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.

The antiviral activity of extracts of the basidial fungus Daedaleopsis confragosa (Bolton) J. Schröt strain F-1368 cultivated under the laboratory conditions has been studied. It was shown that the extracts obtained from 1 L of the culture contained proteins and polysaccharides in the amount of 71±5 and 413±15 mg/L of culture, respectively. Antiviral activity (ED50) against the pandemic influenza virus A/California/07/09 (H1N1)pdm09 was observed at extract concentrations in the range of 0.02-0.12 mg/mL depending on the culture medium. The therapeutic index (CC50/ED50) was at the level of 14-17, which makes it possible to recommend D. confragosa extracts for the development of antiviral agents. It was shown that treatment with hydrolytic enzymes (trypsin, zymolyase) leads to an increase in cytotoxicity and a decrease in the antiviral activity of the extracts. Daedaleopsis confragosa, basidomycetes, extracts, antiviral activity, influenza virus The study was carried out according to the state assignment to FBRI SRC VB «Vector», Rospotrebnadzor, Koltsovo.

Influence of double-stranded RNA (dsRNA) from Saccharomyces cerevisiae yeast upon expression levels of the macrophage genes encoding TLR3 receptor, interferons alpha and beta (IFNα, IFNβ), 2’,5’-oligoadenylate synthetase (OAS) and protein kinase R (PKR) enzymes has been studied in the J774 mouse histiocytic cell culture and in vivo in Balb/c mice. It has been shown that dsRNA exerts a selective activating effect on genes of TLR3 receptor, antiviral proteins IFNα, IFNβ, and OAS, both in vitro and in vivo. With J774 cell culture, the highest induction capacity was observed for the IFNβ gene: 365 to 802-fold. The stimulatory effect was dependent on the dose of dsRNA in the range of 16.9 to 125 μg/ml. The preparation enhanced IFNα gene activity to lesser degree (more than 10-fold), TLR3 and OAS (3 to 4-fold), while the expression levels for these genes were not significantly dependent on the dose of dsRNA. The stimulating effect of dsRNA was dosedependent in murine peritoneal macrophages. The maximum activating effect of the preparation was shown upon administration of the effective antiviral dose (0.5 mg of dsRNA/kg). Five hours after intraperitoneal injection of dsRNA, the highest level of mRNA synthesis was observed for IFNα (54-fold), OAS (43-fold) and TLR3 (28-fold) genes. Expression of the IFNβ gene increased to a lesser degree (9-fold). An increase in the dose of preparation to 1.5 mg/kg led to decrease of the stimulatory effect. Expression levels of the IFNα, TLR3, and OAS genes in that case decreased by 2-4-fold as compared to a lower dose, and the PKR gene expression was 5-fold lower compared to the control. One day after dsRNA administration, a tendency was observed for both experimental groups towards a decreased transcription of macrophage genes, if compared with the 5-hour term. The weakening of gene activity was less pronounced in animals treated with dsRNA at the dose of 1.5 mg/kg. The transcription indices for IFNβ, OAS, and TLR3 genes were much higher during this period (5-10-fold higher than the control values). The dynamics of PKR gene transcription in both experimental systems was significantly different from the expression of other studied genes. The dsRNA preparation at this dose range did not have a pronounced stimulatory effect upon expression of this gene. A moderate increase in PKR gene activity in macrophages of mice was observed only a day following intraperitoneal administration of dsRNA. Concentrations and length of dsRNA molecules are known to be critical factors to the PKR gene activation. An ability to increase the expression of the gene is shown at low dsRNA concentrations (10-7 g/ml and below), while highly polymeric dsRNAs weaken the gene activity. Since the doses and concentrations of dsRNA used in our experiments were significantly different from those mentioned above, it could, in general, affect regulation of PKR gene transcription towards reduction of the stimulatory effect.

— In order to develop a target drug we studied the process of conjugation of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and alendronic acid (ALN). The conjugation reactions were performed both in solution and in the solid phase. The results of the examination of the specific activity of the conjugated GM-CSF confirmed the preservation of biological properties of the protein. The experimental data obtained indicate a higher affinity of GM-CSF-ALN conjugates to hydroxyapatite, an analog of the bone mineral matrix, than of the intact protein.

