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Squalene-based adjuvant compositions that can provide effective induction of specific humoral immune response have been developed. Recombinant receptor-binding domain (RBD) of surface S-protein of SARS-CoV-2 was used to evaluate the properties of the composition. Immunization of mice with the developed squalene-based compositions in combination with RBD allows obtaining high titers of specific antibodies: from 10 5 to 2×10 6 . The blood sera from immunized mice exhibit neutralizing activity against SARS-CoV-2 Delta variant (B.1.617.2) with a titer up to 1:2000., (© 2024. Springer Science+Business Media, LLC, part of Springer Nature.)

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.

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 A 280 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 Ca 2+ 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 Mg 2+ , Zn 2+ , Co 2+ , Ba 2+ , Fe 2+ , Mn 2+ , Ca 2+ , and Cu 2+ 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.

Structure of the chymosin gene of Siberian roe deer (Capreolus pygargus) was established for the first time and its exon/intron organization was determined. Coding part of the chymosin gene of C. pygargus was reconstructed by the Golden Gate method and obtained as a DNA clone. Comparative sequence analysis of the roe deer, cow, and one-humped camel prochymosins revealed a number of amino acid substitutions at the sites forming the substrate-binding cavity of the enzyme and affecting the S4 and S1' + S3' specificity subsites. Integration vector pIP1 was used to construct a plasmid pIP1-Cap in order to express recombinant roe deer prochymosin gene in CHO-K1 cells. CHO-K1-CYM-Cap pool cells were obtained, allowing synthesis and secretion of recombinant prochymosin into the culture fluid. As a result of zymogen activation, a recombinant roe deer chymosin was obtained and its total milk-clotting activity was estimated to be 468.4 ± 11.1 IMCU/ml. Yield of the recombinant roe deer chymosin was 500 mg/liter or ≈468,000 IMCU/liter, which exceeds the yields of genetically engineered chymosins in most of the expression systems used. Basic biochemical properties of the obtained enzyme were compared with the commercial preparations of recombinant chymosins from one-humped camel (Camelus dromedarius) and cow (Bos taurus). Specific milk-clotting activity of the recombinant chymosin of C. pygargus was 938 ± 22 IMCU/mg, which was comparable to that of the reference enzymes. Non-specific proteolytic activity of the recombinant roe deer chymosin was 1.4-4.5 times higher than that of the cow and camel enzymes. In terms of coagulation specificity, recombinant chymosin of C. pygargus occupied an intermediate position between the genetically engineered analogs of B. taurus and C. dromedarius chymosins. Thermostability threshold of the recombinant roe deer chymosin was 55°C. At 60°C, the enzyme retained <1% of its initial milk-clotting activity, and its complete thermal inactivation was observed at 65°C.

The milk-clotting enzyme chymosin is a member of the group of aspartate proteinases. Chymosin is the main component of rennet traditionally obtained from the stomachs of dairy calves and widely used to coagulate milk in the production of various types of cheese. Another source of chymosin, which does not require the killing of animals, is based on recombinant DNA technology. Recombinant alpaca chymosin has a number of valuable technological properties that make it attractive for use in cheese-making as an alternative to recombinant bovine chymosin. The purpose of this work is to study the effect of coexpression of thioredoxin and prochymosin on the refolding of the recombinant zymogen and the activity of alpaca chymosin. To achieve this goal, on the basis of the pET32a plasmid, an expression vector was constructed containing the thioredoxin A gene fused to the N-terminal sequence of the marker enzyme zymogen, alpaca prochymosin. Using the constructed vector, pET-TrxProChn, a strain-producer of the recombinant chimeric protein thioredoxin-prochymosin was obtained. The choice of prochymosin as a model protein is due to the ability of autocatalytic activation of this zymogen, in which the pro-fragment is removed, together with the thioredoxin sequence attached to it, with the formation of active chymosin. It is shown that Escherichia coli strain BL21 transformed with the pET-TrxProChn plasmid provides an efficient synthesis of the thioredoxin-prochymosin chimeric molecule. However, the chimeric protein accumulates in inclusion bodies in an insoluble form. Therefore, a renaturation procedure was used to obtain the active target enzyme. Fusion of thioredoxin capable of disulfide-reductase activity to the N-terminal sequence of prochymosin provides optimal conditions for zymogen refolding and increases the yield of recombinant alpaca chymosin immediately after activation and during long-term storage by 13 and 15 %, respectively. The inclusion of thioredoxin in the composition of the chimeric protein, apparently, contributes to the process of correct reduction of disulfide bonds in the prochymosin molecule, which is reflected in the dynamics of the increase in the milk-clotting activity of alpaca chymosin during long-term storage., Competing Interests: The authors declare no conflict of interest., (Copyright © AUTHORS.)

