Medical academic journal

The journal “Meditsinskiy Akademicheskiy Zhurnal” published since 2001 is an official journal of the Northwest Branch of the Russian Academy Sciences. Its initiator and Editor-in-Chief in 2001-2009 was Academician of the Russian Academy of Medical Sciences Boris Ivanovich Tkachenko whose contribution to the development of the journal is invaluable. The first Deputy Editor-in-Chief of the journal was Academician of RAMS V.A. Nagornev, and its Executive Secretary was Corresponding Member of RAMS N.S. Sapronov.

In September 2009, the position of Editor-in-Chief was taken by Corresponding Member of RAMS I.P. Dudanov, of Deputy Editor-in Chief, by Corresponding Member of RAMS N.S. Sapronov, and of Executive Secretary, by Prof. P.D. Shabanov.

The high publication level and wide appreciation by specialists in the course of formation and development of the journal were ensured by outstanding contributors, including Academicians E.K. Ailamazyan, N.A. Belyakov, Yu.D. Ignatov, Yu.V. Lobzin, V.I. Mazurov, N.A. Maystrenko, and A.A. Totolyan, and Corresponding Members of RAS I.A. Yeriukhin, S.A. Ketlinskiy, M.M. Odinak, Ye.A. Selivanov, and S.A. Simbirtsev.

Since 2010 up to the present time, Editor-in-Chief of the journal is Academician G.A. Sofronov. Since 2012, its Deputy Editor-in-Chief is Academician N.A. Belyakov, and its Executive Secretary is A.B. Dmitriyev PhD. The Editorial and Advisory Boards of the journal include prominent scientists representing the Northwest of Russia and distinguished scientific schools of Saint Petersburg.


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Vol 22, No 2 (2022)

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Analytical reviews

Role of the intestinal microbiota in the pathogenesis of multiple sclerosis. Part 1. Clinical and experimental evidence for the involvement of the gut microbiota in the development of multiple sclerosis
Abdurasulova I.N.

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.

Medical academic journal. 2022;22(2):9-36
pages 9-36 views
M proteins are the major pathogenicity factors of Streptococcus pyogenes
Burova L.A., Suvorov A.N., Totolian A.A.

M proteins are the major pathogenicity factors of the widespread and potentially deadly bacterial pathogen Streptococcus pyogenes. These proteins confer to bacteria resistance against innate and adaptive immune responses. The study of the M proteins of hemolytic streptococci group A and their involvement in pathology clearly indicates that strains of streptococci, for one reason or another devoid of M proteins are unable to multiply in the macroorganism and form a focus of infection. This circumstance in itself once again underlines the leading role of M proteins in the realization of its many properties and in the development of the infectious process. The ability of M proteins to recruit plasma proteins of the macroorganism is their significant pathogenetic properties. The most important is the nonimmune binding by M proteins of human immunoglobulins, because it participates in the suppression of phagocytosis, violations of bacterial opsonization and complement activation along the classical pathway, not to mention the possible involvement of this phenomenon in the genesis of post-infectious complications of autoimmune nature. This review summarizes the current data on the structure M proteins, their functional activity, manifestations of pathogenicity, genetic regulation and methods of emm-typing.

Medical academic journal. 2022;22(2):37-52
pages 37-52 views
Application of herbal medicinal raw material in complex treatment COVID-19
Khaliullina A.S., Shakirova D.K., Aliullina L.A., Morgatskaya O.V.

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.

Medical academic journal. 2022;22(2):53-64
pages 53-64 views

History of medicine

Ways of spread, detection, clinical course of syphilis and leprosy among the population of the north-eastern districts of Eastern Siberia in the 19th century
Gaidarov G.M., Alekseevskaya T.I., Demidova T.V., Sofronov O.Y.

BACKGROUND: In the 19th 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 19th 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 19th 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 19th 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.

