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Vol 21, No 3 (2021)

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Analytical reviews

Infectious complications in patients with chronic lymphocytic leukemia treated with bruton’s tyrosine kinase inhibitors

Torshina Y.S., Serebryanaya N.B.


The aim of this study is to analyze the scientific literature data on the frequency and characteristics of infectious complications during the treatment of patients with lymphoproliferative diseases with a new class of drugs, selective inhibitors of Bruton’s tyrosine kinase (BTK). This work describes the indications for appointing these drugs as well as the participation of BTK in the development and activation of B cells. We have studied the main characteristics of BTK inhibitors used in clinical practice and associated disorders in the activity of off-target tyrosine kinases. The work describes the main types of known infectious complications developing during the treatment with the drugs of this group, the period of their appearance, and characteristic pathogens.

Medical academic journal. 2021;21(3):15-27
pages 15-27 views

Analysis of metabolomic and genomic markers for diagnosing cardiovascular diseases

Zharkova Z.V., Yasenyavskaya A.L., Nikitina I.B., Goretova I.V., Fedoseev I.V., Bashkina O.А., Samotrueva M.A.


Cardiovascular disease is the leading cause of death in the population. Unfortunately, cardiovascular disease and its associated risks are often difficult to diagnose due to the many factors associated with age and other comorbidities that lead to significant uncertainty in diagnostic classification and therapeutic decision making. Therefore, there is a great need to find new biomarkers for more accurate diagnosis, risk assessment and treatment recommendations for both acute and chronic cardiovascular disease. This article presents an analysis of metabolomic and genomic markers used for the diagnosis of cardiovascular disease. The study of the metabolome in combination with the genome and proteome can provide important information about both the pathogenesis of cardiovascular disease and the ability to search for and identify new cardiovascular disease biomarkers. Along with the fundamental data on new cardiovascular disease biomarkers, there is an urgent need for further research confirming their great potential for practical health care.

Medical academic journal. 2021;21(3):29-37
pages 29-37 views

History of medicine

On the history of the study of endogenous antibiotics

Aleshina G.M.


The review presents data on the history of the discovery of the first endogenous antibiotic compounds, on the contribution of Russian scientists, in particular, researchers from the Institute of Experimental Medicine (St. Petersburg), in the study of the structural and functional properties of antimicrobial proteins and peptides – important molecular factors of innate immunity that can act as an alternative to conventional antibiotics in the fight to control pathogenic microorganisms.

Medical academic journal. 2021;21(3):39-47
pages 39-47 views

Original research

Influence of new antimicrobial peptides of the medicinal leech Hirudo medicinalis on the functional activity of neutrophil granule proteins

Grigorieva D.V., Gorudko I.V., Grafskaia E.N., Latsis I.A., Sokolov A.V., Panasenko O.M., Lazarev V.N.


BACKGROUND: Resistance of microorganisms caused dangerous to human health infections to traditional antibiotics is a serious problem for healthcare. In this regard, the development of new effective antimicrobial drugs and therapeutic approaches is an urgent task. Antimicrobial peptides (AMPs) are considered a promising alternative to traditional antibiotic in the fight against resistant microorganisms.

AIM: The aim of this work is to study the effect of new synthesized AMPs of the medicinal leech Hirudo medicinalis (including under conditions of development of oxidative/halogenative stress) on the functional activity of neutrophils granular proteins — the main effector cells of the immune system.

MATERIALS AND METHODS: Myeloperoxidase peroxidase activity was assessed by the rate of o-dianisidine oxidation. Neutrophil elastase activity was determined by the fluorescence method using a specific substrate MeOSuc-AAPV-AMC. Lactoferrin iron-binding activity was assessed spectrophotometrically by the change in absorption of protein solution after addition of Fe3+ salt. Lysozyme activity was determined by the rate of M. lysodeikticus bacterial cells lysis.

RESULTS: Native AMPs 536_1 and 19347_2 inhibited and 12530 increased myeloperoxidase peroxidase activity, this tendency persisted after these AMPs modification by hypochlorous acid (HOCl). In contrast to the native AMP halogenated AMP 3967_1 acquired the ability to enhance myeloperoxidase enzymatic activity. In the presence of AMP 3967_1 neutrophil elastase amidolytic activity increased insignificantly, while AMP 19347_2 inhibited neutrophil elastase activity. After HOCl modification these AMPs retained their ability to regulate neutrophil elastase activity. Synergistic effects (~20%) against gram-positive bacteria M. lysodeikticus were revealed for combination of lysozyme with AMPs 12530 and 3967_1. Inhibition lysozyme antimicrobial activity was observed in the presence of AMPs 19347_2 and 536_1, however the severity of this effect decreased after AMPs modification by HOCl. After HOCl modification AMP 3967_1 increased, while AMP 12530 on the contrary acquired the ability to inhibit lysozyme mucolytic activity.