A proposal is being considered for the use of BAS preparations developed in the Institute of medical biotechnology OF the FBU SSC VB «Vector» in the programs of ensuring the country’s security in the field of medicine, veterinary medicine and obtaining environmentally friendly food products. On the basis of biotechnological methods, a technology for obtaining a complex of biologically active preparations of double-stranded and single-stranded RNA from yeast and phages that can provide a solution to the above problems has been created. The» ideology «of the development consisted in a deep study of the EN-Dogen induction of interferons by the organism itself using evolutionarily formed mechanisms. The basis was taken of inter-veronicaceae mechanisms of action of viruses. Such drugs when injected into the human body and animals can act as viruses, inducing the synthesis of interferons and other proteins-cytokines in the body, but unlike viruses, they do not produce destructive effects. Activation of the endogenous cytokine network in this case at the systemic level can form a state of non-specific resistance against viruses, bacteria and their toxins. Injectable and ointment forms of these drugs based on RNA were developed, tested and approved by the Ministry of health of the Russian Federation and the Department of veterinary medicine of the Russian Federation. Introduction of effective methods of application of the developed preparations allows to use them as etiotropic means of emergency prevention and treatment of viral diseases of the person, calves, pigs, carnivores, birds and fishes, correction of immunodeficiency, strengthening of protective properties of vaccines.

The purpose of the study was to analyze the antitumor effects of the extract of mycelium from Duddingtonia flagrans (strain F-882) on xenografts of human C33a cervical cancer cells. Material and Methods. To evaluate the antitumor effect, we used the absolute values of xenograft volumes and calculated the tumor growth inhibition and the index of tumor growth. Results . At the first stage of the experiment, a 4-week subcutaneous injection of the water extract of F-882 resulted in an almost twofold slowdown in xenograft growth, with the tumor growth inhibition value of 50.6 %. In the second stage of the experiment, a 2.5-week subcutaneous injection followed by a 1.5-week intratumoral injection of F-882 also caused the tumor growth inhibition. After completing F-882 injections, the effect of tumor growth inhibition continued for 2.5 weeks and the tumor growth inhibition value was 58.7 %. Conclusion. The mycelium extract F-882 was shown to have an antitumor effect on subcutaneous xenografts of human C33a cervical carcinoma cells.

Aim: to evaluate the antitumor activity of the drug containing TNF-alpha and high-polymer double-stranded RNA (dsRNA) in the composition of virus-like particles (VLP-TNF-alpha) on B16-F10 melanoma cells. Material and Methods. Analysis of the anti-proliferative effect of VLP-TNF-alpha as well as its components, TNFalpha and dsRNA, was carried out using the MTT -test. Apoptosis of melanoma cells was assessed by flow cytofluorimetry with FITC-annexin V. Results. It was shown that the cytotoxic effect of the drug containing the combination of TNF-alpha and dsRNA on melanoma cells significantly exceeded the total cytotoxic effect of TNF-alpha or dsRNA alone (LD50 for combination drug was 0.05 μg/ml, TNF-alpha – 9.5 μg/ml, dsRNA>20 μg/ml). Conclusion. The drug containing TNF-alpha and dsRNA molecules may be a promising drug for the treatment of malignant tumors, including melanoma.

In experiments on mice, the level of expression of antiviral-response-mediating proteins was studied in peritoneal macrophages, in reaction to the intraperitoneal administration of double-stranded RNA (dsRNA) from the yeast Saccharomyces cerevisiae. Dose (0.5 and 1.5 mg/kg) and time (5 and 24 hours) dependencies of the eff ect of dsRNA on the activity of genes encoding Toll-like receptor 3 (TLR3), IFN-alpha, IFN-beta, 2’,5’-oligoadenylatesynthetase (OAS) and proteinkinase R were determined. The obtained results indicate the increase in the level of transcriptional activity in the cells, most pronounced for TLR3, IFN-alpha and OAS genes. The maximum effect was observed 5 hours after the administration of dsRNA in the effective antiviral dose (0.5 mg / kg).