Medical academic journal. 2022;22(2):65-77
pages 65-77 views

Original research

Attenuation markers of cold-adapted SARS-CoV-2 variants
Gracheva A.V., Korchevaya E.R., Samoilikov R.V., Smirnova D.I., Leneva I.А., Poromov A.A., Pankratov A.А., Trunova G.V., Khokhlova V.А., Nagieva F.G., Svitich O.А., Zverev V.V., Faizuloev E.В.

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.

Medical academic journal. 2022;22(2):79-88
pages 79-88 views
Research of the genetic diversity of human rhinoviruses on the territory of Saint Petersburg 2021–2022
Ksenafontov A.D., Pisareva M.M., Eder V.A., Musaeva T.D., Kiseleva I.V.

BACKGROUND: Respiratory viruses circulate everywhere. Rhinoviruses are the most common cause of human upper respiratory tract infections. Therefore, it is necessary to study circulation of their species and types.

AIM: To study circulation of different species and types of rhinoviruses in Saint Petersburg.

MATERIALS AND METHODS: Detection of rhinoviruses was carried out by real-time PCR using commercial kits AmpliSens ORVI-screen-FL (Central Research Institute of Epidemiology of Rospotrebnadzor, Moscow); the study of the genetic diversity of rhinoviruses was carried out by Sanger sequencing method; alignment of the obtained sequences was carried out in the MAFFT program, the phylogenetic tree was built in the RAxML program, visualization was carried out in FigTree.

RESULTS: The most common species of human rhinoviruses circulating in Saint Petersburg are HRV-A, while the types of rhinoviruses almost never repeat.

CONCLUSIONS: On the territory of St. Petersburg, the genetic diversity of rhinoviruses is very widely represented. From December 2020 to October 2021, 70 rhinoviruses were typed, with HRV-A rhinoviruses being the most common (38 rhinoviruses, or 54%). HRV-C and HRV-B were detected in equal numbers (16 rhinoviruses, or 23% each). Phylogenetic trees of genetic types of rhinovirus species A, B, and C were built. The types of rhinoviruses are highly variable, which complicates their study, as well as the development of vaccines against them. Nevertheless, rhinoviruses significantly affect the epidemiological situation, rhinovirus infections can lead to serious health consequences, so they need to be studied.

Medical academic journal. 2022;22(2):89-96
pages 89-96 views
Development of pyrosequencing-based assay for genotyping of different coronavirus variants
Chistyakova A.K., Stepanova E.A., Isakova-Sivak I.N., Rudenko L.G.

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.

Medical academic journal. 2022;22(2):97-114
pages 97-114 views
Antiviral activity of adamantane derivatives against respiratory syncytial virus
Shtro A.A., Galochkina A.V., Nikolaeva Y.V., Garshinina A.V., Razgulyaeva D.N., Ponomarev K.Y., Mozhaytsev E.S., Suslov E.V., Volcho K.P., Salakhutdinov N.F.

BACKGROUND: Respiratory syncytial infection annually affects large segments of the population around the world. Despite the ease of clinical manifestations in most adults, for children under two years of age, this disease is a serious danger, leading to the development of severe bronchiolitis, even death.

AIM: The aim of this study was to search for drugs with antiviral activity among adamantane derivatives.

MATERIALS AND METHODS: Preparations of the group of adamantane derivatives were synthesized in the Department of Medicinal Chemistry of Natural Compounds of the N.N. Vorozhtsov Novosibirsk Institute of Organic Chemistry SB RAS. The cytotoxicity and antiviral activity of the compounds were studied in HEp-2 cell culture using the MTT test and enzyme immunoassay, respectively.

RESULTS: There were no drugs with high antiviral properties against RSV in this group.

CONCLUSIONS: Despite the absence of drugs with pronounced anti-RSV properties, the information obtained in the course of the work can be used for a targeted search for antitumor substances.