CONCLUSIONS: The use of drugs based on studied AMPs of medicinal leech will have a beneficial effect on the body’s fight against infectious agents due to the antimicrobial action of AMPs themselves. But in addition studied AMPs are capable to modulate the biological activity of own endogenous antimicrobial proteins and peptides: to enhance it, if it is necessary to eliminate pathogen and to inhibit — if it necessary to protect against damage to the body’s own tissues.

Medical academic journal. 2021;21(3):49-62
pages 49-62 views

Clinical research

Psychosomatic disorders (distress, depression, anxiety, somatization) in young patients who have had COVID-19

Ulyukin I.M., Kiseleva N.V., Rassokhin V.V., Orlova E.S., Sechin A.A.


AIM: The mission is to assess possible psychosomatic disorders (in particular, stress as a nervous breakdown, an acute temporal phase of a specific disorder, which is manifested primarily by signs of depression and neurosis) in young patients who have had COVID-19, in the course of rehabilitation, to improve medical and psychological support after their discharge from the hospital.

MATERIALS AND METHODS: 60 convalescents who have had COVID-19 and had practically been healthy before (men aged 19.87 ± 1.64 years) were examined. The main clinical variants and manifestations of COVID-19 in our study were inapparent infection (II) — in 19 cases (31.67%), acute respiratory viral infection (ARVI) — in 21 cases (35.0%), pneumonia without respiratory failure (P) — in 20 cases (33.33%). These are clinical variants and manifestations of mild-to-moderate of COVID-19 severity. The diagnosis of all clinical variants and manifestations of COVID-19, the patients’ examination, treatment and discharge from the hospital were carried out in accordance with regulatory documents. The patients were examined 6–8 month after discharge from the hospital. Psychometric examination of these individuals to separate their clinical manifestations of distress and somatization and manifestations of depression and anxiety was carried out according to the questionnaire “The Four-Dimensional Symptom Questionnaire”, 4DSQ), developed in 1996 by the Dutch specialists B. Terluin. This questionnaire was translation into Russian and adapted by A.B. Smulevich et al. [2014]. Voluntary informed consent was obtained from each of the patients before their participating the study.

RESULTS: Indicators of distress, anxiety, somatization after all the clinical variants and manifestations of COVID-19 have a moderately increased level, which indicates a serious illness that has been suffered, in some cases with an unfavorable outcome. The strongly increased level of depression in our study is probably due to the presence of astheno-neurotic syndrome due to the previous COVID-19 disease. The data on the correlation between the scales of methods indicate the direction of possible psychoprophylactic work with convalescents.

CONCLUSIONS: The results of the study showed that young patients without concomitant diseases who have had COVID-19, even with a mild and low-symptom course, may develop psychosomatic consequences such as distress, anxiety, somatization and some others. The reasons, duration, potential risk factors for their development require further study, however, timely developed preventive and therapeutic and diagnostic measures, taking into account the individual characteristics of the patient, can have a positive effect.

Medical academic journal. 2021;21(3):63-72
pages 63-72 views

Conference proceedings

The features of the course of virus-associated acute lung injury in mice with induced immunosuppression

Aleksandrov A.G.


BACKGROUND: Among all groups of patients with virus-associated acute lung injury with influenza infection, the most severe course is observed in patients with immunosuppression. In this case, despite the studied mechanism of the course of combined pathology, the question of therapy in this group of patients remains unclear.

AIM: To study the features of the course of acute lung injury in influenza infection with secondary immunosuppression in an experiment for the possibility of searching for experimental therapy for this combined pathology.

MATERIALS AND METHODS: The study was performed on 115 outbred female mice. The mouse-adapted pandemic influenza virus A/California/7/09MA (H1N1)pdm09 was used for modeling viral acute lung injury. Experimental immunosuppression was reproduced by administration of methotrexate (1.25 mg/kg intraperitoneally, once every 3 days during 3 weeks before infection). During the experiment, mortality, blood oxygen saturation, the concentration of pro-inflammatory cytokines in the lungs, and the severity of lung injury were measured.

RESULTS: The presence of experimental immunosuppression led to an exacerbation of acute lung injury in infected animals in terms of mortality and lung damage. Changes in the dynamics of proinflammatory cytokines (TNF-á, IL-6, IL-1â) in the lungs were observed during acute lung injury. Retarded recovery of the lungs functional activity was noted.

CONCLUSIONS: The experimental immunosuppression contributed to the exacerbation of acute lung injury and to an increase in the duration of the pathology. These changes could be associated with an altered process of elimination of the pathogen. The reproduced model of combined pathology was used for searching a therapy for these complications.

Medical academic journal. 2021;21(3):75-80
pages 75-80 views

Protective role of silver nanoparticles in influenza infection

Al Farroukh M., Skomorokhova E.A., Magazenkova D.N., Kiseleva I.V.


BACKGROUND: The present study assesses copper metabolism of the host organism as a target of antiviral strategy, basing on the “virocell” concept. This concept suggests that the targets for suppressing viral reproduction can be found in the host’s metabolism.

