Novel coronavirus disease 2019, caused by the SARS-CoV-2, initiate humoral and cellular immune responses against diverse virus antigens. The assessment of SARS-CoV-2-specific is mainly carried out in routine practice by determining specific immunoglobulins. However, high variability in S-protein structure in new genovariants of SARS-CoV-2 virus and the lack of correlation between specific antibodies and CD8+ T-lymphocytes underlie false negative responses, and mass assessment of cellular immunity is complicated due to the complexity of applying ELISPOT and cytofluorometry techniques. To solve this issue, a diagnostic method was developed for assessing SARS-CoV-2-specific cellular immune response, which is based on a skin test followed by evaluating a delayed-type hypersensitivity reaction involving antigen-specific memory T-lymphocytes. A diagnostic preparation CoronaDerm-PS is based on Cord_PS, which is a hybrid recombinant protein consisting of parts of the SARS-CoV-2 structural proteins S, M, N, E. The specific activity of this chimeric antigen was analyzed in cultured T-lymphocyte activation test by assessing interferon-γ production using cytofluorometry. To investigate the chimeric antigen specific activity, a preclinical safety study with CoronaDerm-PS preparation in experimental animals was conducted. A dose-dependent developing skin reaction was observed in 90–100% of guinea pigs vaccinated by EpiVacCorona, CoviVac, Gam-COVID-Vac, which confirms a potential for assessing post-vaccination cellular immunity using CoronaDerm-PS preparation. Upon this, the presence of functionally active T-cell-antigenic epitopes in the recombinant polypeptide allows to evaluate SARS-CoV-2-specific response illustrated by detected response after Gam-COVID-Vac (S-protein) and EpiVacCorona (N-protein) vaccination. Thus, a skin test based on CoronaDerm-PS preparation may be a promising diagnostic tool for rapid mass screening requiring no specialized laboratory equipment for assessing populational SARS-CoV-2-specific immunity. Such a test is distinguished by advantages such as ease of analysis, high specificity and sensitivity. The final decision-making on using this test in a real-world practice may achieved after conducting further clinical safety and effectiveness trials.

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.

New experimental antiviral preparations are developed based on a molecular structure consisting of single-stranded and double-stranded RNAs from different sources and an outer envelope based on dextran with specific isotropic agents. An affection of developed molecular structure preparations on physiological indicators variety in laboratory animals to obtain data on the tolerance of compositions was investigated. It has been shown that administering higher doses of experimental preparations to animals doesn`t cause or lead to short-term and minor deviations in their condition. An application of the method of specific interferon-inducing activity determining in the study of antiviral and other interferon inducers characteristics may be used for estimation of criteria like the minimal effective interferon-inducing dose (MEID) and the modified chemotherapeutical index (mCTI), required for a complete characterisation of drugs.

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.

Проведено исследование генеративной токсичности вакцины ЭпиВакКорона. Вакцину ЭпиВакКорона крысам Вистар вводили внутримышечно в дозах: равной одной вакцинирующей для человека, пересчитанной с учетом разницы в площади поверхности тела — 23 мкг/кг, и в 10 раз ее превышающей — 230 мкг/кг. Контролем служил физиологический раствор. Вакцину вводили 2 раза в неделю самцам в течение 48 дней, самкам — в течение 15 дней до спаривания. Получавших вакцину самок и самцов ссаживали с интактными крысами противоположного пола на 10 – 14 дней. Введение вакцины ЭпиВакКорона в дозах 23 и 230 мкг/кг самкам не влияло на показатели спаривания — индексы покрытия и зачатия, а также на индекс фертильности самок. Введение вакцины ЭпиВакКорона в дозах 23 и 230 мкг/кг самцам не влияло на индекс фертильности самцов и ссаженных с ними интактных самок. При введении вакцины самцам в дозе 230 мкг/кг наблюдали преходящее снижение индекса покрытия самок на 13 % (p << 0,011), связанное c наличием одного нефертильного самца в группе и с отсутствием эструса в течение 12 дней у одной из интактных самок. При этом наблюдали одновременное преходящее увеличение индекса зачатия у самок на 14 % (p << 0,011), предположительно связанное с восстановлением самцов после курса инъекций вследствие более позднего наступления дня первого эструса у ссаженных с ними самок. Количество желтых тел беременности, живых и погибших плодов, мест имплантации, показатели пред- и постимплантационной смертности потомства у вакцинированных самок и интактных самок, спарившихся с получавшими вакцину самцами, не отличались от показателей контрольных животных. Полученные данные свидетельствуют об отсутствии у вакцины ЭпиВакКорона в дозах 23 и 230 мкг/кг признаков генеративной токсичности.

Получено 3 варианта готовых лекарственных форм (ЛФ) рекомбинантного гранулоцитарно-макрофагального колониестимулирующего фактора человека (рчГМ-КСФ) в виде растворов и лиофилизатов с использованием различных вспомогательных веществ. Показано, что введение мышам с цитостатической миелосупрессией полученных образцов ЛФ приводило к увеличению количества нейтрофилов крови на 289 – 800 % при введении жидких ЛФ и на 360 – 860 % — восстановленных до растворов лиофилизатов. Все изученные образцы не проявляли признаков аномальной токсичности. Выбран состав ЛФ рчГМ-КСФ, обладающий наиболее выраженной гемостимулирующей активностью.