Medical academic journal. 2022;22(2):115-123
pages 115-123 views
Comparative study of the pathogenicity of SARS-CoV-2 B.1 AND B.1.617.2 lineages for syrian hamsters
Yakovlev K.S., Mezhenskaya D.А., Sivak K.V., Rudenko L.G., Isakova-Sivak I.N.

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 5th 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.

Medical academic journal. 2022;22(2):125-136
pages 125-136 views

Clinical research

Method for the prevention of hospital infection in Saint Petersburg
Orlov A.V., Pashkevich A.A., Matveev V.S., Vainer L.B., Nachinkina T.A., Kraeva L.A., Romanyuk F.P., Shutova T.A., Kuropatenko M.V., Zhelenina L.A.

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.

Medical academic journal. 2022;22(2):137-146
pages 137-146 views

Conference proceedings

The severity of COVID-19 is accompanied by a change in the expression of the surface marker CD38 in T lymphocytes and NK cells
Vavilova J.D., Boyko A.A., Streltsova M.A., Kust S.A., Yusubalieva G.M., Novikova O.N., Sotnikova A.G., Bychinin M.V., Ischakov R.N., Sapozhnikov A.M., Kovalenko E.I.

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 (CD3CD56+), including CD56bright and CD56dim 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.

Medical academic journal. 2022;22(2):149-156
pages 149-156 views
The effect of heat inactivation of ferrets serum samples on the detection of SARS-CoV-2-specific IgG antibodies in elisa
Goshina A.D., Matyushenko V.A., Donina S.A., Sychev I.A., Isakova-Sivak I.N., Katelnikova A.E., Rudenko L.G.

BACKGROUND: Determination of serum antibody levels to the novel coronavirus SARS-CoV-2 is a necessary tool for assessing humoral immunity in COVID-19 patients or individuals vaccinated with specific vaccines, as well as for studying the immune responses to the viral antigens in animal models. Serum specimens of infected humans and animals are considered potentially infectious material, and therefore heat inactivation of samples at 56 °C for 1 hour is recommended to reduce the risk of infection of personnel during serological studies. However, this procedure may affect the detection of virus-specific IgG and IgM antibodies, making interpretation of the results difficult.

AIM: The goal is to evaluate the effect of heat inactivation of ferret serum samples on the binding of IgG antibodies to the SARS-CoV-2 antigens.

MATERIALS AND METHODS: Serum samples of SARS-CoV-2 naive and immune ferrets were analyzed in three variants: (1) native sera, (2) serum samples than were heated at 56°C for 1 hour, and (3) serum samples that were treated with receptor-degrading enzyme (RDE). The samples were studied in enzyme-linked immunosorbent assay (ELISA) using recombinant RBD protein (receptor-binding domain of SARS-CoV-2 S-protein) as a substrate, followed by determination of specific antibody levels before and after treatments.

RESULTS: It has been shown that heat inactivation of the ferrets’ naive serum samples can lead to false-positive results, while RDE treatment can neutralize the effect of non-specific binding of IgG antibodies to the RBD domain of the SARS-CoV-2 S protein.

CONCLUSIONS: Structural rearrangement of SARS-CoV-2-specific IgG antibodies and the formation of immunoglobulin complexes during heat inactivation of serum samples can affect the avidity of the antigen-antibody complex and lead to false-positive results when performing enzyme immunoassay. One of the possible methods to reduce the risk of “artifacts” is the treatment of blood sera with RDE, which eliminates the effect of heat inactivation.

Medical academic journal. 2022;22(2):157-162
pages 157-162 views
Development of a biosafe ELISA-based platform for assessing immunogenicity in the production of an inactivated whole-virion coronavirus vaccine
Danilov D.V., Shmeleva O.A., Lunin A.S., Kozlovskaya L.I., Piniaeva A.N., Shishova A.A.

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.

Medical academic journal. 2022;22(2):163-169
pages 163-169 views
Mathematical model of the spread of COVID-19 in the rostov region for 2020–2022
Denisenko V.V., Aleshukina A.V.

