Vol 13, No 2 (2023)

An important feature of influenza vaccines, which distinguishes them from other immunobiological preparations, is that they have no fixed composition. Due to the constant influenza virus antigenic variability, production facilities require timely supply with relevant vaccine strains undoable due to the lack of proper method for the convenient, rapid and uninterrupted development of vaccine strains. Among the licensed influenza vaccines, classical inactivated and live influenza vaccines hold a special place. They are based on reassortant vaccine strains obtained by crossing currently circulating influenza virus with the so-called donor strain (cold-adapted attenuation donor for live influenza vaccines or high yield donor for inactivated vaccines). Vaccine strains for licensed live attenuated influenza vaccines are reassortants with the so-called 6:2 genome formula — two genes encoding hemagglutinin and neuraminidase (HA and NA) belong to the current epidemic virus, and six genes encoding internal proteins (PB2, PB1, PA, NP, M and NS) — to cold-adapted master donor virus. There is a very limited number of donors of attenuation. In Russia, there are cold-adapted viruses A/Leningrad/134/17/57 (H2N2) and B/USSR/60/69; in the USA (MedImmune) there are viruses A/Ann Arbor/6/60ca (H2N2) and B/Ann Arbor/1/66ca. MedImmune produces vaccine strains using reverse genetics technique. For other countries, this approach for obtaining vaccines is limited due to the need to purchase a license from the patent holders. In Russia, genetic manipulations with strains for the seasonal live influenza vaccine are not yet allowed; reassortants for the Russian live influenza vaccine are created only by classical reassortment in embryonated chicken eggs. Vaccine candidates for the inactivated influenza vaccine are prepared by the classical reassortment method, the requirements for them are more flexible and allow to use diverse genes combinations from “wild type” virus and master donor virus. High-yielding viruses such as A/PR/8/34 (H1N1), A/Texas/1/77 (H3N2), B/Lee/40 and some others are used as donors of internal genes. Unfortunately, the classical reassortment method does not always allow to promptly obtain a reassortant virus with a 6:2 genome formula. This is hindered by a number of reasons, ranging from the unique properties of a certain epidemic virus ending up with the constellation of genes. The reverse genetics method based on plasmids is an alternative approach to create reassortant vaccine strains allowing to reliably and quickly obtain reassortant viruses of a set 6:2 genome formula. However, this method also has certain weaknesses. This review discusses the advantages and disadvantages of development of conventional influenza vaccine candidates by reverse genetics and classical reassortment in developing chick embryos.

The proteins of many viruses can be assembled into strictly organized structures — virus-like particles bearing antigens of the original viruses and may also be artificially decorated with antigens of other pathogens. These particles contain no viral genome and lack infectivity but can be highly immunogenic and therefore being actively used for vaccine development. Undoubtedly, while designing vaccines, it is necessary to take into account information about the interaction of vaccine components with immune system particularly antigen-presenting cells. This is especially important for virus-like particles because, like other nanometer-sized particles, they can enter antigen-presenting cells using various endocytosis pathways. The latter exploit multiple receptors, generate endocytic vesicles of different sizes, and, most importantly, are associated with varying fates of captured material. Here we review the mechanisms of phagocytosis, macropinocytosis, clathrin-mediated endocytosis, rapid endophilin-mediated endocytosis, and several endocytic pathways associated with lipid rafts. The data are presented on the relationship between various endocytic pathways and sorting of absorbed cargo in early endosomes as well as enzymatic degradation of the late endosomes contents. We also describe the mechanisms of distribution of absorbed antigens within antigen-presenting cells to be loaded onto the class I and II major histocompatibility complex molecules. The data are presented on the endocytosis of various viruses during cell infection, as well as a comparative analysis of the endocytosis pathways for virus-like particles and related viruses. It has been noted that virus-like particles, along with the absorption pathway specific for parent virus, can rely on additional endocytic pathways to be also artificially “targeted” at the selected endocytic receptor and relevant absorption pathway. It allows to select or design particles with optimal endocytosis and antigen presentation to induce a protective immune response upon vaccination. It should be assumed that most prophylactic vaccines require particles that are well engulfed by antigen presenting cells and direct material to endolysosomal degradation, or particles whose uptake directs material to both late and static early endosomes, making antigens available for «direct» and cross presentations. Finally, we discuss virus-like particles for the delivery of drugs or genetically engineered constructs, as well as optimal endocytic pathways that should protect the payload of these particles from endolysosomal degradation.

