[Modification of the Spike Protein for Vaccines against Enveloped RNA Viruses].

Most vaccines work by inducing neutralizing antibodies that target the viral envelope. Enveloped RNA viruses have evolved mechanisms for surface glycoproteins to evade host immune responses, which exhibit substantial variability, even among different strains. Natural infection and vaccines using native forms of surface proteins may induce broadly neutralizing antibodies, yet with low and ineffective levels. Class I membrane-fusion proteins of enveloped RNA viruses, HIV-1, influenza A virus, SARS-CoV-2, yield a stable conformation (so-called "pre-fusion") in providing fusion between viral and host cell membranes. Modified viral surface proteins that are based on these features induce neutralizing antibodies with activity available against a broad spectrum of circulating strains and make it possible to overcome the difficulties associated with escape/variability of viral antigen.

Characteristics of the Airway Microbiome of Cystic Fibrosis Patients.

Microbiota as an integral component of human body is actively investigated, including by massively parallel sequencing. However, microbiomes of lungs and sinuses have become the object of scientific attention only in the last decade. For patients with cystic fibrosis, monitoring the state of respiratory tract microorganisms is essential for maintaining lung function. Here, we studied the role of sinuses and polyps in the formation of respiratory tract microbiome. We identified Proteobacteria in the sinuses and samples from the lower respiratory tract (even in childhood). In some cases, they were accompanied by potentially dangerous basidiomycetes. The presence of polyps did not affect formation of the sinus microbiome. Proteobacteria are decisive in reducing the biodiversity of lung and sinus microbiomes, which correlated with the worsening of the lung function indicators. Soft mutations in the CFTR gene contribute to the formation of safer microbiome even in heterozygotes with class I mutations.

Novel Polycationic Photosensitizers for Antibacterial Photodynamic Therapy.

Antibacterial photodynamic therapy (APDT) is a promising method of treating local infected foci, in particular, surgical and burn wounds, trophic and diabetic ulcers. Photodynamic inactivation (PDI) is able to effectively destroy bacterial cells without them developing resistance in response to treatment.This work was dedicated to the study of photophysical and antibacterial properties of new photosensitizers (PS) based on polycationic phthalocyanines and synthetic bacteriochlorins for photodynamic inactivation of P. aeruginosa bacteria and their biofilms. Gram-negative bacteria P. aeruginosa are often found in infected wounds, presumably in biofilm state and are characterized by rather low susceptibility to APDT, which is a problem. PS were studied for possible aggregation at various concentrations by means of absorption and fluorescence spectroscopy. The results of studies of the ZnPcChol8, (3-PyHp)4BCBr4 and (3-PyEBr)4BCBr4 in water and serum confirm the assumption of a low degree of their aggregation at high concentrations.Consequently, their photodynamic efficiency is high enabling to use these PS at high concentrations to sensitize pathological foci for APDT.It was shown that all the investigated PS had a high efficiency of photodynamic inactivation of Gram-negative bacteria P. aeruginosa, as well as their biofilms. Tetracationic hydrophilic near-infrared photosensitizer (3-PyEBr)4BCBr4 with reduced molecule size had significantly higher efficacy of photodynamic inactivation of P. aeruginosa biofilms compared with other studied photosensitizers.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

Combined Effect of Bone Morphogenetic Protein-2 and Erythropoietin on Regeneration of Cranial Bone Defects in Mice.

Using mouse model of regeneration of critical size cranial defects, we studied combined effect of 1 and 10 µg of BMP-2 of prokaryotic origin and recombinant erythropoietin (Epostim) injected subcutaneously in the area of bone defect in a total dose of 6000 U/kg. Erythropoietin considerably improved quantitative and qualitative characteristics of the bone tissue in the site of implantation when used in combination with BMP-2 in both concentrations.

Resistance of THP-1 Leukemia Cells Infected with Cytomegalovirus to Anti-tumor Antibiotic Doxorubicin and Restoration of the Sensitivity by Inhibitors of the PI3K/AKT/mTOR Molecular Pathway.