BACKGROUND: In the context of the COVID-19 epidemic, there is a need for forecasting tools that enables us to predict the possible epidemic consequences and evaluate the effectiveness of epidemic control. Mathematical modeling can serve as such a tool.

AIM: The Aim is to present a mathematical model of the spread of COVID-19 in the Rostov Region for 2020–2022. To be more precise, the aim is to forecast how the number of people infected, recovered, hospitalized and died from the epidemic of the new coronovirus will change over this period of time.

MATERIALS AND METHODS: The modeling of the epidemics was based on the extended SEIR model proposed by R. Neyer et al. for forecasting the COVID-19 epidemic and implemented as a freely available web application.

RESULTS: The model under consideration made it possible to forecast the volume of hospitalization and the number of deaths in the course of COVID-19 epidemic in the Rostov region for 2020–2022.

CONCLUSIONS: The performed simulations demonstrated the capabilities the considered SEIR model for forecasting the COVID-19 epidemic. It was found that the number of hospitalized patients for the entire period covered by the model does not exceed the capabilities of the health care system of the Rostov Region.

Medical academic journal. 2022;22(2):171-176
pages 171-176 views
Cellular immune response to combined DNA-protein constructs carrying SARS-CoV-2 antigens
Zadorozhny A.M., Borgoyakova M.B., Starostina E.V., Karpenko L.I.

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.

Medical academic journal. 2022;22(2):177-181
pages 177-181 views
Method of obtaining purified SARS-CoV-2 antigen for structural and immunological studies in whole-virion vaccines against coronavirus infection
Zyrina A.N., Tselykh I.O., Bogdan A.S., Kovpak A., Ivin Y.Y., Vasilenko V.E., Piniaeva A.N., Shishova А.A.

BACKGROUND: Despite the large-scale development of vaccines against coronavirus infection, there is still no complete information about the antigenic structure of the SARS-CoV-2 virus particles. This article describes a method of obtaining a pure concentrated whole-virion sample that can be used in various studies.

AIM: The goal is to develop an optimal method of purification of the inactivated SARS-CoV-2 virus in order to obtain a standard with parameters of purity and antigen content sufficient for structural and immunological studies.

MATERIALS AND METHODS: To obtain a pure concentrated virus SARS-CoV-2 we used the sucrose density gradient ultracentrifugation. Fractions with the highest content of viral particles were evaluated based on the concentration of nucleocapsid N and glycoprotein S. The purity of the pooled fractions (the purified sample) was evaluated by the presence of impurity proteins, toxins and bovine serum albumin.

RESULTS: The optimal conditions for obtaining the inactivated purified SARS-CoV-2 whole virus were determined. A standard sample of the inactivated SARS-CoV-2 virus was isolated and characterized.

CONCLUSIONS: The obtained standard sample can be used in enzyme immunoassay to measure the amount of antigen in whole-virion vaccines against coronavirus infection, as well as in various structural studies of SARS-CoV-2 virus particle.

Medical academic journal. 2022;22(2):183-189
pages 183-189 views
Delivery of a DNA vaccine encoding SARS-CoV-2 receptor-binding domain (RBD) by electroporation
Kisakov D.N., Orlova L.A., Sharabrin S.V., Rudometov A.P., Borgoyakova M.B., Starostina E.V., Karpenko L.I., Ilyichev A.A.

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.

Medical academic journal. 2022;22(2):191-196
pages 191-196 views
Surface expression of SARS-CoV-2 epitopes in Enterococcus faecium L3 for live oral vaccine against new coronavirus infection
Kopteva O.S., Desheva Y.A., Ivanova A.N., Vorobyov M.G., Leontieva G.F., Gupalova T.V., Bormotova E.A., Suvorov A.N.

BACKGROUND: Probiotic microorganisms are currently considered as a promising platform for the development of recombinant vaccines expressing viral or bacterial antigens. Probiotic-based mucosal vaccines are easy to produce in large quantities, they have a low cost, provide a fairly long T-cell memory.