The cell wall and membranes of Gram-positive and Gram-negative bacteria provide a physical, osmotic, and metabolic barrier between the internal contents of the bacterial cell and the external environment. Observation of changes in the integrity of the bacterial structure using a scanning electron microscope (SEM) can help elucidate the detailed mechanisms of cell death. The aim of the study was to analyze the morphological changes in microbial cells exposed to new compounds with antimicrobial activity — chlorine-containing derivatives of 5-,6-,7-aminoindoles using SEM. Methods. The present study was carried out using strains of Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli obtained from patients with nonspecific diseases of the respiratory, urinary tract, and intestines with different sensitivities to traditionally used antimicrobial drugs. Results. As a result, the studied chloromethyl-containing compounds of the indole series showed own biological activity, namely antimicrobial. Control cells were morphologically correct and typical. Statistical analysis of cell surface morphometry in control and experimental samples did not reveal significant changes in size after exposure to compounds with laboratory codes T1, T4, T7 and T12. At the same time, compared with control untreated cells of P. aeruginosa, S. aureus and E. coli, treatment with chlorine-substituted derivatives of 5-,6-,7-aminoindoles caused obvious morphological changes, which indicates a deteriorated state of the cell wall. Filamentous cells were observed in P. aeruginosa exposure to T7 and T12. The appearance of long filaments may be associated with the stress experienced by the cell after exposure to the compounds under study. It is believed that the formation of such filaments in bacteria under stress conditions results from defects in cell division, especially in the separation of daughter cells. There are data according to which, when DNA synthesis is suppressed, a bacterium changes its morphology, becomes longer, without reaching cell division. Treatment with T1, T7 and T12 resulted in degradation of the P. aeruginosa cell wall, while treatment with T4 caused the formation of pores on the cell surface. In this study, microscopy showed marked morphological changes in the cell walls of S. aureus, which led to deformation of the cell wall under the influence of T1, T4, T7 and T12. Treatment of E. coli T1, T4, T7 and T12 cells at a concentration of 500 μg/ml caused cell lysis, although normal cells were also found. The appearance of cellular debris around whole E. coli cells indicates membrane damage, which probably leads to a change in osmotic pressure. Conclusion. The results using SEM confirmed the data on the antimicrobial activity of chlorine-substituted derivatives of 5-,6-,7-aminoindoles.

Numerous foreign studies evidence about a pronounced heterogeneity of the Epstein-Barr virus (EBV) populationcirculating throughout the world. Various EBV classifications have been proposed. The attention of Russian researchers has focused on the study of the structural and functional polymorphism of the EBV LMP-1 oncogene in the context of oncological diseases in adulthood. The aim of the work was to assess EBV molecular genetic diversity in children with EBV infection in the Nizhny Novgorod region. There were analyzed blood leukocyte and saliva specimens from children aged 1–17 years with EBV-infectious mononucleosis (n = 69) and sex- and age-matched healthy virus carriers of (n = 32). A total of 178 EBV isolates were studied. For differential detection of EBV-1/EBV-2, we used an optimized one-round PCR variant with electrophoretic detection of amplification products in agarose gel. Nucleotide sequences of the LMP-1gene C-terminal fragment were determined by Sanger sequencing. Bioinformatics data analysis was performed using MEGA X software. As a result, during EBV-infectious mononucleosis, only the EBV-1 type was detected in all children, among healthy virus carriers EBV-1 (93.8±4.3%) and EBV-2 (6.2±4.3%). Based on the EBV classification according to R.H. Edwards et al. the strain affiliation of EBV isolates was determined. A total of five variants of LMP-1 were identified, namely B95-8, China 1, Med–, NC and Alaskan, among which B95-8 dominated. The LMP-1 Med+, China 2, and China 3 variants were not found in any of the studied samples. It has been shown that the region of tandem repeats makes a significant contribution to the genetic diversity of the EBV population. A total of 100 amino acid substitutions were identified, of which the most common in the Nizhny Novgorod region EBV isolates are G212S, S366T, E328Q and S309N. A comparative analysis showed that strains, deletions, repeats, amino acid substitutions in EBV isolates from biological samples in children with infectious mononucleosis had common characteristics with a group of healthy virus carriers. In the active form of EBV infection, the appearance of structurally heterogeneous EBV sequences isolated from blood leukocytes and saliva from a single source was noted. Thus, for the first time, the molecular genetic diversity of EBV in children with various forms of EBV infection was assessed, which is the basis for the prospective development of clinical and epidemiological studies of EBV infection at a new methodological level.

