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INTRODUCTION. Despite existing treatment methods, complete eradication of human immunodeficiency virus (HIV) infection remains an unattainable goal due to the high variability of HIV type 1 (HIV-1). HIV infection necessitates life-long administration of antiretroviral medicinal products, which cause serious adverse drug reactions. The development of gene therapy products based on adeno-associated virus (AAV) vectors encoding broadly neutralising antibodies represents a promising direction for creating long-term therapies capable of countering a wide range of viral variants.AIM. This study aimed to evaluate the protective efficacy of CombiMab-2, a medicinal product consisting of a combination of three AAV vectors (AAV9-VRC07-523, AAV9-10-1074, and AAV9-PGDM1400) encoding broadly neutralising antibodies against HIV-1, in a humanised mouse model.MATERIALS AND METHODS. The study used an HIV infection model based on immunodeficient B-NDG mice humanised with human CD4+ lymphocytes (1.5×107 cells per animal) from a leukoconcentrate of a healthy donor. The experiment used two groups of mice, including a control group (3 animals) receiving saline solution and an experimental group (5 animals) receiving CombiMab-2. The medicinal product was administered into different muscles as three separate components six weeks prior to infection. The CCR5-tropic HIV-1 strain was obtained by transfecting HEK293FT cells with the pNL4-3(AD8) plasmid encoding the full-length virus. The authors monitored viral loads in the plasma of animals by reverse transcription polymerase chain reaction and CD4+ lymphocyte counts in the peripheral blood of animals by flow cytometry for four weeks after infection.RESULTS. Six weeks after CombiMab-2 administration, the levels of broadly neutralising antibodies in the serum of humanised mice ranged from 0.17 μg/mL to 4.0 μg/mL. In the control group, the viral load reached 105 copies/mL one week after HIV-1 infection and continued to rise over the next three weeks. In the treatment group, infection developed only in one mouse, which had the lowest antibody titre before infection. No viral load was detected in the remaining mice of the treatment group, which indicated that the medicinal product was effective if serum concentrations of broadly neutralising antibodies reached 0.5 μg/mL or higher.CONCLUSIONS. The tested medicinal product based on three AAV vectors (AAV9-VRC07-523, AAV9-10-1074, and AAV9-PGDM1400) exhibits protective activity against HIV-1 in humanised mice. The presented data allow the authors to consider CombiMab-2 as a promising antiviral agent that can serve as a basis for further pharmaceutical development.

The aim of the study was to analyze genetic characteristics of strains belonging to group A rotaviruses (RVA) circulating in Moscow in 20152020, including rare strains non-typeable by polymerase chain reaction (PCR). Materials and methods. A total of 289 stool samples were tested; the samples were collected from children aged 1 month to 17 years, hospitalized with acute gastroenteritis. Immunochromatography and real-time reverse transcription-polymerase chain reaction (real-time RT-PCR) assays were used for detection of rotaviruses in the samples. The rotavirus genome sequencing was performed using the Sanger technique and nanopore sequencing. Results and discussion. RVA RNA was detected in 131 clinical samples, and the G/[P] genotype was identified in 125 samples. The general profile showed prevalence of RVA strains with the G9P[8]I1 genotype (37%) followed by G3P[8]I2, G4P[8]I1, G2P[4]I2, G1P[8]I1, and G3P[8]I1 variants (18, 15, 11, 5, and 2%, respectively). Seven (5%) isolates were identified as GxP[8]. In 20152020, the region reported a decline in G4P[8]I1 genotype prevalence (from 39% to 9%) and an increase in the proportion of the G9P[8]I1 genotype (from 6% to 37%) as compared to 20092014. In 20182020, a large number of cases with the previously unknown DS-1-like reassortant strain with the G3P[8]I2 genotype were reported; the above strain has become widely common worldwide in the recent years. Nanopore sequencing was performed to analyze the genome of the G3P[8]I2 strain and the rare G4P[6]I1 strain. It was found that the G4P[6]I1 strain was phylogenetically related to porcine rotaviruses. Conclusion. In the recent years, the genetic diversity of RVA circulating in the Moscow Region has changed significantly. The obtained results prove the importance of continuous monitoring of rotavirus infection and selective sequencing of RVA genes to fine-tune data of the type-specific real-time RT-PCR. The ever-changing genetic composition of the circulating RVA strains calls for regular optimization of RVA genotyping systems based on real-time RT-PCR.