AIM: The aim was to study expression of mRNA fragment of S1 SARS-CoV-2 gene in Enterococcus faecium L3 culture and to confirm the insertion of S1 SARS-CoV-2 protein fragment into the pili of this bacterial strain by immunoelectron microscopy of original (E. faecium L3) and genetically modified strain (L3-SARS) with human sera obtained from patients with SARS-CoV-2.

MATERIALS AND METHODS: mRNA expression was studied by real-time PCR with reverse transcription using primers specific to S1 protein. Immunoelectron microscopy was aimed to study the structure of E. faecium L3 pili with the expression of viral protein SARS-CoV-2. Bacteria were washed three times in PBS by centrifugation at 3500 rpm for 20 min and suspended in 0.1 M NaCl. A 10-fold bacterial concentrate was used. The source of the primary antibodies was a set of polyclonal human sera containing IgG. Labeling was performed using goat IgG conjugated with 18 nm gold particles.

RESULTS: A sharp increase in mRNA amplification of inserted genetic sequence of S1 SARS-CoV-2 gene fragment relatively to the control was demonstrated. These results confirmed that DNA of S1 gene in E. faecium L3 genome is transcribed together with the target pili gene in E. faecium genome. Specific antigens of SARS-CoV-2 on the surface of L3-SARS were determined using electron microscopy, which demonstrated the correct assembly of chimeric molecules of pili on the surface of bacteria.

CONCLUSIONS: Evaluation in expression of SARS-CoV-2 S1 protein after introduction of the corresponding genetic elements into genome of probiotic strain E. faecium L3 allows us to conclude that selected DNA fragments of SARS-CoV-2 were able to direct the synthesis of immunogenic protein S1 that was expressed by the strain E. faecium L3-SARS.

Medical academic journal. 2022;22(2):197-202
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Alphacoronaviruses detected in fecal samples of bats captured in Moscow and Rostov-on-Don in 2021
Korneenko E.V., Samoilov A.E., Artyushin I.V., Yusefovich A.P., Dolotova S.M., Klyuchnikova E.O., Sbarzaglia V.A., Gladkikh A.S., Dedkov V.G., Speranskaya A.S.

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.

Medical academic journal. 2022;22(2):203-208
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Detection of immunoglobulins specific to SARS-CoV-2 virus S-protein in mixed saliva
Lizunkova M.A., Kudar P.A., Koroleva I.V., Desheva Y.A.

BACKGROUND: Since the beginning of the COVID-19 pandemic, scientists have been concerned about finding an effective alternative to blood-based diagnostics of antibodies to SARS-CoV-2. According to the results of numerous studies by domestic and foreign authors, saliva is a valuable diagnostic material. Mixed saliva implies the possibility of painless and non-invasive sampling and opens up great opportunities for diagnosing COVID-19 in vulnerable populations, such as children.

AIM: Development of a method for detecting immunoglobulins specific to the SARS-CoV-2 virus in saliva after infection with COVID-19 and vaccination.

MATERIALS AND METHODS: The study involved 43 people aged 20 to 67 years, divided into 3 groups: recovered from COVID-19; those who believe they have not had COVID-19; vaccinated with various drugs against COVID-19. Samples of unstimulated mixed saliva and capillary blood from a finger were obtained from each subject, which were subsequently examined by methods of qualitative and quantitative enzyme immunoassay.

RESULTS: A high level of correlation between the content of local IgG and IgA in saliva was demonstrated, while the relationship between the content of IgA in blood and saliva was shown to be of medium strength. A higher number of seropositive patients was demonstrated in the group vaccinated with vaccines against SARS-CoV-2, compared with those who recovered from COVID-19, which is consistent with the anamnesis data.