AIM: Evaluation of the effect of copper status indicators on influenza infection in mice.

MATERIALS AND METHODS: Silver nanoparticles (AgNPs) were used as a specific active agent because they reduce the level of holo-ceruloplasmin, the main extracellular cuproenzyme. The mouse model of influenza virus A infection was used with two doses: 1 LD50 and 10 LD50. The following treatment regimens were used: mice were pretreated four days before infection and then every day during infection development until the end of the experiment (day 14).

RESULTS: The mice treated with AgNPs demonstrated significantly lower mortality, the protection index reached 60–70% at the end of the experiment, and mean lifespan was prolonged. In addition, the treatment of the animals with AgNPs resulted in normalization of the weight dynamics. Despite the amelioration of the infection, AgNPs treatment did not influence influenza virus replication.

CONCLUSIONS: This study provides support for the view that silver nanoparticles could be used as protection against influenza.

Medical academic journal. 2021;21(3):81-84
pages 81-84 views

Impact of amino acid substitution T203I in hemagglutinin on growth characteristics in vitro and hemagglutinin thermostability of A/H3N2 influenza viruses

Bazhenova E.A., Stepanova E.A., Kotomina T.S., Larionova N.V., Kiseleva I.V., Rudenko L.G.


Background: Russian live attenuated influenza vaccines are a three-component preparations that contain vaccine strains based on current epidemic influenza A/H1N1, A/H3N2 and B strains. Recent influenza viruses A/H3N2 are most susceptible to drift antigenic changes, and therefore, this component of the live attenuated influenza vaccines must be constantly updated. Current vaccine strains of live attenuated influenza vaccines are obtained by the method of classical reassortment using selective factors in developing chicken embryos. During the process of preparation of live attenuated influenza vaccines strains, single reassortants can acquire various egg-adaptive amino acid substitutions in hemagglutinin and neuraminidase – the genes responsible for the antigenic correspondence of the vaccine strain to the epidemic parent. These amino acid substitutions can affect the biological properties of the vaccine strain, thereby reducing the effectiveness of this component of live attenuated influenza vaccines.

AIM: The aim of the study was to explore the effect of amino acid substitution T203I in hemagglutinin of A/H3N2 influenza viruses on growth characteristics and hemagglutinin thermostability.

MATERIALS AND METHODS: For the study, three pairs of A/H3N2 vaccine reassortants were prepared. Reassortants differed from each other by amino acid Thr or Ile at position 203 in the hemagglutinin. The growth properties of vaccine strains were assessed by titration in eggs at 26–40°C and in a MDCK cell culture at 33°C. The thermostability of the hemagglutinin of studied influenza viruses was assessed by determining their ability to agglutinate 1% erythrocytes after exposure to elevated temperatures in the range of 37–70°C.

RESULTS: The amino acid substitution T203I in hemagglutinin in reassortants obtained on the basis of current influenza A/H3N2 viruses acquired during the preparation of vaccine strains does not affect the temperature sensitivity of viruses. It was shown that viruses with an egg-adaptive substitution T203I in hemagglutinin have more pronounced cold-adapted phenotype and a higher reproductive activity in MDCK cell culture, compared to strains without this mutation. It was found that hemagglutinin of reassortants with 203 Ile is more thermostable than with 203 Thr.

CONCLUSIONS: Our data indicate that the amino acid substitution of T203I in hemagglutinin in current influenza A/H3N2 viruses does not have a negative effect on biological properties, but improves growth characteristics in eggs and MDCK cells, as well as the thermostability of viruses.

Medical academic journal. 2021;21(3):85-90
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Construction of the vaccine strain of the influenza B virus with chimeric hemagglutinin to induce a cross-protective immune response

Baranov K.V., Wong P., Stepanova E.A., Bazhenova E.A., Krutikova E.V., Isakova-Sivak I.N., Rudenko L.G.


BACKGROUND: Influenza viruses cause worldwide epidemics, and the most effective method to prevent influenza disease is regular vaccinations. The development of new generation vaccines is aimed primarily at the formation of an immune response against a wide range of influenza viruses. One of the promising approaches is sequential vaccination with chimeric influenza viruses with identical stem domains of the hemagglutinin surface protein.

AIM: The development of an experimental vaccine strain of influenza B virus with chimeric hemagglutinin consisting of head and stem domains of influenza B viruses belonging to different genetic lineages.

MATERIALS AND METHODS: A chimeric influenza hemagglutinin gene was obtained by genetic engineering from the genetic material of B/Victoria and B/Yamagata influenza strains. The gene was inserted into the vector for the reverse genetics of the influenza virus. The influenza B virus strain with chimeric hemagglutinin was obtained by transfection of Vero cells using an 8-plasmid system. The rest of the genes were obtained from the attenuated influenza B virus with cold-adapted and temperature-sensitive phenotypes. The biological properties of the obtained recombinant strain, its infectious titer in developing chicken embryos and MDCK cell culture were evaluated.