The HIV infection epidemic in Russia continues to evolve, and HIV infection cases have been registered in all territorial entities of the Russian Federation. 2021 Treatment coverage was 82.2% and 56.4% individuals under dispensary observation and living with diagnosed HIV infection. 79.9% receiving ART subjects were shown to achieve undetectable viral load. Highly active antiretroviral therapy (HAART) currently represents a combination of three (less frequently four) antiretroviral drugs targeting pathways involved in various stages of HIV replication in vivo. Treatment failure is a problem facing doctors and patients using HAART. The most common cause of therapeutic failure is the development of HIV drug resistance. The emergence of resistance is associated with processes involving mutation occurring in the viral genome influenced by evolutionary factors. Therefore, it is important clinically and programmatically to learn more about the rate of first-line treatment failure, the rate of switching to a second-line ART regimen, and to identify patients at risk to develop strategies for preventing development of further failure cases. The study was aimed at analyzing ineffectiveness of first-line ART therapy in patients in Northwestern Federal District of the Russian Federation. Materials and methods. Sequencing reactions were performed using the AmpliSens HIV Resist-Seq. Assembly of consensus sequences from fragments obtained during sequencing was carried out using Unipro UGENE software. Isolate genotyping was performed using the MEGA-X software with the Neighbor-joining algorithm. Results. The HIV pol genes in 239 patients with first-line ART failure and 100 naïve patients were sequenced; all sequences genotyped as HIV-1 subsubtype A6. According to analysis, 82% of patients had at least one significant mutation associated with drug resistance for the corresponding viral subtype. In total, we encountered 87 different drug resistance mutations. Conclusion. We have shown increased proportion of patients with first-line ART failure among all patients with treatment failure. The main cause for such changes is probably related to the prevalence of primary drug resistance, estimated here at 8%. Specific differences were found between drug resistance mutation profiles in patients without suppressed viral load and patients with virological breakthrough. The overall results of the study indicate a need to diagnose and characterize HIV drug resistance prior to initiation of therapy in order to avoid ineffective first-line antiretroviral treatment.