Results obtained showed that infection with HCMV prevented the death of THP-1 cells treated with DOX in both active and latent forms of infection. In the presence of mTOR inhibitors (rapamycin and Torin2), the sensitivity of the infected cells to DOX was restored. Rapamycin inhibited the expression of the HCMV protein IE1-p72 and increased sensitivity to DOX. Molecular targets for the creation of new drugs for the treatment of leukemia in patients infected with HCMV were determined.

Recombinant Human Bone Morphogenetic Protein-2 (rhBMP-2) with Additional Protein Domain Synthesized in E. coli: In Vivo Osteoinductivity in Experimental Models on Small and Large Laboratory Animals.

Recombinant human bone morphogenetic protein-2 with an additional s-tag domain (s-tag-BMP-2) synthesized in E. coli is characterized by higher solubility and activity than the protein without additional s-tag domain, which increases the yield during purification and simplifies protein introduction into the osteoplastic materials. The high osteoinductivity of the demineralized bone matrix with s-tag-BMP-2 was shown on the model of regeneration of cranial defects of a critical size in mice and on the model of implantation of porous titanium matrix into defects of femoral and tibial bones in rabbits.

Modern Approaches to Studies of New Osteogenic Biomaterials on the Model of Regeneration of Critical-Size Cranial Defects in Rats.

Osteoinductive characteristics of new osteoplastic materials based on demineralized bone matrix of xenogenic origin with high and controlled degree of purification were studied on the model of regeneration of critical-size cranial defects in rats using modern approaches, including histological analysis, evaluation of morphological parameters of the bone tissue obtained by micro-computed tomography, and estimation of bone tissue growth rate using in vivo fluorochrome label. Demineralized bone matrix and, to a much greater extent, its activated form containing modified recombinant growth factor rhBMP-2 with high content of the dimeric form exhibited osteoinductive activity.

[Regularities of the ubiquitous polyhostal microorganisms selection by the example of three taxa].

The investigation of the bacterial populations' heterogeneity contributes to the control of natural foci, causative agents of nosocomial infections, to the analysis of the microbial evolution. Multilocus sequence typing (MLST) was employed for the analysis of the diversity and features of the distribution of polyhostal ubiquitous microorganisms of the genera Burkholderia, Leptospira, and Listeria, which belong to three bacterial phyla: Proteobacteria, Spirochaetes, and Firmicutes. According to the bacterial samples analysis microbial genotypes prevalent and unique to Russia were identified; their occurrence in different Federal Regions was investigated; their similarity with global spread genotypes was appreciated. Obtained results allowed identifying common regularities of the selection of the microorganisms capable to cause the diseases of human and animals. The formation of genotypes that are most pathogenic for the host was demonstrated for all groups of bacteria. Leptospira spp. and Listeria monocytogenes strains with these genotypes have been circulating for a long time, being supported by natural foci. The formation of a wide variety of genotypes with different pathogenicity was demonstrated in the local geographic areas. In Russia, the zonal difference in all three groups of bacteria is most clearly traced to the Far Eastern Federal Region. The results are thought to contribute to analyzing the factors of selection and the phylogeny of the taxa under study.

Nonthermal plasma affects viability and morphology of Mycoplasma hominis and Acholeplasma laidlawii.

AIM: To study the effects exerted by argon microwave nonthermal plasma (NTP) on cell wall-lacking Mollicutes bacteria. METHODS AND RESULTS: 10(8) CFU ml(-1) agar plated Mycoplasma hominis and Acholeplasma laidlawii were treated with the nonthermal microwave argon plasma for 30-300 s. The maximal 10- and 100-fold drop was observed for A. laidlawii and Myc. hominis, respectively. Similarly treated Escherichia coli and Staphylococcus aureus demonstrated the 10(5) and 10(3) drop, respectively. Removal of cholesterol affected resistance of A. laidlawii. 10 mmol l(-1) antioxidant butylated hydroxytoluene decreased mortality by a factor of 25-200. UV radiation alone caused 25-85% mortality in comparison with the whole NTP. Exogenously added hydrogen peroxide H2O2 did not cause mortality. NTP treatment of Myc. hominis triggered growth of microcolonies, which were several tenfold smaller than a typical colony. CONCLUSIONS: Despite the lack of cell wall, A. laidlawii and Myc. hominis were more resistant to argon microwave NTP than other tested bacteria. Mycoplasma hominis formed microcolonies upon NTP treatment. A role of UV and active species was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: The first study of NTP effects on Mollicutes revealed importance of a membrane composition for bacterial resistance to NTP. New specific Myc. hominis morphological forms were observed. The study confirmed importance of the concerted action of reactive oxygen species (ROS) with UV and other plasma bioactive agents for NTP bactericidal action.