Introduction. Tumor necrosis factor α (TNF-α) is a natural cytokine, characterized by pronounced antitumor properties. A wide range of side effects serves as an obstacle for the use of TNF-α in clinical practice. One of the ways to improve its therapeutic properties is to increase the tropism of the cytokine to the tumor tissue by incorporating it into the targeted delivery system.The aim of the study was to evaluate the antitumor activity of the preparation containing TNF-α as part of the artificial “virus-like particle” (VLP-TNF-α), developed in SRC VB “Vector” as a transport system for delivering proteins to target cells.Materials and methods. The antitumor effect of VLP-TNF-α preparation was evaluated in experimental B16F10 melanoma model by the change of dynamics of tumor growth (volume, mass) and its morphological structure (presence of necrotic processes, blood vessel destruction). The number of the effector immune cells (CD3+, CD11b+) in the tumor tissue was determined by immunohistochemical method.Results. It has been shown that VLP-TNF-α administered intravenously at the doses of 5 × 104 and 1 × 105 IU/mouse inhibits the growth of the primary tumor. The most pronounced and stable effect was observed with a five-fold administration at the dose of 1 × 105 IU/mouse every other day: tumor growth inhibition was 40 % on the 1st day, and 47 % on the 7 th day upon the treatment. Injections of the preparation resulted in the increase of necrosis number, destruction level of the tumor tissue, development of damage and destruction of the tumor blood vessels and its infiltration with immunocompetent cells.Conclusion. The obtained data indicates that TNF-α within the delivery system exerts antitumor activity, which suggests the possibility of its further use for the treatment of malignant neoplasms, in particular, melanoma.The study was performed in accordance with ethical principles adopted by the European Convention for the protection of vertebrate animals used for experimental and other scientific purposes.

The development of the nominal sequence of steps for analyzing the HSI proposed by Landgrebe, which is necessary in the context of the appearance of reference signature libraries for environmental monitoring, is discussed. The approach is based on considering the HSI pixel as a signature that stores all spectral features of an object and its states, and the HSI as a whole - as a two-dimensional signature field. As a first step of the analysis, a procedure is proposed for detecting a linear dependence of signatures by the magnitude of the Pearson correlation coefficient. The main apparatus of analysis, as in Landgrebe sequence, is the method of principal component analysis, but it is no longer used to build classes and is applied to investigate the presence in the class of subclasses essential for the applied area. The experimental material includes such objects as water, swamps, soil, vegetation, concrete, pollution. Selection of object samples on the image is made by the user. From the studied images of HSI objects, a base of reference signatures for classes (subclasses) of objects is formed, which in turn can be used to automate HSI markup with the aim of applying machine learning methods to recognize HSI objects and their states.

Эпидемия ВИЧ-1 является одной из самых острых проблем мирового здравоохранения. По ряду причин эффективной вакцины против этой инфекции на данный момент не создано. В настоящее время важным направлением в разработке вакцины против ВИЧ/СПИДа является конструирование иммуногенов, которые были бы способны индуцировать антитела, нейтрализующие широкий спектр штаммов ВИЧ-1 (bNAbs). Одним из подходов создания таких иммуногенов является конструирование химерных вирусоподобных частиц (VLPs), экспонирующих эпитопы, узнаваемые bNAbs. Целью исследования являлись получение и характеризация химерных VLPs на основе HBcAg, экспонирующих эпитопы, узнаваемые bNAbs 2F5 и 4E10. Материал и методы. Штаммы-продуценты химерных вариантов HBcAg получали путем трансформации клеток Escherichia coli BL21 рекомбинантными плазмидами, несущими гены HBcAg и содержащими встройки, кодирующие эпитопы bNAbs 2F5 и 4E10. Очистку рекомбинантных белков проводили с помощью гель-фильтрации на колонке с сефарозой CL-6B. Способность рекомбинантного HBcAg образовывать вирусоподобные частицы оценивали с помощью электронной микроскопии. Антигенные свойства эпитопов в составе химерных вариантов HBcAg анализировали с помощью иммуноблотинга. Результаты. Получена модифицированная нуклеотидная последовательность гена HBcAg, в состав которой были введены уникальные сайты рестрикции, фланкирующие район главной антигенной детерминанты кора. На основе данной генетической конструкции получены три рекомбинантные плазмиды, кодирующие химерные варианты НВсAg, включающие эпитопы bNAbs 2F5 и 4E10. С помощью иммуноблотинга установлено, что эпитопы, узнаваемые bNAbs, сохраняют свои антигенные свойства в составе химерных НВсAg.