The study of adaptive immunity in COVID-19 convalescent patients is important, because no consensus on whether the disease severity affects formation and durability of COVID-19 immune response has been achieved. A comparative assessment of emergence and durability of sustained cellular and humoral immunity in convalescent patients with COVID-19 of varying severity was carried out. The study involved volunteers with asymptomatic (n = 30), moderate (n = 21) and severe (n = 12) COVID-19. The average age of the subjects was 47.3±12.5 years. The formation of cellular immunity was assessed by increased IFNγ production in response to 16–20 hour-long SARS-CoV-2-derived glycoprotein S (RBD) lymphocyte stimulation. To measure IFNγ level, the Gamma Interferon–IFA-BEST test system manufactured by Vector-Best JSC, Russia, was used. The humoral immune response was recorded by detecting SARS-CoV-2RBD-specific class G antibodies using the “SARS-CoV-2RBD-ELISA-Gamalei” test system (FSBI “NITSEM N.F. Gamalei” of the Ministry of Health of Russia). It was revealed that humoral and cellular immunity against SARS-CoV-2 proteins was formed in all COVID-19 convalescent patients. However, the number of subjects with adaptive immunity to COVID-19 and the duration of its preservation depends on the severity of the infection. A significant decrease in the number of subjects with cellular immunity was revealed in the group of severe COVID-19. Most of the volunteers in this group had class G immunoglobulins before the end of the follow-up. In this group, unlike the other two, no patients were identified in whom only the cellular arm of the immune response was activated. Volunteers who did not retain adaptive immunity to the COVID-19 pathogen appeared only by the end of the follow-up period. Among those recovered after moderate disease 7–8 months later there was a decrease in the number of people with cellular and humoral immunity. This process started earlier than in the group of patients who were asymptomatic and continued until the end of the study. The proportion of individuals with cellular immunity increased, and at later timepoint — with humoral immune response. By the end of the study, a high percentage of volunteers remained asymptomatically infected, having cellular and humoral immunity to SARS-CoV-2. Their number remained significantly higher than in the group of moderate COVID-19, but lower than in severe COVID-19. By the end of the study, an increased number of volunteers with solely cellular immune response was recorded in this group. At the end of the follow-up period, the number of volunteers with humoral immunity against SARS-CoV-2 remained higher compared to those with a cellular immune response.

Introduction. Studies aimed at identifying pathogenetic features of tick-borne natural focal infections depending on etiological agent are relevant to seek out for new associations of biomarkers characterizing the structural and functional phenotype of immune cells significant for the differential diagnosis and prognosis of diseases. The aim of the study was to evaluate the parameters of a leukogram with an expanded profile in relation to the parameters of cytokine status in patients with viral tick-borne encephalitis as well as erythemic tick-borne borreliosis at acute stage of the disease. Materials and methods. The study involved 28 patients with the non-erythemic tick-borne borreliosis and 27 patients with tick-borne encephalitis at the acute stage of the disease, as well as 16 healthy individuals (control group). Venous blood samples were examined using a Sysmex XN1000 analyzer based on reflex testing technology with extended profile allowing to characterize leukocyte reaction during infection and inflammation, including parameters such as the absolute and relative count of immature granulocytes (IG), neutrophil granularity index (NEUT-GI), neutrophil reactivity index (NEUT-RI), count of reactive lymphocytes (RE-LYMP), count of antibody-producing lymphocytes (AS-LYMP). Concentrationof IL-2, IL-4, IL-6, IL-8, IL-10, IFNγ and TNFα in blood serum was determined by enzyme immunoassay using specific reagent kits (Vector-Best, Russia). Results. It was found that changes in the quantitative composition of blood leukocytes in patients with viral tick-borne encephalitis and non-erythemic form of tick-borne borreliosis have a unidirectional tendency characterized by increased count of metabolically active neutrophils (NEUT-RI) and a decreased count of reactive lymphocytes (RE-LYMP) compared to healthy individuals. Changes in the leukogram occur along with high blood concentration of proinflammatory cytokines IL-8 and TNFα and a low level of IFNγ. A positive direct correlation was revealed between the fluorescence intensity parameter characterizing the metabolic activity of neutrophil granulocytes (NEUT-RI) and blood serum IL-8 level both in tick-borne encephalitis (r = 0.422, p < 0.05) and non-erythemic form of tick-borne borreliosis (r = 0.551, p < 0.05). Additionally, in the former, a positive correlation was established between the total leukocyte count (WBC) and concentration of TNFα in the blood serum (r = 0.532, p < 0.05).

