[Intranasal vaccine against COVID-19 based on a recombinant variant of the Sendai virus (Paramyxoviridae: Respirovirus) strain Moscow].

INTRODUCTION: Intranasal vaccination using live vector vaccines based on non-pathogenic or slightly pathogenic viruses is the one of the most convenient, safe and effective ways to prevent respiratory infections, including COVID-19. Sendai virus is the best suited for this purpose, since it is respiratory virus and is capable of limited replication in human bronchial epithelial cells without causing disease. The aim of the work is to design and study the vaccine properties of recombinant Sendai virus, Moscow strain, expressing secreted receptor-binding domain of SARS-CoV-2 Delta strain S protein (RBDdelta) during a single intranasal immunization. MATERIALS AND METHODS: Recombinant Sendai virus carrying insertion of RBDdelta transgene between P and M genes was constructed using reverse genetics and synthetic biology methods. Expression of RBDdelta was analyzed by Western blot. Vaccine properties were studied in two models: Syrian hamsters and BALB/c mice. Immunogenicity was evaluated by ELISA and virus-neutralization assays. Protectiveness was assessed by quantitation of SARS-CoV-2 RNA in RT-PCR and histological analysis of the lungs. RESULTS: Based on Sendai virus Moscow strain, a recombinant Sen-RBDdelta(M) was constructed that expressed a secreted RBDdelta immunologically identical to natural SARS-CoV-2 protein. A single intranasal administration of Sen-RBDdelta(M) to hamsters and mice significantly, by 15 and 107 times, respectively, reduced replicative activity of SARS-CoV-2 in lungs of animals, preventing the development of pneumonia. An effective induction of virus-neutralizing antibodies has also been demonstrated in mice. CONCLUSION: Sen-RBDdelta(M) is a promising vaccine construct against SARS-CoV-2 infection and has a protective properties even after a single intranasal introduction.

Reporter Transgenes for Monitoring the Antitumor Efficacy of Recombinant Oncolytic Viruses.

Accurate measurement of tumor size and margins is crucial for successful oncotherapy. In the last decade, non-invasive imaging modalities, including optical imaging using non-radioactive substrates, deep-tissue imaging with radioactive substrates, and magnetic resonance imaging have been developed. Reporter genes play the most important role among visualization tools; their expression in tumors and metastases makes it possible to track changes in the tumor growth and gauge therapy effectiveness. Oncolytic viruses are often chosen as a vector for delivering reporter genes into tumor cells, since oncolytic viruses are tumor-specific, meaning that they infect and lyse tumor cells without damaging normal cells. The choice of reporter transgenes for genetic modification of oncolytic viruses depends on the study objectives and imaging methods used. Optical imaging techniques are suitable for in vitro studies and small animal models, while deep-tissue imaging techniques are used to evaluate virotherapy in large animals and humans. For optical imaging, transgenes of fluorescent proteins, luciferases, and tyrosinases are used; for deep-tissue imaging, the most promising transgene is the sodium/iodide symporter (NIS), which ensures an accumulation of radioactive isotopes in virus-infected tumor cells. Currently, NIS is the only reporter transgene that has been shown to be effective in monitoring tumor virotherapy not only in preclinical but also in clinical studies.

Iteratively regularized Gauss-Newton type methods for approximating quasi-solutions of irregular nonlinear operator equations in Hilbert space with an application to COVID-19 epidemic dynamics.

We investigate a class of iteratively regularized methods for finding a quasi-solution of a noisy nonlinear irregular operator equation in Hilbert space. The iteration uses an a priori stopping rule involving the error level in input data. In assumptions that the Frechet derivative of the problem operator at the desired quasi-solution has a closed range, and that the quasi-solution fulfills the standard source condition, we establish for the obtained approximation an accuracy estimate linear with respect to the error level. The proposed iterative process is applied to the parameter identification problem for a SEIR-like model of the COVID-19 pandemic.

[Genetic variations in Clupea pallasii herring from okhotsk sea based on microsatellite markers].

The genetic variations among spawning groups of herrings from different spawning grounds of the northwestern part of the Okhotsk Sea was assessed using ten microsatellite loci. All loci were polymorphic with the ex- pected heterozygosity estimates varying at different loci in the range of 0.7-95.0% (with a mean of 68.5%). The degree of genetic differentiation displayed by the herrings from the Okhotsk Sea was not statistically sig nificant (0 = 0.74%). The level of pairwise genetic differentiation F(ST) varied in the range of 0.002-0.014, no was it statistically significant in all comparison pairs between the herring samples.

[Monoclonal antibodies in high-grade gliomas].

