The composition of low-polarity extracts obtained by sequential extraction of the aerial parts of Rhododendron adamsii Rehd. with hexane and methyl tert-butyl ether (MTBE) was investigated using GC-MS. The hexane extract was dominated by non-polar components: squalene, n-alkanes (nonacosane, hentriacontane), sesquiterpenes (trans-nerolidol, spathulenol, β-farnesene), and β-sitosterol. The subsequent MTBE extract was enriched in more polar lipids, primarily free triterpenic acids (ursolic and oleanolic acids). A critical finding was the complete absence of diterpene grayanotoxins in all tested extracts, confirming the safety of the non-polar extraction approach. In bioactivity assays, the total hexane extract demonstrated potent inhibitory activity against the SARS-CoV-2 main protease (3CLpro) with IC50 values of 0.0125–0.025 mg/mL, only one order of magnitude higher than the reference inhibitor disulfiram. Fractionation revealed that the activity was distributed among free acids, bound acids, and the unsaponifiable residue, indicating a multicomponent mechanism. Importantly, none of the samples inhibited HIV-1 protease (IC50 > 0.1 mg/mL), demonstrating selectivity for the cysteine protease 3CLpro over the aspartyl protease of HIV-1. These results highlight that sequential non-polar extraction of R. adamsii provides a grayanotoxin-free lipophilic complex with selective anti-SARS-CoV-2 protease activity, paving the way for bioactivity-guided identification of individual inhibitors.

A cell-based screening system for viral protease inhibitors was developed using firefly luciferase fragment complementation and validated on the SARS-CoV-2 3CLpro model. The optimal luciferase variant incorporating the VLQSGF proteolytic site (Luc III) retained 88% of its native activity. A critical requirement for system performance was the use of an extended nsp4-nsp6 fragment of the viral polyprotein rather than the mature protease, underscoring the importance of the native context for 3CLpro activity. The bicistronic construct pCAG-Luc-III-IRES-nsp4-6 enables coordinated expression of the reporter and protease, thereby increasing assay reproducibility. IC50 values obtained in this system for nirmatrelvir and GC376 correlated with live-virus assay data but differed significantly from those of a cell-free FRET assay, reflecting the impact of cellular barriers. This approach combines simplicity, a standard substrate, and high reproducibility, making it promising for high-throughput screening in basic laboratory settings and adaptable to other viral proteases.

Background: Seasonal influenza remains a significant public health problem, and the constant antigenic drift of viruses requires regular vaccine updates. mRNA vaccines offer a promising platform for the development of new, effective influenza vaccines. Administration of the naked mRNA vaccine using a needle-free jet injection system further enhances its safety, reduces cost, and eliminates the need for lipid nanoparticles, which are traditionally used for mRNA delivery. Lyophilization of naked mRNA allows for long-term storage at +4 °C. Methods: We designed and produced an mRNA vaccine against seasonal influenza, designated mRNA-Vector-Flu, encoding the hemagglutinin (HA) of the A/Wisconsin/67/2022(H1N1)pdm09, A/Darwin/9/2021(H3N2), and B/Austria/1359417/2021 strains. The vaccine was lyophilized and stored for 1 month in a refrigerator (+4 °C). A comparative immunogenicity study was conducted between synthesized immediately before use prepared and lyophilized naked mRNA-Vector-Flu. The preparations were administered to BALB/c mice using a jet needleless injection twice, 3 weeks apart. Immunogenicity was assessed on day 35 of the study. Results: A comparative immunogenicity study of naked mRNA-Vector-Flu demonstrated that both the synthesized immediately before use prepared formulation and the lyophilized form, stored at +4 °C for a month, induced similar levels of virus-specific antibodies and generated a pronounced T-cell immune response. Conclusions: Delivery of the naked mRNA vaccine using a needle-free jet injection ensures a high-level immune response, which improves its safety, reduces its cost, and eliminates the need for lipid nanoparticles traditionally used for mRNA delivery. At the same time, lyophilization of the naked mRNA vaccine preserves its biological activity and ensures its storage for at least a month at +4 °C temperatures. Our results demonstrate that our proposed approach can be considered a promising direction for the development and improvement of the mRNA vaccine platform.