CONCLUSIONS: This work demonstrates and confirms that saliva in the oral fluid is a valuable diagnostic material for the study of local antibodies. The data obtained will allow the development of unique test systems for studying humoral immunity to the SARS-CoV-2 virus, which can become an alternative replacement for the usual determination of virus-specific immunoglobulins in the blood.

Medical academic journal. 2022;22(2):209-214
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Assessment of cell-mediated immune responses to SARS-CoV-2 in Syrian hamsters
Mezhenskaya D.А., Isakova-Sivak I.N., Rudenko L.G.

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.

Medical academic journal. 2022;22(2):215-220
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Modulation of porcine alphacoronavirus pathogenesis by antibodies to SARS-CoV-2
Nefedeva M.V., Titov I.A., Katorkin S.A., Tsybanov S.Z., Lyska V.M., Sereda A.D., Malogolovkin A.S.

BACKGROUND: A variety of antibodies against the SARS-CoV-2 with different functional activities can lead to immune pathology and/or modulation of infection due to antibody-dependent enhancement. An important role in the development of ADE, as exemplified by Dengue fever, is played by low-affinity antibodies, which interacting with the virus, do not neutralize it, but on the contrary forms the virus-antibody complex to immune cells (monocytes/macrophages, B-cells). There are conflicting data on the antibody-dependent enhancement presence in COVID-19 coronavirus infection. Some studies indicate a relationship between the severity of infection in patients infected by SARS-СoV-2 and the level of antibodies to closely related coronaviruses.

AIM: The aim of the study was to understand the possibility of interaction between antibodies to SARS-CoV-2 with closely related porcine coronavirus (alphacoronavirus) and their ability to modulate the development of infection in vitro.

MATERIALS AND METHODS: In the study serums and leukocytes from patients, who are convalescent after COVID-19 infection in 2020, was used. Antibody titers (IgG/IgM) was checked by commercial kit (VektorBest, Russia), based on ELISA method. In the eukaryotic expression system — Chinese hamster ovary cells, monoclonal antibody to SARS-CoV-2 spike protein and its Fab version without Fc domain were obtained. As a model, swine alphacoronavirus- transmissible gastroenteritis virus (TGEV) was used. Replication activity of TGEV was studied in porcine leukocyte cell culture. The ability of this virusto induce the interferon gamma synthesis in leukocytes from convalescents was assessed TigraTest SARS-CoV-2 Enzyme-Linked SPOT analysis (Russia). Virus titration and neutralization assay with sera and monoclonal antibodies were carried out in porcine kidney PK-15 cell line.

RESULTS: Serum samples from 43 donors (59% female, 41% male) aged from 21 to 56 was studied for the presence of class M and G antibodies to SARS-CoV-2 by ELISA method. Class M antibodies were found in only one donor. The highest titers of class G antibodies (>1:400 to >1:1600) were detected among donors 40–50 years old. For four donors with the highest antibody titers, HLA-II type was determined. The DQA1 allele in 3 patients was found to have the *0501 variant, which, according to literature data, is associated with autoimmune thyroid diseases. For the DQB1 allele, two convalescents had exactly the same variants (*0602-8). Among HLA DRB1, all patients had different allele variants (*09, *11, *01,*03,*13,*15,*08,*13).

Among the samples, studied by ELISPOT method, in 45,5% of samples a positive T-cell response was noted after stimulation of macrophages with the S peptides of the SARS-CoV-2 protein and in 22.7% of samples in response to the pool of N peptides of the SARS-CoV-2 protein. Interestingly, infection of macrophages from convalescents with the transmissible gastroenteritis virus caused interferon-gamma expression in 31,8% of cases.

The monoclonal antibody to the Spike protein SARS-CoV-2 and its Fab variant were not able to neutralize the TGEV. Interestingly, serum samples from 16 donor’s convalescent after COVID-19 caused virus neutralization in PK-15 cell culture at dilutions of 1:4-1:8.