RESULTS: A recombinant vaccine strain has been successfully rescued. The head domain of the hemagglutinin of the virus is inherited from the B/Victoria influenza virus, and the stem domain from the B/Yamagata virus. The virus actively replicated in eggs and MDCK cells, with temperature-sensitive and cold-adapted phenotypes identical to classical live attenuated influenza vaccine viruses. The thermal stability of the chimeric hemagglutinin did not differ significantly from the thermal stability of the hemagglutinins of the donor viruses.

CONCLUSIONS: The results obtained indicate the possibility of creating a strain with chimeric hemagglutinin, fragments of which are inherited from different genetic lineages. The growth characteristics and biological properties of the strain make it a promising candidate for the experimental evaluation of the possibility of inducing a cross-protective immune response by sequential vaccination with vaccine strains with identical stem hemagglutinin domains.

Medical academic journal. 2021;21(3):91-96
pages 91-96 views

Receptor-binding domain of SARS-CoV-2 contribution to the neutrophil activation during 100 nm particle-induced immune response in conduction airway mucosa of mice

Bolkhovitina E.L., Vavilova J.D., Bogorodskiy A.O., Okhrimenko I.S., Borshchevskiy V.I., Shevchenko M.A.


BACKGROUND: Airborne pathogens such as virus particles undergo elimination from the respiratory tract by mucociliary clearance and phagocytosis by immune cells. The data about phagocytic cell type infiltration and stimuli that attract phagocytic cells to conducting airway are required for the anti-virus immune response mechanism understanding and the treatment strategy development.

AIM: To detect the role of the receptor-binding domain of SARS-CoV-2 in neutrophil immune response activation in conducting airway mucosa after 100 nm particles application.

MATERIALS AND METHODS: C57BL/6 mice received an oropharyngeal application of fluorescent 100 nm particles suspended in the receptor-binding domain of SARS-CoV-2 solution. 24 hours after, conducting airways of mice were dissected and subjected for immunohistochemistry as whole-mounts. Three-dimensional images of conducting airway regions were obtained using confocal microscopy. Quantitative image analysis was performed to estimate the ingestion activity of neutrophils in conducting airway mucosa.

RESULTS: Neutrophil migration to conducting airway mucosa was detected in case of the application of particles in receptor-binding domain solution, but not in phosphate buffer or bovine serum albumin solution. Receptor-binding domain solution alone also induced neutrophil migration to conducting airway mucosa. Infiltrating conducting airway wall mucosa neutrophils contributed to particles internalization.

CONCLUSIONS: The receptor-binding domain of SARS-CoV-2 can activate the neutrophil-mediated response in conducting airway mucosa.

Medical academic journal. 2021;21(3):97-102
pages 97-102 views

Estimation of 100 nm particle distribution kinetics in mouse lung using confocal laser scanning microscopy

Vavilova J.D., Bolkhovitina E.L., Bogorodskiy A.O., Okhrimenko I.S., Borshchevskiy V.I., Shevchenko M.A.


BACKGROUND: Daily, people inhale airborne viral particles, some of which have a size of about 100 nm, such as particles of SARS-CoV-2. Kinetics of such 100 nm particle distribution in the respiratory tract is important, however, not a properly investigated question.

AIM: To estimate the dissemination of inert viral particles based on the analysis of the spatial distribution of fluorescent 100 nm particles in the mouse lungs at different time points after the application.

MATHERIALS AND METHODS: Fluorescent particles of 100 nm size were applied to C57BL/6 mice. 6, 24, 48 and 72 hours after, lungs were excised and fixed. Lung lobes were stained with immunohistochemistry as whole-mounts and then underwent optical clearance. Three-dimensional images of whole-mount mouse lung lobes were acquired using confocal laser scanning microscopy.

RESULTS: 6 hours after the particle application particles were detected in lungs both as single particles and as particle agglomerates. Particles were both free and internalized by phagocytic cells. 24 hours after the application particles were detected both in bronchial lumen and in the alveolar space. Particles were detected in the mouse lungs up to 72 hours after the application.

CONCLUSIONS: Reaching the respiratory tract of mammalian, inert particles which size equal to SARS-CoV-2 particle size distribute both in bronchi and in alveoli and undergoes internalization of phagocytic cells.

Medical academic journal. 2021;21(3):103-108
pages 103-108 views

Amphotericin b effect on the sensitivity to influenza infection of WI-38 VA-13 cells with IFITM3 gene knockout

Koryabina K.S., Sergeeva M.V., Komissarov A.B., Eshchenko N.V., Stepanov G.A.


BACKGROUND: The application of CRISPR/Cas9 is one of the most rapidly developing areas in biotechnology. This method was used to obtain clones of а human origin cell line with knockout of one or more genes of the IFITM family, representing host restriction factors for influenza infection. Amphotericin B has previously been shown to promote influenza infection by blocking IFITM3 function.