The composition of low-polarity extracts obtained by sequential extraction of the aerial parts of Rhododendron adamsii Rehd. with hexane and methyl tert-butyl ether (MTBE) was investigated using GC-MS. The hexane extract was dominated by non-polar components: squalene, n-alkanes (nonacosane, hentriacontane), sesquiterpenes (trans-nerolidol, spathulenol, β-farnesene), and β-sitosterol. The subsequent MTBE extract was enriched in more polar lipids, primarily free triterpenic acids (ursolic and oleanolic acids). A critical finding was the complete absence of diterpene grayanotoxins in all tested extracts, confirming the safety of the non-polar extraction approach. In bioactivity assays, the total hexane extract demonstrated potent inhibitory activity against the SARS-CoV-2 main protease (3CLpro) with IC₅₀ values of 0.0125–0.025 mg/mL, only one order of magnitude higher than the reference inhibitor disulfiram. Fractionation revealed that the activity was distributed among free acids, bound acids, and the unsaponifiable residue, indicating a multicomponent mechanism. Importantly, none of the samples inhibited HIV-1 protease (IC₅₀ > 0.1 mg/mL), demonstrating selectivity for the cysteine protease 3CLpro over the aspartyl protease of HIV-1. These results highlight that sequential non-polar extraction of R. adamsii provides a grayanotoxin-free lipophilic complex with selective anti-SARS-CoV-2 protease activity, paving the way for bioactivity-guided identification of individual inhibitors.

The hexane extract of Psoralea drupacea Bunge fruits was initially evaluated for antivi-ral activity against SARS-CoV-2 based on GC-MS data indicating high bakuchiol con-tent (87.74%). Unexpectedly, the extract showed no antiviral effect in Vero E6 cells due to cytotoxicity (CC₅₀ = 7.5 μg/mL), while purified bakuchiol demonstrated moderate antiviral activity (IC₅₀ = 6.2 ± 0.8 μg/mL; SI = 2.9). Quantitative NMR revealed that the actual bakuchiol content in the extract was 44.3% — approximately half the GC-MS value — explaining the lack of efficacy at non-cytotoxic concentrations. Both the ex-tract and purified bakuchiol effectively blocked the RBD-ACE2 interaction in a com-petitive ELISA (71.3% inhibition at 50 μM for bakuchiol; IC₅₀ = 18.5 μM). Notably, the extract also inhibited the viral main protease 3CLpro (IC₅₀ = 32.0 ± 3.5 μg/mL), while purified bakuchiol showed no such activity. These findings reveal a dual mechanism: bakuchiol inhibits viral entry via RBD-ACE2 blockade, while other extract components (e.g., angelicin, psoralen) suppress viral replication via 3CLpro inhibition.

This study presents the design, synthesis, and evaluation of a novel series of covalent broad-spectrum inhibitors targeting the coronavirus main protease (3CLpro). The designed compounds feature a tetrahydroquinoline (THQ) scaffold functionalized with a chloroacetamide warhead. The most potent of this series in the primary screening assay, 4bf and 5bf, exhibited low micromolar IC₅₀ values against 3CLpro of SARS-CoV-2, SARS-CoV, and MERS-CoV, thereby demonstrating significant cross-reactivity. Structural analysis via X-ray crystallography confirmed covalent binding to the catalytic Cys145 residue. Complementary molecular dynamics simulations revealed stable binding modes and key interactions, highlighting differences in flexibility and residue contacts between the top inhibitors. While in vitro cytotoxicity was observed in Vero E6 cells, acute toxicity studies in mice revealed an LD₅₀ exceeding 1000 mg/kg for the lead compounds, indicating a promising in vivo safety profile. These findings establish substituted tetrahydroquinolines as a viable scaffold for the development of broad-spectrum anticoronaviral agents.