CONCLUSIONS: We have shown that porcine alphacoronavirus induces (in 31.8% of cases) the synthesis of interferon gamma in macrophages of recovered afterCOVID-19 donors, which may indicate cross-recognition of the antigen of a closely related coronavirus. However, monoclonal antibodies against the Spike protein SARS-CoV-2 did not demonstrate to TGEV neutralization. In turn, the neutralization of the TGEV by sera from recovered COVID-19 donors suggests that not only Spike protein, but also other coronavirus antigens can play a significant role in antigenic imprinting and antibody-dependent enhancement of COVID-19.

Medical academic journal. 2022;22(2):221-228
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COVID-19 and its aftereffect on vestibular function
Pimenova V.M., Gvozdeva A.P., Ryabkova V.A., Gavrilova N.Y.

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.

Medical academic journal. 2022;22(2):229-234
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Optimization of purification conditions and study of antigenic properties of recombinant nucleocapsid protein of different SARS-CoV-2 strains
Rak A.Y., Donina S.A., Isakova-Sivak I.N., Rudenko L.G.

BACKGROUND: In the context of the constant manifestation of new SARS-CoV-2 strains and the need to determine the immunogenicity of new variants of antiviral vaccines, it is necessary to create diagnostic test systems based on conservative viral proteins. The SARS-CoV-2 nucleocapsid protein can be considered as a candidate antigen. However, the relevance of existing test systems based on it for determining the titer of antibodies produced in response to infection by recently emerging strains is unknown.

AIM: The goal is to optimize the conditions for obtaining recombinant N proteins of various SARS-CoV-2 strains and to analyze the possibility of creating ELISA test systems based on them.

MATERIALS AND METHODS: Bacterial strains producing N proteins were obtained by amplifying the corresponding genes and ligating them into the pETDuet-1 expression vector. Expression was induced at 20 or 37°C for 1, 2, 4, or 20 h using inducer (IPTG) concentrations of 0.1 mM or 0.5 mM with or without the addition of 3% ethanol. Proteins were purified from biomass by metal affinity chromatography and used as antigens for the detection of antiviral antibodies by ELISA.

RESULTS: It was found that the concentration of the inductor sufficient for the expression of recombinant proteins is 0.1 mM, the induction time is 1 h, and the required temperature is 37 °C. The influence of the presence of ethanol as an expression-stimulating reagent was not revealed. When determining the titers of antiviral antibodies using the obtained proteins, cross-reactivity of serums of COVID-19 convalescents was established regarding to antigens of various SARS-CoV-2 strains.

CONCLUSIONS: The possibility of effective induction of protein synthesis at a minimum concentration of the inducer and cultivation time indicates the economy of its production, and antigen recognition by antiviral antibodies indicates a native structure. Cross-reactivity of the blood sera of convalescents indicates the slow character of the evolution of the antigenic properties of the SARS-CoV-2 N protein. Thus, the purified proteins can be used as a basis for development of diagnostic test systems.

Medical academic journal. 2022;22(2):235-241
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Immunogenicity of mRNA encoding RBD SARS-CoV-2 in complex with a polycationic carrier
Rudometov A.P., Sharabrin S.V., Borgoyakova M.B., Volosnikova E.A., Rudometova N.B., Orlova L.A., Ilyichev A.A., Karpenko L.I.

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.

Medical academic journal. 2022;22(2):243-248
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Generation and characterization of SARS-CoV-2 pseudoviruses
Rudometova N.B., Shcherbakov D.N., Karpenko L.I.

BACKGROUND: Pseudovirus technology is a versatile and valuable tool for both fundamental and applied virological research. Pseudotyped viruses provide the same cell entry mechanism as SARS-CoV-2 and are widely used to investigate the virus entry mechanism, cell tropism, and virus neutralization assays.

AIM: The aim of the work is to obtain pseudotyped SARS-CoV-2 viruses and evaluate their transducing activity.

