Vol 22, No 2 (2022)

The review discusses the complex role of the intestinal microbiota in the pathogenesis of multiple sclerosis, summarizes data from studies of changes in the composition of the intestinal microbiome in patients with multiple sclerosis, and provides evidence of the involvement of the intestinal microbiota in the development of experimental autoimmune encephalomyelitis in animals, a valid model of multiple sclerosis.

COVID-19 is an acute respiratory viral infection caused by the coronavirus SARS-CoV-2 (2019-nCoV). Currently, approaches to coronavirus infusion are mostly confined to pathogenetic and symptomatic therapy. New treatment strategies include research to find new molecul candidates for COVID-19 treatment, as well as the repositioning of existing medicinal products. Recently, medicinal plants have been actively studied as potential candidates for COVID-19 treatment, showing high levels of antiviral activity and anti-inflammatory activity. This review focuses on medicinal plants whose biologically active substances are used or can be used for the treatment and the supportive therapy for a new coronavirus infection.

BACKGROUND: In the 19^th century, the process of annexation of lands in the north-east of the Russian Empire was completed. The influx of newcomers, political exile, criminal hard labor created the prerequisites for an aggravation of the epidemiological situation in the newly annexed territories. The morbidity of the population began to acquire an epidemic character. Syphilis, which spread in the 19^th century, leprosy struck foreign communities in the northern and eastern regions of the Pacific Ocean, the Sea of Okhotsk, the Kamchatka Peninsula and the alien population from other territories.

AIM: Systematize, as well as identify certain trends in the prevalence and forms of manifestation of syphilis and leprosy among the population of the north-eastern districts of Eastern Siberia of the 19^th century. according to the reporting materials of doctors and authorities of that time. Let us explain: in this article, the authors quote from the documents, preserving (if possible) the style and punctuation of the originals, and also use geographical names and names of territories adopted in the Russian Empire in the 19^th century.

MATERIALS AND METHODS: On the basis of archival materials, the article discusses the ways of importation and spread of syphilis among representatives of individual foreign communities, the clinical course and forms of manifestation. The multiplicity of ways of penetration of syphilis to the outskirts of Eastern Siberia is noted. The reports of seconded staff physicians, doctors, paramedics during the examination of the population and the treatment of patients in hospitals provide the first statistical information on the incidence of men, women, their nationality, and the outcome of treatment. The reports of seconded staff physicians, doctors, paramedics during the examination of the population and the treatment of patients in hospitals provide the first statistical information on the incidence of men, women, their nationality, and the outcome of treatment.

RESULTS: The article highlights the issues of studying the role of factors contributing to a high level of the spread of syphilis and leprosy among “inorodtsy” (indigenous dwellers), describes the symptoms of manifestation, and especially chronic syphilis, and notes the role of unsanitary living conditions and household arrangements in the spread of diseases.

CONCLUSIONS: Observation of the clinical manifestations of syphilis allowed doctors to isolate leprosy as an independent form of the disease, and to investigate its causes. These observations were the basis for the development of measures aimed at stopping the spread of the disease among the population. The beginning of the emergence of medical and police supervision aimed at identifying patients, registering them and treating them is noted.

BACKGROUND: Unprecedented anti-epidemic measures and the widespread use of vaccines against COVID-19 have reduced the rate of hospitalization and mortality from the disease, but have not stopped the SARS-CoV-2 pandemic spread. The development of live vaccines against COVID-19, capable of providing the formation of a long-term humoral and cellular immune response and cross-protection against new SARS-CoV-2 variants of concern, is relevant. Previously at the I.I. Mechnikov Research Institute of Vaccines and Sera SARS-CoV-2 cold-adapted (ca, cold-adapted) variants were obtained. This work is aimed to search for methodological approaches that allow in vitro screening studies to assess the attenuation (att) phenotype of ca SARS-CoV-2 variants.

MATERIALS AND METHODS: The SARS-CoV-2 laboratory strain Dubrovka and its variants were cultured in Vero and Calu-3 cells. Quantitation of the virus was carried out by titration in Vero cells and by real-time RT-PCR. The attenuation (att) phenotype of SARS-CoV-2 variants was determined on an animal model of COVID-19 on Syrian hamsters.