The composition of the lipophilic components of Centaurea scabiosa L. has been studied. The raw material was subjected to extraction with hexane and methyl tert-butyl ether (MTBE) using both exhaustive and sequential schemes for a detailed characterization. The resulting extracts were fractionated into acidic and neutral components via treatment with alkali solutions. The acidic compounds were converted into methyl esters for subsequent gas chromatography–mass spectrometry (GC-MS) analysis, while the neutral unsaponifiable fractions were separated into groups of different polarities using column chromatography on silica gel. This approach enabled the identification of a complex profile of lipophilic substances. In the acidic fractions, aliphatic acids with chain lengths from C10 to C32, including unsaturated variants, were characterized. The neutral fractions revealed over compounds, encompassing n-alkanes, substantial levels of the unsaturated branched hydrocarbon squalene, and a diverse array of oxygenated terpenoids. The latter were mainly represented by highly active triterpene alcohols and ketones belonging to the ursane, oleanane, lupane, and cycloartane types. The sterol composition was dominated by β-sitosterol and accompanied by cholesterol, campesterol, stigmasterol, stigmast-7-en-3-β-ol, fucosterol, and stigmastan-3-β-ol. Bioactivity screening demonstrated that several of the obtained lipophilic extracts, particularly those of lower polarity, exhibited high inhibitory activity against the main protease of SARS-CoV-2, underscoring the potential of C. scabiosa as a valuable source of anti-coronavirus agents.

A series of new heterocyclic ursane and 28-norursane hybrids - derivatives of 5-amino-1,2,3,4-thiatriazole, 1-tetrazole-5-thione, and 1-tetrazole-5-amines were prepared. Reacting triterpenoids holding NCS groups at different distances from the pentacyclic backbone with hydrazine hydrate resulted in ursane-derived hydrazinecarbothioamides. Subsequent nitrosation afforded terpenoid derivatives of 5-amino-1,2,3,4-thiatriazole. Heterocyclization of amino-thioureas with 3β-acetoxyurs-12-en-28-yl substituent under the action of Hg(OAc)2-NaN3 led to hybrids of 1-tetrazole-5-amines. 1-Tetrazole-5-thiones with different positions of heterocycle relative to the triterpene skeleton were prepared by coupling sodium azide with triterpene isothiocyanates. The activity of the new heterocyclic derivatives as inhibitors of 3CLpro of SARS-CoV-2 was investigated. Remarkable inhibition was observed for the 1-tetrazole-5-thione hybrids of triterpenoids. The highest activity among the studied compounds was provided by the combination of a 1-tetrazole-5-thione moiety at the C(28)H2 group of the ursane frame having a free OH group at the 3-position. Molecular docking assumed the covalent binding of 3CLpro via the formation of a disulfide bond between the thiol groups of the catalytic Cys145 and the tetrazole heterocycle of the new hybrid compounds. The triterpenoid backbone provided multiple external hydrophobic contacts essential for the stability of the complex. The results demonstrate the potential of heterocyclic thione hybrids as non-peptidomimetic covalent inhibitors targeting 3CLpro protease (3-Chymotrypsin-like Protease).

Background: Since the emergence of SARS-CoV-2 in the human population, the virus genome has undergone numerous mutations, enabling it to enhance transmissibility and evade acquired immunity. As a result of these mutations, most monoclonal neutralizing antibodies have lost their efficacy, as they are unable to neutralize new variants. Antibodies that neutralize a broad range of SARS-CoV-2 variants are of significant value in combating both current and potential future variants, making the identification and development of such antibodies an ongoing critical goal. This study discusses the strategy of using heterologous antigens in biopanning rounds. Methods: After four rounds of biopanning, nanobody variants were selected from a phage display library. Immunochemical methods were used to evaluate their specificity to the S protein of various SARS-CoV-2 variants, as well as to determine their competitive ability against ACE2. Viral neutralization activity was analyzed. A three-dimensional model of nanobody interaction with RBD was constructed. Results: Four nanobodies were obtained that specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 spike glycoprotein and exhibit neutralizing activity against various SARS-CoV-2 strains. Conclusions: The study demonstrates that performing several rounds of biopanning with heterologous antigens allows the selection of nanobodies with a broad reactivity spectrum. However, the fourth round of biopanning does not lead to the identification of nanobodies with improved characteristics.