Pelviс varicose veins (PVV) in women is of extremely high relevance due to a close relationship with reproductive disorders and disease relapse. Despite on research in the field, the results of the analysis on immune reactivity in PVV are rather contradictory, and mainly relate to general mechanisms. The study of cell-mediated and humoral arms of adaptive immunity will allow us to assess an intensity and dynamics of PVV progression as well as a potential for using immune status parameters to optimize diagnosis and immunological correction. The aim of this study was to assess characteristics of cellular and humoral immunity in peripheral (ulnar) and local (ovarian) venous pools in women with mild and moderate PVV. The study involved 142 women of reproductive age (mean age — 37.2±7.1 years) with diagnosed PVV — mild (Group 1) (n = 79) and moderate (Group 2) (n = 63) forms. Data from 30 apparently healthy women (mean age 33.5±6.3 years) were in control group. Flow cytometry was used to identify lymphocyte subsets. The functional state of the humoral immunity was assessed by measuring concentration of immunoglobulins IgA, IgG, IgM by using radial immunodiffusion in the gel. According to our data obtained, patients in group 1 had changes found only in the local blood flow such as prominent lymphocytopenia (decreased level of CD3⁺ and CD4⁺ lymphocytes). Patients in group 2 were featured with more pronounced changes: increased lymphocyte level, CD3⁺, CD4⁺/CD8⁺, IgA and lower levels of IgM, IgG in peripheral blood; high average leukocyte and lymphocyte count, IgA and reduced levels of CD3⁺, CD4⁺, CD4⁺/CD8⁺, IgM and IgG in the local area. It can be concluded that altered parameters in the local blood flow are of primary origin in mild PVV being characterized by disturbed cellular immune arm. Along with increasing disease severity, the host compensatory capabilities decline, which is manifested by pronounced combined immunodeficiency at the level of local blood flow less evident at peripheral level. These results may contribute to a more accurate assessment of intensity and dynamics of PVV progression to optimize diagnostics and immunological correction.

The aim of the study was to identify differences in the immune response in chronic opisthorchiasis with or without clinical manifestations of hepatobiliary system diseases. A comparison of laboratory parameters of immune system functioning (lymphocyte phenotypes detected by flow cytometry, concentrations of immunoglobulins and cytokines, indicators of nonspecific resistance) in patients with chronic opisthorchiasis (caused by Opisthorchis felineus) lacking clinical manifestations and with manifestations of cholecystitis, cholangitis, pancreatitis, gastritis, gastroduodenitis, confirmed by ultrasound and FGDS. It was found that the immunological parameters of the innate immune response were increased in all patients with chronic opisthorchiasis compared with the control group of apparently healthy subjects, which indicates activated macrophage-phagocytic arm in patients with opisthorchiasis invasion. The indicator of the stimulated NBT test is maximal in chronic opisthorchiasis without hepatobiliary system diseases, in case of comorbidity with such diseases it declines, but remains significantly higher than the values of the control group. This may be due to the exhaustion in the reserve capabilities of bactericidal neutrophil systems in patients with symptoms of damage to the hepatobiliary system due to prolonged inflammation. Indicators of the cellular arm in patients with chronic opisthorchiasis without clinical manifestations of hepatobiliary system diseases demonstrate a decrease in the absolute and relative level of cytotoxic T-lymphocytes. In the group with picture of hepatobiliary system disorders, an even deeper imbalance of indicators is revealed: the relative and absolute level of lymphocytes, the absolute count of T-lymphocytes are reduced, which may indicate about involvement of cellular immunity in the pathogenesis of complications of chronic opisthorchiasis. In all examined patients with chronic opisthorchiasis, the humoral immune response was also activated — the total immunoglobulin E was significantly increased; at the same time, in patients with hepatobiliary system lesions, this parameter increases even more and a decrease in B-lymphocytes is detected. In all patients with chronic opisthorchiasis, there is also an increased level of proinflammatory cytokine IL-8, which rises even more in the group with hepatobiliary system diseases. The data obtained indicate the involvement of all arms of the immune response in a prominent inflammatory process when chronic opisthorchiasis is complicated by clinically overt hepatobiliary system diseases: the degree of deviation in the indicators of the innate and adaptive immune response from the norm and from those in uncomplicated opisthorchiasis increases.