Despite the long history of use and the knowledge of the genetics and biochemistry of E. coli , problems are still possible in obtaining a soluble form of recombinant proteins in this system. Although, soluble protein can be obtained both in the cytoplasm and in the periplasm of the bacterial cell. The latter is a priority strategy for obtaining soluble proteins. The fusion protein technology followed by detachment of the fusion protein with proteases is used to transfer the target protein into the periplasmic space of E. coli . We have continued for the first time to use the main viral protease 3CL of the SARS-CoV-2 virus for this purpose. We obtained a recombinant 3CL protease and studied its complex catalytic properties. The authenticity of the resulting recombinant enzyme, were confirmed by specific activity analysis and activity suppression by the known low-molecular-weight inhibitors. The catalytic efficiency of 3CL (0.17 ± 0.02 µM-1-s-1) was shown to be one order of magnitude higher than that of the widely used tobacco etch virus protease (0.013 ± 0.003 µM-1-s-1). The application of the 3CL gene in genetically engineered constructs provided efficient specific proteolysis of fusion proteins, which we demonstrated using the receptor-binding domain of SARS-CoV-2 spike protein and GST fusion protein. The solubility and immunochemical properties of RBD were preserved. It is very important that in work we have shown that 3CL protease works effectively directly in E. coli cells when co-expressed with the target fusion protein, as well as when expressed as part of a chimeric protein containing the target protein, fusion partner, and 3CL itself. The results obtained in the work allow expanding the repertoire of specific proteases for researchers and biotechnologists., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Belenkaya, Merkuleva, Yarovaya, Chirkova, Sharlaeva, Shanshin, Volosnikova, Vatsadze, Khvostov, Salakhutdinov and Shcherbakov.)

We performed a search for nanoantibodies that specifically interact with the receptor-binding domain (RBD) of the SARS-CoV-2 surface protein. The specificity of single-domain antibodies from the blood sera of a llama immunized with RBD of SARS-CoV-2 surface protein S (variant B.1.1.7 (Alpha)) was analyzed by ELISA. Recombinant trimers of the SARS-CoV-2 spike protein were used as antigens. In this work, a set of single-domain antibodies was obtained that specifically bind to the RBD of the SARS-CoV-2 virus., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Despite the rapid development and approval of several COVID vaccines based on the full-length spike protein, there is a need for safe, potent, and high-volume vaccines. Considering the predominance of the production of neutralizing antibodies targeting the receptor-binding domain (RBD) of S-protein after natural infection or vaccination, it makes sense to choose RBD as a vaccine immunogen. However, due to its small size, RBD exhibits relatively poor immunogenicity. Searching for novel adjuvants for RBD-based vaccine formulations is considered a good strategy for enhancing its immunogenicity. Herein, we assess the immunogenicity of severe acute respiratory syndrome coronavirus 2 RBD conjugated to a polyglucin:spermidine complex (PGS) and dsRNA (RBD-PGS + dsRNA) in a mouse model. BALB/c mice were immunized intramuscularly twice, with a 2-week interval, with 50 µg of RBD, RBD with Al(OH) 3 , or conjugated RBD. A comparative analysis of serum RBD-specific IgG and neutralizing antibody titers showed that PGS, PGS + dsRNA, and Al(OH) 3 enhanced the specific humoral response in animals. There was no significant difference between the groups immunized with RBD-PGS + dsRNA and RBD with Al(OH) 3 . Additionally, the study of the T-cell response in animals showed that, unlike adjuvants, the RBD-PGS + dsRNA conjugate stimulates the production of specific CD4+ and CD8+ T cells in animals.

Conjugates of the natural alkaloid (a R ,7 S )-colchicine with bicyclic monoterpenoids and their derivatives were synthesized for the first time. Molecular docking of the synthesized agents in the active site of the main viral protease of the SARS-CoV-2 virus was carried out. The cytotoxic properties of the agents against different cell lines and the ability to inhibit the main viral protease 3CLPro were studied., (© Springer Science+Business Media LLC 2023.)

Based on the data obtained by molecular modeling of the non-covalent interaction of non-symmetric N -benzylbispidin-9-ol amides with the active site of the main protease 3CLpro of the SARS-CoV-2 virus, a series of compounds was synthesized, and their inhibitory activity against 3CLpro was studied and compared with that of the known inhibitor ML188 (IC 50 = 1.56±0.55 µmol L -1 ). It was found that only compound 1g containing the 1,4-dihydroindeno[1,2- c ]pyrazole fragment showed moderate activity (IC 50 = 100±5.7µmol L -1 ) and was characterized by the highest calculated binding energy among the studied bispidine derivatives according to molecular docking data., (© Springer Science+Business Media LLC 2023.)