The interaction of the ectodomain of the human coronavirus spike protein with the phospholipid monolayers formed on the aqueous subphase surface has been investigated. The changes in the molecular organization of monolayers of two neutral phospholipids—dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylethanolamine—after the injection of a protein solution underneath the monolayer have been analyzed. Experiments were performed with a recombinant ectodomain of the S-protein, expressed in a CHO-K1 cell strain. Electron microscopy data showed that the protein is trimerized. Grazing incidence diffraction measurements were performed to study the influence of the trimer ectodomain of the S-protein on the structure of the dipalmitoylphosphatidylcholine monolayer. It is found that protein injection under the monolayer does not induce disturbance of the monolayer crystal structure. The experimental results obtained in X-ray studies and compression isotherm measurements indicate that the interaction with the S-protein does not result in destabilization of the monolayer for both phospholipids.

Unlike most attempts to find compounds with activity against SARS-CoV-2 aimed at reusing approved drugs, our goal was to find compounds with antiviral activity among the 1,2-dithioloquinoline derivatives we synthesized. These compounds exhibit pleiotropic effects, having a novel structure compared to known pharmaceuticals. In this work, we diversified the structure of 1,2-dithioloquinoline by introducing various substituents (chlorine atom, ether, amide, sulfonamide groups, pharmacophore heterocycles) into different positions of this tricyclic framework. Using docking with further quantum chemical post-processing of calculations of the created virtual library of 404 structures, more than ten compounds of the aryl(4,4-dimethyl-4,5-dihydro-1H-[1,2]dithiolo[3,4-c]quinoline-1-ylidene)amine series were discovered, synthesized and experimentally studied. The bioactive screening of newly synthesized compounds revealed that six of them demonstrate suppression of SARS-CoV-2 replication in Vero E6 cell culture in the micromolar range of EC50 values (from 0.27 to 98.48 mu M). The best compound, in the structure of which fragments of tricyclic dithioloquinoline and streptocide are combined, demonstrated a highest ability to protect Vero E6 cells from SARS-CoV-2 with EC50 = 0.27 mu M, as well as low cytotoxicity with a selectivity index SI > 370, exceeding all reference values compounds used in the study (remdesivir, GC376, ML188).

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a multifunctional cytokine with therapeutic applications in oncology and neurodegenerative diseases. However, its clinical use is limited by the high cost of eukaryotic production systems. Here, we developed a cost-effective Escherichia coli-based platform for high-yield production of biologically active recombinant human GM-CSF (rhGM-CSF) using SUMO fusion technology. The engineered pET-SUMO-GM plasmid enabled expression of a 33 kDa fusion protein, accounting for 23-25% of total cellular protein, though it primarily accumulated in inclusion bodies. A multi-step purification strategy-including nickel affinity chromatography, Ulp protease cleavage, and hydrophobic chromatography-yielded >99.5% pure rhGM-CSF. In vitro functional assays demonstrated equivalent activity to the WHO international standard (ED50: 0.045 vs. 0.043 ng/mL in TF-1 cell proliferation). In vivo, the preparation significantly restored neutrophil counts (3.4-fold increase, p ≤ 0.05) in a murine cyclophosphamide-induced myelosuppression model. Our results establish a scalable, prokaryotic-based method to produce functional rhGM-CSF, overcoming solubility and folding challenges while maintaining therapeutic efficacy. This approach could facilitate broader clinical and research applications of GM-CSF, particularly in resource-limited settings.