[The role of technogenic and biological factors in spread of foodborne bacterial infections].

Epidemiologic aspects of ecology of foodborne infection causative agents that have taken root in contemporary technogenic loci created by humans in urbocenoses which include agrocomplexes of animal breeding, vegetable growing in open and closed ground are discussed. Soil and water sources with a wide transport network where microorganisms dwell in the environment under favorable for vital functions and growth conditions (humus deposits, temperature regimen, optimal pH, associations with hydrobionts, biofilm formation) are potent secondary reservoirs of infection causative agents. Some molecular-genetic mechanisms of polyhostality of Gram negative bacteria and listeria are examined.

Broadly Reactive Nanobody Targeting the H3 Hemagglutinin of the Influenza A Virus.

Monoclonal antibodies and recombinant antibody fragments are a very promising therapeutic tool to combat infectious diseases. Due to their unique paratope structure, nanobodies (VHHs) hold several advantages over conventional monoclonal antibodies, especially in relation to viral infections. Influenza A viruses (IAVs) remain a major threat to public health. The hemagglutinin (HA) protein is the main protective and immunodominant antigen of IAVs. In this study, three broadly reactive nanobodies (D9.2, E12.2, and D4.2) to H3N2 influenza strains were isolated and Fc-fusion proteins (VHH-Fcs) were obtained and characterized in vitro. This modification improved the nanobodies' binding activity and allowed for their interaction with a wider range of strains. The D9.2-Fc antibody showed a 100% protection rate against mortality in vivo in a mouse lethal model. Furthermore, we demonstrated that the observed protection has to do with Fc-FcγR interactions. These results indicate that D9.2-Fc can serve as an effective antiviral agent against the H3N2 influenza infection.

[Experimental whooping cough of nonhuman primate].

Despite considerable success in study of Bordetella pertussis virulence factors, pathogenesis of whooping cough, duration of B. pertussis bacteria persistence, types and mechanisms of immune response are still keep underinvestigated. It can be explained by the absence ofadequate experimental animal model for pertussis study. Our study estimates clinical and laboratory parameters of whooping cough in non-human primates of the Old World in the process of intranasan infection by virulent B. pertussis bacteria. Also the duration of B. pertussis bacteria persistence in animals was investigated. 14 animal units of 4 species of non-human primates of the Old World were used for intranasal infection. The examination of infect animals included: visual exploration of nasopharynx, thermometry, clinical and biochemical blood analyses, identification ofB. pertussis, using microbiologic and molecular genetic analyses, estimation of innate and adoptive immune factors. The development of infectious process was accompanied by generation of B. pertussis bacteria, catarrhal inflammation of nasopharyngeal mucosa, leucocytosis, hypoglycemia specific for pertussis, and activation of innate and adaptive immunity for all primates regardless of specie were seen. While repeated experimental infection in primates single bacterial colonies were registered during only first week after challenge. It occurs like the absence of inflammation of nasopharyngeal mucosa and the lack of laboratory marks of whooping cough, recorded after first challenge. The evident booster effect of humoral immunity was observed. As a model for investigation of B. pertussis bacteria persistence and immune response against whooping cough we suggest the usage of rhesus macaque as more available to experiments.

[Bactericidal action of non-thermal plasma on biofilms formed in vitro and within a root channel].