The problems of bacterial infections in medicine and veterinary medicine require careful study and rapid solution. Due to continuous and, in some cases, irrational use of antibiotics, the efficiency of their effect on host has been noticeably decreasing; moreover, resistance to antibacterial drugs is steadily growing, antibiotic-resistant strains emerge, which are not amenable to conventional medical treatment. The unprecedented rise of pathogenic bacteria resistance to antibiotics requires generation of new drugs to combat them. One of the ways to increase the effectiveness of antibiotic therapy is to use combination drugs. Combination dosage forms provide an increased therapeutic effect and should not be toxic to the body. To overcome the microbial resistance reducing host burden of antibiotics, we proposed a combination preparation based on antibiotic, cluster silver and specific bacteriophage for treatment of infectious diseases caused by S. aureus, including MRSA strains. Each component has already proven in the treatment of pathogen-caused infectious diseases. But while using this combination agent, it became possible to reduce the amount of antibiotic and get rid of antibiotic-resistant and phage-resistant forms of bacteria. The study showed the effectiveness of the combination preparation on S. aureus MRSA bacteria, while reducing the amount of antibiotics in proposed composition by 2–4 times compared to use of antibiotic alone. The efficacy of the preparation containing was as follows: 10 mg/ml gentamicin, 7 mg/ml cluster silver and 10⁶ BOV/ml bacteriophage, on S. aureus MRSA bacteria in suspension is comparable to the effectiveness of gentamicin with a concentration of 40 mg/ml. For S. aureus 209 and S. aureus ssp. bacteria with reduced resistance to antibiotics, the use of two-component compositions (antibiotic + cluster silver; antibiotic + bacteriophage) also allowed to reduce the antibiotic concentration by 2–3 times. It should be noted that with a constant amount of antibiotic, increasing the amount of cluster silver by 4–5 times, there is a rise in bactericidal properties of the resulting preparations. Repeated introduction of drug dose allows to achieve a marked decrease in level of pathogenic microorganisms in the suspension studied. This drug has no limitations depending on the state and degree of microbial resistance.

Patient A.I., 58 years old, on September 6, 2022 was hospitalized within several hours in the infectious diseases department of the Novocheboksarskaya City Hospital of the Ministry of Health of Chuvashia. The patient in serious condition was admitted being accompanied by ambulance team, according to his son, from the night of 09/05/2022 until the afternoon of 09/06/2022 the patient complained of hectic temperature, diffuse pain in the abdominal cavity. In addition, the patient noted cough, feeling of air lack. As a result, an ambulance was called in. At admission, computed tomography revealed signs of bilateral viral interstitial pneumonia, with a lesion area of 51% and areas of consolidation. The diagnosis of COVID-19 was confirmed by PCR in a nasopharyngeal swab. History: Dyscirculatory encephalopathy of degrees 1–2, arterial hypertension, chronic indurative pancreatitis. Objective examination: on admission the body temperature was 38.8°C. SaO₂ — 56%. Blood pressure — 93/50 mm Hg. Art. Deep palpation revealed diffuse soreness and tension in the muscles of the anterior abdominal wall. The assessment of the condition on the NEWS2 scale comprised 11 points. Treatment was started, but a sudden death occurred. A clinical diagnosis was made: a new coronavirus infection COVID-19, virus verified. Competing disease: Acute parapancreatitis. Abscess of the mesentery of the small intestine. Complications of the underlying disease: Bilateral viral interstitial pneumonia. Acute respiratory distress syndrome. Septicemia (SOFA — 2 points, procalcitonin = 2 ng/ml). Post-mortem sectional examination revealed signs of viral-bacterial pneumonia. In the brain — pericellular and perivascular edema, heart — prominent edema of the interstitium, muscle fibers fragmented, kidneys — necronephrosis, pancreas — periductal, interlobular and intralobular sclerosis of the pancreas, between the islets of neutrophilic infiltrates. Hyperplasia was noted in the lymphoid and hematopoietic tissue, vasculitis, signs of interstitial inflammation in the parenchymal organs were determined. According to the results of microbiological examination of lung tissue, abundant growth of Klebsiella pneumoniae was found. Conclusion. The cause of death of the patient A.I. 58 years old was a novel coronavirus infection COVID-19, which caused bilateral viral pneumonia, in parallel with bacterial microflora, complicated by acute respiratory failure. There was also a generalized spread of infection with developed septicemia and multiple organ failure (pulmonary, cerebral, renal).