The review is devoted to a relatively young direction in therapy of malignant gliomas, which is based on applying monoclonal antibodies against tumour-associated antigens. The current data on efficacy of main therapeutic agents in clinical practice or clinical trials concerning high-grade gliomas, especially glioblastoma multiforme, is summarized. Of particular interest is bevacizumab, a humanized monoclonal antibody against vascular endothelial growth factor (anti-VEGF), which is widely used in glioblastoma. Major clinical trials devoted to bevacizumab monotherapy and combinations of bevacizumab with other therapeutic modalities in primary and recurrent glioblastoma conducted since 2006 till now are reviewed. The results ofexperimental and clinical application ofmonoclonal antibodies against epidermal growth factor receptor (EGFR) and its mutant variant EGFRvIII are analyzed, showing the most significant clinical effectiveness of nimotuzumab--a humanized monoclonal antibody. Significant part of the review is devoted to discussion of experimental and clinical data concerning efficacy of antibodies against VEGF receptor 2, platelet-derived growth factor receptor α, hepatocyte growth factor and its receptor c-Met. Unbiassed analysis of clinical trials on monoclonal antibodies does not allow us to conclude that passive immunotherapy directed against antigens listed above can significantly improve the overall survival of patients suffering from glioblastoma multiforme. This finding has encouraged us to mention several alternative approaches to passive immunotherapy and to list several prospective antigens for developing monoclonal antibodies.

[Analysis of microsatellite loci variations in herring (Clupea pallasii marisalbi) from the White Sea].

The genetic diversity among spawning groups of herring from different parts of the White Sea was assessed using ten microsatellite loci. All loci were polymorphic with the expected heterozygosity estimates varying in the range of 12.7-94.1% (mean was 59.5%). The degree of genetic differentiation displayed by White Sea herring was statistically significant (theta = 2.03%). The level of pairwise genetic differentiation F(ST) was 0-0.085, and it was statistically significant in most of the comparison pairs between the herring samples. A hierarchical analysis of molecular variance (AMOVA) revealed the statistically significant differentiation of White Sea herring. 96.59% genetic variation was found within the samples and 3.41% variation was found among the populations. The main component of interpopulation diversity (1.85%) falls at the differences between two ecological forms of herring, spring- and summer-spawning. Within the spring-spawning form, the presence of local stocks in Kandalaksha Gulf, Onega Bay, and Dvina Bay was demonstrated.

[Comparative analysis of genetic variability of White Sea Cod (Gadus morhua marisalbi) at allozyme and microsatellite markers].

Variability of cod spawning and feeding schools from Kandalaksha Bay of the White Sea, was examined at six allozyme and eight microsatellite loci. The degree of genetic differentiation at allozyme loci constituted 0 = 0.36% [95% bootstrap interval 0.0458; 0.6743]. The differentiation estimates obtained using microsatellite markers were higher, 0 = 1.33% [0.057; 3.11]. It was demonstrated that the level of genetic diversity in the White Seacod was lower than that established for the Atlantic cod from Barents Sea using the same set of al- lozyme and microsatellite markers. The genetic data obtained support the opinion that the White Sea cod is a reproductively independent group formed as a result of the Holocene dispersal of Atlantic cod.

[On differentiation of cod (Gadus morhua L.) groups in Baltic Sea].

Using the AGP*, PGI-1*, PGI-2*, LDH*, IDH*, and PGM* allozyme markers, the differentiation of cod groups during the spawning period in Baltic Sea was evaluated. It was shown that cod samples from the eastern and western parts of Baltic Sea (being statistically significantly different from the cod samples from the Norwegian Sea, Barents Sea, and the Skagerrak Strait) demonstrated a high degree of identity (I = 0.99-1.0), which could be associated with the observed from the beginning of the present millennium increased migration of cod from eastern Baltic regions to the waters of Bornholm basin, and the distribution of the part of migrants from Bornholm basin to Arkona basin.

[The chemiluminescent detection of activity in peripheral blood neutrophils under allergic diseases of lungs in children with fungous sensibilization].

The high mobilization activity of neutrophils as a capacity of momentary feed-back reaction to factually any external stimuli, make it possible to use them in various diagnostic systems in vitro. Among them is the automated test system of chemiluminescent detection of functional activity of peripheral blood neutrophils in parallel response to luminol, opsonizing zymosan and different antigens. This approach makes it possible to evaluate the initial and induced activity of phagocytes and its dynamics in case of adding in vitro to test system of one or another allergen. The application of micro technique of chemiluminescent detection of functional activity of peripheral blood neutrophils revealed the stimulating effect of allergens Aaspergillus fumigatus, Alternaria alternata, Cladosporum herbatum, Candida albicans and Penicillinum notatum. This is an additional evidence of high sensitivity of children with allergic diseases of lungs to fungi and hence one more factor of diagnostics and treatment.