Introduction. During the COVID-19 pandemic, the development of preventive vaccines, including those based on new platforms, became extremely relevant. One such platform is vaccines, which combine, for example, DNA and protein components into a single vaccine. The aim of this study was to investigate the immunogenicity of a DNA vaccine encoding a polyepitopic T-cell immunogen of the SARS-CoV-2 virus, combined with the recombinant RBD protein (the receptor-binding domain of the SARS-CoV-2 virus S protein, Wuhan-Hu-1 strain) conjugated to a polycationic carrier – polyglucin-spermidine (PGS), and to assess the contribution of individual components to the development of an immune response in BALB/c mice. Materials and methods. To create the DNA vaccine (pBSI-COV-Ub), we used a strategy of designing an artificial polyepitope immunogen consisting of conserved immunodominant fragments of various structural proteins of the SARS-CoV-2 virus, containing a large number of T-lymphocyte epitopes: helper and cytotoxic. The recombinant RBD protein was conjugated with the polycation PGS, and upon mixing it with DNA, it formed the vaccine complex CCV–BSI, whose immunogenic properties were investigated in this work. Results. Immunization of BALB/c mice with the CCV–BSI combined construct resulted in the induction of high antibody titers with neutralizing activity against live SARS-CoV-2 virus, as well as the formation of a virus-specific T-cell response, as demonstrated by ELISA, neutralization assay and ELISpot. It has been shown that the protein component contributes to the humoral immune response, while DNA contributes to the cellular immune response. Administration of the recombinant RBD protein led to the induction of only antibodies, administration of the DNA vaccine led to the induction of only a T-cell response, and administration of the combined preparation led to the induction of both a humoral immune response and specific T cells. Conclusion. The unique combination of DNA and protein within a single vaccine construct allows for overcoming the limitations of each of these vaccine types and leads to the induction of both arms of immunity. The protein component can be replaced according to the current viral strain, and a universal T-cell immunogen can provide a response to a wide range of circulating variants. This platform can be further used to develop vaccines against various highly variable viruses.

West Nile fever is an infectious disease caused by the West Nile virus (WNV), which is transmitted by mosquitoes. Epidemiological surveillance confirms the potential risk of WNV infection in human populations. The lack of specific antiviral therapeutics and vaccines against WNV underscores the urgent need to develop effective therapeutic approaches. In this study, a recombinant chimeric monoclonal antibody (mAb) 900 was generated based on the broadly neutralizing and protective murine mAb 9E2. The antigen-binding regions of the murine mAb were fused with the constant domains (CH2-CH3) of human IgG1. Two key amino acid clusters, M252/S254/T256 and H433/N434, were introduced into the CH2-CH3 domains to enhance the affinity of mAb 900 for the neonatal Fc receptor (FcRn). The engineered mAb 900 was produced in CHO cells and purified to high homogeneity. Biophysical characterization confirmed its stability and correct dimeric assembly. Comparative analysis demonstrated that mAb 900 retained the high antigen-binding affinity and potent virus-neutralizing activity of its murine predecessor. Most importantly, mAb 900 demonstrated significant protective efficacy in a lethal mouse model of WNV infection. These results establish the proof of concept for mAb 900 as a promising candidate for further preclinical development against WNV infection.

Antibodies are protein molecules whose primary function is to recognize antigens. However, recent studies have demonstrated their ability to hydrolyze specific substrates, such as proteins, oligopeptides, and nucleic acids. In 2023, two separate teams of researchers demonstrated the proteolytic activity of natural plasma antibodies from COVID-19 convalescents. These antibodies were found to hydrolyze the S-protein and corresponding oligopeptides. Our study shows that for antibodies with affinity to recombinant structural proteins of the SARS-CoV-2: S-protein, its fragment RBD and N-protein can only hydrolyze the corresponding protein substrates and are not cross-reactive. By using strict criteria, we have confirmed that this proteolytic activity is an intrinsic property of antibodies and is not caused by impurities co-eluting with them. This discovery suggests that natural proteolytic antibodies that hydrolyze proteins of the SARS-CoV-2 virus may have a positive impact on disease pathogenesis. It is also possible for these antibodies to work in combination with other antibodies that bind specific epitopes to enhance the process of virus neutralization.