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.

During the COVID-19 pandemic, the development of prophylactic vaccines, including those based on new platforms, became highly relevant. One such platform is the creation of vaccines combining DNA and protein components in one construct. For the creation of DNA vaccine, we chose the full-length spike protein (S) of the SARS-CoV-2 virus and used the recombinant receptor-binding domain (RBD) of the S protein produced in CHO-K1 cells as a protein component. The immunogenicity of the developed combined vaccine and its individual components was compared and the contribution of each component to the induction of the immune response was analyzed. The combined DNA/protein vaccine possesses the advantages of both underlying approaches and is capable of inducing both humoral (similar to subunit vaccines) and cellular (similar to DNA vaccines) immunity., (© 2023. Springer Science+Business Media, LLC, part of Springer Nature.)

Identification of factors behind the level and duration of persistence of the SARS-CoV-2 antibodies in the blood is assumed to set the direction for studying humoral immunity mechanisms against COVID-19, optimizing the strategy for vaccine use, antibody-based drugs, and epidemiological control of COVID-19. Objective: This study aimed to study the relationship between clinical and demographic characteristics and the level of IgG antibodies to the RBD of SARS-CoV-2 spike protein after COVID-19 in the long term. Residents of the Altai Region of Western Siberia of Russia, Caucasians, aged from 27 to 93 years (median 53.0 years), who recovered from COVID-19 between May 2020 and February 2021 ( n = 44) took part in this prospective observational study. The titer of IgG antibodies to the RBD of SARS-CoV-2 spike protein was measured repeatedly in the blood at 4-13 months from the beginning of the clinical manifestation of COVID-19 via the method of enzyme-linked immunosorbent assay. The antibody titer positively correlated with age ( p = 0.013) and COVID-19 pneumonia ( p = 0.002) at 20-40 and 20-24 weeks from the onset of COVID-19 symptoms, respectively. Age was positively associated with antibody titer regardless of history of COVID-19 pneumonia (beta regression coefficient p = 0.009). The antibody titer decreased in 15 (34.1%) patients, increased in 10 (22.7%) patients, and did not change in 19 (43.2%) patients from the baseline to 48-49 weeks from the onset of COVID-19 symptoms, with seropositivity persisting in all patients. Age and COVID-19 pneumonia are possibly associated with higher IgG antibodies to the spike protein RBD of SARS-CoV-2 following COVID-19 in the long term. Divergent trends of anti-RBD IgG levels in adults illustrate inter-individual differences at 4-13 months from the onset of COVID-19 symptoms.

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.

Recombinant chymosins (rСhns) of the cow and the camel are currently considered as standard milk coagulants for cheese-making. The search for a new type of milk-clotting enzymes that may exist in nature and can surpass the existing "cheese-making" standards is an urgent biotechnological task. Within this study, we for the first time constructed an expression vector allowing production of a recombinant analog of moose chymosin in the expression system of Escherichia coli (strain SHuffle express). We built a model of the spatial structure of moose chymosin and compared the topography of positive and negative surface charges with the correspondent structures of cow and camel chymosins. We found that the distribution of charges on the surface of moose chymosin has common features with that of cow and camel chymosins. However, the moose enzyme carries a unique positively charged patch, which is likely to affect its interaction with the substrate. Biochemical and technological properties of the moose rChn were studied. Commercial rСhns of cow and camel were used as comparison enzymes. In some technological parameters, the moose rChn proved to be superior to the reference enzymes. Сompared with the cow and camel rСhns, the moose chymosin specific activity is less dependent on the changes in CaCl2 concentration in the range of 1-5 mM and pH in the range of 6-7, which is an attractive technological property. The total proteolytic activity of the moose rСhn occupies an intermediate position between the rСhns of cow and camel. The combination of biochemical and technological properties of the moose rСhn argues for further study of this enzyme., (Copyright © AUTHORS.)

Aptamer selection against novel infections is a complicated and time-consuming approach. Synergy can be achieved by using computational methods together with experimental procedures. This study aims to develop a reliable methodology for a rational aptamer in silico et vitro design. The new approach combines multiple steps: (1) Molecular design, based on screening in a DNA aptamer library and directed mutagenesis to fit the protein tertiary structure; (2) 3D molecular modeling of the target; (3) Molecular docking of an aptamer with the protein; (4) Molecular dynamics (MD) simulations of the complexes; (5) Quantum-mechanical (QM) evaluation of the interactions between aptamer and target with further analysis; (6) Experimental verification at each cycle for structure and binding affinity by using small-angle X-ray scattering, cytometry, and fluorescence polarization. By using a new iterative design procedure, structure- and interaction-based drug design (SIBDD), a highly specific aptamer to the receptor-binding domain of the SARS-CoV-2 spike protein, was developed and validated. The SIBDD approach enhances speed of the high-affinity aptamers development from scratch, using a target protein structure. The method could be used to improve existing aptamers for stronger binding. This approach brings to an advanced level the development of novel affinity probes, functional nucleic acids. It offers a blueprint for the straightforward design of targeting molecules for new pathogen agents and emerging variants., (© 2022 Wiley-VCH GmbH.)