RESULTS: In experiments on Syrian hamsters, the presence of the att phenotype in the ca variants of the virus was established. Animals infected with virus ca variants had significantly less weight lost, had less viral load in the lungs and brain and less pronounced pathological changes in the lungs compared to infection with the virulent strain. In vitro experiments on Vero and Calu-3 cells revealed probable attenuation markers of the virus ca variants for syrian hamsters: (1) ability to reproduce at low temperature (ca phenotype); (2) inability to reproduce at 39 °C (ts phenotype); (3) changes in the species and tissue specificity of the virus.

CONCLUSIONS: The developed methodological approaches to the identification of SARS-CoV-2 attenuation markers are a valuable tool for monitoring the stability of the phenotype of candidate vaccine strains.

BACKGROUND: The rapid spread of SARS-CoV-2 virus, which caused the COVID-19 pandemic, and the emergence of new co-circulating antigenic variants require the development and update of subtyping kits and protocols. Pyrosequencing-based protocols are promising approach for detection of single nucleotide polymorphisms.

AIM: In this study we designed the assays for genotyping Variants of Concern of the SARS-CoV-2 coronavirus using polymerase chain reaction, followed by determination of the virus variant in the sample by pyrosequencing.

MATERIALS AND METHODS: Pyrosequencing assays were designed based on alignment of SARS-CoV-2 sequences. Testing was performed using RNA of SARS-CoV-2 viruses of different lineages (alpha, beta, gamma, delta, and omicron). Pyrosequencing was performed using the PyroMark Q24 system.

RESULTS: The protocols of sample preparation and pyrosequencing were developed and tested for sequencing of regions encoding substitutions in amino acid positions: L18F, T19R, T20N; A67V, Δ69-70; G142D, Δ143-145; Δ156-157, R158G; Δ242-244; K417N/T; L452R; S477N, T478K, E484A/K/Q; H655Y; N679K, P681H/R. The specificity of the system was also evaluated in reactions with a negative control sample (RNA isolated from human nasal swab).

CONCLUSIONS: In this study, we developed and initially tested protocol for detecting coronavirus variants (alpha, beta, gamma, delta, and omicron) from samples collected from cell culture, based on the PCR technique, followed by genotyping of the variants by pyrosequencing with PyroMark Q24. The developed protocols may be adjusted to the current epidemiological situation by increasing the number of detectable sites.

BACKGROUND: Syrian hamsters are the most sensitive model for studying the pathogenesis of a new coronavirus infection and testing prophylactic and therapeutic drugs against SARS-CoV-2. Accordingly, it is important to identify pathomorphological indicators of tissue damage in coronavirus-infected animals, which would correlate with the severity of the disease.

AIM: Comprehensive assessment of the pathogenicity of SARS-CoV-2 viruses of B.1 and B.1.167.2 lineages on the model of Syrian hamsters to identify the most sensitive criteria that correlate with the clinical manifestation of the disease.

MATERIALS AND METHODS: Intranasal infection of animals with SARS-CoV-2, followed by the assessment of the clinical picture of the disease and detailed pathomorphological studies of various organs collected on the 5^th day after infection.

RESULTS: The SARS-CoV-2 Delta virus (B.1.617.2) was shown to be less pathogenic for Syrian hamsters compared to the ancestral strain that circulated during the first wave of the COVID-19 pandemic (B.1). The histopathological characterization of lung tissue sections of infected animals revealed the most sensitive morphometric indicator that correlates with the severity of SARS-CoV-2-induced pathology, namely, the alveolar wall thickness.

CONCLUSIONS: The use of this indicator makes it possible to determine even slight differences in the severity of virus-induced pathology in the Syrian hamster model, which can be critical in the preclinical evaluation of prophylactic and therapeutic drugs for COVID-19.

BACKGROUND: Cystic fibrosis is one of the most frequent monogenic diseases, in which, in addition to medical measures, measures for the prevention of the infectious process in the lungs are of great importance, which is provided by a clear organization of the work of any medical institution where patients are observed and where medical and rehabilitation measures are carried out.

AIM: The article describes the experience of the children’s Center in St. Petersburg to prevent cross-infection of patients with cystic fibrosis who have the different luge microbiome.