The main problems of using TNF-alpha in antitumor therapy are its rapid degradation in the bloodstream and the limited selectivity of accumulation in the tumor tissue. The SRC VB «Vector» developed a biodegradable molecular construct that provides protection against proteases and ensures targeted delivery of proteins to the tumor tissue. This construct was used to create an antitumor drug containing recombinant human TNF-alpha (rhTNF-alpha).The aim of the study was to analyse rhTNF-alpha pharmacokinetics in the delivery system after a single administration.Materials and methods: the rhTNF-alpha drug carried by the delivery system was intravenously administered to female outbred ICR (СD-1) mice only once at two effective antitumor doses, 2.55 μg and 5.1 μg / 20 g of body weight. The concentration of TNF-alpha in the serum and supernatants of organ homogenates, obtained at different time points after administration, was analysed by immunoenzyme assay.Results: the obtained curves of TNF-alpha concentration in the blood were satisfactorily described by the equation for the twocompartment model without absorption. The rapid phase of elimination from the blood took 0–4 h, the slow one — 4–24 h. The highest specific content of protein was observed in the skin, spleen, and kidneys tissue. The calculation of pharmacokinetic parameters demonstrated that the highest values of tissue availability fT were obtained for the kidneys and skin; the drug was retained for longer periods of time in the kidneys, liver and skin (according to the MRT data). As a rule, complete elimination of the drug was observed by the end of the first day after administration.Conclusions: rhTNF-alpha carried by the delivery system was quickly eliminated from the blood and distributed in the internal organ tissues after a single intravenous administration to mice in the effective doses range. The main organs in which rhTNF-alpha was distributed were skin, kidneys, and spleen. The elimination of the drug from the blood was a two-phase process which was generally over by the end of the first day.

A suggested method for obtaining recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) includes the accumulation of the producer strain biomass enriched with the target product up to 30% of total protein content, its isolation and purification. The later consists of the following stages: ultrasound cell disintegration, washing of inclusion bodies with buffer solutions, GM-CSF solubilization from inclusion bodies by 6 M urea, denaturation-renaturation of protein molecules and purification by chromatography on DEAE-Sepharose and combined chromatography on CM-Sepharose and Q-Sepharose followed by dialysis. The proposed method makes it possible to yield up to 10 mg of the protein preparation from 1 g of wet cells with the purity of 98% and high activity shown on the human erythroleukemia cell line. granulocyte-macrophage colony-stimulating, GM-CSF, producer strain, cultivation, chromatographic purification. The work was performed in the framework of the State Assignment «Adjustment of the Technology of Preparative Obtaining and Purification of Recombinant Proteins» (no. 13/18).

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.

Conjugates of recombinant human tumor necrosis factor alpha (TNFα) and alendronic acid linked through the protein sulfhydryl, carboxyl, and amino groups were obtained with crosslinking agents of different types. The conjugation reactions were conducted in solution and on a solid phase. Unlike the conjugation reactions in solution, the method involving immobilization of active components on a hydroxyapatite column was shown to result in the conjugates with a specified stoichiometry and a high degree of homogeneity. The TNFα conjugates retained the specific cytolytic activity and demonstrated the higher affinity to hydroxyapatite, an analogue of the bone mineral matrix, than TNFα.

An osteotropic agent based on the human recombinant tumor necrosis factor alpha (TNF-alpha) has been designed for treatment of bone metastases. It represents a molecular construct containing yeast double- stranded ribonucleic acid (dsRNA) covered by the conjugate of polyanion dextran with TNF-alpha and bisphosphonate alendronic acid. This construct is characterized by the combination of substances possessing antitumor activity (TNF-alpha, dsRNA) and a vector molecule (bisphosphonate) providing tropism to hydroxyapatite, the main mineral component of the bone tissue matrix. The conjugation conditions were optimized and the conjugates of TNF-alpha and alendronic acid with dextran were synthesized. The molecular constructs were obtained by self-assembly, and the resultant complexes were separated by gel filtration on Sepharose CL-6B. The electrophoretic analysis has shown decreased mobility of dsRNA in the complex with the conjugate as compared to mobility of the original dsRNA. This confirms formation of the designed structures. Transmission electron microscopy confirmed the presence of particles with sizes of 30–40 nm in the drug preparation. Evaluation by the sorption/desorption method showed a higher affinity of TNF-alpha conjugates to hydroxyapatite as compared to original TNF-alpha molecules (from 1.0–1.8 mol/L vs. 0.3 mol/L of potassium phosphate buffer for desorption, respectively).