MATERIALS AND METHODS: Using genetic engineering methods, a genetic construct carrying the SARS-CoV-2 glycoprotein S gene was obtained, as well as the pLenti-Luc-GFP reporter plasmid encoding the green fluorescent protein (GFP) and firefly luciferase genes. Pseudovirus particles were generated by transfection of eukaryotic cells. The transducing activity of pseudoviral particles displaying SARS-CoV-2 glycoprotein S on their surface was studied using HEK293, HEK293-hACE2, and HEK293-hACE2-TMPRSS2 (t) cell cultures.

RESULTS: Based on the second-generation lentiviral platform, pseudoviruses were obtained that exhibit SARS-CoV-2 S glycoprotein on their surface. It was found that the pseudoviruses penetrate more efficiently into HEK293-hACE2-TMPRSS2 cells than into HEK293-hACE2. Pseudoviruses have been shown to be sensitive to neutralization by recombinant monoclonal antibodies that interact with the receptor-binding domain (RBD) of the SARS-CoV-2 S glycoprotein.

CONCLUSIONS: The pseudoviruses can be used both to search for antiviral drugs that would be able to block the penetration of SARS-CoV-2 into the target cell, and to evaluate the effectiveness of the developed monoclonal antibodies and vaccines against SARS-CoV-2.

Medical academic journal. 2022;22(2):249-253
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Analysis of the course of the new coronavirus infection COVID-19 in pregnant women under treatment in the Infection Department of Clinical Hospital no. 1 in Smolensk
Skvortsova A.A., Zakharova A.I., Kostina S.A., Rozinkova O.S., Melnikova A.B., Simakina E.N.

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.

Medical academic journal. 2022;22(2):255-259
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The impact of SARS-CoV-2 peptides on activation of NK cells
Ustiuzhanina M.O., Britanova O.V., Kovalenko E.I.

BACKGROUND: NK cells, alone with T lymphocytes, have a high antiviral activity. Exploring the contribution of NK cells in fighting SARS-CoV-2 infection may promote the development of appropriate treatments for COVID-19. Previously, NK cell response was considered nonspecific, provided by a combination of signals from activating and inhibitory receptors. Currently, the existence of certain subpopulations of antigen-specific, or adaptive, NK cells has been shown.

AIM: To evaluate the functional response of NK cells induced by SARS-CoV-2 peptides.

MATERIALS AND METHODS: The functional response of NK cells to SARS-CoV-2 peptides was determined by their degranulation (surface CD107a expression) and IFNγ production levels, and by the activation degree (HLA-DR expression level). Volunteers who recovered from COVID-19 participated in the study, and immune cells from a healthy volunteer without SARS-CoV-2-specific antibodies were used as controls.

RESULTS: NK cells from individuals who had recovered from COVID-19, in contrast to a donor who had not been infected, showed a higher level of IFNγ production in response to SARS-CoV-2 peptides, compared with control samples. The level of degranulation of NK cells from donors previously infected with SARS-CoV-2 was higher than in the corresponding control. The proportion of activated NK cells obtained from recovered donors was also higher in samples stimulated with SARS-CoV-2 peptides.

CONCLUSIONS: We have demonstrated the activation of NK cells obtained from people who had previously recovered from COVID-19 in response to SARS-CoV-2 peptide antigens in cultures of peripheral mononuclear cells in vitro. This study reveals the possibility for further investigation of antigen-specific NK cells in COVID-19 disease. The use of such cells could help develop treatments for SARS-CoV-2 infection.

Medical academic journal. 2022;22(2):261-268
pages 261-268 views
Activation and modulation of Ire1-Xbp1 pathway in SARS-CoV-2 infected Vero cells (B.1.1.7 Alpha, B.1.617.2 Delta, B.1.1.529 Omicron variants)
Shishova A.A., Baryshnikova V.S., Ermakova M.Y., Turchenko Y.V., Dereventsova A.V., Fominykh K.V.

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.

Medical academic journal. 2022;22(2):269-276
pages 269-276 views

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