AIM: Evaluation of efficiency of non-thermal plasma as bactericidal agent affecting biofilms formed in vitro and on walls of a root channel. MATERIALS AND METHODS: The multiple antibiotic resistant strain Staphylococcus epidermidis isolated from pulpitis was used. Biofilms formed in vitro on the plastic surface and ex vivo at the walls of the root canal were treated with plasma torch formed by argon:air (9:1) mixture eradiated with 100 kHz electrtomagnetic field. Bacterial viability was determined by plating and by differential Live/Dead labeling. RESULTS: The dose-dependent decrease in living bacteria was demonstrated. The three-step kinetics ofbacterial killing was observed. Total elimination ofup 10(9) CFU/sample was reached at exposition of 240 s or more. CONCLUSION: The non-thermal plasma effectively destroyed bacterial biofilms within root channels.

[The express diagnostic of microorganisms affecting respiratory tract of patients with mucoviscidosis].

The shared bacteria Burkholderia capacia complex and Achromobacter sp. infect the respiratory tract of patients with mucoviscidosis brining on disorders of respiratory patency. Burkholderia capacia complex is characterized by transmissivity and higher lethality of patients infected by Burkholderia. Hence, the importance of differentiation of these phenotypically similar microorganisms is obvious. The developed express technique of diagnostic includes the separation of DNA from phlegm amplification and sequenation was fragments of genes recA, gltB, gyrB, 16S rDNA. The evaluation of products of amplification of genes recA, gltB makes it possible to differentiate Burkholderia capacia complex and Achromobacter sp. The analysis of successions of recA, gltB, gyrB makes it possible to identify genotype of Burkholderia capacia complex on the basis of data of allele profiles of strains of Burkholderia capacia complex circulating in Russia. The succession of gene 16S rDNA makes it possible to determine the taxonomic position of microorganism dominating in phlegm and not belonging to Burkholderia capacia complex or Achromobacter sp. The real time polymerase chain reaction in presence of intercalating dye Sybr Green I, DMSO and D(+)-trehalose makes it possible to differentiate Burkholderia capacia complex from other microorganisms infecting respiratory tract of patients with mucoviscidosis. This approach provides additional reduction of diagnostic duration and decrease possibility of contamination.

[Vaccines to prevent Ebola virus disease: current challenges and perspectives].

RELEVANCE: Ebola virus disease (EVD) is an acute infectious disease with an extremely high case fatality rate reaching up to 90%. EVD has become widely known since 2014-2016, when outbreak in West Africa occurred and led to epidemic, which caused travel-related cases on the territory of other continents. There are two vaccines against EVD, prequalified by WHO for emergency use, as well as a number of vaccines, approved by local regulators in certain countries. However, even with the availability of effective vaccines, the lack of data on immune correlates of protection and duration of protective immune response in humans and primates is limiting factor for effectively preventing the spread of EVD outbreaks. AIMS: This review highlights experience of use of EVD vaccines during outbreaks in endemic areas, summarizes data on vaccine immunogenicity in clinical trials, and discusses perspectives for further development and use of effective EVD vaccines.

Fluorothiazinon inhibits the virulence factors of uropathogenic Escherichia coli involved in the development of urinary tract infection.

Uropathogenic Escherichia coli (UPEC) is the most common pathogenic bacterium associated with urinary tract infection. Due to the development of antibiotic resistance and MDR, UPEC infection has become a serious problem in the last decade. In order to combat resistance, it is necessary to develop innovative antimicrobial agents that act by different mechanisms than conventional antibiotics. Among the new therapeutic strategies, suppression of pathogen virulence has become a promising alternative, since it fundamentally reduces selective pressure and the development of resistance. In our study, we showed that the compound Fluorothiazinon suppressed UPEC's ability to form biofilms and to move using the flagellum, as well as to penetrate into cells. Prophylactic use with subsequent treatment of FT in rodent models led to an improvement in survival and significantly reduced the bacterial load in the organs of the urinary system, thereby inhibiting the development of ascending infection and preventing the development of pathological changes in prostate tissues. These results suggest that FT affects several UPEC virulence factors at once and if similar results can be found in clinical trials it can potentially be used as a new drug against UPEC.

A novel antivirulent compound fluorothiazinone inhibits Klebsiella pneumoniae biofilm in vitro and suppresses model pneumonia.