Background. Polysaccharides are known to possess adjuvant properties, they are biodegradable, safe, and are of low-labor production. In this regard, the development of polysaccharide-based adjuvants is an urgent task.The aim. To develop a method for obtaining mannans from the cell walls of Saccharomyces cerevisiae yeast and to study their adjuvant properties using subunit vaccine model.Materials and methods. The preparation of mannans was obtained from the Saccharomyces cerevisiae yeast by enzymatic and alkaline hydrolysis. Its adjuvant properties were assessed in BALB/c mice immunized with the recombinant receptor-binding domain (RBD) of the SARS-CoV-2 (S) protein (Delta (B.1.617.2)). The titers of specific antibodies in the blood sera were determined by ELISA assays using the recombinant RBD (Wuhan-Hu-1 and Delta), and the recombinant (S) protein (Wuhan-Hu-1, Delta and Omicron) as antigens. The titers of virus-neutralizing antibodies were determined using virus-neutralization tests with the SARS-CoV-2 virus strains Wuhan – hCoV19/Australia/VIC01/2020 (Wuhan-Hu-1), Delta – hCoV-19/Russia/PSK-2804/2021 (Delta (B.1.617.2)), and Omicron 1 – hCoV-19/Russia/Moscow171619-031221/2021 (Omicron (B.1.1.529)).Results. The developed scheme allowed for obtaining up to 200 mg of mannans from 10 g of yeast cell debris. Double, with a two-week interval, immunization with RBD (50 μg) in combination with mannans (40 μg and 10 μg) induced the production of specific antibodies in titers from 1:2477330 to 1:188360. The titer of virus-neutralizing antibodies to the Delta – hCoV-19/Russia/PSK-2804/2021 was 1:485 (40 μg of mannans per mouse).Conclusions. We developed a scheme for obtaining a low-toxic preparation of mannans from the Saccharomyces cerevisiae yeast. The highest adjuvant activity was achieved when using mannans at the dose of 40 µg per mouse. Blood sera obtained from the immunized animals neutralized both homologous and heterologous SARS-CoV-2 strains.

To increase the effectiveness and immunogenicity of modern vaccines, especially subunit ones, it is required to use adjuvants. Polysaccharides, due to their safety and biocompatibility, are desirable candidates for the creation of vaccine adjuvants. The aim of our study was to develop a method for obtaining beta-Glucans from the yeast Saccharomyces cerevisiae cell wall, and evaluate their adjuvant properties. The high purity and non-toxicity of the resulting preparation was achieved by using enzyme complexes of cellulase and protease in combination with ultrasound (22 kHz) at the purification stage. The developed scheme allows for the yield of beta-Glucans up to 2 g from 100 g of the biomass of wet cells. The adjuvant properties of beta-Glucans were studied in 50 male BALB/c mice, weighing 16–18 g. Immunization was performed twice, with a 14-day interval, intramuscularly, 200 μl per animal. The recombinant receptor-binding domain (RBD) of the surface S protein of the SARS-CoV-2 virus (Wuhan-Hu-1 and B.1.617.2 (Delta)) was used as an antigen, at a dose of 50 μg per animal. A positive control group was administered with the antigen combined with aluminum hydroxide. As a negative control, mice injected with the saline solution were used. The titers of specific antibodies in the blood sera were determined by ELISA assays. RBD (Wuhan-Hu-1 and Delta), and S protein (Wuhan-Hu-1, Delta and Omicron) were used as antigens. The titers of virus-neutralizing antibodies were measured in neutralization tests using SARS-CoV-2 virus strains Wuhan-Hu-1, Delta (B.1.617.2) and Omicron (B.1.1.529). The results of the study have shown that beta-Glucans have the ability to enhance the production of specific and virus-neutralizing antibodies in mice immunized with RBD. The titers of specific and virus neutralizing antibodies are comparable to their levels in the group immunized with RBD and Al(OH)₃. It has been found in the experiments in white outbred ICR mice that the preparation belongs to practically non-toxic substances. Therefore, it can be concluded that the use of beta-Glucans could become a preferable alternative to the conventional adjuvants based on aluminum salts, being biocompatible, biodegradable and non-toxic substances of low labor-intensive production.

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 105 to 2×106. 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.

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.