Aim. Study of biological properties of attenuated variants of the virulent A/WSN/33 strain of influenza virus, obtained by the site-specific mutagenesis of PB2-gene. Materials and methods. Site-specific mutants of A/WSN/33 of influenza virus, having in PB2-gene ts-mutations from genome of cold-adapted (CA) master-strains: A/Ann Arbor/6/60 (H2N2); A/Leningrad/134/17/57 (H2N2); A/Krasnodar/101/35/59 (H2N2) were obtained with help of reverse genetics methods. The ts-phenotype, att-phenotype, immunogenicity and protective efficacy in homologous and heterologous control infections were studied in the obtained site-specific mutants. Results. It was shown that the inclusion in the PB2-gene of the virulent A/WSN/33 strain as single mutations and a combination of mutations from the genomes of CA donor-strains leads to a change in the ts-phenotype and att-phenotype of the mutants obtained. These mutants had high protective efficacy in homologous and heterologous control infection. Conclusion. The results obtained allow us to consider the site-specific mutants of influenza virus as possible candidates for live influenza vaccines.

Aim. Study the possibility of obtaining attenuated variants of influenza virus by including specially selected site-specific mutations into a conservative sequence of PA-gene (terminal segment of COOH-domain of the PA-gene) of a virulent strain. Materials and methods. А/ WSN/33 - a virulent strain of influenza virus was used in the study. I nclusion of site-specific mutations into PA-gene of the A/WSN/33 virulent strain was carried out using a two-step mutation PCR. Cloning was carried out using GoldenGate reaction. 8-plasmid transfection system based on pHW2000 vector was used. Transformation was carried out in rubidium competent bacterial cells of DH5a strain. Transfection was done using Lipofectamine LTX (Invitrogen) reagent in a 293T and MDCK cells’ co-culture. Results. Transfectants with F658A substitution in the COOH-domain of the PA-gene were shown to acquire ts-phenotype and sharply reduce the ability to reproduce in mice lungs. Introduction of F658A substitution into COOH-domain of the PA-gene in combination with introduction of ts-mutations from ca influenza virus strains into the genome of the virulent strain resulted in obtaining transfectants that have phenotypic characteristics typical for live influenza vaccine candidates. Conclusion. The ability to obtain attenuated variants of influenza viruses by introducing specially selected site-specific mutations into conservative sequence of the PA-gene is shown.

Aim. Establish genetic characteristics, carry out phylogenetic analysis and determination of molecular markers of resistance to etiotropic preparations against influenza A/H3N2 and В viruses that had circulated in Russia in 2013 - 2015. Materials and methods. 80 biological samples containing influenza A/H3N2 virus RNA and 31 samples containing influenza В virus RNA were studied. Sequencing of PCR fragments was carried out in ABI-3100 PRIZMTM GeneticAnalyzer (AppliedBiosystems, USA) and using MiSeq (Illumina, USA). Data treatment and analysis was carried out using CLC v.3.6.5., DNASTAR and BioNumerics v.6.5. programs. Results. In 2013 - 2014 A/Texas/50/2012-like clade 3C.3 influenza A/H3N2 viruses dominated, 10% belonged to subclade 3C.2a and 10% - to ЗС.ЗЬ. Most of the viruses (81%) of 2014 - 2015 were of 3C.2a clade, the portion of viruses belonging to ЗС.ЗЬ and ЗС.За was 9 and 10%. Yamagata-like viruses predominated among the studied influenza В viruses, only 1 virus of 2014 - 2015 belonged to Victoria lineage, 1 reassortant of Yamagata and Victoria lineages was detected. Rimantadine-resistance mutation S31N (М2 protein) was detected in all the influenza A/H3N2 viruses. Mutations determining resistance to oseltamivir (NA gene) were not detected in influenza A/H3N2 and В viruses. Conclusion. Increase of influenza morbidity in 2014 - 2015 was determined by the emergence of influenza A/H3N2 and В viruses, antigenically distinct from those that had circulated previously and those included into the vaccine, thus resulting in the WHO decision to change А/ H3N2 and В components of the 2015 - 2016 vaccine. Simultaneous circulation of 2 lineages of influenza В virus and emergence of their reassortants gives evidence on the necessity of use of quadrivalent vaccines, containing both lineages.