The problematic treatment of infections caused by multiple-resistant Klebsiella, especially in ICU, is the leading cause of prolonged hospitalization and high mortality rates. The use of antibiotics for the prevention of infections is considered unreasonable as it may contribute to the selection of resistant bacteria. In this regard, the development of drugs that will be effective in preventing infection during various invasive procedures is extremely necessary. We have shown that the developed innovative antibacterial compound fluorothiazinone (FT) that suppresses the formation of biofilms is effective in the prevention of a model pneumonia caused by a multi-resistant clinical K. pneumoniae isolate. Prophylactic use followed by treatment with FT in mice with acute pneumonia modulates the local innate immune response without suppressing protective properties in the early stages of infection, while contributing to a decrease in the bacterial load in the organs and preventing lethal pathological changes in the lungs at later stages of K. pneumoniae infection. Further development of such antivirulence drugs and their use will reduce morbidity and mortality in nosocomial infections, as well as reduce the number of antibiotics used.

[Mathematical model for assessing the level of cross-immunity between strains of influenza virus subtype H3N2].

INTRODUCTION: The WHO regularly updates influenza vaccine recommendations to maximize their match with circulating strains. Nevertheless, the effectiveness of the influenza A vaccine, specifically its H3N2 component, has been low for several seasons. The aim of the study is to develop a mathematical model of cross-immunity based on the array of published WHO hemagglutination inhibition assay (HAI) data. MATERIALS AND METHODS: In this study, a mathematical model was proposed, based on finding, using regression analysis, the dependence of HAI titers on substitutions in antigenic sites of sequences. The computer program we developed can process data (GISAID, NCBI, etc.) and create real-time databases according to the set tasks. RESULTS: Based on our research, an additional antigenic site F was identified. The difference in 1.6 times the adjusted R2, on subsets of viruses grown in cell culture and grown in chicken embryos, demonstrates the validity of our decision to divide the original data array by passage histories. We have introduced the concept of a degree of homology between two arbitrary strains, which takes the value of a function depending on the Hamming distance, and it has been shown that the regression results significantly depend on the choice of function. The provided analysis showed that the most significant antigenic sites are A, B, and E. The obtained results on predicted HAI titers showed a good enough result, comparable to similar work by our colleagues. CONCLUSION: The proposed method could serve as a useful tool for future forecasts, with further study to confirm its sustainability.

[Evaluation of the dynamics of detection of viable SARS-CoV-2 (Coronaviridae: Betacoronavirus: Sarbecovirus) in biological samples obtained from patients with COVID-19 in a health care setting, as one of the indicators of the infectivity of the virus].

INTRODUCTION: The study of the mechanisms of transmission of the SARS-CoV-2 virus is the basis for building a strategy for anti-epidemic measures in the context of the COVID-19 pandemic. Understanding in what time frame a patient can spread SARS-CoV-2 is just as important as knowing the transmission mechanisms themselves. This information is necessary to develop effective measures to prevent infection by breaking the chains of transmission of the virus. The aim of the work is to identify the infectious SARS-CoV-2 virus in patient samples in the course of the disease and to determine the duration of virus shedding in patients with varying severity of COVID-19. MATERIALS AND METHODS: In patients included in the study, biomaterial (nasopharyngeal swabs) was subjected to analysis by quantitative RT-PCR and virological determination of infectivity of the virus. RESULTS: We have determined the timeframe of maintaining the infectivity of the virus in patients hospitalized with severe and moderate COVID-19. Based on the results of the study, we made an analysis of the relationship between the amount of detected SARS-CoV-2 RNA and the infectivity of the virus in vitro in patients with COVID-19. The median time of the infectious virus shedding was 8 days. In addition, a comparative analysis of different protocols for the detection of the viral RNA in relation to the identification of the infectious virus was carried out. CONCLUSION: The obtained data make it possible to assess the dynamics of SARS-CoV-2 detection and viral load in patients with COVID-19 and indicate the significance of these parameters for the subsequent spread of the virus and the organization of preventive measures.