MATERIALS AND METHODS: The measures taken included a system for monitoring and dynamic control of the seeded pathogenic microflora with a strict separation of patients with cystic fibrosis, who had the isolation and carriage of various pathogens. The system provided for the separation of flows of patients with cystic fibrosis and the prevention of cross-infection, as well as a number of activities related to the disinfection of equipment.

RESULTS: The methods adopted and put into practice have ensured the prevention of cross-infection of patients with cystic fibrosis, excreting various pathogenic bacteria from sputum. The patient management system made it possible to halve the number of hospitalizations of patients with Pseudomonas aeruginosa infection per CF patient per year: from 1.3 in 2001 to 0.6 in 2021, reduce the risk of cross-infection, reduce the number of relapses, hospitalizations and the average length of stay of a patient in a hospital.

CONCLUSIONS: The developed system of assistance has shown its effectiveness and can be implemented in one form or another in the work of cystic fibrosis treatment centers in the Russian Federation.

BACKGROUND: COVID-19 mediates activation of immunocompetent cells. Little is known about the phenotypic marker CD38 on the surface of T and NK cells and its association with the severity of COVID-19.

AIM: To analyze the distribution of the surface marker CD38 in subsets of T and NK cells at the different stages of their differentiation in the groups of patients with COVID-19 and convalescents, and to reveal the relationship between the level of CD38 expression in subpopulations of T and NK cells with the severity of COVID-19.

MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) from patients and donors were isolated using ficoll density gradient followed by cytofluorimetric analysis of the surface markers: CD3, CD56, CD38.

RESULTS: In PBMC samples the level of CD38 expression on NK cells (CD3^–CD56⁺), including CD56^bright and CD56^dim subsets, as well as on T lymphocytes, differentiating conventional CD56^–(CD3⁺CD56^–) and NKT-like (CD3⁺CD56⁺) cells, were analyzed. Among CD56^– T cells and NKT-like cells the highest levels of CD38 expression were registered in the groups of patients of moderate severity and convalescents, and, in contrast, the decreased CD38 levels were detected in the group of patients from the intensive care unit.

CONCLUSIONS: The findings suggest that the level of CD38⁺ T cell in COVID-19 may be of use as diagnostic value.

BACKGROUND: SARS-CoV-2 vaccine immunogenicity is evaluated in neutralization test with live virus. It is performed in a biosafety level 3 zone because requires live virus stage. Therefore, control laboratories should be certified for this class of work. The development of technology based on enzyme-linked immunosorbent assay as an analogue of the neutralization reaction makes it possible to create an immunobiological product in a shorter time and in conditions without special requirements for control laboratories.

AIM: Development of an enzyme-linked immunosorbent assay for assessing SARS-CoV-2 vaccine immunogenicity by measuring neutralizing antibodies production in immunized animals.

MATERIALS AND METHODS: Recombinant receptor-binding domain fused to a С-terminal hexahistidine sequence was produced in Escherichia coli cells and purified via metal-affinity chromatography on WorkBeads NiMAC (Bio-Works). Purified protein was used in enzyme-linked immunosorbent assay as an antigen for sorption. The sera of mice immunized with the vaccine preparation were tested for neutralizing activity against the SARS-CoV-2, as well as in the developed enzyme-linked immunosorbent assay.

RESULTS: Sera with high neutralizing titers showed a high degree of binding to recombinant receptor-binding domain fused to a С-terminal hexahistidine sequence in enzyme-linked immunosorbent assay, while sera from non-immunized animals or sera with neutralization titers less than 1:8 were not reactive in enzyme-linked immunosorbent assay. The Spearman and Pearson correlation coefficients for neutralization test titers and optical density in enzyme-linked immunosorbent assay were 0.759 and 0.76, respectively. The developed assay can be used as a semi-quantitative method for assessing the immunogenicity of a vaccine against coronavirus infection.

CONCLUSIONS: The developed platform makes it possible to reliably assess the immunogenicity of an inactivated coronavirus vaccine under conditions that do not require a high biosafety conditions.