Cloning and expression of brain-specific anion transporter BSAT1 and isolation of monoclonal antibodies to its extracellular fragment.

Brain-specific anion transporter BSAT1 extracellular fragment (451-557) cDNA was cloned in a vector for prokaryotic expression, a producer E. coli strain was obtained, and recombinant extracellular fragment BSAT1(451-557)was purified and used for immunization of BALB/c mice. Splenic cells from mice with verified immune response were used for hybridoma generation. Several hybridoma clones producing monoclonal antibodies to BSAT1 extracellular fragment were selected. Antibody specificity was confirmed by ELISA, immunoblotting with recombinant BSAT1(451-557), and immunofluorescent BSAT1 assay on rat brain sections and cultured HEK293 cells. It was demonstrated that the obtained antibodies specifically bind native rat and human BSAT1 and can be used in both fundamental studies of structures forming the blood-brain barrier and development of targeted transport of diagnostic preparations and drugs across the blood-brain barrier.

[Preliminary data on variation of four microsatellite loci in Pacific herring Clupea pallasii].

Variation of microsatellite loci Cpa 10, Cpa 113, Cpa4, and Cpa7 was for the first time examined in Pacific-type herring Clupea pallasii from the White Sea (CI. pallasii marisalbi), the Kara Sea (CI. pallasii suworowi), the Sea of Okhotsk, and Lake Nerpich'e, Kamchatka Bay, northwestern Pacific (CI. pallasiipallasii). All loci exhibitedhigh genetic diversity. The estimates of expected heterozygosity varied from 41.5 to 95.6% (mean, 82%). The level of pairwise genetic differentiation Fst at all microsatellite loci varied from 0.005 to 0.076 (0.019, on average) and t was statistically significant (P < 0.05) in most of the pairs of herring samples. Estimates of genetic differentiation among the herring of one subspecies were lower than between the groups belonging to different subspecies.

Cloning and expression of human neuron-specific enolase cDNA in Escherichia coli.

cDNA fragment encoding neuron-specific enolase was amplified from the cDNA library of human brain. Then the fragment was cloned for expression in E. coli using the vector pET28-a. High level of neuron-specific enolase expression was confirmed by SDS-PAAG electrophoresis and immunochemical identity by immunoblot analysis. The constructed producer strain is the cheapest source of neuron-specific enolase suitable for the use in diagnostic applications.

[Analysis of microsatellite variation in the rainbow trout (Parasalmo (Oncorhynchus) mykiss) from Kamchatka].

Genetic variation of Kamchatka rainbow trout Parasalmo (O.) mykiss was examined using 10 microsatellite DNA loci, and phylogeographic comparison with other representatives of the species across the distribution range was performed. It was demonstrated that Kamchatka populations differed from other geographic groups of rainbow trout in a number of microsatellite loci. These populations also displayed distinct clustering and were characterized by lower genetic diversity. Analysis of a set of 26 different microsatellite loci (personal and literature data) demonstrated that most of the populations within the Kamchatka region were separated from one another, characterized by marked geographic differentiation, and affiliation to certain river basins. In Kamchatka rainbow trout, with high degree of probability, three geographic clusters (northwestern, southwestern, and eastern) were identified. In general, analysis of microsatellite DNA supported the data on low genetic diversity of the Kamchatka group Parasalmo (O.) mykiss, based on the variation estimates for a number of genes of nuclear and mitochondrial DNA, and allozyme loci.

[Analysis of microsatellite DNA of rainbow trout (Parasalmo (Oncorhynchus) mykiss) of Kamchatka: selection of loci and optimization of the method].

The variation of a sample of rainbow trout (Parasalmo (Oncorhynchus) mykiss) from natural populations of several rivers of the Kamchatka Peninsula with respect to 43 microsatellite DNA loci has been studied. These loci were earlier used for analysis of Asian populations of closely related salmonids. Ten of them may be regarded as markers and seen promising for further studies on intraspecific relationships of rainbow trout of Kamchatka. Their use in studies on more numerous samples from different localities and populations of Parasalmo (O.) mykiss in the Asian part of the species range will ensure efficient population genetic analysis of the Kamchatka population group of this species.

[Mono- and associated microflora during wound infection in patients with burns].