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.

The development of preventive vaccines became the first order task in the COVID-19 pandemic caused by SARS-CoV-2. This paper reports the construction of the pVAX-RBD plasmid containing the Receptor-Binding Domain (RBD) of the S protein and a unique signal sequence 176 which promotes target protein secretion into the extracellular space thereby increasing the efficiency of humoral immune response activation. A polyglucine-spermidine conjugate (PGS) was used to deliver pVAX-RBD into the cells. The comparative immunogenicity study of the naked pVAX-RBD and pVAX-RBD enclosed in the PGS envelope showed that the latter was more efficient in inducing an immune response in the immunized mice. In particular, RBD-specific antibody titers were shown in ELISA to be no higher than 1 : 1000 in the animals from the pVAX-RBD group and 1 : 42000, in the pVAX-RBD-PGS group. The pVAX-RBD-PGS construct effectively induced cellular immune response. Using ELISpot, it has been demonstrated that splenocytes obtained from the immunized animals effectively produced INF-y in response to stimulation with the S protein-derived peptide pool. The results suggest that the polyglucine-spermidine conjugate-enveloped pVAX-RBD construct may be considered as a promising DNA vaccine against COVID-19.

Docking and quantum-chemical methods have been used for screening of drug-like compounds from the own database of the Voronezh State University to find inhibitors the SARS-CoV-2 main protease, an important enzyme of the coronavirus responsible for the COVID-19 pandemic. Using the SOL program more than 42000 3D molecular structures were docked into the active site of the main protease, and more than 1000 ligands with most negative values of the SOL score were selected for further processing. For all these top ligands, the protein-ligand binding enthalpy has been calculated using the PM7 semiempirical quantum-chemical method with the COSMO implicit solvent model. 20 ligands with the most negative SOL scores and the most negative binding enthalpies have been selected for further experimental testing. The latter has been made by measurements of the inhibitory activity against the main protease and suppression of SARS-CoV-2 replication in a cell culture. The inhibitory activity \of the compounds was determined using a synthetic fluorescently labeled peptide substrate including the proteolysis site of the main protease. The antiviral activity was tested against SARS-CoV-2 virus in the Vero cell culture. Eight compounds showed inhibitory activity against the main protease of SARS-CoV-2 in the submicromolar and micromolar ranges of the IC50 values. Three compounds suppressed coronavirus replication in the cell culture at the micromolar range of EC50 values and had low cytotoxicity. The found chemically diverse inhibitors can be used for optimization in order to obtain a leader compound, the basis of new direct-acting antiviral drugs against the SARS-CoV-2 coronavirus.

The development of preventive vaccines became the first order task in the COVID-19 pandemic caused by SARS-CoV-2. This paper reports the construction of the pVAX-RBD plasmid containing the Receptor-Binding Domain (RBD) of the S protein and a unique signal sequence 176 which promotes target protein secretion into the extracellular space thereby increasing the efficiency of humoral immune response activation. A polyglucine-spermidine conjugate (PGS) was used to deliver pVAX-RBD into the cells. The comparative immunogenicity study of the naked pVAX-RBD and pVAX-RBD enclosed in the PGS envelope showed that the latter was more efficient in inducing an immune response in the immunized mice. In particular, RBD-specific antibody titers were shown in ELISA to be no higher than 1 : 1000 in the animals from the pVAX-RBD group and 1 : 42 000, in the pVAX-RBD-PGS group. The pVAX-RBD‒PGS construct effectively induced cellular immune response. Using ELISpot, it has been demonstrated that splenocytes obtained from the immunized animals effectively produced INF-γ in response to stimulation with the S protein-derived peptide pool. The results suggest that the polyglucine-spermidine conjugate-enveloped pVAX-RBD construct may be considered as a promising DNA vaccine against COVID-19., Competing Interests: Conflict of interest. The authors declare they have no conflict of interest., (© Pleiades Publishing, Inc. 2021, ISSN 0026-8933, Molecular Biology, 2021, Vol. 55, No. 6, pp. 889–898. © Pleiades Publishing, Inc., 2021.Russian Text © The Author(s), 2021, published in Molekulyarnaya Biologiya, 2021, Vol. 55, No. 6, pp. 987–998.)

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 Escherichia coli [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.