BACKGROUND: One approach that makes it possible to create vaccines relatively quickly and inexpensively is the creation of DNA vaccine constructs. They are of particular interest for the prevention of COVID-19, as they induce both types of immune response – humoral and T cell. Previously, we have created two DNA vaccines based on the pVAX vector: one encodes the full gene of the S protein of the SARS-CoV-2 virus, the second only encodes the receptor-binding domain (RBD) of the S protein of the SARS-CoV-2 virus. Next, the “naked” DNA was wrapped in a polycationic polyglucin-spermidine complex conjugated with the RBD protein. The resulting combined DNA-protein constructs were named CCV-RBD and CCV-S, respectively.

AIM: To investigate the induction of the T cell immune response of the developed combined DNA protein candidate vaccines in an animal model.

MATERIALS AND METHODS: BALB/c mice were immunized with constructs CCV-RBD and CCV-S, after which their spleens were removed from which splenocytes were isolated. The cellular response was assessed by the ability of splenocytes to secrete cytokines in response to stimulation with viral peptides. The intensity of the response was recorded using the intracellular cytokine staining (ICS) method using flow cytometry.

RESULTS: BALB/c mice were immunized twice with an interval of three weeks with a dose of 100 μg of DNA (8 animals per group): 1) CCV-RBD 2) CCV-S and 3) intact animals. It has been shown that both T helper lymphocytes (CD4⁺) and cytotoxic lymphocytes (CD8⁺) of animals immunized with CCV-S and CCV-RBD respond with the release of cytokines in response to stimulation with viral peptides.

CONCLUSIONS: In the case of CCV-RBD, a trend towards a higher response in both CD4⁺ and CD8⁺ was observed compared to the CCV-S group. Possibly, this difference may be due to more efficient synthesis of the RBD protein than the S protein, providing a DNA vaccine.

BACKGROUND: Nucleic acid-based prevention tools provide a promising platform for developing vaccines, including those against COVID-19. Previously, we developed the pVAXrbd DNA vaccine encoding the receptor-binding domain (RBD) of SARS-CoV-2, which, when administered intramuscularly to animals, induced a relatively weak immune response. The next stage of the study is to increase the immune response, in particular, using electroporation as one of the methods for increasing the immunogenicity of DNA vaccines.

AIM: The aim of this article is to evaluate the immune response using electroporation in mice after immunization with pVAXrbd.

MATERIALS AND METHODS: BALB/c mice were immunized with pVAXrbd using direct and reverse polarity square wave direct current electroporation with three pulses of 12 V for 30 ms and an interval of 950 ml with a current limit of 45 mA.

RESULTS: BALB/c mice were immunized twice with an interval of three weeks with a dose of 100 μg of DNA. Using ELISA, the titers of RBD-specific antibodies in the group of animals immunized with pVAXrbd using electroporation were 1:109350, which is 16 times higher than in the group of animals that received the DNA vaccine only intramuscularly (titers 1:6750). IFNγ ELISpot analysis showed that the largest number of cells (2434 spots/splenocytes, million) producing IFNγ in response to stimulation with peptides from the RBD protein was registered in the group of animals immunized with pVAXrbd using electroporation. For comparison, in the control group, the number of cells is 6.5 times lower: 380 spots / splenocytes, mln.

CONCLUSIONS: Administration of the pVAXrbd DNA vaccine to laboratory animals by electroporation significantly enhances both the humoral and cellular specific immune response compared to intramuscular administration of the naked DNA vaccine.

BACKGROUND: Bats are a reservoir of a large number of viruses, including coronaviruses. Monitoring viruses in bats is an important task.

AIM: To detect viruses belonging to Coronaviridae family in bats which habitat in European part of Russia.

MATERIALS AND METHODS: We used PCR amplification of viral genome fragments, followed by high-throughput sequencing.

RESULTS: RdRp gene fragments of at least two different alphacoronaviruses (Bat coronavirus, Coronaviridae) were revealed in four bats of three species.

CONCLUSIONS: Our results demonstrate the presence of viruses of the Alphacoronavirus genus in 4 of 17 bats. Coronavirus-positive animals were captured in 2021 in Moscow, Moscow Region and Rostov-on-Don, these four animals have been found to be carriers of different isolates of the same alphacoronavirus, which allows us to suggest the possibility of transmission of this virus between animals between different species. One animal was found as carrier of genome fragments of two different alphacoronaviruses.