AIM: The aim of this study was to evaluate the effect of amphotericin B on the sensitivity of IFITM knockout cells to influenza A virus infection.

MATERIALS AND METHODS: WI-38 VA-13 cells and mutant clones with IFITM3 knockout (F3 clone) or IFITM1, IFITM3 knockout (clone E12) were infected with influenza virus A/PR/8/34 (H1N1) in the presence or absence of amphotericin B. Forty-four hours after infection, the culture medium was taken to determine the infectious activity of the virus by titration in the MDCK cell culture, as well as the hemagglutinating activity of the virus. The infected cells were stained with fluorescently labeled antibodies against the viral NP protein, and the number of NP-positive cells was determined by flow cytometry.

RESULTS: The addition of amphotericin B increased the hemagglutinating and infectious activity of the virus in WI-38 VA-13cells, while the difference was insignificant for clones with IFITM gene knockout. A similar dependency was obtained for the percent of infected cells.

CONCLUSIONS: Mutant cells with a knockout of one or several genes of the IFITM family were equally susceptible to influenza infection regardless of the addition of amphotericin B, which confirms the crucial importance of a defect in the IFITM3 protein in increasing the permissiveness of cells to influenza A virus.

Medical academic journal. 2021;21(3):109-112
pages 109-112 views

Comparing of level of circulation of respiratory viruses during epidemic seasons 2015–2020 and COVID-19 pandemic in Saint Petersburg

Ksenafontov A.D., Pisareva M.M., Eder V.A., Musaeva T.D., Timofeeva M.M., Kiseleva I.V.


BACKGROUND: Respiratory viruses circulate everywhere. Problem of pandemic respiratory virus SARS-CoV-2 is especially relevant. The understanding of level of circulation of different viruses can help in developing a strategy of respiratory viruses combat.

AIM: To compare circulation of respiratory viruses during different seasons.


RESULTS: The most common viruses before the pandemic were influenza and respiratory syncytial viruses. COVID-19 pandemic season 2020/2021 had significant difference from previous epidemic seasons. Influenza viruses have largely disappeared, but the circulation of seasonal coronavirus and metapneumovirus has increased. The circulation of rhinovirus remained at the same level.

CONCLUSIONS: The emergence of pandemic SARS-CoV-2 virus had a significant impact on some respiratory viruses’ circulation, such as influenza or respiratory syncytial viruses.

Medical academic journal. 2021;21(3):113-117
pages 113-117 views

Respiratory viral infections including caused by coronaviruses in oncological and oncohematological patients

Kuleshova A.V., Iskova I.P., Kiseleva E.E., Chebotkevich V.N.


BACKGROUND: Patients with cancer are prone to developing infectious complications. They significantly aggravate the course of the underlying disease and worsen the quality of life of patients. The emergence of infections is largely promoted by immunosuppression associated with the use of cytostatic drugs, high-dose chemotherapy and hematopoietic stem cell transplantation in hematological cancer patients. Among infectious agents, respiratory viruses, especially influenza viruses, play an important role. The urgency of this problem has increased many times in connection with the development of the COVID-19 pandemic.

AIM: To study the features of the course of respiratory infections, including coronavirus (seasonal and COVID-19), infections in cancer and oncohematological patients during hospitalization and in outpatient settings.

MATERIALS AND METHODS: The study of the frequency and course of infectious complications in cohorts of patients with oncological diseases, who were under dispensary supervision in the polyclinic in Kirovsk, Leningrad region. Retrospective analysis of the frequency and characteristics of the course of coronavirus infection caused by HCoVs (OC43, 229E, NL63, HKU1) in patients treated at the clinics of the Russian research institute of Hematology and Transfusionology. Prospective study of the coronavirus COVID-19 of patients hospitalized at the Russian research institute of Hematology and Transfusionology and at the Kirov interdistrict hospital in Leningrad Region during the period of its conversion to specialized infection diseases hospital.

RESULTS: Coronavirus infection caused by HCoVs (OC43, 229E, NL63, HKU1), occurs in hematological cancer patients more often in association with other respiratory viruses. In the cases of detection of the SARS-CoV-2 in hospitalized patients, they need to be transferred to specialized infectious hospitals.

CONCLUSIONS: Respiratory viral infections are risk factors in cancer and oncohematological patients. Outpatient oncological and oncohematological patients require constant dispensary observation and special attention during the COVID-19 pandemic, outbreaks of influenza and other viral infections.

Medical academic journal. 2021;21(3):119-122
pages 119-122 views

Etiology of severe acute respiratory infections in Ekaterinburg in different epidemic seasons

Lelenkova E.V., Markaryan A.Y.


BACKGROUND: Acute respiratory viral infections are ubiquitous. Part of the cases are severe and require hospital treatment.

AIM: Studying the etiology of severe acute respiratory infections in patients of Ekaterinburg hospitals in different epidemic seasons (from 2017 to 2020).