AIM: To study etiologic role of microorganisms during wound infection and elucidation of their populations interaction in patientswith burns. MATERIALS AND METHODS: 2137 patients with wound infection treated in burn center were studied. 3179 samples of wound exudates and 6501 strains of microorganisms were studied. Assessment of microorganism's etiologic role was carried out using results of calculation of various statistic indexes, including indexes of ecological similarities of microorganisms in the wound (indexes of contamination, prevalence or persistence, incidence). Ecological similarity of wounds' microflora was determined by calculation of the coefficient Kj. RESULTS: It was established that Staphylococcus aureus is the most common cause of mono- and mixed wound infections. It was determined that Kj value for common agents points to antagonism or associative interaction. During particular time periods of the study values of Kj significantly differed and depended from species. Specifically, for Pseudomonas aeruginosa the values of Kj were equal or exceeded 30% during several years. It was the first time when correlation between persistence and ecological similarity indexes was studied in agents of wound infections. CONCLUSION: In order to reveal factors influencing on microbial etiology of mixed wound infections it is reasonable to consider the nature of interspecies interactions of causative agents.

[Elimination of neurospecific proteins from CNS: pathogenetic and methodical aspects].

Appearance of neurospecific proteins (NSP) outside the brain plays a certain pathogenetic role in the development of autosensitization occurring in many kinds of CNS injuries and diseases. Analysis of modern views of cerebrospinal fluid (CSF) exchange allows us to suppose that NSP are eliminated from the brain tissue within CSF, moving from the subarachnoid space into cranial veins, and by lymphatic way, into deep cervical lymph nodes. Elevation of NSP level in CSF indicates an actual neudegenerative process. Serum levels of NSP are determined by the balance between the elimination of NSP from the brain, on the one hand, and their metabolism and the response of the immune system to the appearance of these autoantigenes in the blood stream, on the other. Basing on their own data on the dynamics of NSP (NSE, GFAR, and MBP) concentrations and the proportions of these proteins in CSF and serum (coefficient of elimination) in rats after ischemic, hypoxic, and autoimmune cerebral lesions, the authors offer an algorithm of pathogenetic evaluation, including, on the one part, a conclusion on the presence or absence of a neurodegenerative process, and, on the other, a conclusion on a normal or lowered rate of NSP elimination (metabolism). The results of such an analysis may have a clinical significance in terms of the development of a pathogenetic therapy, including, in every individual case, not only neuroprotectors, but also pharmaceuticals directed towards correction of the functional condition of the immune system.

[Production and characteristics of PEGylated immunoliposome transport vectors specific for neural tissue astrocytes].

PEGylated (stealth) immunoliposomes covalently linked to antibodies against human gliofibrillary acidic protein (GFAP) were prepared by coupling the thiolated monoclonal anti-GFAP antibodies, D4, with a maleimide derivative of the phosphatidyl ethanolamine of the liposomal membrane. Depending on the initial protein-to-lipid ratios, the immunoliposomes prepared (with a diameter of about 70 nm) were coupled with 60 to 240 molecules of the antibodies. In vitro experiments with cultures of the embryonic rat brain astrocytes demonstrated a specific binding of these immunoliposomes, which could be inhibited by the preincubation of the cells with free non-coupled D4 but not with non-specific antibodies. Administered intravenously into rats, the immunoliposomes exhibited a kinetic behaviour typical for the PEGylated liposomes: after 24 h, approximately 20% of the doses injected were still present in the blood; the elimination rate constants were 0.05-0.09 h(-1), the elimination half-lives were 8-15 h. With such a systemic longevity, as well as with such a specificity, these immunoliposomes, non-penetrating through intact blood-brain barrier (BBB), should be useful in delivering pharmacological agents to glial brain tumours (which continue to express GFAP) or to other pathological loci in the brain with a partially disintegrated BBB.

[The role of nitric oxide in fertility disorders in men].

The aim of the study was investigation of NO influence on fertility properties of ejaculate in the diseases of male sex organs. A total of 73 males aged 20-55 years with different sexual disorders were examined. Potentiometric assay for nitrates in ejaculate, size and shape of spermatosoa were studied. NO3 was present in all sperma samples. In the control group nitrates levels were 0.662 +/- 0.147 mmol/l. In men with sexual diseases nitrates levels in ejaculate ranged from 0.114 mmol/l to 1.530 mmol/l). In normozoospermia nitrates occurred in "mean" levels (0.295 mmol/l to 0.900 mmol/ 1). In pathospermia nitrates level was high. NO produces a protective action if it was in adequate quantity. Spermatozoid concentration and a relative number of progressive-mobile forms correlated negatively with nitrates of spermatic plasma (r = -0.40) while dead spermatozoa percentage correlated positively. Thus, low NO3 content in ejaculate may result from microcirculatory disorders and endothelial dysfunction in testicular parenchyma and epididimus. NO hyperproduction occurs in inflammatory urogenital diseases. It probably induces pathospermia via direct toxic effects of NO and stimulation of lipid peroxidation.