BACKGROUND: The pandemic caused by the SARS-CoV-2 virus at the end of 2019 remains to be a serious healthcare problem. Constant antigenic drift of the pathogen led to a decrease of licensed COVID-19 vaccines effectiveness. And the development of broad-spectrum vaccines with high effectiveness rate against evolutionarily divergent SARS-CoV-2 variants remains an urgent issue. Unlike virus-specific antibodies with limited spectrum of action, T-cell immunity has a wider cross-protective potential. Syrian hamsters are the most appropriate model for preclinical evaluation of new vaccine candidates, since these animals are susceptible to SARS-CoV-2 infection and show clinical symptoms of the disease. However, study of T-cell vaccine response in hamsters is complicated by the lack of available reagents and test systems for adequate assessment of the virus-specific cellular immunity levels after vaccination.

AIM: In this work, we report an optimized protocol of stimulation of Syrian hamsters’ immune cells with a live SARS-CoV-2 virus to assess virus-specific T-cell responses.

MATERIALS AND METHODS: Intranasal infection of animals with SARS-CoV-2 virus followed by stimulation of immune cells with different doses of whole live coronavirus and counting of IFNγ-producing cells by ELISpot method.

RESULTS: Stimulation of spleen and lung cells with SARS-CoV-2 at a dose 0.1 TCID₅₀/cell is the most optimal viral concentration for detecting maximum of cytokine-producing cells in SARS-CoV-2-infected animals. Stimulation of cells with whole virus revealed greater number of virus-specific cells compared to a stimulation with pools of SARS-CoV-2 lyophilized peptides (S and N proteins).

CONCLUSIONS: Overall, the new methodology allows assessment of the immunogenicity of COVID-19 T-cell vaccines more accurately in preclinical studies using the Syrian hamsters model.

BACKGROUND: The pandemic has led to a development of various immunological complications of COVID-19 including the chronic fatigue syndrome and fibromyalgia. These syndromes, which often manifest themselves on a background of autoimmune diseases, may develop with an entrainment of vestibular function into the pathological process.

AIM: Aim of the study was to estimate vestibular function in groups of patients with autoimmune dysfunctions accompanied by the chronic fatigue syndrome and fibromyalgia, who have had COVID-19 and those have not had COVID-19 in their anamnesis, and to compare it with vestibular function of healthy volunteers.

MATERIALS AND METHODS: A functional investigation of vestibular system called “Vestibular passport” and the anamnesis taking by a standard questionnaire were performed in patients with an implied autoimmune dysfunction and presence of the chronic fatigue syndrome, fibromyalgia and/or postural orthostatic tachycardia, and also for a control group of healthy participants.

RESULTS: Patients who have had COVID-19 in their anamnesis demonstrated significantly higher percentage of cases of vestibulopathy than healthy volunteers (33 and 6 %, respectively). In patients without COVID-19 in their anamnesis the percentage of vestibulopathy cases did not differ significantly from the corresponding percentage in healthy volunteers (14 and 6 %, respectively). Patients’ complaints which indicate a vestibular pathology were confirmed in 2/3 of all cases.

CONCLUSIONS: Patients with chronic fatigue syndrome and fibromyalgia who have had COVID-19 were more prone to vestibulopathies than patients without COVID-19 in their anamnesis, whose vestibular indices did not differ from that in healthy volunteers. The data obtained shoul be considered as preliminary.

BACKGROUND: RBD, receptor-binding domain, a key region of the SARS-CoV-2 surface glycoprotein for virus binding to host cell receptors and one of the targets of virus-neutralizing antibodies. That is why RBD is a promising immunogen for the development of vaccines that can provide protection against COVID-19. Vaccine mRNA is one of the new and rapidly developing vaccine platforms, and the delivery system is a very important component of it.

AIM: The aim of this work was to present the results of a study of the antigenic properties of mRNA encoding the receptor-binding domain of SARS-CoV-2 when administered in combination with a polycationic carrier.

MATERIALS AND METHODS: Dynamic and electrophoretic light scattering were used to characterize mRNA complexes with a polyglucin-spermidine conjugate. To assess the immunogenicity of mRNA were immunized BALB/c mice. The specific activity of the sera was assessed using enzyme immunoassay.