MATERIALS AND METHODS: 1,132 cases of severe acute respiratory infection were assessed. The structure of laboratory-confirmed cases was determined.

RESULTS: In the assessed seasons, the proportion of respiratory viruses in the etiological structure of severe acute respiratory infections was 56.0% on average. B/Yamagata lineage of influenza viruses was predominant in the season of 2017/2018 (23.9% from the total number of respiratory viruses), — influenza А (H1N1)pdm09 viruses were predominant in the season of 2018/2019 (27.7%), and influenza A and B viruses were identified in 2019/2020 (39.4% and 31.7%, respectively).

СONCLUSIONS: The obtained results confirm a key role of influenza viruses in the etiology of severe acute respiratory infections among the hospital patients in different epidemic seasons.

Medical academic journal. 2021;21(3):123-126
pages 123-126 views

Optimization of M2e cassette amino acid composition for the development of universal influenza vaccine

Mezhenskaya D.А., Isakova-Sivak I.N., Katelnikova A.E., Rudenko L.G.


The development of a universal influenza vaccine with a wide spectrum and duration of action is one of the serious public health problems. This study is dedicated to optimization of an immunogen covering the M2e epitopes of influenza A viruses circulating in the natural reservoir, as well as the creation of a prototype of a universal influenza vaccine with subsequent quantitative and qualitative assessment of the induced anti-M2e responses in ferrets.

Medical academic journal. 2021;21(3):127-130
pages 127-130 views

Destructive effect of RNаse a on the SARS-CoV-2 virus (in vitro research)

Morozov I.A., Godovalov A.P., Oborin D.A.


BACKGROUND: For today, the most important and discussed issue in the professional medical community is the problem of prevention and treatment of a new coronaviral infection (COVID-19). The main reason for the non-decreasing increase in morbidity and mortality is the absence of an etiotropic drug. In our study, it is proposed to use a previously available drug for the treatment of tick-borne encephalitis, ribonuclease A, obtained from the pancreas of cattle.

AIM: The aim of investigation was to study the antiviral activity of RNаse A against SARS-CoV-2 in in vitro experiments.

MATERIALS AND METHODS: The experiment used samples of 50 patients with a confirmed (by PCR) primary diagnosis of a new coronoviral infection COVID-19. The preparation for the study was served by ribonuclease A (neoFroxx GmbH, Germany) at a concentration of 0.5; 1; 5 and 10 mg/ml, incubated at 4 and 37°C, the exposure was 20 minutes, 20 hours. A set of reagents OTT-PCR-RV-SARS-CoV-2 (Syntol, Russia) was used as test systems.

RESULTS: of the current study is the revealed antiviral activity of ribonuclease A at a minmal concentration of 0.5 mg/ml during incubation from 20 minutes to 20 hours, in the temperature range of 4–37°C.

CONCLUSIONS: The data obtained in the in vitro study confirmed the ability of ribonuclease A to destroy viral RNA, which suggests the possible use of the drug both for the treatment of patients and for the treatment of environmental objects.

Medical academic journal. 2021;21(3):131-134
pages 131-134 views

Rational selection of hemagglutinin variants of H3N2 influenza viruses in preparation of live influenza vaccine strains to optimize growth characteristics

Stepanova E.A., Bazhenova E.A., Krutikova E.V., Larionova N.V., Kiseleva I.V., Rudenko L.G.


BACKGROUND: Up to date Russian live attenuated influenza vaccines are produced in developing chicken embryos. During passaging in embryos, the virus isolated from the human respiratory tract undergoes adaptation to the receptors in embryos. The population of the virus, at any passage in chicken embryos, is heterogeneous and contains variants of viruses with one or another set of adaptive substitutions. Before preparing the vaccine strain, the population of the epidemic virus is cloned and the genetic sequence of the hemagglutinin and neuraminidase clones is analyzed. The growth characteristics of the vaccine strain and its antigenic properties depend on the correct choice of the variant of the virus.

AIM: The aim of the study was to select the variant of the H3N2 subtype virus for the preparation of a vaccine reassortant based on data on the composition of the population and an assessment of its growth properties.

MATERIALS AND METHODS: Viruses were cloned in developing chicken embryos, sequencing of the hemagglutinin and neuraminidase genes of the clones was performed. On the basis of the clones selected based on the results of the analysis of the population, strains of a live influenza vaccine were obtained by the reassortment in the chicken embryos. The growth characteristics of the strains, the phenotype in eggs, and the antigenic properties by hemagglutination inhibition test were evaluated.

RESULTS: The influenza virus A/Kansas/14/2017 recommended by WHO for the epidemic season 2019-2020 acquired a pair of D190N + N246T substitutions dominating in the population at the 7th passage in eggs. From the population of A/Kansas/14/2017-like strain A/Brisbane/34/2018, from the third passage in the eggs, it was possible to obtain a variant of the virus with substitutions G186V + S219Y in hemagglutinin. The growth characteristics of the strain based on A/Kansas/14/2017 (passage E7) were significantly inferior to the characteristics of the strain based on A/Brisbane/34/2018 (passage E3), in the absence of differences in antigenic properties.

