SARS-CoV-2, (+)-3e demonstrated pronounced antiviral activity against all the strains.