The aim of the work is to evaluate the hemostimulating activity of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) conjugates with alendronic acid (ALN) in the model of cytostatic myelosuppression and the dynamics of rhGM-CSF accumulation as a part of the conjugate in the bone tissue and bone marrow of mice.Materials and methods. The conjugates obtained by a solid-phase synthesis using 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide or periodate oxidation, were used. A hemostimulating activity was evaluated in a model of a cytostatic myelosuppression induced by the administration of cyclophosphamide to CBA/Calac mice. RhGM-CSF preparations were injected subcutaneously for 4-5 days at the dose of 90 µg/kg. After the injections cycle had been completed, the total leukocyte and segmented neutrophil counts were carried out in the blood samples, and the total karyocyte count was carried out in the bone marrow samples.The tissue distribution of rhGM-CSF preparations was assessed in outbred CD-1 mice after a single intravenous administration at the effective dose. The content of rhGM-CSF in blood, femoral tissue and bone marrow was determined by enzyme immunoassay.Results. RhGM-CSF conjugates with ALN have been shown to retain the ability of the original protein to increase the number of leukocytes, segmented blood neutrophils, and bone marrow karyocytes under the action of conjugates. The stimulation of the neutrophil production used to be observed at earlier times than in the case of rhGM-CSF. The increase in the total number of bone marrow cells after the introduction of all three conjugates was more pronounced compared to the original protein (by 34%). The increased hemostimulatory effect of the AEG conjugate was accompanied by a more intense accumulation of rhGM-CSF in the bone tissue and bone marrow of mice. The rhGM-CSF introduced into the conjugate was detected in the bone tissue for 24 h and it circulated in the bloodstream for a longer time compared to the original protein.Conclusion. The data obtained make it possible to conclude that further work on the development of effective hemostimulating drugs based on rhGM-CSF conjugates with ALN, is promising.

Interferon inducers are of interest as the first line of defense against viral infections, including influenza. Double-stranded ribonucleic acid (dsRNA) for intranasal administration has been developed, the excipients and drug components were selected at the Institute of Medical Biotechnology, a branch of the State Research Center of Virology and Biotechnology «Vector». This work presents the results of the study on antiviral activity and pharmacological safety of a new form of dsRNA. The antiviral activity of the preparation was studied in BALB/c mice infected with influenza A/Aichi/2/68 (H3N2) or Bishkek/01/2009 (H1N1pdm09) viruses. A safety study was performed with a single administration of intranasal dsRNA into white outbred ICR mice. The study showed that the administration of therapeutic and prophylactic regimen of intranasal dsRNA at a dose of 2.5 mg/kg increases the survival and average life expectancy of mice infected with the mentioned strains of influenza virus. The protective effect of the preparation in mice infected with a lethal dose of Bishkek/01/2009 virus was comparable to the effect of Tamiflu. The absence of toxic effects of intranasal dsRNA at a pharmacological dose in laboratory mice, the functional state of their physiological systems, as well as main types of metabolism, established in the experiments, lead to a conclusion concerning pharmacological safety of the preparation and the prospects for further work to complete pharmaceutical development of a new antiviral drug.

The hemostimulating properties of granulocyte-macrophage colony-stimulating factor (GM-CSF) make possible its clinical use in alleviating side effects of anti-cancer radio- and chemotherapy, in bone marrow transplantation, and in the treatment of some primary immunodeficiency conditions associated with leukopenia. The State Research Center of Virology and Biotechnology “Vector” of the Federal Service for Surveillance on Consumer Rights Protection and Human Wellbeing has developed a high-performance technology for production of recombinant human GM-CSF (rhGM-CSF) based on a recombinant E. coli strain.The aim of the study was to assess hemostimulating activity of the rhGM-CSF preparation obtained using the new developed technology, as observed in cell culture and in the mice model of myelosuppression induced by cyclophosphamide administration.Materials and methods: in vitro evaluation of rhGM-CSF hemostimulating activity was performed by MTT assay in the commercial HL-60 promyelocytic leukemia cell culture with preliminary suppression of cell growth rate by adding a low concentration of dimethyl sulfoxide to the medium. In vivo studies were carried out in CBA/CaLac mice with cyclophosphamide-induced myelosuppression. The hemostimulating properties of the drug were evaluated after subcutaneous administration of 1–175 µg/kg doses for 4–5 days, following administration of a cytostatic agent. The total number of leukocytes and the content of their morphological forms were determined in blood samples taken at different time points after the drug administration. The statistical processing of the experimental data was based on analysis of variance using Statgraphics v. 5.0 software.Results: the proliferative activity of HL-60 cells incubated with the rhGM-CSF preparation for 72 hours was shown to be dose-dependent. The highest values of the increase in proliferative activity associated with an increase in the drug dose were observed in the concentration range from 0.04 to 0.64 ng/mL (proliferative activity increased by 11–18% when the dose was increased twofold). The experiments in mice demonstrated a two-phase pattern of the dose-dependent effect. The drug showed the highest hemostimulating effect at the dose of 90 µg/kg.Conclusions: the rhGM-CSF preparation obtained using the new developed technology has a pronounced hemostimulating activity confirmed by both in vitro and in vivo test systems.

The method of obtaining and purification of the double-stranded RNA of bacteriophage p6 was developed. It was shown that the created method makes it possible to obtain high purity dsRNA preparations possessing interferon-inducing and phagocyte-stimulating activity.

Hemostimulating activity of Panagen substance representing the nucleoprotein complex extracted from the human placenta was studied using the model of cyclophosphan-induced myelosuppression in mice. Panagen was found to have hemostimulating properties comparable with activity of Derinat drug.