CONCLUSIONS: The variant of egg adaptation of hemagglutinin G186V in strains of clade 3c.3a is preferable for the preparation of live influenza vaccine strains; variant N246T is not optimal. When preparing strains, it is necessary to analyze the composition of the virus population by cloning and choose the most optimal option for preparing strains. The persistence of egg-adaptive substitutions in passaged variants of the virus is not always optimal for strains of live influenza vaccine, and therefore it is preferable to use the population as close as possible to the initial variant to start work on the strain.

Medical academic journal. 2021;21(3):141-146
pages 141-146 views

Growth characteristics of experimental live influenza vaccine strains with modified NP and NS genes

Prokopenko P.I., Matyushenko В.A., Isakova-Sivak I.N., Rudenko L.G.


BACKGROUND: Vaccination is the most effective means of fighting influenza epidemics, but the immunogenicity of licensed influenza vaccines is not always satisfactory. One of the ways to increase the immunogenicity of an attenuated live influenza vaccine is to shorten the open reading frame of the NS1 protein, a modulator of innate antiviral immunity. In addition, the T-cell response to vaccination can be optimized by including the NP gene from the epidemic parental virus into the genome of vaccine strains.

MATERIALS AND METHODS: The open reading frame of the NS1 protein of the master donor virus A/Leningrad/134/17/57 was truncated to 126 amino acids by site-directed mutagenesis. The HA, NA, and NP genes of the model virus A/Anhui/1/2013 (H7N9) were cloned into the pCIPolISapIT vector. The rescue of recombinant influenza viruses was performed by transfection of Vero cells with a desired set of plasmids. The growth properties of the recombinant viruses were determined in embryonated chicken eggs incubated at different temperatures, as well as in the tissues of the respiratory tract of mice (nasal turbinates, lungs).

RESULTS: Experimental live influenza vaccine strains of subtype H7N9 with genome compositions 6:2 and 5:3 and carrying a full-length or truncated NS1 gene were actively replicated in eggs under optimal conditions, while maintaining the temperature-sensitive and cold-adapted phenotypes characteristic of classical live influenza vaccine strains. All viruses lacked the ability to grow in the lungs of C57BL/6J mice, which confirms the attenuated phenotype of the viruses. In the nasal passages of mice, only viruses with the full-length NS1 gene replicated, while viruses expressing the truncated NS1 protein were not detected in the respiratory tract of animals.

CONCLUSIONS: The results indicate that modification of the NS1 gene of the vaccine virus and the inclusion of wild-type NP gene in its genome does not affect its growth characteristics in eggs. A decrease in the activity of viral replication in the upper respiratory tract of mice with a shortening of the NS1 open reading frame indicates an increase in the attenuating properties of modified vaccines, which opens up prospects for the use of new vaccines in children under three years of age.

Medical academic journal. 2021;21(3):135-139
pages 135-139 views

Prospects of using conservative linear B-cell epitopes of influenza virus A neuraminidase for induction of cross-protective immune response

Sychev I.А., Kopeikin P.M., Tsvetkova E.V., Shamova O.V., Desheva Y.A., Isakova-Sivak I.N.


BACKGROUND: Influenza is a dangerous, widespread infectious disease that takes thousands of lives during annual epidemics, and also causes significant damage to the country’s economy. The most effective means of fighting the influenza virus is vaccination of the population. Due to the variability of influenza viruses, the strain composition of influenza vaccines must be updated annually. In this regard, an urgent task is to improve the existing influenza vaccines in order to expand their spectrum of action. One of the promising approaches is the targeted induction of the humoral immune response to the conservative linear epitopes of influenza A virus neuraminidase.

AIM: This project is aimed at assessing the immunogenicity and cross-protective activity of conserved neuraminidase epitopes in order to select promising targets for the targeted design of broad-spectrum influenza vaccines.

MATERIALS AND METHODS: Peptides corresponding to linear B-cell epitopes of neuraminidase were chemically synthesized de novo. The peptides were conjugated with keyhole limpet hemocyanin. CBA mice were immunized and challenged with A/PR/8/34 (H1N1) and A/Philippines/2/1982 (H3N2) viruses at a dose of 3 LD50. The survival rate of the animals was assessed within 14 days after infection. The immunogenicity of the peptides was assessed in a standard enzyme-linked immunosorbent assay using the recombinant neuraminidase proteins of the viruses A/California/07/2009 (H1N1) and A/Hong Kong/4801/2014 (H3N2) as antigen.

RESULTS: Immunization of neuraminidase with peptides MNPNQKIITIGS and ILRTQESEC, but not DNWKGSNRP, protected mice from lethality caused by the H1N1 and/or H3N2 virus. The protective potential of the peptides correlated with the levels of antineuraminidase antibodies after immunization.