RESULTS: As a result, the sizes and surface charge of the RBD-encoding mRNA complexes with the polyglucin-spermidine conjugate were determined. It has been shown that wrapping mRNA in a polyglucin-spermidine conjugate shell leads to an increase in the induction of RBD-specific antibodies in BALB/c mice compared to naked mRNA.

CONCLUSIONS: An mRNA encoding the receptor-binding domain of SARS-CoV-2 has been obtained. It has been shown that the packaging of mRNA into the polyglucin-spermidine conjugate shell leads to an increase in immunogenic properties.

BACKGROUND: The physiological features of the pregnant woman’s body include her in the risk group for a more severe course of the disease, therefore, the management and treatment of such patients requires the most careful monitoring, as well as the coordinated work of a gynecologist and an infectious disease specialist.

AIM: To evaluate the variants of the course of the disease in pregnant women with confirmed coronavirus infection.

MATERIALS AND METHODS: The medical records of patients who were treated in the infectious diseases department of the Clinical Hospital No. 1 in Smolensk from May 2020 to September 2021 were analyzed, the necessary data were included in the protocols of our study. Such indicators as the main diagnosis and severity of the course of the disease, complaints at admission, the presence and stage of fever, age, gestational age, the presence of concomitant pathology and a complicated obstetric and gynecological history were evaluated.

RESULTS: Analysis of the course of the disease according to the medical history showed that a moderate form of a new coronavirus infection dominates pregnant women.

CONCLUSIONS: A trend towards a moderate course of a new coronavirus infection COVID-19 has been established. The probability that this is due to the presence in most patients with a history of diseases of the cardiovascular system, kidneys, liver, as well as diabetes, obesity, anemia.

BACKGROUND: The emergence of new variants of the SARS-CoV-2 virus at the end of 2020, which became a source of increased risk for global public health, prompted the study of their molecular biological characteristics and pathogenic effects. It is known that one of the reasons for the viruses’ pathogenic action is their interaction with the defense mechanisms of the cell. Ire1 (inositol-requiring enzyme 1)-mediated splicing of Xbp1 mRNA (X-box binding protein 1) is a protective mechanism that is activated in response to the accumulation of misfolded proteins in the cell, a situation that occurs due to uncontrolled synthesis of viral proteins during infection. Studying the interaction of different variants of the SARS-CoV-2 virus with this protective mechanism will help to shed light on various aspects of the pathogenesis of a new coronavirus infection.

AIM: Study of Ire1-Xbp1 defense mechanism activation and modulation in SARS-CoV-2 infected Vero cells.

MATERIALS AND METHODS: We studied the activation of the Ire1 enzyme in Vero cells infected with various variants of the SARS-CoV-2 virus using immunoblotting and antibodies to various forms of this protein in the cell. In addition, we studied the activation of Xbp1 mRNA splicing under conditions of infection with various variants of the SARS-CoV-2 virus in a PCR reaction with specific primers.

RESULTS: Reproduction of B.1.1.529 (Omicron) strain in Vero cells is slower than B.1.1.7 (Alpha) and B.1.617.2 (Delta) strains of SARS-CoV-2. The whole reproduction cycle is 48 hours.

Ire1-dependent defense mechanism is activated after 12 hours of SARS-CoV-2 infection with either of three variants. However, despite the activation of the Ire1 endonuclease domain, there is no Xbp1 mRNA splicing in SARS-CoV-2 infected cells. Inhibition of Xbp1 mRNA splicing occurs slower in Vero cells infected with the B.1.1.529 — Omicron variant.

CONCLUSIONS: The paper describes the reproduction of various variants of the SARS-CoV-2 virus in Vero cell culture and the activation of the Ire1-Xbp1 defense mechanism during infection. The Ire1 endonuclease is phosphorylated, however, mRNA splicing of the Xbp1 transcription factor is impaired in SARS-CoV-2 infected cells. A decrease in the rate of inhibition of this protective mechanism in Vero cells infected with the Omicron (B.1.1.529) variant of the SARS-CoV-2 virus may be the reason for its lower pathogenicity described in various studies.