CONCLUSIONS: The presence of a cross-protective potential in two conserved linear B-cell epitopes of influenza A neuraminidase (MNPNQKIITIGS and ILRTQESEC) allows them to be recommended as a target for the development of a broad-spectrum influenza vaccine.

Medical academic journal. 2021;21(3):147-151
pages 147-151 views

Generation and in vitro characterization of engineered cold-adapted influenza A strains with modified NS gene

Chistyakova A.K., Prokopenko P.I., Krutikova E.V., Stepanova E.A., Isakova-Sivak I.N., Rudenko L.G.


BACKGROUND: The high variability of influenza strains and the emergence of new variants of viruses lead to the need for constant updating of the composition of influenza vaccines. One of the options for solving this problem is the development of vaccines with enhanced cross-protection against a wide range of influenza strains. Genetically engineered preparations based on live influenza vaccine can be used for targeted stimulation of the cellular immune response. It has been experimentally established that CTL epitopes inserted into the NS gene of the live influenza vaccine strain cause the activation of lymphocytes and the formation of a pool of resident memory T-cells in the lungs of model animals. It is optimal to use experimentally confirmed immunogenic regions for insertion.

AIM: The aim of this study was to rescue a panel of experimental cold-adapted live attenuated influenza vaccine strains with a modified NS gene using A/Leningrad/134/17/57 backbone and recent influenza strains of H1N1, H3N2 and H7N9 subtypes, and evaluate their properties in vitro.

MATERIALS AND METHODS: A cassette encoding immunogenic, conserved among a wide range of influenza strains T-cell epitopes of the influenza virus PB1 protein restricted by common HLA-allotypes was inserted into the gene encoding the NS1 protein. The modified NS gene was cloned into the pCIPolISapIT influenza virus reverse genetics vector. Chimeric influenza viruses were rescued by transfection of Vero cells by electroporation using a standard 8-plasmid system. The growth characteristics of viruses were assessed in developing chicken embryos.

Results: Three strains were successfully obtained based on the live influenza vaccine master donor virus A/Leningrad/ 134/17/57 with a modified NS gene and influenza viruses of the H1N1, H3N2, H7N9 subtypes. Thus, modification of NS gene by insertion of immunogenic PB1 epitopes did not affect the viability and replicative activity of the rescued chimeric live influenza vaccine strains, regardless of the composition of the surface proteins. The strains replicated well at an optimal temperature, had temperature-sensitive phenotype and were able to grow at low temperature.

CONCLUSIONS: The strains will be further studied as candidates for influenza prophylaxis as an experimental universal influenza vaccine.

Medical academic journal. 2021;21(3):153-158
pages 153-158 views

The role of coinfection with influenza viruses in the pathogenesis of severe infection in patients with COVID-19

Shvedova T.N., Kopteva O.S., Kudar P.A., Lerner A.A., Desheva Y.A.


BACKGROUND: Despite the continuing global spread of the coronavirus infection COVID-19 caused by the SARS-CoV-2 coronavirus, the mechanisms of the pathogenesis of severe infections remain poorly understood. The role of comorbidity with other seasonal viral infections, including influenza, in the pathogenesis of the severe course of COVID-19 remains unclear.

MATERIALS AND METHODS: The present study used sera left over from ongoing laboratory studies of patients with varying degrees of severity of COVID-19. The study was approved by the Local Ethics Committee of the Federal State Budgetary Scientific Institution “IEM” (protocol 3/20 from 06/05/2020). We studied 28 paired samples obtained upon admission of patients to the hospital and after 5–7 days of hospital stay. Paired sera of patients with COVID-19 were tested for antibodies to influenza A and B viruses. The presence of IgG antibodies specific to the SARS-CoV-2 spike (S) protein was studied using an enzyme-linked immunosorbent assay (ELISA). The serum concentration of C-reactive protein and the neutrophil-lymphocyte ratio on the day of hospitalization were also assessed.

RESULTS: At least a 4-fold increase in serum IgG antibodies to SARS-CoV-2 S protein was found both in patients with PCR-confirmed SARS-CoV-2 infection and without PCR confirmation. It was shown that out of 18 patients with moderate and severe forms of COVID-19 infection, six of them showed at least a 4-fold increase in antibodies to influenza A/H1N1, in one to influenza A/H3N2 and in two cases to the influenza B. Laboratory data in these two groups were characterized by significant increases in serum C-reactive protein and neutrophil-lymphocyte ratio concentrations compared with the moderate COVID-19 group.

CONCLUSIONS: Serological diagnostics can additionally detect cases of coronavirus infection when the virus was not detected by PCR. In moderate and severe cases of COVID-19, coinfections with influenza A and B viruses have been identified. The results obtained confirm the need for anti-influenza immunization during the SARS-CoV-2 pandemic. Influenza virus screening can significantly improve patient management because recommended antiviral drugs (neuraminidase inhibitors) are available.

Medical academic journal. 2021;21(3):159-164
pages 159-164 views

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